THE ROLE OF INSULIN-LIKE GROWTH FACTORS

IN LONG-TERM MEMORY ENHANCEMENT

by

SARAH A. STERN

A DISSERTATION SUBMITTED TO GRADUATE FACULTY OF THE

GRADUATE SCHOOL OF BIOLOGICAL SCIENCES, NEUROSCIENCE
DOCTORAL PROGRAM, IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY,
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI

2014
 UMI Number: 3644313

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ii
iii
 ABSTRACT

THE ROLE OF INSULIN-LIKE GROWTH FACTORS IN LONG-TERM MEMORY

ENHANCEMENT

by

SARAH STERN

ADVISOR: CRISTINA M. ALBERINI, PHD.

Both short-term and long-term memories allow organisms to adapt their actions to

changing environments. However, over time, memories may become less strong and be

eventually forgotten. Furthermore, the loss of long-term memory that accompanies a

number of diseases can be devastating. Clinically, there is an urgent need to find

interventions that enhance and preserve memory function in both a healthy and disease

state.

In this thesis, I have studied the role of a system of growth factors known as the Insulin-

Like Growth Factor (IGF) system in long-term memory enhancement.

First, I have studied the effects of the IGFs on both hippocampal and amygdala

dependent tasks. I have found that IGF-II enhances memories of hippocampus, but not

amygdala, dependent tasks significantly and persistently, and that Insulin also enhances

those same forms of memory, but transiently. In contrast, I found that IGF-I does not

enhance either type of memory.

iv
Secondly, I have elucidated the role of IGF-II in the enhancement of remote memories. I

found that IGF-II, but not IGF-I or Insulin, enhances 2-week old IA memories when

injected into the rat anterior cingulate cortex (aCC) immediately after or 2 weeks after

training, but does not enhance recent long-term memory (up to 9d after training) when

injected immediately or 48h after training. When IGF-II is injected immediately after a

reactivation, the period of enhancement can be extended to 4 weeks, but not 2 months,

after training.

Lastly, I characterized the types of memories enhanced by systemic treatment of IGF-II

in both young adult C57Bl/6J (B6) mice and in a mouse model of autism spectrum

disorder (ASD), BTBR T+ Itpr3tf/J (BTBR), In B6 mice, I showed that systemically

injected IGF-II enhances both aversive and non-aversive memories, as well as working,

short-term and long-term memories, yet produces no adverse effects in mice and does not

restrict memory flexibility. In BTBR mice, which exhibit profound behavioral

phenotypes characteristic of ASD, I found that IGF-II reverses social recognition deficits,

repetitive behaviors and memory deficits.

Together, my studies indicate that IGF-II is a powerful enhancer of long-term

hippocampus dependent memories that is potentially useful for clinical therapeutics.

v
 DEDICATION

This thesis is dedicated to two special people in my family who passed away over the

course of my graduate studies.

To my grandfather, Abe Harris, who always pushed me to aim higher than I thought I

should but was always immensely proud of me no matter what I actually attained, and to

my brother-in-law, Asher Strobel, whose legacy continues to inspire me and everyone

who knew him.

ACKNOWLEDGEMENTS

A number of people must be acknowledged for their contributions, without which this

thesis would not have been possible.

It goes without saying that my largest debt of gratitude is to my mentor, Dr. Cristina

Alberini. From the very start of my rotation, she trusted me to work on high-impact

projects and facilitated my growth from a fresh-faced college graduate with barely any

background in the hard sciences to an independent scientist capable of asking and

answering the “next big question.” I have learned an enormous amount from her, and

hope to continue to learn from her even as I graduate from the lab.

I would be remiss if I did not thank many of the past and present Alberini lab members

who made my time in graduate school both extremely rewarding, as well as extremely

fun. Akinobu Suzuki and Dillon Chen invited me to work on their projects when I joined

vi
the lab and patiently taught me many techniques. Dillon especially took me under his

wing and willingly dispensed sound advice on numerous scientific and non-scientific

matters. Without his initial discovery that IGF-II enhanced IA memory, this thesis would

certainly be quite different! Gabriella Pollonini and Virginia Gao have been wonderful

colleagues, but more importantly, wonderful friends throughout the years. They are the

greatest comfort when data is hard to come by and the greatest cheerleaders when the

newest piece of the puzzle is added. Dhananjay Bambah-Mukku has been an inspiration

to me – his enthusiasm for science bubbles over onto everyone he speaks to, and I thank

him for keeping my own enthusiasm high as well and for always answering my many

questions. I thank Amy Khotz, with whom I worked closely on two of the projects in the

lab for her help and expertise in working with mice. To the other past and present

members of the Alberini Lab during my time – Carmen Inda, Elizavieta Muravieva,

Melissa Noel, Amy Arguello, Xiaojing Ye, Michael Garelick, Sarah Johnson, Reto Bisaz,

Allesio Travaglia, Charles Finsterwald, Susan Sheng and Nelson Humula – I thank for

their invaluable discussions and help around the lab. To my committee, Deanna Benson,

Bob Blitzer, Charles Mobbs, Mark Baxter and Steven Salton, I thank for their many

valuable insights during my thesis proposal and committee meetings, and for their

wonderful “after hours” advice as well.

I thank the graduate school, and especially Lily Recanati, for their help throughout my

studies, and especially during my first year of graduate school. Without their help and

support, joining the Alberini lab might not have been a possibility. I also thank the Grants

vii
and Contracts Office for the work they have done with my NRSA fellowship applications

and renewals.

I must thank my family and friends for their unwavering support over the years. My

parents, Zev and Madelyn, have supported my endeavors unflinchingly over the years,

provided many meals and laundry, and managed not to take it personally when I snapped

at them during stressful times. In return, I hope they can enjoy telling all of their friends

about their daughter, “the Doctor.” I thank my brother, Nehemia, for always keeping me

on my toes thinking, and my in-laws, Ron and Diane and my brothers-in-law Aryeh and

Joey, who have been a great support and are always interested to hear about the world of

science.

Last, but certainly not least, I thank my husband, Josh Strobel. From the moment

we met, he has been my biggest fan and my greatest champion through this journey. He’s

endured far more than most spouses would, learned to cook because I never got home in

time to make dinner, delayed a honeymoon for two years, and listened to me vent (and

vent and vent some more), without ever complaining. I can never thank him enough for

all of the support that he has given me over the years and continues to give me, and I

hope that the next 5 years brings us as many wonderful adventures as the first five.

viii
 PREFACE

Life is all memory, except for the one present moment that goes by you so quickly you
hardly catch it going. ~ Tennessee Williams

Memories are a fundamental part of our identity. As highlighted by the quote

above, memories guide our behavior at every moment by reminding us of our past actions

and their outcomes. For those individuals whose capacity to form memories is disrupted,

life becomes increasingly difficult and isolating. The cognitive dysfunction associated

with many debilitating diseases, including Alzheimer’s disease (AD), diabetes,

Parkinson’s disease (PD), and even in normal aging, demands effective therapies that can

lead to recovery of memory functions or memory enhancement.

One strategy that may lead to the identification of memory enhancers is to

capitalize on the knowledge gained by the biological study of long-term memory

formation and storage. In the last few decades, extraordinary progress has been made in

the understanding of the cellular and molecular mechanisms underlying memory

formation in several different species and types of learning. Results from these studies

have left us with core knowledge about the role of conserved gene expression pathways

required for memory formation, such as those regulated by the cAMP response element-

binding-CCAAT enhancer binding protein (CREB-C/EBP) transcription factors

(Alberini, 2009). In parallel, cellular and electrophysiological investigations have led to

the discovery of long-term potentiation (LTP) and long-term depression (LTD) which

provide cellular models for testing mechanisms of plasticity associated with memory

formation (Bliss and Collingridge, 1993). Disruption of these mechanisms has pointed to

useful approaches and targets for the development of therapies that attenuate obtrusive

ix
memories, such as those contributing to post-traumatic stress disorder (PTSD), phobias,

and drug addiction (Ressler and Mayberg, 2007). On the other hand, the identification of

mechanisms that can amplify, enhance and/or strengthen synaptic plasticity would

provide potential therapeutic tools for enhancing adaptive memories and counteracting

the onset and progression of disorders of cognitive functions.

The discovery that memory could be strengthened was made through some of the

first studies of memory consolidation, the process by which memory becomes stable and

resistant to disruption. These seminal studies showed that a number of endogenous stress

hormones, such as epinephrine and corticosterone and the exogenous agonists that bind

their receptors, could dose-dependently enhance memory when administered post-

training (McGaugh, 2000b). The distinction between pre- and post-training injections

enables a clear dissociation between the learning process itself vs. the processes that

consolidate memory once acquired. Thus, a compound that impairs or enhances memory

via pre-training injections may have the effect due to impairment or enhancement of the

acquisition of the memory, whereas post-training injections can only exert their effects

only by altering the post-learning, consolidation phase. For this reason, although genetic

models can provide great insight into biological mechanisms, even conditional knockouts

do not allow for a timeframe short enough to examine those kinds of differences, and

therefore, the establishment of compounds that enhance memory consolidation is most

likely with short-term manipulations that can target specific temporal phases.

A number of animal models and tasks have been classically studied in the field of

the biological basis of memory, and most are utilized in this thesis. The rodent is an ideal

model organism in which to study memory consolidation and enhancement, because

x
much like humans, they are able to form robust long-term memories that can last a

lifetime. For example, rats exhibit a sophisticated repertoire of behaviors with which to

test memory functions, and mice are particularly useful for the number of inbred strains

and transgenic models that exhibit particular disease phenotypes.

A wide variety of tasks have been utilized in rodent models to study memory

functions, and most have capitalized on the robust memories that are formed after fear-

based experiences. IA and Classical (or Pavlovian) Fear Conditioning (FC) are two of the

most common tasks used in these studies. Each has its particular advantages – IA is a

single-trial learning task, requiring both the hippocampus and the amygdala (Berman et

al, 1978; Lorenzini et al, 1996), in which an animal learns to avoid entering a context in

which it previously received a footshock. Training occurs within seconds, and thus the

task provides a very precise starting point from which to follow molecular changes in the

brain. FC, which measures the fear response (ie. freezing) of an animal to a context or

tone which was previously paired with a shock, is useful because animals can be tested

for a contextual component (contextual fear conditioning, CFC) and an auditory

component (auditory fear conditioning, AFC), requiring both hippocampus and amygdala

in the case of CFC, but only the amygdala in the case of AFC (Phillips and LeDoux,

1992). Thus, the utilization of both IA and FC can provide complementary insights into

whether a particular compound is effective in enhancing different memories, as well as

provide information as to what brain regions are critical for the enhancement to occur.

Because of their one-trial nature, these tasks are also ideal for establishing the

mechanisms by which memory enhancement occurs. Other tasks can be extremely useful

for establishing a number of translationally relevent parameters of memory enhancement.

xi
These include non-aversive tasks, such as object location and novel object recognition,

which are based on rodents’ preference for novelty and assesses their memory for the

objects themselves and/or their location, as well as tests of memory flexibility, such as

extinction and reversal learning, which test the animal’s ability to modify their learned

behavioral responses over time.

Using these models and tasks, the search for effective memory enhancers is

ongoing. The identification of the CREB-C/EBP gene cascade led originally to a number

of studies showing that increasing CREB function could lead to increased memory

performance (Brightwell et al, 2007; Josselyn et al, 2001; Lee et al, 2001). These studies

were informative and interesting, but beg the question as to what proteins downstream of

this pathway are responsible for these effects, and whether increasing some of those

targets specifically might also lead to a similar, or even better, memory enhancement.

One downstream target of C/EBPβ that is an attractive candidate as a memory enhancer

is IGF-II, which itself is induced in the hippocampus 20h after IA training (Chen et al,

2011).

IGF-II is part of the Insulin-Like Growth Factor System, which is comprised of

three growth factors (Insulin, IGF-I and IGF-II), 6 receptors (IGF-IR, IGFII-R, Insulin

Receptor A, Insulin Receptor B, and Insulin/IGF1-R hybrids), and 7 binding proteins that

serve to regulate bioavailability of IGF-I and IGF-II (IGFBP1-7) (Jones and Clemmons,

1995). In the central nervous system (CNS), Insulin and the Insulin-like growth factors

have only recently begun to be studied, and there is currently very little known about

what roles they play in cognitive functions.

xii
 In this dissertation, I will focus on the three growth factors that are part of the IGF

system, and more specifically the role of IGF-II, in enhancing memory. Because a

discussion of memory enhancement necessitates an understanding of memory processes,

I will first summarize the basic knowledge of mechanisms underlying learning and

memory and outline the various stages/forms of learning and memory storage that can be

targeted for enhancement. I will then briefly review selected mechanisms of memory

enhancement, and I will summarize specifically what is known about the IGF system,

both in the periphery, as well as in the central nervous system (CNS), especially in

relation to memory and cognitive functions.

I will next describe my results relating to the IGF system and memory

enhancement. I will briefly describe the studies that led us to examine IGFs in memory

enhancement, and will then discuss my studies examining the specific role of each IGF in

memory enhancement over both recent and remote timepoints. Lastly, I will discuss

studies looking at the role of systemic treatments of IGF-II in enhancing memory in both

normal adult mice and an autism mouse model.

xiii
TABLE OF CONTENTS
CHAPTER I: BACKGROUND………..………………………………………………………. 1

1.1: Memory formation and storage involve multiple phases: memory

consolidation and

reconsolidation

1.2: Memory is made up of multiple memory systems

1.3: Memory consolidation involves the reorganization of brain circuits over time.

1.4: Mechanisms of memory enhancement: the CREB-C/EBP pathway

1.5. The Insulin-like growth factor system: Discovery and peripheral functions

1.6 Insulin-like growth factors: role in CNS and cognition

1.6.1: Insulin

1.6.2. IGF-I

1.6.3. IGF-II

CHAPTER II: RESULTS

Insulin-Like Growth Factors differentially enhance hippocampal and amygdala-

dependent memories ........................................................................................…. 25

CHAPTER III: RESULTS

The role of IGFs in remote memory enhancement …………………….………. 43

CHAPTER IV: RESULTS

Enhancement of memories by systemic administration of Insulin-Like Growth

Factor II ….……………….……………………………………………………. 55

CHAPTER V: RESULTS

Systemic treatments of IGF-II rescue autism phenotypes in BTBR mice ……… 80

xiv
CHAPTER VI: DISCUSSION ………………………………………………………………. 95

REFERENCES …………………………………………………………………………... 125

xv
 LIST OF FIGURES

FIGURE A: Multiple memory systems as targets of memory enhancement ……………... 5

FIGURE B. The reorganization of brain circuits over time ………………………………. 7
FIGURE C. Schematic representation of the CREB-C/EBP pathway targeted for memory
enhancement …………………………………………………………………………….. 9
FIGURE D: Insulin and IGF receptors and their downstream signaling cascades ………. 15
FIGURE E: Organization of IGF prepropeptides and conservation of IGF-II amino acid
sequence ………………………………………………………………………………... 18
FIGURE 1: IGF-II enhances hippocampal-dependent memory via the IGF-IIR ………... 29
FIGURE 2: IGF-II does not enhance AFC memory ……………………………………... 31
FIGURE 3: Insulin transiently enhances hippocampal dependent memory ……………... 34
FIGURE 4: Insulin does not enhance memory when injected into BLA ………………... 35
FIGURE 5: IGF-I does not enhance memory when injected into hippocampus ………… 37
FIGURe 6: IGF-I does not enhance memory when injected into BLA ….......................... 38
FIGURE 7: IGF-II does not enhance memory when injected into aCC immediately after
training …………………………………………………………………………………. 45
FIGURE 8: Insulin does not enhance memory when injected into aCC immediately after
training …………………………………………………………………………………. 46
FIGURE 9: IGF-I does not enhance memory when injected into aCC immediately after
training …………………………………………………………………………………. 47
FIGURE 10: IGF-II enhances remote, but not recent, long-term memory via the aCC … 48
FIGURE 11: IGF-II enhancement of remote memory is temporally limited and extended by
reactivation ……………………………………………………………………………... 50
FIGURE 12: Insulin and IGF-I do not enhance remote memory ………………………... 51
FIGURE 13: Systemic IGF-II enhances contextual fear, but not auditory fear, memory .. 59
FIGURE 14: Systemic IGF-II does not lead to adverse effects …………………………. 61
FIGURE 15: Systemic treatment with IGF-II enhances memory via hippocampal IGF-IIR
and increases the expression of immediate early genes in the brain …………………… 65
FIGURE 16: IGF-II increases strength and/or persistence of non-aversive memories ….. 69
FIGURE 17: IGF-II potentiates memory when given with reactivation ………………… 71
FIGURE 18: IGF-II enhances memory without altering flexibility ……………………... 73

xvi
FIGURE 19: IGF-II rescues social recognition deficits in BTBR mice …………………. 86
FIGURE 20: IGF-II reverses a repetitive behavior in BTBR mice ……………………… 88
FIGURE 21: IGF-II rescues memory deficits in BTBR mice ............................................ 89
FIGURE 22: IGF-II does not alter anxiety responses in BTBR mice …………………… 91

xvii
LIST OF TABLES
TABLE 1: General motor and sensory responses of vehicle- and IGF-II-injected mice ... 62

TABLE 2: Detailed statistics for Chapter II ……………………………………………. 108

TABLE 3: Detailed statistics for Chapter III …………………………………………... 111

TABLE 4: Detailed statistics for Chaper IV …………………………………………… 113

TABLE 5: Detailed statistics for Chapter V …………………………………………… 122

xviii
LIST OF ABBREVIATIONS

2-DG: 2-Deoxy-D-glucose

aCC: Anterior cingulate cortex

AD: Alzheimer’s disease

APP: Amyloid precursor protein

Arc (Arg3.1): activity-regulated cytoskeleton-associated protein

ASD: Autism spectrum disorder

AFC: Auditory fear conditioning

B6: C57Bl/6

BBB: blood-brain barrier

BDNF: brain derived neurotrophic factor

BLA: basolateral amygdala

BTBR: BTBR T+ Itpr3tf/J

CaMKII-α: Ca2+/calmodulin-dependent protein kinase II-alpha

cAMP: Cyclic adenosine monophosphate

C/EBP: CCAAT enhancer binding protein

CFC: Contextual fear conditioning

CI-MPR: cation independent mannose-6-phosphate receptor

CNS: central nervous system

CREB: cAMP response element-binding protein

CS: conditioned stimulus

Dgcr8: DiGeorge syndrome chromosomal region 8

ERK: extracellular signal-regulated kinase

xix
FC: Fear conditioning

FoXo3: Forkhead box O3

GABA: γ-Aminobutyric acid

GH: growth hormone

GLUT4: glucose transporter type 4

GPCR: G-Protein coupled receptor

Hs-dCREB2-b: heat shock-inducible CREB repressor isoform

IA: Inhibitory avoidance

IEG: immediate early gene

IGF: Insulin like growth factor

IGF-I (IGF-1): Insulin like growth factor I

IGF-II (IGF-2): Insulin like growth factor II

IGF-IR (IGF-1R): Insulin like growth factor I receptor

IGF-IIR (IGF-2R): Insulin like growth factor II receptor

IGFBP: IGF binding protein

IKK: IκB kinase

IR: Insulin receptor

IR-A: Insulin receptor-A

IR-B: Insulin receptor-B

kDA: kilodalton

LTD: Long-term depression

LTM: long-term memory

LTP: Long-term potentiation

xx
L-LTP: Late-long term potentiation

MAPK: mitogen-activated protein kinase

MeCP2: methyl CpG binding protein 2

MPEP: Methyl-6-phenylethynyl-pyridine

mRNA: messenger RNA

MSA: multiplication stimulating activity

MTL: medial-temporal lobe

mU: milliunit

nAChR: Nicotinic acetylcholine receptor

NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells

nOR: novel object recognition

NR2A: N-methyl D-aspartate receptor subtype 2A

NR2B: N-methyl D-aspartate receptor subtype 2B

NSILA: non-suppressible insulin-like activity

OP: object placement

PD: Parkinson’s disease

PI3K: phosphoinositide 3-kinase

PKA: protein kinase A

PKCα: protein kinase C-alpha

PS1: Presenilin-1

PTSD: Post-traumatic stress disorder

RNA: Ribonucleic acid

STM: short-term memory

xxi
TGF-β1: transforming growth factor-beta

US: unconditioned stimulus

Zif268 (Egr-1): zinc finger protein (early growth response protein 1)

xxii
 CHAPTER I.
BACKGROUND

1.1. Multiple phases of long-term memory: memory consolidation and reconsolidation

Around the turn of the 20th century, Hermann Ebbinghaus, followed by Georg Müller and

Alfons Pilzecker, conducted their famous studies in memorizing nonsense syllables (Ebbinghaus,

1913), discovering what ultimately became known as memory consolidation, the notion that

memory first exists in a labile state that gradually strengthens and becomes resistant to disruption

(Lechner et al, 1999). The notion that memory stabilizes over time mapped on closely to Ribot’s

clinical findings in amnesiac patients that cerebral trauma often leads to selective loss of recent,

but not remote memory (Ribot, 1882). Since the publication of these seminal studies, it has been

universally recognized that memory consists of multiple phases, including acquisition/encoding,

working memory/short-term memory, long-term memory/consolidation, memory retrieval, and

reconsolidation. In the context of memory enhancement, it is critical to differentiate between

these different stages of memory. Cognitive dysfunction can result from impairments in one or

more of these stages, and it is thus critical to identify the stages can be selectively targeted for

the most efficacious cognitive therapy.

Acquisition/Encoding is the initial stage of memory formation in which the subject learns

something new (ie. acquires information). This process requires that the subject be attentive, and

that there be no retroactive or proactive interference that could occlude learning of the current

information (Lechner et al, 1999). Injections of compounds/agents before this stage may

therefore affect the initial learning of a task, whereas injections given post-acquisition can be

considered independent of this stage.

1
 Most models consider working memory and short-term memory to be distinct entities

(Baddeley, 2003). While working memory refers to the holding of information online in order to

keep processing additional information, short-term memory is the ability to hold the given

information in mind for a given period (Baddeley, 2012). Thus, working memory is often

classified as “working attention” because attention is required in order to keep adding new

information to what is pre-existing. Indeed, this process can be thought of as the bridge from

encoding to short-term memory. Short-term memory itself can be classified in a number of ways.

Most often, short-term memory is considered to be a small amount of information held in mind

for a relatively short period of time (ie. remembering a phone-number until you can write it

down). This differs very obviously from long-term memory, which can hold information for long

periods of time, without a predefined limit on the quantity of information held.

Long-term memories (LTM), in fact, can last for days, months, years, even a lifetime.

This process of consolidation that accompanies the transition from a labile memory to one that is

stable and resistant to disruption requires a number of molecular, cellular and structural changes

that occur over time, with some completing over hours or days and others extending over weeks,

months or perhaps even years. Once those events are completed, the memory is considered

stable, or consolidated (McGaugh, 2000a). If any of the phases underlying consolidation is

disrupted, memory is lost.

Once consolidated, a memory can be retrieved, or recalled. In experimental studies,

retrieval is elicited through testing for memory of the task, but retrieval may also occur via

internal replay, as is thought to occur during sleep. Importantly, both the number and modality of

retrievals can influence memory consolidation and storage.

2
 In the 1960’s, and later again around the year 2000, it was shown that retrieval of a

memory that has become resilient to the initial molecular interference initiates another round of

consolidation as, in fact, this retrieved memory becomes again temporarily labile, and undergoes

a re-stabilization process in order to be maintained (Nader and Einarsson, 2010). This re-

stabilization is known as reconsolidation.

1.2 Multiple memory systems.

Memories can be classified not only by their temporal stages, but also according to their

type and function, as well as by which brain regions underlie their formation and processing

(Squire, 1992). The view that memories could be anatomically localized was long disputed until

the landmark discovery of Brenda Milner, who studied the memory deficits of a patient known as

H.M.. Due to severe epilepsy, H.M. underwent a bilateral medial temporal lobe (MTL) resection

and, while he emerged fully treated from the epilepsy, he developed profound memory deficits

that were mostly restricted to the formation of long-term explicit memories (Squire and Wixted,

2011). Studies conducted with H.M. and other patients with selective brain damage suggested

that there are numerous memory types that can exist independently, but often interact (Corkin et

al, 1997) (Figure A). Explicit – or declarative – memories, which were disrupted in H.M., can be

either semantic (referring to memories of facts or concepts ie. your date of birth) or episodic

(referring to memories of events ie. your birthday party), and are subserved primarily by the

MTL, particularly by the hippocampus. Moreover, there are implicit – or non-declarative – types

of memories, the memories of how to do things, which critically involve motor learning, such as

skiing, playing piano, or riding a bicycle. One type of implicit memory is the memory formed as

a consequence of simple associative learning; when the associations are emotional, (i.e. between

3
a neutral stimulus and a stimulus with an emotional or motivational valence, either aversive or

rewarding), the learning relies primarily on the amygdala. When the associations produce

reflexive skeletal responses, as in the case of delay eyeblink conditioning, the learning relies

primarily on the cerebellum. A more gradual type of implicit learning that results in the

formation of motor skills and habits relies primarily on the dorsal striatum.

Importantly, these memory systems are not mutually exclusive in nature, acting alone and

on one type of memory, but rather, they coexist and often interact. For example, the IA and CFC

tasks that are commonly employed to study memory in rodents involve multiple brain regions

and multiple types of memories. In fact, these tasks require both an emotional, implicit

component, mediated by the amygdala, and a contextual, explicit component mediated by the

hippocampus (Phillips and LeDoux, 1992). Similarly, by shifting certain parameters such as

cueing specific arms or providing enriched contextual cues, the same radial arm maze with food

reward can be used to train animals to visit specific arms by very different strategies, mediated

by either amygdala, hippocampus, or striatum (White and McDonald, 2002). Thus, depending on

task demands, various structures may be engaged to process what seems, at first, like identical

information (ie. that a number of arms contain food) in different ways, producing different kinds

of memories.

For the purposes of developing memory enhancers, it is important to note that in

cognitive impairments associated with AD or aging-related cognitive decline, the most

vulnerable memories are those same memories that were lost in H.M. Implicit memories are very

often spared, or at the very least, impaired at the last stages of the disease. Thus, there is an

urgent need to identify therapies that target impairments of hippocampal-dependent memories.

At the same time, impairments associated with PD are often linked to working memory and

4
executive control, and thus discovering mechanisms of enhancement for this disorder is equally

important.

Figure A. Multiple memory systems as targets of memory enhancement. Memory consists of multiple stages
and types. A number of different stages (in blue) are involved in acquiring, storing, and retrieving a memory. A
number of cognitive disorders (in red) have symptoms that are associated with deficits in specific stages, while
others may have deficits that are more general or unclear in nature (such as those in cognitive decline over aging).
Long-term memory can be subdivided into a number of different types which rely on different brain regions (in
purple). Impairments of these different memories are also associated with different disorders. Putative memory
enhancers may be associated with the improvement of a specific stage or memory type, which will therefore affect
the clinical population that will receive therapeutic benefit. WM, working memory; STM, short-term memory;
LTM, long-term memory. (Adapted with permission from (Squire, 1992). Copyright 1992 American Psychological
Association). The use of APA information does not imply endorsement by APA.

1.3 Memory consolidation involves the reorganization of brain circuits over time

Another view of memory involves the reorganization of brain circuits. When viewed at

the systems level, the consolidation of declarative memories undergoes a time-dependent

reorganization across brain regions (Frankland and Bontempi, 2005). As alluded to above,

lesions of the MTL produce not only a differential effect on declarative vs. non-declarative

memories, but also a graded effect in the temporal realm. MTL lesions produce retrograde

5
amnesia – H.M., for example, could no longer form new memories, and could not remember

recently formed memories from before his surgery, but older memories from his childhood were

spared (Squire et al, 2011). This, and other similar clinical observations, led to the theory that

declarative memories were initially consolidated in the hippocampus, and over time, became

independent of the hippocampus and reliant on cortical regions (Figure B). Many lesion studies

followed in animal models and confirmed that the hippocampus is required for a limited time

post-acquisition, although recent findings suggest that for extremely rich, episodic memories, the

hippocampus may remain perpetually involved (Dudai, 2012; Teng and Squire, 1999).

A number of studies have confirmed the importance of cortical areas, and in particular

areas of the prefrontal cortex, in maintaining memories after they become hippocampal-

independent. Mice that are heterozygous for a null mutation of Ca2+

/calmodulin-dependent protein kinase II-alpha (CaMKII-α), which is expressed predominantly in

forebrain, have impaired cortical plasticity, but normal hippocampal plasticity. Behaviorally,

these mice show normal memories at early timepoints (1-3 days post-training), but have impaired

remote memories (10-50 days post-training) (Frankland et al, 2004; Frankland et al, 2001).

Similarly, mice overexpressing a dominant-negative form of p21-activated kinase, which

regulates spinogenesis in culture, show altered synaptic structure and spines specifically in

cortical areas, and behaviorally have normal water-maze memory 1 day after training, but

impaired memory 21 days later (Hayashi et al, 2004). More recently, a number of studies have

focused specifically on a region of the brain known as the anterior cingulate cortex (aCC) and,

through a combination of 2-DG and immediate early gene (IEG) mapping and

lesions/inactivations, have shown that this region is critical for the retrieval of remote memories

(Bontempi et al, 1999; Frankland et al, 2004; Maviel et al, 2004). The nature of how exactly the

6
anterior cingulate or other cortical areas contribute to remote memory, as well as whether they

are merely involved in the retrieval or also the storage of remote memories, is not currently

known.

Most rodent studies complete long-term memory testing at a 24h or 48h timepoint after

training, yet, there is a clear need for enhancers that extend memory to longer-timepoints. As the

mechanisms underlying memory enhancement become elucidated, it will be even more critical to

study these late stages of memory.

Figure B. The reorganization of brain circuits over time. Reprinted from (Frankland et al, 2005).

1.4 Mechanisms of Memory Enhancement: Historical Perspectives

The number of compounds found to enhance memories in animal models are far too numerous to

detail in this thesis. Instead, I focus an important mechanism of memory enhancement that is

7
particularly relevant in the context of Insulin-Like Growth Factors, namely that of the CREB-

C/EBP pathway.

1.4.1. Gene Expression: The CREB-C/EBP Pathway. One of the first mechanisms identified

as critical for long-term memory consolidation (both explicit and implicit) is the requirement for

de novo protein and ribonucleic acid (RNA) synthesis (Davis and Squire, 1984). Similar

requirements have been found for long-term plasticity, including long-term facilitation in Aplysia

californica and a phase of LTP that lasts more than one hour, known as late-LTP (L-LTP)

(Alberini, 2009), though not all late forms of LTP appear to share this requirement (Villers et al,

2012). In addition to pharmacological approaches, molecular and genetic methods have provided

evidence that transcription and translation are essential for memory, findings which led to the

subsequent discovery of important, evolutionarily conserved pathways required for long-term

memory formation: those regulated by the transcription factors CREB and C/EBP (Figure C).

CREB is activated by numerous signal transduction pathways implicated in memory

consolidation and enhancement, from growth factor stimulation of tyrosine kinase receptors

coupled to the activation of Ras and extracellular signal-regulated kinase (ERK); to G-Protein

Coupled Receptor (GPCR) activation coupled to activation of adenylyl cyclase, increase in

cyclic adenosine monophosphate (cAMP), and activation of protein kinase A (PKA); to the

activation of stress pathways and glutamate release leading to release of intracellular calcium and

related downstream events. All of these pathways can lead to the activation of an activator form

of CREB, CREB1. This activation in turn can lead to the transcriptional regulation of a number

of target genes, including immediate early genes (IEG). A number of IEGs are transcription

factors, which regulate the expression of effector genes that are thought to participate in

8
downstream synaptic changes underlying synaptic plasticity. This is, for example, the case with

the IEG/transcription factor C/EBPβ, a CREB-regulated target gene required for memory

consolidation (Alberini, 2009).

Figure C. Schematic representation of the CREB-C/EBP pathway targeted for memory enhancement.
Reprinted with permission from (Alberini and Chen, 2012) (copyright Elsevier).

9
Disrupting CREB1 results in memory deficits, while augmenting CREB1 activation results in

memory enhancement. The first demonstrations that CREB plays an essential role in both the

disruption and enhancement of memory consolidation came from studies in invertebrates,

namely Aplysia californica and Drosophila melanogaster. In Aplysia, Bartsch et al.(Bartsch et al,

1995) reported that the disruption of ApCREB1, the Aplysia homolog of mouse CREB1 leads to

an impairment in long-term facilitation (a cellular model of long-term memory in Aplysia), and

that the interference of a repressor form of CREB, ApCREB2, induces a long-term facilitation

response from training protocols that would normally only produce short-term facilitation. In

parallel studies, Yin et al.(Yin et al, 1995) using transgenic Drosophilae with a heat shock–

inducible CREB repressor isoform (hs-dCREB2-b) showed that flies with the transgene on have

impaired memory. Further studies in mice and rats extended these conclusions by showing that

while the knockout or knockdown of CREB1 results in long-term memory impairment, viral

overexpression of CREB1 in amygdala and hippocampus enable LTM formation from massed

training protocols of either cued-fear conditioning or place learning, which normally produce

only short-term memory (STM) (Brightwell et al, 2007; Josselyn et al, 2001). In Aplysia,

knockdown of C/EBP impairs long-term memory, while overexpression of C/EBP leads to

memory enhancement, similar to that produced by overexpression of CREB1, as short-term

facilitation is converted to long-term facilitation (Alberini et al, 2012; Lee et al, 2001).

Manipulations of C/EBP also lead to memory disruption or enhancement in rats(Alberini et al,

2012) or mice, as hippocampal knockdown impairs memory, while forebrain expression of a

general dominant-negative inhibitor of the C/EBP/ATF family (E-GFP/AZIP), which

presumably relieves their inhibition, results in enhanced spatial memory (Chen et al, 2003).

10
 From the seminal studies that directly manipulated CREB1 levels, many other findings

followed and have identified memory-enhancing effects that correlate with increases in the

activation of the CREB pathway. A natural direction of these studies has been to target activators

that are upstream of CREB, such as cAMP, PKA or ERK/mitogen-activated protein kinase

(MAPK) (Xia et al, 2008), though it is interesting to note that in certain cases in which neuronal

excitability is altered, a decrease in cAMP signalling may actually be preferable (Wang et al,

2011). Others have theorized that lifting the inhibition of CREB activation might be an effective

route for memory enhancement. For example, in mice, releasing the inhibition provided by

calcineurin, a Ca2+/calmodulin-dependent phosphatase which regulates pCREB levels, also leads

to memory enhancement, via CREB-dependent mechanisms, of both short-term and long-term

hippocampal-dependent memories, without changing working memory(Malleret et al, 2001). In

Aplysia, inhibition of calcineurin facilitates intermediate-term and LTM, but not STM (Sharma et

al, 2003). These studies have been extremely important in gaining an understanding of how

memories can be enhanced in an experimental setting by manipulation of CREB levels and/or

activity, and imply that the activation of the CREB-dependent pathway might be an effective

target for promoting and enhancing hippocampal-dependant LTMs.

There have been a number of attempts to screen for drugs that via CREB activation could

potentially be used as cognitive enhancers in humans. These screenings led to the identification

of a number of candidate drugs, including rolipram, which inhibits phosphodiesterase type 4

(PDE4), an enzyme that catalyzes hydrolysis of cAMP (Tully et al, 2003; Xia et al, 2008).

Additionally, CREB-C/EBP target genes may be valuable candidates for memory enhancement.

One C/EBPβ-target gene upregulated after learning in the hippocampus and required for memory

consolidation is IGF-II. As I will detail below, administration of recombinant IGF-II

11
immediately after learning or memory retrieval significantly enhances memory and prevents

forgetting (Chen et al, 2011). In general, it is important to recognize that, because CREB has

important functions throughout the body and diverse effects throughout the brain, an effective

treatment based on manipulating CREB or its target genes may require the development of

strategies that enhance selectivity and specificity (Barco et al, 2003).

1.5. The Insulin-Like Growth Factor System: Discovery and peripheral functions

Charles Best and Frederick Banting are credited with the discovery of insulin, a hormone

present in some form in both vertebrates and invertebrates, for which the Nobel Prize was

awarded in 1924 (but see (Friedman, 2010) for insight into prior studies that established that the

pancreas was the site of such a hormone). Insulin is cleaved to its active final form from its

precursor pro-insulin, which itself is cleaved from preproinsulin. The active hormone is

composed of two polypeptide chains (A and B) linked by disulfide bonds and totaling 51 amino

acids.

Insulin in the periphery is synthesized in the β-cells of the islets of Langerhans in the

pancreas. Upon the detection of external stimuli such as increased blood glucose levels, insulin is

released from the pancreas and performs its main role by stimulating glucose uptake into cells.

Interestingly, insulin release is inhibited by increased noradrenaline, leading to increased blood

glucose levels during stress. In addition, insulin facilitates the conversion of glucose to fatty

acids and glycogen in adipose tissue and liver, respectively, and promotes the uptake of amino

acids to make proteins in muscle. Deficits in producing insulin leading to excessively high blood

sugar are called Type I Diabetes (also known as juvenile diabetes because patients are frequently

diagnosed during childhood), whereas a deficit in the cellular response to insulin, insulin

12
resistance, is known as Type II Diabetes (or adult-onset diabetes, as it is frequently diagnosed in

adulthood).

IGF-I and IGF-II were originally discovered in the late 1950’s as factors mediating

somatotropin (growth hormone) actions, and were therefore originally called somatomedin C and

A, respectively, as somatomedin B was subsequently removed from the group (Daughaday et al,

1972; Salmon and Daughaday, 1957). Soon after, work in cell culture led to the discovery of a

group of factors that promoted cell growth and division, and these were termed multiplication

stimulating activity (MSA) (Dulak and Temin, 1973). A third group of investigators then

discovered a group of factors that led to “insulin-like activity”, and these were named non-

suppressible insulin-like activity (NSILA) I and II because their activity was not abated by

antibodies to insulin (Zapf et al, 1978). Subsequent work identified all three of these studies as

isolating the same factors, and they were soon renamed Insulin-Like Growth Factors after their

amino acid sequences became known and were found to be remarkably similar to insulin. IGF-I

and IGF-II share 62% of their amino acids in common, and both share 42% with human insulin.

(Herington et al, 1983) Mature IGF-I has 70 amino acids and a molecular weight of 7.5

kilodaltons (kDa), and similar to insulin, has an A and B chain connected by disulphide bonds.

Mature IGF-II has 67 amino acids, a molecular weight of 7.5 kDa and also has conserved the

three interchain disulphide bridges of insulin.

Unlike insulin, which is produced primarily in the pancreas, IGF-I and II are produced

primarily in the liver, but are also secreted locally from other tissues where they act in a

paracrine manner. IGF-I is essential for embryonic development and in general for cell growth,

differentiation and survival (Jones and Clemmons, 1995). Peripheral IGF-I is mainly known for

its role in regulating growth along with pituitary Growth Hormone (GH). Specifically, when GH

13
binds its receptor on hepatocytes, IGF-I synthesis is stimulated. Once released, IGF-I acts on

targets such as bone to stimulate growth. In reality, IGF-I acts on almost every tissue in the body,

and is involved not only in body growth, but in CNS development, liver regeneration,

gametogenesis, kidney and cardiovascular development and immune modulation (Puche and

Castilla-Cortazar, 2012). The regulation of IGFs in the periphery is extremely tight – 98% of

IGF-I is bound by IGFBPs in serum (80% by IGFBP3 alone), conferring IGF-I a half-life of

many hours. Laron syndrome, which is characterized by dwarfism, is caused by a deletion of the

GH receptor, leading to lack of IGF-I production, as well as of IGFBP3. In mice as well,

knockout of IGF-I leads to a reduction in size by 40-45% (Laron, 2001).

Unlike IGF-I, the role of IGF-II in the periphery is much less characterized. Although

IGF-I shows highest expression in the periphery during adulthood, IGF-II has a more

complicated regulation. In mice, IGF-II maintains its peak expression during fetal development

(O'Dell and Day, 1998) and decreases dramatically postnatally in peripheral tissues, while

remaining relatively high in the brain. In humans, IGF-II mRNA is also reduced in the adult

compared to fetal levels, however serum levels do not correlate with this, and are actually higher

in the adult, suggesting that IGF-II is rapidly produced and degraded in the fetus. IGF-II also

shows high expression in a number of tumors, most notably Wilm’s tumor.

Though each IGF has a homologous receptor, there is a degree of promiscuity in the

binding. For example, although IGF-II binds with highest affinity to IGF-IIR, it can also bind to

the IGF-IR and IR (with particular affinity for the IR-A isoform), and a similar situation is true

of IGF-I and its homologous receptor. Insulin binds with highest affinity to IR, and binds with

lower affinity to IGF-IR, but cannot bind to IGF-IIR (See Figure D).

14
Figure D: Insulin and IGF receptors, binding affinities and downstream cascades. Reprinted from
(Fernandez and Torres-Aleman, 2012b)

Despite the structural similarity of insulin, IGF-I and IGF-II, their homologous receptors

are not all functionally alike. The insulin and IGF-I receptors (IR and IGF-IR, respectively) are

both tyrosine kinase receptors, with two alpha and beta subunits linked by disulfide bonds.

Binding of the alpha subunit by a ligand stimulates autophosphorylation of the intracellular beta

subunit and leads to downstream signaling cascades that are quite similar between the two,

involving phosphoinositide 3-kinase (PI3K) and Akt or Ras and MAP kinase, which mediate

responses to many other cellular stimuli (Adamo et al, 1989; Siddle et al, 2001). The question of

specificity of responses at the different receptors is assumed to be due to differences in ligand

15
binding and intrinsic signalling capacities of the receptors. Adding to this complexity, the IR

actually has two isoforms, A and B, which confer different biological responses despite being so

similar. IR-A, which lacks Exon 11 and thus 12 amino acids, is predominantly expressed on

neurons in the brain, whereas IR-B is predominantly expressed on astrocytes in the brain and is

the predominant isoform peripherally as well (Belfiore et al, 2009). It is not currently known

how the different isoforms of IR lead to different functional outcomes, yet the accumulating

evidence suggests that IR-B mediates predominantly metabolic signaling, whereas IR-A

mediates growth-related signaling, similar to IGF-I (Belfiore et al, 2009). Lastly, hybrid

receptors can be formed between either IR isoform and the IGF-IR, and show similar affinity for

IGFs as IGF-IR, but lower affinity for insulin. The downstream consequences of ligand binding

at this receptor are presently unknown (Siddle et al, 2001).

In contrast to IGF-IR and IR, the IGF-IIR, which is also known as the cation independent

mannose-6-phosphate receptor (CI-MPR), is a single transmembrane polypeptide with no

intrinsic kinase activity, made up of a large extracellular domain with 15 repeats and a short,

hydrophilic cytoplasmic domain, which includes several tyrosine, threonine and serine

phosphorylation sites. The IGF-IIR/CI-MPR is most widely known for is role in lysosomal

enzyme targeting and is most abundant at the trans-Golgi, with only 10% of the receptor on the

plasma membrane (Hawkes and Kar, 2003), which continuously cycle between the membrane

and endosomal compartments (Nielsen, 1992). From the IGFs, IGF-IIR binds IGF-II with

highest affinity – this appears to be due to a number of residues in the A domain of IGF-II that

are recognized by the receptor (Phe48-Arg49-Ser50 and Ala54-Leu55) that, when replaced,

drastically reduce binding (Figure E). In IGF-I, the Ala54-Leu55-Leu56 are replaced with

Asp54-Leu55-Arg56, which may account for its low affinity for the IGF-IIR (Nielsen, 1992).

16
The downstream effects of ligand binding to the IGF-IIR are not well understood. Until recently,

it was believed that because the receptor has no intrinsic catalytic actibity, that it had no signal

transduction capacity and served only to endocytose IGF-II and other ligands (50

phosphomannosylated lysosomal enzymes, as well as retinoic acid, transforming growth factor-

beta (TGF-β1) precursor, renin, prolactin, and other glycoproteins) in order to send them to

lysosomal degradation. A number of studies have shown, however, that thre are indeed

biological responses mediated by the IGF-IIR, such as calcium influx, amino acid uptake,

glycogen synthesis, cell motility and growth, and acetylcholine release (Nielsen, 1992). The

manner in which the receptor can stimulate these effects while lacking a kinase domain is

unclear, but a growing number of studies suggest that the IGF-IIR can interact with a

heteromeric G-protein, as activation of the IGF-IIR by Leu27IGF-II, an IGF-II analog that

preferentially binds to the IGF-IIR, potentiates Ach release from the adult rat hippocampus by a

G protein-sensitive, PKCα-dependent pathway (Hawkes et al, 2007; Hawkes et al, 2006). IGF-

II-mediated activation of the IGF-IIR was also found to regulate hypertrophy of the cardiac cells

via Gq-mediated increased phosphorylation of protein kinase C-alpha (PKCα) and CaMKII (Chu

et al, 2009).

In addition to the receptors, there are also 6 binding proteins that regulate the

bioavailability of IGF-I and IGF-II peripherally and in the brain, but do not bind insulin. In the

brain IGFBP2 is the most abundant and modulates IGFII bioavailability by sequestering IGF-II,

preventing its interaction with the IGF-IR (Rorive et al, 2008). In contrast to IGFBP-3 in the

periphery, IGFBP-2 and IGFBP-5 have much greater affinity for IGF-II than IGF-I (Jones et al,

1995), whereas IGFBP-4 does not exhibit a preference. Both IGFBP-4 and 5 can be found in the

hippocampus, among other regions, whereas IGFBP-2 is predominantly found in the cerebellum.

17
The IGFBPs form complexes with IGFs, limiting their actions until they are cleaved by proteases

into forms that have little to no affinity for the IGFs (Werner and Leroith, 2014).

Figure E: Organization of IGF prepropeptides and conservation of IGF-II amino acid sequence.
Top: Homologous domains of B, C, A, D and E domains of IGF prepropeptides are indicated. Signal peptides are
shown in grey boxes, and arrows indicate sites of cleavage or proteolyic processing.
Bottom: Amino acid sequence of mature IGF-II, given as single letter amino acid codes and differences in sequence
between human and other species shown in alignment. Adapted from(Nielsen, 1992).

1.6 Insulin-Like Growth Factors: Role in CNS and cognition

It is now known that IGF-I and II are both synthesized locally in the brain (Haselbacher

et al, 1985) (Caelers et al, 2003) (Ayer-le Lievre et al, 1991; Holzenberger et al, 1997; Sara et

al, 1986) but controversy remains concerning whether or not Insulin can also be produced in the

brain (Adamo et al, 1989). A number of studies suggest that Insulin is indeed synthesized within

the brain (Banks, 2004; Schwartz et al, 1992; Woods et al, 2003), but it is likely that peripheral

18
transport via a receptor-mediated active transport system contributes greatly to Insulin content in

the brain. Though both IGF-I and IGF-II are present at relatively high levels during prenatal

development (Ayer-le Lievre et al, 1991), postnatal levels of IGF-II are much higher in CNS

compared to IGF-I (Valentino et al, 1990). In particular, IGF-II itself is synthesized in the

choroid plexus, and thus IGF-II is available in the brain via local synthesis, choroid plexus

synthesis, as well as peripheral synthesis that crosses the blood-brain barrier (BBB) (Duffy et al,

1988; Pardridge, 1986; Reinhardt and Bondy, 1994).

1.6.1: Insulin

Regardless of whether Insulin is transported to or produced in the CNS, Insulin has

functions in the brain that extend past metabolic regulation. Insulin receptors are distributed

widely throughout the brain (Adamo et al, 1989), particularly in the olfactory bulbs, arcuate

nucleus of the hypothalamus, amygdala and hippocampus, suggesting that regardless of whether

it is transported from the periphery or synthesized centrally, Insulin has important functions in

the brain. Unlike the periphery, where Insulin functions to regulate glucose homeostasis, it

appears to have much more diverse functions within the CNS. The particular brain regions in

which the receptor is found, for example hippocampus, suggest also that Insulin plays a role in

higher cognition. Accordingly, in addition to its role in maintaining weight and controlling food

intake, Insulin is also involved in cognitive functions and its receptor levels have been linked to

the cognitive declines observed in Type 2 diabetes patients (Zhao et al, 2004), as well as the

neurodegeneration associated with Alzheimer’s Disease (Watson and Craft, 2003). In both

rodent models and human sporadic AD patients, levels of both Insulin and its receptor are

19
decreased, and studies implementing intranasal adminstration of Insulin have had promising

results (Craft et al, 2012).

In physiological conditions, there is an emerging role of Insulin in the enhancement of

memory. Studies injecting Insulin systemically are difficult to interpret because of the

subsequent alteration of blood glucose levels, ie. hypoglycemia (Zhao et al, 2004). However, a

number of studies have looked specifically at the role of Insulin in the brain itself. Notably, i.c.v.

or intrahippocampal infusions of 4 milliunits (mU) Insulin immediately after IA training leads to

increased memory 24h later, suggesting that Insulin acts on consolidation mechanisms to

increase memory retention (Babri et al, 2007; Park et al, 2000). Similarly, high doses of Insulin

(12 to 24 mU) administed i.c.v. 30 minutes before Morris water maze training leads to increased

memory retention at a probe trial (Haj-ali et al, 2009; Moosavi et al, 2006), and low doses of

Insulin (100 uU and 1 mU) injected unilaterally into the hippocampus 10 minutes before

spontaneous alternation testing led to increased working memory performance on that task

(McNay et al, 2010). This suggests that different tasks are differentially sensitive to relative

amount of Insulin – long-term memory tasks might require higher doses to potentiate memory

than working memory tasks. The Insulin receptor is upregulated after a spatial learning task in

the hippocampus (Zhao et al, 1999), suggesting its involvement in the increase in memory with

insulin administration.

1.6.2: IGF-I

In the CNS, IGF-I expression is high during prenatal development, but lowers

dramatically postnatally (Ayer-le Lievre et al, 1991). IGF-I has been less studied than Insulin in

the context of cognitive enhancement. No studies have directly examined the role of IGF-I in a

20
direct regulation of memory function or cognitive enhancement. However, a number of studies

suggest that IGF-I may be involved in regulating brain functions such as hippocampal

neurogenesis and exercise-induced protection against cognitive impairment (Aberg et al, 2000;

Carro et al, 2001; Liu et al, 2001a; Liu et al, 2001b; O'Kusky et al, 2000; Trejo et al, 2001).

IGF-I is increased in the hippocampus of rats that had exercised compared to non-exercised rats,

and blockade of IGF-I with an IGF-I antibody blocks the beneficial effects of exercise on

cognition, via a decrease in brain derived neurotrophic factor (BDNF) and downstream signaling

(Ding et al, 2006). When administered s.c. for 10 days, IGF-I led to increased expression of N-

methyl D-aspartate receptor subtype 2B (NR2B) messenger RNA (mRNA) and decreased

expression of N-methyl D-aspartate receptor subtype 2B (NR2A) mRNA in the hippocampus (Le

Greves et al, 2005).

IGF-I has also been studied in a number of disease models. It has been suggested that

IGF-I may be beneficial in the treatment of AD via inactivation of the Forkhead box O3- nuclear

factor kappa-light-chain-enhancer of activated β cells (FoXo3/Nf-kB) pathway (Fernandez et al,

2012a); however studies looking at IGF-I treatment in AD models have shown mixed results

(Carro et al, 2002; Cohen et al, 2009; Freude et al, 2009). In a rat model of diabetes, learning

deficits in the Morris water maze are reversed by systemic administration of IGF-I, suggesting

that IGF-I may have therapeutic effects for certain diseases (Lupien et al, 2003). Similarly, a

truncated IGF-I peptide partially restores a Rett syndrome phenotype in methyl CpG binding

protein 2 (MeCP2) knockout mice, suggesting that IGF-I and/or other IGFs may be useful as a

potential autism therapy (Tropea et al, 2009).

1.6.3: IGF-II

21
 Because the prevailing hypothesis for many years has been that IGF-II is primarily

degraded by the IGF-IIR, and exerts its actions only through the IGF-IR in a similar manner to

IGF-I (Werner et al, 2014), it has been the least studied IGF in the brain.

The IGF-IIR is found throughout the brain, with particular concentration in striatum,

deeper layers (layers IV and V) of the cortex, pyramidal and granule cell layers of the

hippocampal formation, selected thalamic nuclei, Purkinje cells of the cerebellum, pontine

nucleus and motoneurons of the brainstem as well as in the spinal cord (Hawkes et al, 2003). The

same study found that the receptor is also present on cholinergic neurons throughout the brain.

Prenatal choline supplementation increases IGF-II mRNA and protein expression in the frontal

cortex and hippocampus (Mellott et al, 2007) and IGF-II potentiates Ach release in the

hippocampus (Kar et al, 1997). A more recent study has also shown the IGF-IIR is present on (γ-

Aminobutyric acid) GABA-ergic neurons, and IGF-II can attenuate GABA release in the

hippocampus through the IGF-IIR (Amritraj et al, 2010).

It has also been recently shown that IGF-II can impact cognitive functions. We have

recently shown that IGF-II mRNA and protein are increased in the hippocampus following IA

training, and that IGF-II is required for IA memory consolidation. As I will detail below in

Chapters 2 and 4, we have also shown that IGF-II enhances IA and CFC memories when injected

into the hippocampus (Chen et al, 2011) and when injected systemically (Stern et al, 2014).

Interestingly, contextual fear extinction also leads to increased hippocampal IGF-II, and IGF-II

facilitates extinction and promotes hippocampal neurogenesis when injected directly into the

hippocampus (Agis-Balboa et al, 2011). IGF-II also appears to regulate synapse formation, as

mice with IκB kinase-Nf-kb (IKK/Nf-kb) deficient neurons in the forebrain have reduced spine

density and decreases in a number of postsynaptic proteins, as well as a spatial learning

22
impairment in the Morris water maze. IGF-II restores synapse density and promotes spine

maturation in these mice, which depends on IGF-IIR and MEK/ERK activation (Schmeisser et

al, 2012).

Lastly, a number of recent studies have also suggested that IGF-II is dysregulated in

disease models. IGF-IIR and a number of lysosomal enzymes are increased in the hippocampus

and frontal cortex of the AD model, amyloid precursor protein+presenilin-1 (APP+PS1)

transgenic mice compared with wild-type controls, and beta-amyloid plaques were

immunopositive for these lysosomal enzymes (Amritraj et al, 2009). Galantamine, an

acetylcholinesterase inhibitor used in the treatment of AD, rapidly increased IGF-II mRNA

levels 3h to 12h after injection, and this increase was blocked by co-injections of an α7 Nicotinic

acetylcholine receptor (nAChR) antagonist (Kita et al, 2013). Mice that are heterozygous for

DiGeorge syndrome chromosomal region 8 (Dgcr8), a candidate gene for 22q11.2 deletion-

associated schizophrenia, show decreased IGF-II mRNA levels, and IGF-II administration

rescues both the proliferation of adult neural stem cells and their spatial working memory deficits

(Ouchi et al, 2013).

23
RESULTS

24
 CHAPTER II.
DIFFERENTIAL EFFECTS OF INSULIN-LIKE GROWTH FACTORS ON
HIPPOCAMPAL AND AMYGDALA-DEPENDENT MEMORIES

ABSTRACT

Recent work has implicated the polypeptide system of Insulin-Like Growth Factors, in cognitive

functions, and specifically in memory enhancement. Here, we tested whether the three members

of the IGF system, IGF-II, IGF-I and Insulin, enhance a number of different memories when

injected into brain regions known to be critical for long-term memory functions, including the

hippocampus and amygdala. I found that in contrast to IGF-II, which persistently enhances

hippocampal-dependent, but not amygdala-dependent, memory in rats, Insulin also enhances

only hippocampal-dependent memories, but does so transiently. IGF-I does not enhance either

hippocampal- or amygdala-dependent memories. I conclude that out of the three IGFs, IGF-II

has the most potent effect as a memory enhancer.

25
INTRODUCTION

The Insulin-Like Growth Factor system is comprised of three structurally similar

polypeptides, Insulin, IGF-I and IGF-II. They are classically known for their involvement in

growth and development during the lifespan (Jones et al, 1995). Recent work, however, has

implicated these factors in cognitive functions, and a number of studies have demonstrated their

potential to enhance memory and alleviate symptoms of cognitive disorders. These factors may

prove clinically beneficial for the treatment of a number of cognitive disorders, and indeed, both

Insulin and IGF-I have begun to be tested in clinical trials (Babri et al, 2007; Freiherr et al, 2013;

Khwaja et al, 2014; Park et al, 2000). Recent studies shown that when injected immediately after

IA training, insulin enhances memory, tested 24h after training (Babri et al, 2007; Park et al,

2000). Another study has shown that Insulin enhances spatial maze learning when injected

intracerebroventricularly (Haj-ali et al, 2009) or intrahippocampally (McNay et al, 2010;

Moosavi et al, 2006). IGF-I has been less studied for its specific role in memory enhancement

thus far, yet has been shown to be involved in memory enhancement provided by other

substances, namely as mediating the cognitive enhancement provided by increased exercise and

the resulting increase in neurogenesis (Aberg et al, 2000; Carro et al, 2001; Carro et al, 2002), as

well as a truncated IGF-I peptide which partially rescues the Rett syndrome-like phenotypes in

MeCP2 knockout mice (Tropea et al, 2009).

Previous work in our lab has shown that IGF-II, the least well characterized of the IGF’s

in the brain, is a target of C/EBPβ, which binds in vivo to a C/EBPβ consensus sequence in the

promoter region of the rat IGF-II exon 1 (Chen et al, 2011). Hippocampal levels of IGF-II are

increased 20h after IA training, and IGF-II is required for IA memory consolidation (Chen et al,

26
2011). Thus, it is possible that administration of exogenous IGF-II might lead to memory

enhancement.

These growth factors are attractive candidates for cognitive enhancement as they can be

easily administered to clinical populations. However, to date, there has been no comprehensive

study in well-established animal models testing the efficacy of the three members of the system

in enhancing different types of memories.

A number of brain regions are known to be critical for long-term memory functions,

including the hippocampus, amygdala and anterior cingulate cortex. The hippocampus is

required for contextual/declarative memories, as lesions or inactivation of the hippocampus

critically impair these memories, which can be tested in rats via the contextual-based fear tasks,

IA and CFC (Lorenzini et al, 1996). These memories, due to their emotional, associative

component, also require a functional amygdala, known for its role in non-declarative, emotional

memories (Berman et al, 1978; Phillips et al, 1992). In contrast, the auditory fear conditioning

task requires a functional amygdala, but not hippocampus (Phillips et al, 1992). This provides an

attractive route to determine which brain regions are critical for a memory enhancement effect,

as they can easily be elucidated using the different tasks/injections into different brain regions.

I thus tested whether Insulin, IGF-I and IGF-II, enhance IA, AFC and CFC memories

when injected into either hippocampus, amygdala or anterior cingulate immediately after

training. Detailed statistics for the results presented in this chapter can be found in Table 2.

27
 RESULTS

Locally delivered IGF-II enhances hippocampal, but not amygdala, dependent memories

via hippocampal injections.

Because IGF-II is required in the hippocampus for IA memory, I first tested whether

intrahippocampal injections of IGF-II immediately after IA training would modulate memory

strength. Rats were tested 24h and 7d after training, and those that were injected with IGF-II

showed significantly enhanced memory relative to vehicle-injected animals (Figure 1a). This

enhancement was dose-dependent, with a dose a 250ng showing the maximal effect (Figure 1b).

Moreover, the effect was long-lasting, as memory was still enhanced in IGF-II injected rats when

tested three weeks after training, a time when vehicle-injected rats have low latencies indicative

of forgetting (Figure 1c).

IGF-II binds most strongly to its cognate receptor, but can bind to other IGF receptors as

well, with lower affinity. To determine which receptor mediates the memory enhancement effect

of IGF-II, I tested the effect of IGF-I and IGF-II receptor (R) selective inhibitors. Specific

inhibitors of IGF-IIR (anti-IGF-IIR antibody) but not IGF-IR (JB1) co-injected with IGF-II

completely abolished the memory enhancement compared to respective controls (Figure 1d). The

inhibitors alone did not affect memory retention (Figure 1d).

I next determined whether IGF-II would similarly enhance other fear tasks, CFC and

AFC. I injected vehicle or IGF-II into the hippocampus immediately after CFC/AFC training and

tested them for contextual memory 24h after training, and auditory fear memory 48 after

trianing. Rats injected with IGF-II displayed significantly more freezing during the contextual

fear test, but did not show any difference in auditory fear memory (Figure 1e), suggesting that

IGF-II might selectively enhance hippocampal-dependent memories.

28
 Hippocampus
IA
A Train Test 1 Test 2 B Train Test 1 C Train Test 1

24h 7d 24h 3w

1000 1000 800

** *** ***
800 * 800 ***

Mean Latency (s)

Mean Latency (s)
Mean Latency (s)

600
*
600 600
400
400 400
200
200 200

0 0 0
Test 1 Test 2 Veh 2.5ng 25ng 250ng Test 1

Veh IGF-II Veh IGF-II

IA CFC AFC
CFC AFC
D Train Test 1 E Tr Test 1 Test 1

24h 24h 48h

Veh
1000 * ** 60
*
100
IGF-II
800 IGF-II/AntiIGF-IIR 80
Mean Latency (s)

IGF-II/JB1
% Freezing
% Freezing

40
600 Anti-IGFIIR 60

JB1
400 40
20
200 20

0 0 0
Test 1 context tone

Veh IGF-II
Fig. 1 IGF-II enhances hippocampal-dependent memory via the IGF-IIR. Experimental schedules are shown
above each graph. (a) Memory retention is expressed as mean latency ± SEM of rats injected with vehicle or IGF-II
immediately after IA training and tested 24h and 7d later. (b) Memory retention is expressed as mean latency ± SEM
of rats injected with vehicle or IGF-II (2.5ng, 25ng or 250ng) immediately after IA training and tested 24h later. (c)
Memory retention is expressed as mean latency ± SEM of rats injected with vehicle or IGF-II immediately after IA
training and tested 3w later. (d) Memory retention is expressed as mean latency ± SEM of rats injected with vehicle,
IGF-II, IGF-I inhibitor JB1 or IGF-IIR blocking antibody immediately after IA training and tested 24h later. (e)
Memory retention is expressed as mean % freezing ± SEM of rats injected with vehicle or IGF-II immediately after
CFC/AFC training and tested 24h (context, CFC Test1) and 48h (tone, AFC Test 1) later. Experiment shown in Fig.
1A was conducted by Dillon Chen. Experiment shown in Figures 1C-1E were conducted in collaboration with
Dillon Chen.

29
IGF-II does not enhance AFC memory via amygdala injections.

To test directly whether IGF-II enhances amygdala-dependent memories I first injected

IGF-II into the basolateral amygdala (BLA) immediately after IA training. A test given at 24h

after training revealed no differences between IGF-II- and vehicle-injected rats (Fig. 2a),

indicating that although IA requires both a functional hippocampus and amygdala, only

hippocampal injections of IGF-II are effective in enhancing the memory. I then sought to

confirm that IGF-II does not enhance amygdala-dependent memory by testing whether IGF-II

enhances AFC memory when injected into the BLA. Groups of rats were trained in AFC with

one tone-shock pairing and were injected immediately after with vehicle or IGF-II in the BLA.

Consistent with our prior results, I found that IGF-II did not enhance AFC memory when

injected into the BLA at 24h (Test 1) after training (Fig. 2b).

30
 


 

















  

Fig. 2. IGF-II does not enhance AFC memory. Experimental schedule is shown above graph. Memory retention is
expressed as the % freezing ± SEM of rats given bilateral amygdala injections (arrow) of vehicle or IGF-II
immediately after AFC training and tested 24h (Test) later (n=9).

31
Insulin transiently enhances hippocampal-dependent memories via hippocampal injections.

I next tested whether Insulin, like IGF-II, enhances IA and/or CFC memory when

injected into the dorsal hippocampus immediately after training. Groups of rats were trained in

IA and injected immediately after with vehicle or Insulin. They were tested 24h (Test 1) and 7d

later (Test 2). Consistent with previous reports (McNay 2010, Haj-ali 2009), a two-way ANOVA

followed by a Bonferonni post-hoc test revealed that insulin-injected rats had enhanced memory

at a 24h IA test, as compared to vehicle-injected. However, when tested for memory persistence

at 7d after training, memory retention of insulin-injected rats had decreased to control levels,

indicating that insulin-mediated memory enhancement is transient (Fig. 3a).

I then asked whether Insulin has a similar effect on CFC and/or AFC. I trained animals

on a combined fear conditioning protocol in order to examine both CFC and AFC memory from

one training protocol. Rats were trained with two tone-shock pairings and injected immediately

after with vehicle or Insulin. Rats were tested for CFC memory 24h after training, AFC memory

48h after training, and were retested for both 6d after the first tests, respectively. Confirming the

results seen with IA, Insulin-injected rats showed a trend toward increased CFC memory at Test

1 compared to controls, but memory of Insulin-injected animals significantly declined at Test 2

and their freezing levels did not differ from vehicle-injected (Fig. 4b, left). There was no

difference in AFC memory between groups at either test (Fig. 3b, right), suggesting that, like

IGF-II, Insulin enhances hippocampal-dependent memory selectively, but does so only

transiently.

However, when testing with the combined CFC/AFC experiment, I observed that CFC

memory declined in control animals between Test 1 and Test 2, likely due to extinction, and

freezing levels on the AFC test were close to ceiling, complicating our interpretation of the data

32
(Fig. 3b). To confirm our results, I therefore repeated these tests, conducting CFC and AFC

training and testing separately.

I first tested the effect of bilateral Insulin injections into the dorsal hippocampus on CFC

memory. Rats were trained in CFC with one unsignalled footshock, injected immediately

afterwards with vehicle or Insulin, and tested 24h (Test 1) and 7d (Test 2) later. As expected,

Insulin-injected rats displayed significantly enhanced memory compared to vehicle-injected

controls at Test 1. At Test 2, however, Insulin-injected rats displayed significantly lower memory

than they had shown at Test 1, which was similar to control levels, confirming that the memory

enhancement is, indeed, transient (Fig. 3c).

I next confirmed that hippocampal injections of Insulin do not enhance AFC. I tested rats

using an AFC protocol with one tone-shock pairing. Indeed, injections of Insulin into the

hippocampus immediately after AFC training did not enhance memory at either a 24h (Test 1) or

7d (Test 2) test (Fig. 3d).

One possibility that could explain the decline in memory in the Insulin-injected rats is

that memory extinguishes faster during the 24h test (Test 1) in the rats injected with Insulin

compared to controls. Thus, to exclude the possibility that exposure to the context during Test 1

contributes to the decline in memory performance at Test 2 in Insulin-injected rats, groups of rats

were injected with vehicle or Insulin immediately after IA training and tested only at 7d. Rats

injected with Insulin showed similar IA memory retention to vehicles, further confirming that

Insulin does not persistently enhance hippocampal-dependent memory (p=0.7593, Student’s t-

test) (Fig. 3e).

33
 ##" #'%
  
   
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Fig. 3. Insulin transiently enhances hippocampal dependent memory.

Experimental schedule is shown above figure. (a). Memory retention is expressed as the mean latency ± SEM (in
seconds, s) of rats given bilateral hippocampal injections (arrow) of vehicle or Insulin immediately after IA training
and tested 24h (Test 1) and 7d (Test 2) later (n=7). (b). Memory retention is expressed as the mean % Freezing ±
SEM of rats given bilateral hippocampal injections (arrow) of vehicle or Insulin immediately after CFC/AFC
training and tested 24h (Test 1, CFC), 48h (Test 1, AFC), 7d (Test 2, CFC), and 8d (Test 2, AFC) later (n=7). (c).
Memory retention is expressed as the mean % freezing ± SEM of rats given bilateral hippocampal injections (arrow)
of vehicle or Insulin immediately after CFC training and tested 24h (Test 1) and 7d (Test 2) later (n=8). (d).
Memory retention is expressed as the mean % freezing ± SEM of rats given bilateral hippocampal injections (arrow)
of vehicle or Insulin immediately after AFC training and tested 24h (Test 1) and 7d (Test 2) later (n=5-6). (e).
Memory retention is expressed as the mean latency ± SEM (in seconds, s) of rats given bilateral hippocampal
injections (arrow) of vehicle or Insulin immediately after IA training and tested 7d (Test) later (n=5-6). *p<0.05;
***p<0.001. Experiment shown in Figure 4B was conducted in collaboration with Dillon Chen.

34
Insulin does not enhance memory via amygdala injections.

I next tested whether Insulin affects memory when injected into the BLA. Groups of rats

were trained in either IA or AFC, both tasks that require a functional amygdala, and were

bilaterally injected immediately after with vehicle or insulin into the BLA. Rats were then tested

at 24h (Test 1) and 7d (Test 2) after training. I saw a trend towards memory enhancement at both

Test 1 and Test 2 in both tasks, however this was not statistically significant for either IA (Fig.

4a) or AFC (Fig. 4b). Furthermore, injections of a higher dose of Insulin into the BLA reversed

any trend of memory enhancement in the AFC task, as memory at both Test 1 (24h after training)

and Test 2 (7d after training) was similar to vehicle-injected rats (Fig. 4c).


 
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Fig. 4: Insulin does not enhance memory when injected into BLA.
Experimental schedule is shown above figure. (a). Memory retention is expressed as the mean latency ± SEM (in
seconds, s) of rats given bilateral BLA injections (arrow) of vehicle or Insulin immediately after IA training and
tested 24h (Test 1) and 7d (Test 2) later (n=8-9). (b and c). Memory retention is expressed as the mean % freezing ±
SEM of rats given bilateral BLA injections (arrow) of vehicle or Insulin immediately after AFC training and tested
24h (Test 1) and 7d (Test 2) later (b: n=5-8; c: n=5-8).

35
IGF-I does not enhance memory via hippocampal injections

We have previously shown that hippocampal injections of IGF-I do not enhance IA

memory (Chen et al, 2011); I confirmed here that IGF-I does not enhance hippocampal-

dependent memories using both the combined CFC/AFC protocol and the separate protocols for

each task. Groups of rats were first injected with vehicle or IGF-I immediately after combined

CFC/AFC training and were tested 24h (CFC Test 1), 48h (AFC Test 1), 7d (CFC Test 2) and 8d

(AFC Test 2) later. There was no difference in memory latency between vehicle- and IGF-I-

injected rats at either test, confirming that IGF-I does not enhance hippocampal-dependent fear

memories (Fig. 5a).

Similar experiments were then conducted to investigate whether IGF-I enhances CFC and

AFC memory when tested separately. Rats were trained in CFC with one unsignalled footshock,

injected immediately afterwards with vehicle or IGF-I, and were then tested 24h (Test 1) and 7d

(Test 2) later. I saw no difference in memory retention between vehicle- or IGF-I-injected rats at

either test (Fig. 5b). Similar results were obtained when IGF-I was injected into the hippocampus

immediately after AFC training. There was no difference between vehicle- and IGF-I-injected

rats at either Test 1 or Test 2 (Fig. 5c).

36
 !! !%#
   
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Fig. 5: IGF-I does not enhance memory when injected into hippocampus.
(a). Memory retention is expressed as the mean % Freezing ± SEM of rats given bilateral hippocampal injections
(arrow) of vehicle or IGF-I immediately after CFC/AFC training and tested 24h (Test 1, CFC), 48h (Test 1, AFC),
7d (Test 2, CFC), and 8d (Test 2, AFC) later (n=6-7). (b). Memory retention is expressed as the mean % freezing ±
SEM of rats given bilateral hippocampal injections (arrow) of vehicle or IGF-I immediately after CFC training and
tested 24h (Test 1) and 7d (Test 2) later (n=6). (c). Memory retention is expressed as the mean % freezing ± SEM of
rats given bilateral hippocampal injections (arrow) of vehicle or Insulin immediately after AFC training and tested
24h (Test 1) and 7d (Test 2) later (n=5-6). Experiment shown in Figure 7A was conducted in collaboration with
Dillon Chen.

37
IGF-I does not enhance memory via amygdala injections.

I next tested whether IGF-I enhances memory when injected into BLA. Groups of rats

were trained in either IA or AFC, and were bilaterally injected immediately after with vehicle or

IGF-I into the BLA. Rats were then tested at 24h (Test 1) and 7d (Test 2) after training. I saw no

difference in memory retention between vehicle- or IGF-I-injected rats with when tested on

either IA (Fig. 6a) or AFC (Fig. 6b).

 
A "! #$
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Fig. 6: IGF-I does not enhance memory when injected into BLA.
Experimental schedule is shown above figure. (a). Memory retention is expressed as the mean latency ± SEM (in
seconds, s) of rats given bilateral BLA injections (arrow) of vehicle or IGF-I immediately after IA training and
tested 24h (Test 1) and 7d (Test 2) later (n=5-8). (b). Memory retention is expressed as the mean % freezing ± SEM
of rats given bilateral BLA injections (arrow) of vehicle or IGF-I immediately after AFC training and tested 24h
(Test 1) and 7d (Test 2) later (b: n=4-6).

38
MATERIALS AND METHODS

Animals: Adult male, Long Evans rats weighing between 200-250 g served as subjects in all

experiments. Animals were all singly housed and maintained on a 12h light-dark cycle.

Experiments were performed during the light cycle. All rats were allowed ad libitum access to

food and water and were handled for 3 min per day for 5d prior any procedure. All protocols

complied with the National Institutes of Health Guide for the Care and Use of Laboratory

Animals and were approved by the Mt. Sinai School of Medicine and New York University

Animal Care Committees.

Cannulae Implants and hippocampal injections: Rats were anesthetized with ketamine (65

mg/kg, i.p.) and xylazine (7.5 mg/kg, i.p.), and stainless-steel guide cannulae (22-gauge for

hippocampus, 28-guauge for basolateral amygdala, BLA, and anterior cingulate cortex, aCC)

were stereotactically implanted to bilaterally target the hippocampus (4.0 mm posterior to the

bregma; 2.6 mm lateral from midline; and 2.0 mm ventral) (Taubenfeld et al, 2001) or BLA

(2.8 mm posterior to bregma; 5.3 mm lateral from midline; and 6.25 mm ventral). The rats were

returned to their home cages and allowed to recover from surgery for 7 days. Immediately after

training, rats received bilateral injections of compounds as specified. All injections are indicated

by arrow in the experimental schedule. All hippocampal injections were carried out in 1μl per

side, and BLA injections were carried out in 0.5μl per side. Hippocampal injections used a 28-

gauge needle that extended 1.5 mm beyond the tip of the guide cannula and connected via

polyethylene tubing to a Hamilton syringe. Amygdala injections used a 33 gauge needle that

extended 1.5 mm beyond the top of the guide. The infusions were delivered at a rate of 0.33

μl/min using an infusion pump. The injection needle was left in place for two min after the

39
injection to allow complete dispersion of the solution.

To verify proper placement of cannula implants in hippocampus, at the end of the behavioral

experiments, rats were killed and their brains were postfixed with 10% buffered formalin. For

examination of BLA placements, rats were perfused with 4% paraformaldehyde in PBS and

postfixed in the same solution. Forty-micrometer coronal sections were cut through the relevant

brain region, and examined under a light microscope. Rats with incorrect cannula placement

were discarded from the study.

Inhibitory Avoidance: Inhibitory avoidance was carried out as previously described

(Taubenfeld et al, 2001). The IA chamber (Med Associates. Inc., St. Albans, VT) consisted of a

rectangular Perspex box divided into a safe compartment and a shock compartment. The safe

compartment was white and illuminated and the shock compartment was black and dark. Foot

shocks were delivered to the grid floor of the shock chamber via a constant current scrambler

circuit. The apparatus was located in a sound-attenuated, non-illuminated room. During training

sessions, each rat was placed in the safe compartment with its head facing away from the door.

After 10 seconds, the door separating the compartments was automatically opened, allowing the

rat access to the shock compartment; the rats usually enter the shock (dark) compartment within

10-20 seconds of the door opening. The door closed 1 second after the rat entered the shock

compartment, and a 2-second 0.6mA footshock was administered. Latency to enter the shock

compartment was taken in seconds as acquisition. The rat was then returned to its home cage and

tested for memory retention at the designated time-point(s). Retention tests were done by placing

the rat back in the safe compartment and measuring its latency to enter the shock compartment.

40
Footshocks were not administered on the retention tests, and testing was terminated at 900

seconds. All behavioral tests were done in blind.

Contextual and auditory fear conditioning (CFC and AFC)

Rats were conditioned in contextual fear conditioning chamber, which consisted of a rectangular

Plexiglass box (30.5 × 24.1 × 21.0 cm) with a metal grid floor (Model ENV-008 Med

Associates).

For combined CFC/AFC protocol, fear conditioning was carried out as described previously38.

All rats were pre-exposed to this chamber for 5 min. The next day, rats were placed in the CFC

chamber for 120 s and then presented with 30 s of the auditory cue consisting of a 5 kHz 75 dB

tone that co-terminated with a 0.6 mA 2s foot shock. One hundred twenty seconds after the first

foot shock, another 30s auditory cue was presented that also co-terminated with another 0.6 mA

2s foot shock. Rats were returned to their home cage 120 s after the second foot shock. Twenty-

four hour later, rats were placed back in the CFC chamber and their freezing levels recorded for

5 min and scored. Twenty-four hours after CFC test, rats were placed in a different context (the

illuminated IA box) for 120 s before being presented with three 30 s auditory cues. The three 30 s

auditory cues were separated by 120 s. Freezing levels during the cue presentations was recorded

and scored by an experimenter who was blind to the treatment conditions.

During CFC-only training, rats were placed in the chamber for 120 s and then presented an

unsignalled 0.6mA footshock for 2 s. One minute later, rats were removed. Testing occurred at

designated time-points in the same chamber, wherein the rat was placed for 3 minutes. No

footshock was delivered during testing.

41
During AFC-only training, rats were placed in the chamber for 120s and then presented with 30s

of the auditory cue consisting of a 5 kHz 75 dB tone that co-terminated with a 0.6 mA 2 s foot

shock. Rats were returned to their home cage 1 minute after the second foot shock. Testing

occurred at designated timepoints by placing rats into a distinct context (the illuminated IA box)

and after 120s presenting three 30s tones with an inter-trial interval of 120s. Testing ended 60s

after the last tone presentation. No footshock was delivered during testing.

Sessions were recorded and freezing, defined as lack of movement except for breathing, was

scored blindly.

Drug and Oligonucleotide Injections: Recombinant mouse IGF-I and IGF-II were purchased

from R&D (Minneapolis, MN), dissolved in 0.1% bovine serum albumin (BSA) in 1xPBS, and

injected at 0.25μg/injection/side (Chen et al, 2011). Recombinant human Insulin was purchased

from Sigma Aldrich (St. Louis, MO), dissolved in 1xSaline, and injected at 2mU or

8mU/injection/side. At the lower dose, Insulin has been used to enhance memory when injected

into the hippocampus (Babri et al, 2007). The IGF-I receptor antagonist JB1 (Bachem

Biosciences) was dissolved in PBS. JB1 was injected at 20ng/μl. Anti-IGF-IIR antibody (R&D)

was dissolved in 1x PBS and injected at 5ng/μl. This concentration blocked 95% of IGF-II

receptor in an in vitro binding assay (R&D).

Statistical Analysis: One- or two-way analysis of variance (ANOVA) followed by either the

Newman-Keuls, or Bonferroni post-hoc test, or Student’s t-test was used for statistical analyses.

42
 CHAPTER III.
THE ROLE OF IGFS IN REMOTE MEMORY ENHANCEMENT

INTRODUCTION

The standard model of systems consolidation posits that over time memory becomes less

dependent on the hippocampus and more dependent on cortical areas, as hippocampal lesions at

later time points do not impair memory retention (Frankland et al, 2005). The prefrontal cortex is

suggested to be involved in the regulation of remote memories, specifically a region known as

the anterior cingulate cortex (aCC).

The first studies to show the involvement of the aCC in remote memory recall looked at

2-Deoxy-D-glucose (2-DG) uptake (Bontempi et al, 1999) as well as IEG induction (Frankland et

al, 2004; Maviel et al, 2004) after 5 and 8 arm mazes and CFC, respectively. All three studies

found activation of the hippocampus at early timepoints after retention testing, and activation of

prefrontal areas, including the aCC at remote timepoints after retention testing. Furthermore,

lidocaine inactivation of the aCC before retrieval impaired memory at remote, but not recent

timepoints (Frankland et al, 2004). This is correlated with an increase in the number of apical

and basal dendrites, as well as dendritic density in the aCC at remote timepoints (Restivo et al,

2009; Vetere et al, 2011). A recent study showed that remote memories may be retained in the

aCC by methylation, as pharmacologic inhibition of methylation at 1 month after CFC training

impaired memory (Miller et al, 2010).

Interestingly, however, a number of studies have shown a role for the aCC in recent long-

term memory as well. Arc is induced in the aCC 1hr after IA training, and 48h memory is

impaired when Arc antisense is injected into the aCC around the time of training (Holloway and

McIntyre, 2011). Another study similarly found Arc and c-fos induction in the aCC 90m after IA

43
training, and that protein synthesis is required in the aCC after training (Zhang et al, 2011).

Finally, a muscarinic cholinergic agonist injected into aCC before training dose-dependently

enhances 24h IA memory.

Therefore, the role of the aCC in recent and remote memory consolidation is not yet

clear, and it remains to be seen whether enhancement of recent and remote memory can be

achieved via the aCC.

When injected bilaterally into the dorsal hippocampus immediately after training, IGF-II

enhances memory for at least three weeks. However, injections of IGF-II into the hippocampus

24h after training has no effect on memory, indicating that the effect of IGF-II into the

hippocampus is temporally limited (Chen et al, 2011). Here, I asked whether IGF-II, IGF-I and

Insulin enhance memory, particularly at remote timepoints, when injected into the aCC. Detailed

statistics for the results presented in this chapter can be found in Table 3.

RESULTS

IGFs do not enhance recent memory when injected into anterior cingulate (aCC).

I first tested whether IGF-II enhances recent long-term memory when injected into the aCC, a

cortical area involved in long-term IA memory (Malin et al, 2007; Zhang et al, 2011),

immediately after training. Groups of rats were trained in IA and injected immediately after with

vehicle or IGF-II. When tested 24h (Test 1) and 7d (Test 2) later, both groups showed similar

memory latencies. Thus, IGF-II does not enhance IA memory via the aCC (Fig. 7).

44
 

  !
  !


  






!"
 








 !
  !

 

Fig. 7. IGF-II does not enhance memory when injected into aCC. Experimental schedule is shown above graph.
Memory retention is expressed as the mean latency ± SEM (in seconds, s) of rats given bilateral aCC injections
(arrow) of vehicle or IGF-II immediately after IA training and tested 24h (Test 1) and 7d (Test 2) later (n=10-11).

45
 I next tested whether Insulin enhances memory when injected into the aCC. Groups of

rats were trained in IA and injected immediately after with vehicle or Insulin. Tests at 24h (Test

1) and 7d (Test 2) after training revealed no difference in memory retention between groups,

indicating that, similar to IGF-II, Insulin does not enhance IA memory via the aCC (Fig. 8).



 
 


 




!












 

  

Fig. 8: Insulin does not enhance memory when injected into aCC. Experimental schedule is shown above graph.
Memory retention is expressed as the mean latency ± SEM (in seconds, s) of rats given bilateral aCC injections
(arrow) of vehicle or Insulin immediately after IA training and tested 24h (Test 1) and 7d (Test 2) later (n=9-11).

46
 I next tested whether IGF-I enhances memory when injected into the aCC. Groups of rats

were trained in IA and injected immediately after with vehicle or Insulin. Tests at 24h (Test 1)

and 7d (Test 2) after training revealed no difference in memory between groups, showing that

similar to IGF-II and Insulin, IGF-I does not enhance IA memory via the aCC (Fig. 9).

aCC
IA
Train Test 1 Test 2

24h 7d
900
800
Mean latency (s)

700
600
500
400
300
200
100
0
Test 1 Test 2

Veh IGF-I

Fig. 9: IGF-I does not enhance memory when injected into aCC. Experimental schedule is shown above graph.
Memory retention is expressed as the mean latency ± SEM (in seconds, s) of rats given bilateral aCC injections
(arrow) of vehicle or IGF-I immediately after IA training and tested 24h (Test 1) and 7d (Test 2) later (n=9-11).

47
IGF-II enhances remote memories when injected into the aCC.

IGF-II did not enhance recent long-term memory when injected into aCC immediately

after training. I asked here whether injections of IGF-II into the aCC enhance memory when

administered at later time points or when tested at later timepoints, and whether these effects

were temporally limited. I first trained rats in IA and 48h later injected vehicle or IGF-II

bilaterally into the aCC. Rats were then tested at 24h (Test 1) and 7d (Test 2) after injection, and

there was no difference in memory retention between groups at either test (Figure 10a),

suggesting that IGF-II indeed does not enhance recent long-term memory via the aCC.

A separate group of rats were trained, injected with vehicle or IGF-II immediately

afterwards, but then tested 2w and 4w later. In contrast to tests at early timepoints, here, I found

that IGF-II-injected rats showed enhanced memory at the 2w test and a trend towards

enhancement at the 4w test, compared to vehicle-injected rats (Figure 10b), suggesting that in the

cortex, IGF-II may enhance remote, but not recent, memory.

  
 
   
 


 
  

 
**















 

 
 
 


  

48
Figure 10: IGF-II enhances remote, but not recent long-term memory via the aCC. Experimental schedule is
shown above graph. Memory retention is expressed as the mean latency ± SEM (in seconds, s) of rats given bilateral
aCC injections (arrow) of vehicle or IGF-II (a) 48h after IA training and tested 24h (Test 1) and 7d (Test 2) later or
(b) immediately after IA training and tested 2w and 4w later (n=9-11). **p<0.01

I next asked whether injections IGF-II into the aCC at remote time-points would also lead

to memory enhancement. Rats were trained in IA, and two weeks later were injected with vehicle

or IGF-II. Rats were tested 24h (Test 1) and 7d (Test 2) after the injection. Here, I saw that IGF-

II-injected rats displayed significantly higher memory retention than vehicle-injected rats at both

Test 1 and Test 2 (p<0.001, two-way ANOVA with Bonferonni post-hoc test, Figure 11a). When

I injected IGF-II after 4 weeks, however, I did not see a significant enhancement at a test 24h

after injected (Test 1) or 7d later (Test 2) (Figure 11b).

Reactivation can re-engage consolidation mechanisms in the hippocampus, hence,

although IGF-II does not enhance memory when injected 24h after training, it does enhance

memory when injected at the same time-point in concert with a reactivation (Chen et al. 2011). I

therefore asked whether reactivating the memory would enable IGF-II to enhance remote

memory. Rats were trained and two, four, or eight weeks later were injected with vehicle or IGF-

II into the aCC immediately after reactivation. Rats were then tested 24h (Test 1) and 7d (Test 2)

after the reactivation. IGF-II enhanced IA memory when given in concert with reactivation at

both 2 weeks (Figure 11c), and 4 weeks (p<0.05, p<0.01, Figure 11d). When IGF-II was injected

2 months after training, there was a non-significant trend towards memory enhancement,

suggesting that the ability to enhance remote memories via IGF-II is time-limited (11e).

49
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Figure 11. IGF-II enhancement of remote memory is temporally limited and extended by reactivation.
Experimental schedule is shown above graph. Memory retention is expressed as the mean latency ± SEM (in
seconds, s) of rats given bilateral aCC injections (arrow) of vehicle or IGF-II (a) 2w or (b) 4w after IA training and
tested 24h (Test 1) and 7d (Test 2) after injection or immediately after a reactivation given (c) 2w (d) 4w or (e)
2months after IA training and tested 24h and 7d after injection (n=9-11).*p<0.05, **p<0.01, ***p<0.001.

50
 I followed up by determining whether Insulin and IGF-I can also enhance remote

memories via aCC injections. I trained rats and two weeks later injected vehicle, IGF-I or insulin

into the aCC immediately after, or in the absence of, a reactivation. Rats were tested 24h (Test 1)

and 7d (Test 2) after the injection, and there was no effect of IGF-I or insulin on memory

performance in either case (Figure 12a).

B    !
  !


#   

#   








!$
 

  "

 
 
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  !
  !


Figure 12: Insulin and IGF-I do not enhance remote memory. Experimental schedule is shown above graph.
Memory retention is expressed as the mean latency ± SEM (in seconds, s) of rats given bilateral aCC injections
(arrow) of vehicle, Insulin or IGF-I in the absence of, or immediately after, a reactivation given 2w after training and
tested 24h (Test 1) and 7d (Test 2) after injection (n=9-11).

51
MATERIALS AND METHODS

Animals: Adult male, Long Evans rats weighing between 200-250 g served as subjects in all

experiments. Animals were all singly housed and maintained on a 12h light-dark cycle.

Experiments were performed during the light cycle. All rats were allowed ad libitum access to

food and water and were handled for 3 min per day for 5d prior to any procedure. All protocols

complied with the National Institutes of Health Guide for the Care and Use of Laboratory

Animals and were approved by the Mt. Sinai School of Medicine and New York University

Animal Care Committees.

Cannulae Implants and hippocampal injections: Rats were anesthetized with ketamine (65

mg/kg, i.p.) and xylazine (7.5 mg/kg, i.p.), and stainless-steel guide cannulae (28-gauge for aCC)

were stereotactically implanted to bilaterally target the aCC (2.7 mm anterior to the bregma; 0.6

mm lateral from midline; and 1.3 mm ventral) (Miller et al, 2010). The rats were returned to their

home cages and allowed to recover from surgery for 7 days. Immediately after training, rats

received bilateral injections of compounds as specified. All injections are indicated by arrow in

the experimental schedule. All aCC injections were carried out in 0.5μl per side. aCC injections

used a 33-guage needle that extended 1.2 mm beyond the top of the guide and connected via

polyethylene tubing to a Hamilton syringe.. The infusions were delivered at a rate of 0.33 μl/min

using an infusion pump. The injection needle was left in place for two min after the injection to

allow complete dispersion of the solution.

To verify proper placement of cannula implants in aCC, at the end of the behavioral experiments,

rats were killed and their brains were postfixed with 10% buffered formalin. Forty-micrometer

52
coronal sections were cut through the aCC, and examined under a light microscope. Rats with

incorrect cannula placement were discarded from the study.

Inhibitory Avoidance: Inhibitory avoidance was carried out as previously described

(Taubenfeld et al, 2001). The IA chamber (Med Associates. Inc., St. Albans, VT) consisted of a

rectangular Perspex box divided into a safe compartment and a shock compartment. The safe

compartment was white and illuminated and the shock compartment was black and dark. Foot

shocks were delivered to the grid floor of the shock chamber via a constant current scrambler

circuit. The apparatus was located in a sound-attenuated, non-illuminated room. During training

sessions, each rat was placed in the safe compartment with its head facing away from the door.

After 10 seconds, the door separating the compartments was automatically opened, allowing the

rat access to the shock compartment; the rats usually enter the shock (dark) compartment within

10-20 seconds of the door opening. The door closed 1 second after the rat entered the shock

compartment, and a 2-second 0.6mA footshock was administered. Latency to enter the shock

compartment was taken in seconds as acquisition. The rat was then returned to its home cage and

tested for memory retention at the designated time-point(s). Retention tests were done by placing

the rat back in the safe compartment and measuring its latency to enter the shock compartment.

Footshocks were not administered on the retention tests, and testing was terminated at 900

seconds. All behavioral tests were done in blind.

Insulin and IGF Injections: Recombinant mouse IGF-I and IGF-II were purchased from R&D

(Minneapolis, MN), dissolved in 0.1% bovine serum albumin (BSA) in 1xPBS, and injected at

0.25μg/injection/side (Chen et al, 2011). Recombinant human Insulin was purchased from Sigma

53
Aldrich (St. Louis, MO), dissolved in 1xSaline, and injected at 2mU or 8mU/injection/side. At

the lower dose, Insulin has been used to enhance memory when injected into the hippocampus

(Babri et al, 2007).

Statistical Analysis: One- or two-way analysis of variance (ANOVA) followed by either the

Newman-Keuls, or Bonferroni post-hoc test, or Student’s t-test was used for statistical analyses.

54
 CHAPTER IV.
ENHANCEMENT OF MEMORIES BY SYSTEMIC ADMINISTRATION OF INSULIN-LIKE
GROWTH FACTOR II

ABSTRACT
To treat cognitive disorders in humans, new effective therapies that can be easily delivered

systemically are needed. Previous studies showed that a bilateral injection of insulin-like growth

factor II into the dorsal hippocampus of rats or mice enhances fear memories and facilitates fear

extinction. Here, I report that, in mice, systemic treatments with IGF-II given before training

significantly enhance the retention and persistence of several types of working, short-term and

long-term memories, including fear conditioning, object recognition, object placement, social

recognition and spatial reference memory. IGF-II-mediated memory enhancement does not alter

memory flexibility or the ability for new learning, and also occurs when IGF-II treatment is

given in concert with memory retrieval. Thus, IGF-II may represent a potentially important and

effective treatment for enhancing human cognitive and executive functions.

55
INTRODUCTION

Memory and cognitive impairments are associated with numerous diseases or deficits, including

Alzheimer’s disease, aging, dementias, anxiety/stress-related disorders and autism spectrum

disorder, and the need for novel therapies that may lead to memory/cognitive enhancement is

extremely high. Furthermore, for rapid translation into clinical therapies, it is critical to identify

effective and successful treatments that can be easily administered through systemic delivery.

IGF-II is a polypeptide belonging to the insulin system that plays an important role in normal

somatic growth and development, tissue repair and regeneration (Roth, 1988; Russo et al, 2005;

Werther et al, 1998). An increase in IGF-II expression in the hippocampus is required for rat

inhibitory avoidance memory formation (Chen et al, 2011) and mouse extinction learning (Agis-

Balboa et al, 2011), and a bilateral hippocampal injection of recombinant IGF-II enhances fear

memory retention, prevents its forgetting (Chen et al, 2011) and facilitates extinction (Agis-

Balboa et al, 2011). Although these findings are important as proofs of principle, to progress in

the direction of establishing novel effective clinical treatments it is essential that the effect of

IGF-II is tested with systemic routes of administration and on different types of memories,

particularly those that are lost or impaired in aging, cognitive disorders and neurodegeneration.

IGFs, including IGF-II, have been shown to cross the blood-brain barrier (BBB) in

several species (Duffy et al, 1988; Reinhardt et al, 1994), and IGF-II receptors are located on

BBB capillaries, indicating a receptor-mediated transport system for IGF-II transcytosis across

the BBB (Rosenfeld et al, 1987). Here, I used C57BL/6J mice to test the effect of an acute

systemic injection of IGF-II on several types of short and long-term memories, working

memories, as well as memory flexibility. I utilized both fear-based/passive tasks that measure

inactive behaviors (e.g. freezing), as well as non-aversive/active tasks that measure approach

56
behaviors (e.g. object investigation), to determine if the effects of IGF-II are applicable to

multiple types of temporal lobe-dependent memory. I also investigated the effect of

systemically-delivered IGF-II on extinction, working memory and memory flexibility. Finally, I

established the safety of the treatment and the effects on metabolic parameters. Detailed statistics

for the results presented in this chapter can be found in Table 4.

RESULTS

IGF-II enhances contextual but not auditory fear conditioning. First, I determined whether

an acute, systemic treatment (subcutaneous injection, s.c.) of IGF-II, like an injection given

directly into the rat hippocampus (Chen et al, 2011), affects aversive memories in mice. S.c.

injections were chosen because previous studies have successfully employed this route of

administration of IGFs in different species to affect general functions without profoundly

changing glucose metabolism or body weight (Douglas et al, 1991; Guler et al, 1989; Spencer et

al, 1996; Zhuang et al, 1996), even when the IGFs were injected continuously for up to two

weeks (Conover et al, 2002; Zhuang et al, 1996). Detailed statistical analyses for all experiments

can be found in Table S1.

To establish a dose-response curve, mice were injected with 5, 15, 30, 45, 60 or 100

μg/kg of IGF-II or vehicle 20 min before contextual fear conditioning training. Fifteen, 30 and

45 μg/kg of IGF-II, but not the other doses, significantly enhanced CFC memory, tested 24h after

training (Figure 13a). Thirty mg/kg of IGF-II showed the strongest effect on CFC memory,

whereas 15 and 45 mg/kg produced intermediate, although significant, memory enhancement. I

further investigated the effect of 15 and 30 μg/kg of IGF-II on CFC retention at 24h and 7d after

training. Fifteen μg/kg of IGF-II significantly increased memory retention at 24h and produced a

57
very strong trend toward memory enhancement at 7d (Figure 13b), while 30 μg/kg of IGF-II

resulted in a more robust and significant enhancement at both 24h and 7d retention tests (Figure

13c). Thus, for all subsequent experiments I used the more effective dose of 30 μg/kg, injected

s.c., unless otherwise indicated. Furthermore, IGF-II injected 20 min before CFC training

significantly enhanced short-term memory tested at 1h after training (Figure 13d), indicating that

IGF-II injected before training may enhance not only memory consolidation, the process that

following learning makes memory long-lasting and resilient to interferences (McGaugh, 2000b),

but also learning and/or short-term memory. As IGF-II injected immediately after CFC training

significantly enhanced memory retention at 24h (Figure 13e), I concluded that IGF-II also affects

memory consolidation, in agreement with our previous findings obtained with IGF-II

hippocampal injections (Chen et al, 2011). The effect of post-training IGF-II treatment was

transient, as 7d later, the IGF-II effect was lost (Figure 13e). Compared to vehicle, the post-

training injection of IGF-II did not affect short-term memory retention tested 1h after training

(Figure 13f). Thus, to produce a stronger, and more persistent effect on memory retention with

systemic injections, IGF-II should be administered before training.

IGF-II injected 20 min before auditory fear conditioning training did not change retention

either before or after the onset of the tone given at testing 24h later (Figure 13g). This indicates,

in agreement with our previous data with IGF-II injections directly into the rat basolateral

amygdala (Chen et al, 2011), that IGF-II does not enhance amygdala-dependent auditory fear

conditioning and shows that freezing per se is not affected by IGF-II administration. Thus, IGF-

II appears to target memories known to involve hippocampal and/or cortical regions, but not

those that are more strictly amygdala-dependent, suggesting specific regional or network

mechanisms for IGF-II. These results indicate that IGF-II may be particularly suitable for

58
targeting medial temporal lobe-dependent memories and cognitive and executive functions rather

than amygdala-driven, Pavlovian conditioning.

a Tr Test 1 b Tr Test 1 Test 2 c Tr Test 1 Test 2

24h 24h 7d 24h 7d

80 80 80
***

% Freezing
** *

% Freezing
% Freezing

60
* ** 60 * 60

40 40 40

20 20 20
0 0 0
Veh 5 15 30 45 60 100 Test 1 Test 2 Test 1 Test 2
Concentration (ѥg/kg) Veh IGF-II Veh IGF-II
15 ѥg/kg 30 ѥg/kg

d Tr Test e Tr Test 1 Test 2 f Tr Test g Tr Test 1

1h 24h 7d 1h 24h

** **
60 * 80 60 80
% Freezing

% Freezing
% Freezing

45 60 45 % Freezing 60
30 40 30 40
15 20 15 20
0 0 0 0
Test 1 Test 2 Pre-Tone Tone
Veh IGF-II Veh IGF-II Veh IGF-II Veh IGF-II

Figure 13. Systemic IGF-II enhances contextual fear, but not auditory fear memory. Experimental schedules
are shown above each panel. Tr: Training. (a-f) Data are expressed as mean (± s.e.m.) percentage (%) freezing. Mice
were injected () with vehicle (Veh) or IGF-II 20 min before or immediately after training. (a) 15, 30 and 45 μg/kg,
but not other doses, of IGF-II enhances contextual fear conditioning memory (n=6-9). (b) 15μg/kg IGF-II enhances
memory at 1d, but not 7d, after training (n=7-8). (c) 30μg/kg enhances memory at both 1 and 7d after training (n=8-
11). (d) IGF-II enhances short-term memory, 1h after training (n=7). (e) IGF-II injected immediately after training
enhances memory at 24h, but not 7d, after training (n=7-8). (f) IGF-II injected immediately after training does not
enhance short-term memory (n=6) (g) IGF-II has no effect on auditory fear conditioning memory (n=6). *P<0.05,
**P<0.01, ***P<0.001. Experiments shown in Figure 1a and 1d were conducted in collaboration with Amy Kohtz.

59
Acute IGF-II treatment does not produce aversive effects. To determine if the IGF-II

treatment produces adverse effects, I assessed the safety of the s.c. IGF-II injection by

conducting a standard observational battery of tests that included physical, behavioral, and

sensorimotor evaluations (Paylor et al, 1998). As shown in Table 1, no differences were found

between vehicle and IGF-II injected mice at 30 min, 24h or 7d after injection. Furthermore,

systemic IGF-II treatment did not affect general locomotor activity (Figure 14a), or the open

field locomotion/anxiety test assessed at either 20 min or 24hr after injection (Figure 14b, Figure

14c). Consistent with previous reports (Conover et al, 2002; Zapf et al, 1985; Zhuang et al,

1996), IGF-II did not affect blood glucose levels tested 30 min, 12h, 24h and 7d after IGF-II

injection (Figure 14d). Additionally, IGF-II did not affect body weight measured immediately,

24h and 7d after training (Figure 14e), time points at which memory enhancement was found.

I concluded that acute systemic treatments with IGF-II are safe and that IGF-II targets

memory formation and retention rather than motor or anxiety responses.

60
 a Tr Test 1 b Tr Test 1

24h 24h

Total line crossings

300 40 50 250

# Center entries

Time in center (s)

# Beam breaks
40 200
30
200
30 150
20
20 100
100 10 10 50
0 0 0 0

Veh IGF-II
Veh IGF-II

c Tr Test 1

24h

Total line crossings

40 40 300
# Center entries

Time in center (s)

30 30
200
20 20
100
10 10

0 0 0

Veh IGF-II

d e
Veh IGF-II
30m 12h 24h 7d 25
 24
Weight (g)


 

 23
22

21

20
 19
 h) )
Tr 4h d)
-24 r (2 r (7
30m 12h 24h 7d e-tr ( o st-t o st-t
Pr P P

Figure 14. Systemic IGF-II does not lead to adverse effects. IGF-II has no effect on (a) locomotor activity,
expressed as mean (± s.e.m.) beam breaks of mice injected with vehicle (Veh) or IGF-II 24h before testing (n=5-6),
(b-c) Open field test, expressed as mean (± s.e.m.) center entries (Left) or time spent in the center (in seconds, s)
(Middle) or number of total line crossings (Right) in the open field arena at (b) 20 min and (c) 24h after injection of
vehicle or IGF-II (n=6), (d) mean (± s.e.m.) of blood glucose concentration (mg/dl) 30 min, 12h, 24h, and 7d after
injection of Veh or IGF-II (n/6), or (e) mean (± s.e.m.) weight (g) of mice injected with either Veh or IGF-II 20 min
before CFC training and tested 24h before (Pre), immediately after (Tr), 24 h and 7 days after training (Post, n=7).

61
Table 1: General motor and sensory responses of vehicle- and IGF-II-injected mice

30 min Test 24h Test 7d Test

Veh IGF-II Veh IGF-II Veh IGF-II

Physical Characteristics
23.0 24.83 22.8 24.0 24.0 25.67
Weight
(±0.40) (±0.48) (±0.53) (±0.52) (±0.65) (±0.71)
38.14 38.23 38.32 37.88 38.36 38.0
Temp
(±0.11) (±0.12) (±0.1) (±0.33) (±0.15) (±0.21)
Whiskers (% with) 100 100 100 100 100 100
Bald Patches (% with) 0 0 0 0 0 0
0 0 0 0 0 0
Palpebral Closure (% with)
Exophthalmos (% with) 0 0 0 0 0 0
Piloerection (% with) 0 0 0 0 0 0
General Behavioral Observations
(% subject displaying response)
Wild Running 0 0 0 0 0 0
Freezing 0 0 0 0 0 0
Sniffing 100 100 100 100 100 100
Licking 0 0 0 0 0 0
Rearing 100 100 100 100 100 100
Jumping 0 0 0 0 0 0
Defecation 0 0 0 0 20 33.33
Urination 0 16.67 0 0 20 16.67
Movement around cage 100 100 100 100 100 100
Sensorimotor Reflexes (% subjects
showing normal response)
Righting 100 100 100 100 100 100
Whiskers 100 100 100 100 100 100
Eye-blink 100 100 100 100 100 100
Ear-twitch 100 100 100 100 100 100
Cage Movement 100 100 100 100 100 100
Motor Responses
26.0 29.83 54.0 47.5 50.6 33.67
Wire Suspension (s)
(±8.34) (±8.66) (±3.38) (±9.1) (±4.28) (±7.57)
10.6 11.67 10.8 12.67 13.2 13.67
Pole Test
(±1.12) (±0.67) (±1.29) (±1.02) (±0.45) (±0.61)
Tail Suspension (% subjects
showing normal response) 100 100 100 100 100 100

62
Systemically delivered IGF-II enhances memory via the IGF-IIR expressed in the brain

and increases brain expression of immediate early genes (IEGs).

IGF-II has been shown to cross the BBB (Duffy et al, 1988; Reinhardt et al, 1994).

Here, I asked whether IGF-II delivered systemically affects memory via IGF-IIR expressed in

the brain, as indicated previously by intra-hippocampal injections (Chen et al. 2011). IgG or a

functionally blocking IGF-IIR antibody (anti-IGF-IIR) was injected into the dorsal hippocampus

of mice, followed, immediately after, by a vehicle or IGF-II s.c. injection, 20 minutes before

CFC training (Figure 15a). Testing, 24h later, revealed that anti-IGF-IIR completely blocked

memory enhancement elicited by the s.c. delivery of IGF-II, without itself affecting memory

retention. Together, these data show that s.c. administered IGF-II rapidly biodistributes in the

blood and brain regions, and that itsexerts its effect on memory occurs via IGF-IIR expressed in

the brain.

To begin identifying the mechanisms by which a systemic injection of IGF-II affects

cognitive functions, and to determine whether the s.c. route of administration influences cellular

responses in the brain, I investigated whether the IGF-II injection changes the expression of

IEGs known to be regulated by neural activity and/or plasticity (Guzowski, 2002). Specifically, I

evaluated the effect of a 30 μg/kg s.c. injection of IGF-II on the expression levels of activity-

regulated cytoskeleton-associated protein (Arc/Arg3.1) and zinc finger protein Zif268 (also

known as early growth response protein 1, Egr-1) in the hippocampus and prefrontal cortex of

naïve and trained mice. Both Arc and Zif268 are known to play an essential role in long-term

plasticity and memory (Guzowski, 2002).

Quantitative western blot analysis revealed that compared to vehicle, an injection of IGF-

II significantly augmented the expression levels of both Arc (Figure 15b) and Zif268 (Figure

63
15c) in the hippocampus, but not in the prefrontal cortex, of naïve mice (Figure 3e and 3f),

although there was a trend toward an increase in cortical Zif268. Furthermore, in agreement

with previous studies (Alberini, 2009; Bramham, 2007; Holloway et al, 2011; Zhang et al,

2011), training significantly elevated both Arc and Zif268 levels in the hippocampus (Figure

15d-e) and led to a significant induction of Arc and a trend toward an increase of Zif268 in the

prefrontal cortex; however, IGF-II did not further change this increase (Figure 15e-f). I

concluded that systemic treatments with IGF-II modify the activity-related molecular activation

of neural cells in brain regions known to critically process medial temporal lobe-dependent

memory formation.

Interestingly, when I examined the expression of plasticity markers, pCREB and

pCAMKII in the prefrontal cortex of naïve or trained mice, injected with vehicle or IGF-II, I

found that while there was no change in induction between vehicle-injected, naïve and trained

mice, IGF-II-injected mice showed a dramatic decrease in both of these markers 1hr after

training (15f-g).

64
 a

Tr Test 1

24h

* IgG/Veh
100
* * Anti-IGFIIR/Veh
80

% Freezing
IgG/IGF-II
60
Anti-IGFIIR/IGF-II
40
20
0

Hippocampus
b c




Arc 
Zif268 




Actin Actin 


** *
* **
200 ** 200
**
% of Naive-Veh

% of Naive-Veh
150 150
100 100
50 50
0 0

Prefrontal Cortex
d e




Arc 
Zif268 




Actin Actin 


200 * 

% of Naive-Veh

% of Naive-Veh

150



100
50 

0

f pCREB g pCAMKII
*** ***
150 150
** *
% of Naive-Veh
% of Naive-Veh

** *
100 100

50 50

0 0

Naive-Veh Trained-Veh Naive-IGF-II Trained-IGF-II

65
Figure 15. Systemic treatment with IGF-II enhances memory via hippocampal IGF-IIR and increases the
expression of immediate early genes (IEGs) in the brain.
(a). Experimental schedule is shown above each panel. Tr: Training. Data are expressed as mean (± s.e.m.)
percentage (%) freezing. Mice were injected (éé) with IgG or anti-IGF-IIR intrahippocampally and immediately
after with vehicle (Veh) or IGF-II s.c., 20 min before CFC training. They were tested 24h later. (b-g. Examples and
quantification of western blots of hippocampal and prefrontal cortex total extracts from naive and trained mice
injected () with either vehicle (Veh) or IGF-II 20 min before CFC training and euthanized 1h after training.
Values were normalized to actin. Data are expressed as mean percentage (± s.e.m) of naive mice injected with Veh
(Naïve-Veh). In naïve mice, compared to Veh, IGF-II injection (Naïve-IGF-II) increases the levels of (b) Arc and (c)
Zif268 in the hippocampus, but not prefrontal cortex (d, Arc: e, Zif268). Training significantly increases the levels
of Arc in the hippocampus and prefrontal cortex and of Zif268 in the hippocampus (Trained-Veh); IGF-II treatment
does not further increase the levels of either IEGs following training (Trained-IGF-II), but does decrease the levels
of (f) pCREB and (g) pCAMKII in the prefrontal cortex. (n=7-10), *P<0.05, **P<0.01, ***P<0.001.

IGF-II enhances several types of non-aversive memories and fear extinction.

Because many types of memory impaired in psychopathologies are non-aversive, it is important

to determine the effect of IGF-II on different non-aversive tasks relevant for potential translation

into clinical applications. Toward this end, I examined the effect of systemic IGF-II treatment

on the novel object recognition (nOR) and object placement (OP) tasks (Dix and Aggleton,

1999), which model hippocampal and cortical-dependent episodic types of memories (Antunes

and Biala, 2012), and are known to be impaired in Alzheimer’s disease (Dere et al, 2007). These

tasks are based on rodents’ innate preference for novelty. Compared to vehicle, injection of IGF-

II 20 min before nOR training did not affect nOR memory retention at 4h after training (Test 1),

but significantly enhanced it at 24h after training (Test 2). In fact, at Test 2, the vehicle group

performed at chance level (i.e. 50%), while the IGF-II-injected mice showed significant memory

(Figure 16a). Compared to vehicle, IGF-II significantly enhanced OP memory at both 4h and 24h

after training (Figure 16b). The IGF-II effect was not due to changes in motivation to explore, as

both IGF-II- and vehicle-injected mice spent similar amounts of time exploring the objects in

either task at both training and testing (Figure 16c-f).

I then investigated the effect of IGF-II on a social recognition memory, a fundamental

and adaptive memory in numerous species, which can be measured by examining the ability of

66
the animal to differentiate between a social and non-social stimulus (i.e. a mouse vs. an object),

and between a novel and familiar mouse (Satoh et al, 2011). Mice were injected s.c. with either

IGF-II or vehicle 20 min before testing their interaction with either a novel object or a novel

mouse (Test 1). Five minutes later, they were tested for their immediate memory by assessing

their interaction with either the previously encountered mouse or a novel mouse (Test 2). Long-

term memory was subsequently tested 24h after Test 2 by recording the animals’ interaction

between the previously encountered mouse vs. another novel mouse (Test 3). C57Bl/6 mice are

highly social and prefer to interact with a social stimulus (i.e. the mouse) compared to a non-

social stimulus (i.e. the object), and IGF-II did not change the preference of the mice toward a

mouse vs. an object (Test 1, Figure 16g). IGF-II also did not affect the short-term memory of the

mice of a novel vs. the previously encountered mouse (Test 2, Figure 16g). However, at the 24h

test, when vehicle-injected mice did not show a preference for the mouse that they have

previously encountered, IGF-II-injected mice spent more time interacting with the novel mouse,

indicating that their social interaction memory persisted (Test 3, Figure 16g). There was no

change between the two groups in total exploration time during any of the test phases (Figure

16h).

Finally, as a previous study showed that hippocampal injections of IGF-II enhance

contextual fear extinction in mice (Agis-Balboa et al, 2011) and because extinction learning is a

therapeutically important behavioral approach in cognitive-behavioral therapies (Ressler and

Mayberg, 2007), I tested whether extinction is enhanced by systemic administration of IGF-II.

Extinction is the process by which an experience (conditioned stimulus or CS; e.g. context

exploration) that was previously paired with an aversive stimulus (unconditioned stimulus, or

US; e.g. footshock) gradually becomes weaker following repeated re-exposures to the CS in the

67
absence of a US. Compared to vehicle, IGF-II injected 20 min before each daily extinction trial

(CS exposure, Figure 4i-k) significantly facilitated extinction, as shown by the lower freezing

levels (Figure 16i), and the higher rate of extinction (Figure 4j) in the IGF-II-injected mice. A

comparison of the last extinction trial confirmed that IGF-II significantly promotes extinction

(Figure 16k).

68
 a b c Tr Test 1 Test 2 d Tr Test 1 Test 2
Tr Test 1 Test 2 Tr Test 1 Test 2
4h 24h 4h 24h

Total exploration (s)

4h 24h 4h 24h
* ** 60
80 ** *** 80 90

% Preference
80
% Preference

% Exploration
70 40
70 70 60
50
60 60 40 20
30
20
50 50 10 0
Test 1 Test 2 Test 1 Test 2 Tr Test 1 Test 2
Veh IGF-II Veh IGF-II Veh IGF-II Veh IGF-II

e f g Test 1 Test 2 Test 3 h

Tr Test 1 Test 2 Tr Test 1 Test 2 Test 1 Test 2 Test 3

4h 24h 4h 24h 20m 25m 24h 20m 25m 24h

Total exploration (s)

Total exploration (s)

90 40 80 *** 150
80 **
% Preference
Exploration

70
60
30 *** 100
70
50 20
40 50
30 10 60
%

20
10 0 50 0
Tr Test 1 Test 2 Test 1 Test 2 Test 3 Test 1 Test 2 Test 3
Veh IGF-II Veh IGF-II Veh IGF-II Veh IGF-II

i-k Tr T1 E1 E2 E3 E4
24h 24h 24h 24h 24h
*
i j k ***
60 *** 80 ***
Rate of change (%)

% Freezing of Test 1

100
% Freezing

40
60 *
40 **
50
20 **
20
0 0 0
T1 E1 E2 E3 E4 T1 E1 E2 E3 E4 T1 E4
Veh IGF-II Veh IGF-II Veh IGF-II

Figure 16. IGF-II increases strength and/or persistence of non-aversive memories. Experimental schedules are
shown above each panel. (a) IGF-II enhances nOR memory, expressed as mean (± s.e.m.) % preference for a novel
object (n=9-10) and (b) enhances OP memory, expressed as mean (± s.e.m.) % preference for a displaced object
(n=6-8) in mice injected () with vehicle (Veh) or IGF-II 20 min before training, (c, e) without altering baseline
preference expressed as the mean (± s.e.m.) % exploration of the object that will be replaced/displaced or (d,f) total
exploration time expressed as the mean (± s.e.m.) time (s) spent exploring object. (g) IGF-II does not affect social
recognition 20 min after injection (Test 1) or immediate social memory tested 5m later (Test 2). However, it
enhances social learning memory tested at 24h (Test 3). Data are expressed as mean (± s.e.m.) % preference for a
novel mouse (n=10). (h). Total exploration time is not altered in social recognition task. Data are expressed as mean
(± s.e.m.) total exploration (s) of both test stimuli (n=10). (i-k). Schematic representation of the behavioral
extinction protocol. Tr: Training; T1: Test 1; E1-E4: Extinction trials 1-4. (i) IGF-II facilitates contextual fear
extinction, expressed as mean (± s.e.m.) % freezing (n=9-10). (j) IGF-II increases the rate of extinction, expressed
as mean (± s.e.m.) % rate of change from Test 1. Mice were injected with Veh or IGF-II 20 min before each
extinction trial. (k) Freezing levels at E4, expressed as mean (± s.e.m.) % freezing of T1. *P<0.05, **P<0.01,
***P<0.001. Experiments conducted in Figure 4a-h were conducted in collaboration with Amy Kohtz.

69
IGF-II potentiates memory when given with reactivation. Memory strength can be modulated

not only around the initial phase of training, but also by targeting retrieval or reactivation and the

post-retrieval process known as reconsolidation (Chen et al, 2011; Frenkel et al, 2005; Inda et al,

2011; Tronson and Taylor, 2007). I have previously shown that memory enhancement can also

be achieved by injecting IGF-II into the rat hippocampus immediately after inhibitory avoidance

retrieval (Chen et al, 2011). Using the nOR task, I asked whether IGF-II injected systemically

can enhance memory when given before memory reactivation. IGF-II or vehicle were injected 20

min before a nOR test (reactivation) given 4h after training (when vehicle-injected controls still

show memory). A control group of mice were injected with IGF-II in the absence of reactivation.

As shown in Figure 3g, IGF-II significantly enhanced memory retention 24h after training in

mice that underwent reactivation but not in those that did not. These results indicate that memory

reactivation re-engages mechanisms that enable IGF-II to enhance memory retention (Figure

17a), and suggest that multiple temporal windows for intervention exist, which are not limited to

new learning and initial memories. No changes in total exploration time during training or

testing were found (Figure 17b).

70
 Tr Reactivation Test
a

4h 24h
100 *** ***
% Preference 75

50
25 Veh
0 Tr Reactivation Test IGF-II
b noR-IGF-II
Total exploration (s)

25
20
15
10
5
0
Tr Reactivation Test

Figure 17. IGF-II potentiates memory when given with reactivation. (a) Mean % (± s.e.m.) preference for the
novel object in mice injected with either Veh or IGF-II 20 min before a reactivation given 4h after Tr. There was no
effect of IGF-II in the absence of reactivation (noR-IGF-II) (n=6). (b) There was no change in total exploration time,
expressed as the mean (± s.e.m.) time (s) spent exploring novel objects. ***P<0.001.

IGF-II enhances working memory, without changing behavioral flexibility. One concern

about memory enhancing treatments is whether the enhanced memory becomes inflexible

(Floresco and Jentsch, 2011 ). Hence, I determined whether the IGF-II-dependent memory

enhancement affected behavioral flexibility.

I first tested the rate of contextual extinction of mice that were injected with 15 μg/kg of

IGF-II before training and had an enhanced CFC memory, compared to that of vehicle-injected

controls (Figure 18a-c). Although IGF-II-injected mice maintained significantly higher freezing

throughout the extinction protocol (Figure 18a), both groups of animals extinguished at a similar

rate (Figure 18b-c), indicating that the IGF-II-enhanced CFC memory remains flexible and

similarly susceptible to extinction as a normal, non-enhanced memory.

71
 Furthermore, I employed a Y-maze paradigm to assess working memory, learning, long-

term memory, and reversal learning. Working memory can be evaluated by quantifying

spontaneous alternation, which is considered to be an index of active retrograde working

memory, because in a Y-maze, mice generally explore the least recently visited arm and thus

tend to alternate their visits between the three arms. Many brain regions, including the

hippocampus, septum, basal forebrain, and prefrontal cortex, play an important role in this task

(Lalonde, 2002). Compared to vehicle, an injection of IGF-II 20 min before spontaneous

alternation testing significantly increased the alternation rate, indicating that IGF-II enhances

working memory (Figure 18d). I then assessed reinforced spatial reference memory and reversal

learning, which measures the ability to adapt to changes and inhibit the previously learned

response (a form of behavioral inhibition). During habituation to the Y-maze, mice showed no

difference in preference for any one arm (Habituation, Figure 18e). Training began one day later

and consisted of two blocks of 5 trials per day, given once/day for 2 days with one arm of the Y-

maze baited with a food reward. Mice were injected with IGF-II or vehicle 20 min before the

first training session. Compared to vehicle, IGF-II significantly enhanced acquisition of the first

training session (A1, Figure 18e) and a significant enhancement persisted on the second day (A2,

Figure 18e). One day after the end of training, reversal learning was assessed by switching the

location of the food reward to the previously unbaited arm. Mice were exposed to this reversal

protocol for 2 blocks of 5 trials per day for 2 days, and both vehicle- and IGF-II injected mice

showed similar acquisition of the new location (R1 and R2, Figure 18e), confirming that IGF-II

enhances working, reference and long-term memories, without changing memory flexibility.

72
 a-c Tr T1 E1 E2 E3 E4 E5 E6 E7 E8

24h 24h 24h 24h 24h 24h 24h 24h 24h

a 80
b c **
** 80
** 140 *

% Freezing of Test 1
120

Rate of change (%)

60 60
100
% Freezing

n.s 80
40 40
60

20 *** 20 40
20
0 0 0
T1 E8
T1
E1
E2
E3
E4
E5
E6
E7
E8

T1
E1
E2
E3
E4
E5
E6
E7
E8
Veh IGF-II
Veh IGF-II Veh IGF-II

Test Habituation A1 A2 R1 R2
d e
24h 24h 24h 24h
20m
80 100
* 90
70 Veh
80 ***
% Alternation

IGF-II
% Correct

60 70
60
50
50

40 40
30 A1 A2 R1 R2
30 20
n

10

10
10

10
5
5

5
Tr 1-5
io

1-
1-

1-

Veh
6-

6-
6-

6-

IGF-II
at

ls
ls

ls
ls
itu

ls

ls
ls

ls
ia
ia

ia
ia

ia

ia
ia

ia
ab

Tr
Tr

Tr
Tr

Tr

Tr
Tr
H

Figure 18. IGF-II enhances memory without altering flexibility. (a-c) Schematic representation of the
behavioral schedule; Tr: Training; T1:Test 1; E1-E8: Extinction trials 1-8. (a) CFC memory retentions in IGF-II- or
vehicle (Veh)–injected (éé) mice extinguish at similar rates. Data are expressed as the mean (± s.e.m.) % freezing
(n=8). (b) No difference in the rate of extinction (%) expressed as the mean (± s.e.m.) rate of freezing change: [E-
T1]/T1 (c) or as the % freezing of E8/T1. (d) IGF-II enhances working memory (% alternation) in the spontaneous
alternation task, expressed as the mean (± s.e.m.) % alternation. Mice were injected with Veh or IGF-II 20 min
before being placed into the Y-Maze (n=7-8). (e) Spatial reference task in Y maze. Data are expressed as the mean
(± s.e.m.) % correct entry choice of mice in the Y maze (baited arm). A1-A2: Acquisition sessions 1-2; R1-R2:
Reversal sessions 1-2; Each Acquisition or Reversal session refers to a block of 5 trials (n=7-8) (E). *P<0.05,
**P<0.01, ***P<0.001. Experiments shown in Figure 6d-e were conducted in collaboration with Amy Kohtz.

73
MATERIALS AND METHODS

Animals. Adult male C57Bl/6J mice (6-10 weeks of age, Jackson Laboratory, Maine, USA)

were group housed (except for mice that underwent hippocampal cannulation, who were singly

housed following surgery) on a 12 hr light/dark cycle, with ad libitum access to food (except for

the Y-Maze experiment) and water. Experiments were performed during the light cycle. All

protocols complied with the NIH Guide for the Care and Use of Laboratory Animals and were

approved by the New York University Animal Welfare Committee.

Drugs. Recombinant mouse IGF-II (R&D, Minnesota, USA cat # 792-MG) was dissolved in

0.1% bovine serum albumin-phosphate-buffered saline (0.1% BSA-PBS, pH 7.4), and

administered subcutaneously (s.c.) in 0.3 ml. Anti-IGF-II receptor antibody (anti-IGFIIR) or IgG

control (both from R&D) were dissolved in 1× PBS and injected bilaterally into the

hippocampus at 20ng/μl. This concentration blocks 90% of binding to the IGF-II receptor in an

in vitro binding assay (R&D).

Contextual/Auditory Fear Conditioning (CFC and AFC) and Extinction. Mice were handled

for 2-3 minutes (min) per day for 5 days (d) before training. The conditioning chamber consisted

of a rectangular Perspex box (30.5 × 24.1 × 21.0 cm) with a metal grid floor (Model ENV-008

Med Associates, St. Albans, Vermont, USA) through which foot shocks were delivered via a

constant current scrambler circuit. Freezing, defined as lack of movement besides heart beat and

respiration, was recorded every 10th second (sec) by trained observers blind to the experimental

conditions. The number of scores indicating freezing were calculated as a percentage of the total

number of observations (Schrick et al, 2007).

74
CFC was carried out as described (Guedea et al, 2011). An unsignalled 2-sec 0.7 mA footshock

was delivered after two minutes in the chamber, after which the mouse remained for one more

minute. During testing, the mouse was placed back in to the conditioning chamber for 3 min in

the absence of a footshock. AFC procedure was modified from Han et al. (Han et al, 2009). After

2 min in the chamber, mice received one tone-shock pairing (30-sec,tone coterminating with a

0.7 mA footshock) and one minute later, were returned to the homecage. Memory was tested in

an alternate context. During testing, after one min in the new context (pre-tone) the tone was

played for 2 min. A blind experimenter scored all of the experiments and memory was measured

as the % time spent freezing during the 2-min tone, or during the pre-tone. CFC extinction was

performed as described (Guedea et al, 2011). One day following CFC training, mice were placed

into the conditioning chamber for 3 min each day for 5 or 9 days.

Spontaneous Alternation. Carried out as previously described (Mandillo et al, 2008). Y-Maze

consisted of 3 black polycarbonate arms (7.62 x 12.7 x 38.1 cm) with wells at the end of 2 arms

for food rewards (1.93 cm diameter x 1.27 cm deep). Mice were allowed to freely explore from

the center of the maze for 10 min. Spontaneous alternation was defined as successive entries into

each of the three arms on overlapping triplet sets (e.g., ABC, BCA, CAB, etc.). Percentage

alternation was defined as the ratio of actual alternations (total alternations) to possible

alternations (total arm entries − 2) × 100.

Spatial Reference Memory in the Y-Maze. Carried out as previously described (Van der

Borght et al, 2007). Mice were single-housed and food restricted for one week prior to and

during testing by giving ½ food pellet (Purina Lab Diet® 5001, Missouri USA) plus 1 fruit loop

75
(Kellogg’s) each day. Habituation was identical to spontaneous alternation. Acquisition – one

arm (counterbalanced between animals) was designated as the “correct” arm and baited with a

fruit loop, which the mouse could eat. Mice were enclosed in the start arm for 1 min, and then

permitted to choose between the two arms. Acquisition consisted of two blocks of 5 trials (10

trials total) per day for two days. Learning and memory were calculated as the % correct arm

chosen over each block of trials. Reversal was identical to acquisition, with the “correct” arm

switched. An observer blind to conditions scored all of the experiments.

Novel-Object Recognition (nOR) and Object Placement (OP). Adapted from (Dix et al,

1999). Mice were handled for 2-3 min each day for 5d before training. Objects were

counterbalanced during training and testing. Mice were trained in a clean square homecage, free

of bedding, containing two identical objects (Mega Bloks 120), and were permitted to interact

freely for 3 min (nOR) or 5 min (OP). Four and 24 hours (h) later, mice were placed back into

the homecage, and one object was replaced with a novel object (nOR) or one object was moved

to an alternate location (OP). In both cases, one object remained constant throughout all of

training and testing, while the other was replaced. Memory was measured as the % time spent

interacting with the novel object over 3 min (nOR) or with the object in a new location over 5

minutes (OP). An observer blind to conditions scored all of the experiments.

Social Interaction in the Open-Field. Carried out as previously described (Kwon et al, 2006;

Satoh et al, 2011). Mice were handled for 2-3 min a day for 5d prior to testing. On each side of a

clean square cage a rectangular wire-holder was placed that would contain either a novel object

or a stimulus mouse (C57Bl/6J). Stimulus mice were habituated to the wire-holders the day

76
before testing to prevent excessive movement. On testing day, test mice were habituated to the

cage with empty wire-holders for 10 min. During each subsequent phase the test mouse was

placed into the cage and allowed to interact freely for 5 min. The order of presentation was as

follows. Test 1: a mouse (novel mouse 1) and novel object. Test 2: The object was immediately

replaced with a novel mouse (novel mouse 2). Test 3: Twenty-four hours later: novel mouse 1

and a third novel mouse (novel mouse 3). Sociability was measured as the % time spent

interacting with the mouse vs. the object. Immediate and long-term social memories were

measured as the % time spent interacting with the novel mouse. An observer blind to conditions

scored all of the experiments.

Open Field. Mice freely explored an open field arena (43.2 x 43.2 x 30.5 cm) from Med

Associates (ENV-515), lit at 210 lux and divided into 16 quadrants, for 5 min. Total quadrant

entries were calculated as locomotion in the open field. The # of entries in the four center

quadrants, as well as the time spent (in seconds) in the four center quadrants were taken as

measures of anxiety. An observer blind to conditions scored all of the experiments.

Locomotor Activity. Mice freely explored a rectangular (20.3 cm x 15.9 cm x 21.3 cm) Perspex

Med-Associates box (ENV-010MD) with 8 infrared beams for 15 min. The number of beam

breaks was recorded throughout the session automatically.

Observational Battery. Carried out as previously described (Crawley and Paylor, 1997; Paylor

et al, 1998) at designated time-points after injection (30 min, 24h and 7d). Body temperature was

taken with a digital rectal probe (Harvard Apparatus) and physical characteristics were recorded.

77
The mouse was then observed in an empty cage for 1 min, where general behavioral observations

were recorded. Sensorimotor reflexes and simple motor responses were then tested in the order

described in Table 1. The wire suspension test measured the latency of a mouse to fall when

suspended upside-down about 20 mm over an empty cage from a wire cagetop. In the vertical

pole test, a mouse was placed facing up on a cloth-tape-covered pole (1.5 cm diameter, 40 cm

long), which was lifted to a vertical position for 1 minute. The pole test score was calculated as

follows: Fell before the pole reached 45° or 90° angle: 0 or 1, respectively; fell in 0-10 sec: 2,

11-20 sec: 3, 21-30 sec: 4, 31-40 sec: 5, 41-50 sec: 6, 51-60 sec: 7; stayed on 60 sec and climbed

halfway down: 8; climbed to lower half of pole: 9; climbed down and off in: 51-60 sec: 10, 41-

50 sec: 11, 31-40 sec: 12, 21-30 sec: 13, 11-20 sec: 14, 1-10 sec: 15. An observer blind to

conditions scored all of the experiments.

Glucose Measurements. Tail blood was taken at designated time-points after injection (30 min,

12h, 24h and 7d). Glucose was measured using the Accu-Check Aviva Plus monitoring system

(Roche, San Francisco, USA).

Cannula Implants and Drug Injection. Performed as described previously (Fernandez et al,

2008). Mice were anesthetized with ketamine (100mg/kg) and xylazine (75mg/kg) and guide

cannula (C232GC; 22 gauge; Plastics One) were directed toward the hippocampus (−1.7 mm

posterior to bregma, ±1.5 mm lateral to midline, −2.3 mm ventral to skull surface) using a

stereotaxic apparatus (Kopf Instruments). Mice recovered for at least 5d before testing. Drugs

were delivered in 0.25 μl over 45 seconds via injection cannula (26 gauge, extending 0.8 mm

beyond the 1.5 mm guide) attached to polyethylene tubing (PE50) connected to a 10 μl Hamilton

78
syringe and controlled by a microinfusion pump (Harvard Apparatus). Cannulae placement was

verified at the end of the behavioural experiments, following fixation of the brains in 10%

formalin. Forty-micrometer coronal sections were cut through the hippocampus, stained with

cresyl violet and examined under a light microscope. All surgeries correctly targeted the

hippocampus.

Western Blot Analysis. Western blot analysis was done as reported previously (Chen et al,

2011). Protein extracts from dorsal hippocampus and prefrontal cortex (including anterior

cingulate cortex, prelimbic cortex and infralimbic cortex) were obtained. Equal amounts of total

protein (10-20μg per lane) were resolved on denaturing SDS–PAGE gels and transferred to

Immobilon-FL membranes (Millipore) by electroblotting. Primary antibodies: anti-Arc (1/1,000,

Synaptic Systems), anti-Zif268/EGR1 (1/1,000, Cell Signaling) or anti-actin (1/10,000, Santa

Cruz Biotechnology). Secondary antibodies: anti-rabbit IRDye800CW and anti-mouse

IRDye680 (1/10,000, Li-Cor). Membranes were scanned on the Li-Cor Odyssey imager under

non-saturating conditions. Data were quantified using pixel intensities with Odyssey software

according to the protocols of the manufacturer (Li-Cor, Nebraska, USA).

Statistical analysis. One- or two-way analysis of variance (ANOVA) followed by either the

Dunnetts, Newman–Keuls, or Bonferroni post hoc test or Student’s t-test were used for statistical

analyses.

79
 CHAPTER V.

SYSTEMIC TREATMENTS OF IGF-II RESCUE AUTISM PHENOTYPES IN BTBR MICE.

ABSTRACT
Autism spectrum disorder is a developmental disability characterized by altered social

interaction and cognitive functions as well as increased repetitive behavior. The etiology of ASD

is still unknown and several genetic variations and mechanisms have been proposed to be

associated with this disorder, possibly because the diagnosis of ASD is mainly based on

symptoms. Thus far, there is no efficacious therapy that can ameliorate most of the major ASD

symptomatology and the identification of potential novel targets and therapies is urgently

needed. Here I show that the polypeptide insulin like growth factor II, which acts as cognitive

enhancer in physiological conditions, rescues all major deficits in a well-characterized mouse

model of ASD. Indeed, social interaction and cognition, long-term memory and repetitive

behavioral deficits are all rescued by systemic IGF-II treatment. The treatment does not affect

locomotor or anxiety-like behaviors. I propose that IGF-II represents a novel potential therapy

for treating ASD.

80
INTRODUCTION

Autism, generally defined as autism spectrum disorder, is a neurodevelopmental disorder

characterized by impaired in social interactions, deficits in communication, and restrictive and

repetitive patterns of behaviors and interests (McFarlane et al, 2008; Volkmar et al, 2009). While

only about half of the children with an autism spectrum disorder also show cognitive

impairments with an IQ score under 70 (Charman et al, 2011), autistic subjects generally have

severely impaired social cognition and social cognitive abilities, as well as problems in dividing

attention and processing the “whole”, and instead focus more on details (Lord et al, 2000;

Volkmar et al, 2009); Subjects with ASD rarely make eye contact or show joint attention. They

also have low imitation skills, and an increased attention to inanimate objects rather than social

ones (Davies et al, 2011; McPartland et al, 2011)

Social cognition, and in general social cognitive abilities, are based on learning through

observation, and include an understanding of the intentions and emotions of others during social

interactions, suggesting that ASD subjects are impaired in learning and cognition (Frith and

Frith, 2012). Hence a number of studies suggest that autism is characterized by deficits in

learning and cognitive processing, and indicate a need for cognitive enhancement as a

therapeutic intervention for ASD. Cognitive enhancers, in fact, may also provide rescuing of

attentional abilities, the ability to understand social cues, and executive functions. Unfortunately

cognitive enhancers or other efficacious treatments for ASD are still lacking; thus, preclinical

and clinical investigations that may identify novel therapies are greatly needed.

In order to identify novel treatments, preclinical investigations in animal models of ASD

are critical. Several mouse models exhibiting one or more behavioral deficits characteristic of

autism have been generated by either forward or reverse genetic approaches, (Provenzano et al,

81
2012; Silverman et al, 2010b). Among these, one inbred strain, the BTBR mice, recapitulates all

core behavioral deficits of ASD and is thus considered a representative model for translational

evaluations (MacPherson et al, 2008; McFarlane et al, 2008; Moy et al, 2007; Roullet and

Crawley, 2011). BTBR mice exhibit normal general health and physical abilities (Moy et al,

2007) but show cognitive impairments, which include deficits in fear conditioning, object

recognition, spatial learning and probabilistic reversal learning. They also have social deficits

including reduced social approach, social interaction, juvenile play and social transmission of

food preference, reduced ultrasonic vocalizations in social settings, and high levels of repetitive

behaviors including self-grooming and marble burying (McFarlane et al, 2008; Pobbe et al,

2011; Scattoni et al, 2008; Silverman et al, 2010a). BTBR mice lack a corpus callosum, have

anatomical alterations of the hippocampus, and spontaneous deletion of the disrupted in

schizophrenia-1 (DISC-1) gene (Stephenson et al, 2011; Wahlsten et al, 2003).

Because the lack of corpus callosum is fully penetrant, BTBR also offers a unique model

for investigating the influence of callosal abnormalities. Evidence suggests that callosal

abnormalities are associated with ASD. Human callosal abnormalities are fairly frequent, having

a reported frequency in the range of 0.7 to 5.3% in the USA. Although a portion of these patients

is asymptomatic, the rest of the subjects with callosal abnormalities have reported cognitive and

social impairments (Badaruddin et al, 2007; Paul et al, 2007). Moreover, neuroimaging studies

reveal that reduction in the volume of the corpus callosum is one of the most consistent finding

autistic patients (Frazier and Hardan, 2009).

Recent studies suggest that some symptoms of ASD deficits can be reversed by

behavioral intervention and/or pharmacotherapy (Gould et al, 2011; Silverman et al, 2012;

Silverman et al, 2010a; Yang et al, 2009). Although behavioral therapies have been the most

82
promising avenue thus far, they require chronic management and thus carry a significant

financial load for the families and society. Furthermore, most patients will continue to exhibit

symptoms throughout their lives (Myers and Johnson, 2007). Most current medication is

prescribed off-label, including antidepressants given for repetitive behaviors and social anxiety,

antipsychotics for severe behavioral problems such as aggression or self-injurious behaviors, or

psychostimulants to combat hyperactivity (Leskovec et al, 2008). None of these have been

effective at relieving all of the core autism symptoms, and many exacerbate other aspects of the

disease which limit usefulness, for example, increased tics in patients given psychostimulants.

Hence, thus far, no treatment appears to be identified that can effectively reverse all major

autism symptoms.

We have recently found that the administration of the growth factor IGF-II, which

belongs to the insulin system and remains relatively highly expressed in the brain throughout

adulthood if compared to the other members, dramatically enhances contextual fear conditioning

memory if injected directly into the hippocampi of rats (Chen et al, 2011). Injection of IGF-II in

the hippocampus of mice enhances fear extinction (Agis-Balboa et al, 2011). Furthermore, a

systemic injection of IGF-II significantly enhances several cognitive functions, including

executive functions as well as social recognition memory in C57BL/6J (B6) mice (see previous

Chapter). The effect is rapid and persistent. Importantly, as IGF-II crosses the BBB, it provides a

unique opportunity for potential, rapid translational applications. Because of the striking effects

as a cognitive and social learning enhancer, here I asked whether systemically administered IGF-

II affects, or even perhaps ameliorates, behavioral functions of ASD. Toward this end, I tested

the effects of a subcutaneous (s.c.) administration IGF-II to either adult male BTBR or, as

controls, B6 mice, which is a standard inbred strain of mice that have normal social behavior,

83
low repetitive behaviors, as well perform highly on most cognitive tasks. The dose and time of

IGF-II administration were chosen on the basis of the studies in the previous chapter in B6 mice

showing a dose-response curve and profound cognitive enhancing effects. Detailed statistics for

the results presented in this chapter can be found in Table 5.

RESULTS

IGF-II rescues social deficits in a mouse model of ASD. One of the hallmarks of ASD is a

deficit in social skills (Lord et al, 2000). BTBR mice faithfully model this phenotype, as they

exhibit deficits in both sociability and social memory (McFarlane et al, 2008). BTBR and B6

mice were injected s.c. with 30 μg/kg of IGF-II 20 minutes before testing their social preference.

This dose was selected because it emerged as the optimal dose able to significantly enhance

memory and executive function in B6 mice. In agreement with previous literature, vehicle-

injected B6 mice spent significantly more time with the stranger mouse compared to the object,

whereas vehicle-injected BTBR mice did not show a preference and spent similar amounts of

time investigating both targets (McFarlane et al, 2008) (Test 1, Figure 19a). This social deficit in

BTBR did not reflect an olfactory impairment or inability to detect novelty, because 5m after

Test 1 the same BTBR mice spent significantly more time investigating a novel mouse compared

to the one just seen (Test 2, Figure 19a). Notably, IGF-II completely rescued the social deficit of

BTBR mice: in fact, the IGF-II-injected BTBR mice spent significantly more time with the

stranger mouse compared to the novel object (Test 1, Figure 19a). Furthermore, both vehicle-

injected B6 and BTBR mice had no preference for the mouse they had encountered earlier when

exposed to a novel vs. a previously encountered mouse 24hr after Test 1. In contrast, IGF-II-

injected BTBR mice still displayed significant social recognition memory, as did IGF-II injected

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B6 mice, as was shown in the previous chapter (Test 3, Figure 19a). The effect was not due to

changes in motivation or other general abilities to explore because the total exploration times for

all groups were similar a at both Test 1 and Test 3 where differences among preferences were

found (Figure 19b). Hence, IGF-II rescues the social interaction deficits and increases social

memory in BTBR mice.

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 Test 1 Test 2 Test 3

20m 25m 24h

a 
***
***
*** *** **
**
*** *** *
**


% Preference





Test 1 Test 2 Test 3
***
b ** **



Total exploration (s)









Test 1 Test 2 Test 3

B6 -Veh B6 -IGF-II BTBR-Veh BTBR-IGF-II

Figure 19. IGF-II rescues social recognition deficits in BTBR mice. Experimental timeline is shown above
graphs. (a) IGF-II rescues social recognition deficits (Test 1) and enhances social memory (Test 3) in BTBR mice
injected (arrow) with IGF-II 20m before social recognition testing compared to vehicle (Veh). IGF-II does not
change % preference in BTBR mice 5 min after Test 1 (Test 2). Data are expressed as the mean (± s.e.m.) %
preference for a novel mouse (n=7-8). (b). IGF-II does not affect total exploration time during the social recognition
task. Data is expressed as the mean (± s.e.m.) total time in seconds (s) spent with stimuli in BTBR mice injected (éé)
with vehicle (Veh) or IGF-II or B6 mice injected with vehicle 20m before social recognition testing. *P<0.05,
**P<0.01, ***P<0.001. Experiments shown in Figure 1 were conducted in collaboration with Amy Kohtz.

86
IGF-II reduces repetitive behaviors in BTBR mice.

Restricted and repetitive behaviors are another core symptom of ASD (Veenstra-VanderWeele

and Blakely, 2012). Because IGF-II can affect behavioral responses in a short-term temporal

scale, as revealed by our findings that short-term memory and working memory are significantly

enhanced by treatment (see Chapter 4) I tested whether 30 μg/Kg of a s.c. injection of IGF-II

twenty minutes before tasting could affect repetitive behavior. I therefore investigated

stereotyped repetitive behaviors in BTBR and B6 mice using the marble-burying task. As

previously established, vehicle-injected BTBR mice engage in repetitive behaviors, as they spent

more time burying and buried significantly more marbles than B6 mice (Amodeo et al, 2012).

IGF-II did not change the time spent burying or the number of marbles buried by B6 in a marble

burying test 20m after vehicle or IGF-II injection, but did attenuate the time spent burying in

BTBR mice tested at the same time. When tested again 24h later, there was no effect of IGF-II

on B6 mice, but a significant reduction in both the time spent burying marbles (Figure 2a) and

the number of marbles buried (Figure 2b) of BTBR mice, which became similar to those of B6

controls, showing that IGF-II reduces repetitive behavior in BTBR mice.

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 Test 1 Test 2

20m 24h

a b **
400 8
*** *** ** ** *

Marbles buried (#)

*** *** *** ** **
6
Burying time (s)

300

200 4

100 2

0 0
Test 1 Test 2 Test 1 Test 2
B6-Veh B6-IGF-II BTBR-Veh BTBR-IGF-II

Figure 20. IGF-II reduces a repetitive behavior in BTBR mice. Experimental timeline is shown above graphs.
IGF-II reduces excessive marble burying of BTBR mice (n=5-7). BTBR and B6 mice were injected (éé) with Veh or
IGF-II and tested 20m (Test 1) and 24h (Test 2) later on the marble burying task. (a) Data are expressed as the mean
(± s.e.m.) time spent burying in seconds (s). (b) Data are expressed at the mean (± s.e.m.) number of marbles buried.
*P<0.05, **P<0.01, ***P<0.001. Experiments shown in Figure 2 were conducted in collaboration with Amy Kohtz.

88
IGF-II rescues memory deficits in BTBR mice.

Approximately 1/3 of children diagnosed with ASD have a comorbid intellectual

disability (Rorive et al, 2008). To determine whether IGF-II can rescue cognitive deficits in a

mouse model of ASD, I next tested the effect of IGF-II on the known deficits in contextual fear

conditioning of BTBR mice (MacPherson et al, 2008). Mice received a s.c. injection of IGF-II

20 minutes before training and were tested 24 h after training. In line with previous findings, we

found that vehicle-injected BTBR mice had impaired long-term contextual fear conditioning

memory retention compared to vehicle- and IGF-II-injected B6 (Figure 3). Notably, an injection

of IGF-II 20m before training completely rescued the memory deficit, measured at 24h after

training (Figure 3), and IGF-II injected BTBR mice displayed similar memory retention at

vehicle-injected B6 mice.

Tr Test

24h

*
*
80 ** *** B6-Veh
** B6-IGF-II
60
% Freezing

BTBR-Veh
40 BTBR-IGF-II

20

Figure 21. IGF-II rescues memory deficits in BTBR mice. Experimental timeline is shown above graph. IGF-II
rescues CFC memory deficit. Data are expressed as the mean (± s.e.m.) % freezing in BTBR mice injected (arrow)
with vehicle (Veh) or IGF-II and B6 mice injected with Veh 20m before training (Tr) and tested 24h after training
(n=6). *P<0.05.

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IGF-II does not alter anxiety levels in BTBR mice.

It has been suggested that altered anxiety contributes to the BTBR behavioral phenotypes (Pobbe

et al, 2011), and it is controversial whether or not the autistic-like effects of BTBR are due to

altered stress and anxiety (Silverman et al, 2010c). Hence, I investigated the effects of a s.c

injection of 30 μg/Kg of IGF-II on open field and elevated plus maze, tests that investigate the

natural aversion of rodents to open space and are routinely used to determine anxiety behavior in

rodents (Bailey and Crawley, 2009).

Compared to vehicle-injected B6 mice, vehicle-injected BTBR mice made less center

entries (Figure 4a) and spent less time in the center of the open field (Figure 4b). Vehicle-

injected BTBR mice also showed a strong trend towards increased total entries in the open field

(Figure 4c). These results suggest that BTBR mice have a higher level of anxiety behavior as

detected by open field. Compared to vehicle, IGF-II did not change number of entries into the

center, time spent in the center or total entries (Figure 4a-c) of BTBR mice in the open field

arena, indicating that it had no effect on exploratory locomotion or general anxiety aspects.

Compared to vehicle-injected mice, vehicle-injected BTBR mice and IGF-II injected

BTBR mice spent more time in the open arms of the elevated plus maze (Figure 4d). These

results would suggest that the BTBR mice are less anxious than B6 mice if anxiety is measured

according to an elevated plus maze. However, as there was no difference between vehicle- and

IGF-II-injected BTBR mice, indicating that IGF-II did not alter their general anxiety this task.

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 Test

20m

a ** b c
20 ** 40 * 300
% Center entries

Center time (s)

30

Total Entries
15 200

10 20
100
5 10

0 0 0

d
150 **
Time in open arms (s)

** B6-Veh
100 BTBR-Veh
BTBR-IGF-II
50

Figure 22. IGF-II does not alter anxiety responses in BTBR mice. Experimental timeline is shown
above graphs. There was no change in anxiety-like behavior of BTBR mice in open field (n=5-6) or in the elevated
plus maze (n=10-13). BTBR mice were injected (arrow) with vehicle (Veh) or IGF-II, and B6 mice were injected
with Veh 20m before open-field or elevated plus maze testing. (a) Data is presented as the mean (± s.e.m.) number
of entries into the center of the open field. (b) Data is presented as the mean (± s.e.m.) time in seconds (s) spent in
the center of the open field. (c) Data is presented as the mean (± s.e.m.) total # of entries in the open-field. (d). Data
is presented as the mean (± s.e.m.) time in seconds (s) spent in the open arms of the elevated plus maze. *P<0.05,
**P<0.01. Experiments shown in Figure 4 were conducted in collaboration with Amy Kohtz.

91
MATERIALS AND METHODS

Animals

Adult male C57Bl/6J or BTBR T+ Itpr3tf/J mice (6-10 weeks of age, Jackson laboratories, Maine

USA) were maintained, group housed, on a 12 hr light/dark cycle, with ad libitum access to food

and water. Experiments were performed during the light cycle. All protocols complied with the

NIH Guide for the Care and Use of Laboratory Animals and were approved by the NYU

University Animal Welfare Committee.

Drugs

Recombinant mouse IGF-II (R&D, Minnesota, USA) was dissolved in 0.1% bovine serum

albumin-phosphate-buffered saline (0.1%BSA-PBS, pH 7.4), and administered at 30 mmg/Kg

subcutaneously in 0.3 ml.

Contextual Fear Conditioning (CFC)

Mice were handled for 2-3 minutes per day for 5 days before training. The conditioning chamber

consisted of a rectangular Perspex box (30.5×24.1×21.0 cm) with a metal grid floor (Model

ENV-008 Med Associates, St. Albans, Vermont) through which foot shocks were delivered via a

constant current scrambler circuit. CFC training was carried out as described (Guedea et al,

2011). After two minutes in the chamber, an unsignalled 2-sec 0.7 mA footshock was delivered.

After an additional minute in the chamber, the mouse was returned to its homecage. Freezing

was measured at 24h and 7d after training by placing the mouse back in to the conditioning

chamber for 3 minutes in the absence of a footshock. Freezing, defined as lack of movement

besides heart beat and respiration, was recorded every 10th second by trained observers unaware

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of the experimental conditions. Freezing scores were calculated and expressed as a percentage of

the total number of observations.

Social Interaction in the Open-Field

Carried out as previously described (Satoh et al, 2011). Mice were handled for 2-3 minutes a day

for 5 days prior to social interaction testing. On each side of a clean square cage a rectangular

wire-holder was placed that would contain either a stimulus mouse (C57Bl/6) or a novel object.

Stimulus mice were habituated to the wire-holders the day before testing to prevent excessive

movement. On testing day, test mice were habituated to the cage with empty wire-holders for 10

minutes. During each subsequent phase the test mouse was placed into the cage and allowed to

interact freely for 5 minutes. Phase 1: a mouse (novel mouse 1) and novel object were placed

under the wire holders. Phase 2: The object was immediately replaced with a novel mouse (novel

mouse 2). Phase 3: Twenty-four hours later, novel mouse 1 was placed under a wire-holder with

a third novel mouse (novel mouse 3). Sociability was measured by a blind observer as the % time

spent interacting with the mouse vs. the object. Immediate and long-term social memories were

measured by a blind observer as the % time spent interacting with the novel mouse.

Marble Burying

Carried out as previously described (Deacon, 2006). Empty homecages were filled with 5cm of

bedding, on top of which 12 marbles were placed in 4 rows of 3 marbles each. The number of

successfully buried marbles was counted after the mice had spent 15m in the cage under a red

light. Digging was defined as coordinated movements of fore or hind limbs that displace the

substrate. A marble was defined as “buried” when <25% of the marble was visible. Video

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sessions were recorded, and both the time spent burying and number of buried marbles were

scored by a blind observer.

Open Field

Mice were allowed to freely explore an open field arena (43.2 cm x 43.2 cm x 30.5 cm) from

Med Associates (ENV-515) designated into 16 quadrants for 5 minutes. Total quadrant entries

were calculated as locomotion in the open field. Anxiety was assessed as the # of entries in the

four center quadrants, as well as the time spent (in seconds) in the four center quadrants.

Elevated Plus Maze

Statistical analysis

One- or two-way analysis of variance (ANOVA) followed by either the Dunnetts, Newman–

Keuls, or Bonferroni post hoc test or Student’s t-test was used for statistical analyses.

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 CHAPTER VI.
DISCUSSION

Cognitive enhancement remains a critical challenge in the treatment of many disease

populations, as well as for young and aging adults. In this thesis, I have focused on a particular

group of growth factors known as IGFs that could prove clinically beneficial, and have studied

their effectiveness in enhancing a number of types of memories over different timespans.

6.1. IGF-II persistently enhances hippocampal-, but not amygdala-, dependent memory.

IGFs have been well established to play a role in the CNS and have recently begun to be

studied as potential mediators of cognitive functions (Agis-Balboa et al, 2011; Alberini et al,

2012; Chen et al, 2011; Fernandez et al, 2012b; Russo et al, 2005). However, very little was

known about the specific role that each IGF plays in the enhancement of different types of

memories (ie. hippocampal vs. amygdala dependent, aversive vs. non-aversive), as well as how

long-lasting those effects would be. I tested the effects of IGF-II, Insulin and IGF-I on both

hippocampal and amygdala-dependent memories and found that when injected into the

hippocampus, IGF-II had a robust and persistent effect on hippocampal-dependent memories

such as IA and CFC that lasted up to three weeks. In contrast, hippocampal injections of insulin

led to only a transient increase in memory retention that disappears between 1 and 7 days after

training. Interestingly, neither insulin nor IGF-II had any effect on amygdala-dependent

memories, and IGF-I had no effect on either hippocampal- or amygdala-dependent memories.

These results are interesting in light of the different receptors that are targeted by the

IGFs. Each IGF targets its own cognate receptor with highest affinity, but can bind some or all of

the other receptors with lower affinity (See Fig. E). The fact that the different IGFs lead to

95
different effects on behavior suggests that they do not all bind to the same receptor to produce

these effects, and that the behaviors are likely a result of the different downstream signaling

cascades from the different receptors. I found that IGF-II mediated memory enhancement

requires the IGF-II receptor, but not the IGF-I receptor. This, however, does not rule out a

possible partial contribution of the Insulin receptors to IGF-II mediated memory enhancement.

IGF-II binds with relatively high affinity to IR-A, which is preferentially expressed on neurons

and mediates growth-related responses in a similar manner to IGF-I and binds with low affinity

to IR-B, the typical IR, which is preferentially expressed on glia and mediates metabolic

responses (Belfiore et al, 2009). At least one study has shown that IGF-II and Insulin lead to

divergent effects upon binding to IR-A, mitogenic and metabolic, respectively (Frasca et al,

1999; Morrione et al, 1997), indicating that it is possible that IGF-II may lead to memory

enhancement via IR-A, as well as via the IGF-IIR. At this time there are no specific inhibitors to

the different IR isoforms, and thus this question requires further study. Nevertheless, the

extension of memory enhancement by IGF-II to longer timepoints, whereas insulin leads to

transient responses, suggests that at least the later components of the memory enhancement are

mediated specifically by the IGF-IIR.

None of the IGFs that I tested had a significant enhancing effect on amygdala-dependent

memories, even when injected directly into the amygdala. This suggests that IGF-II is more

effective in enhancing hippocampal/cortical types of declarative memories and executive

functions rather than amygdala-dependent, Pavlovian types of responses. However, the manner

by which IGF’s exert this selectivity is currently unknown. Most studies indicate that all of the

IGF receptors are detected in the amygdala, albeit with slightly lower levels than in the

hippocampus (Hawkes and Kar, 2004). However, it is already known that divergent mechanisms

96
exist in the hippocampus and amygdala to regulate memory functions. For example, amygdala,

but not hippocampus, is required for AFC memory (Phillips et al, 1992). Protein synthesis is

required in the amygdala, but not the hippocampus, for IA reconsolidation (Taubenfeld et al,

2001; Tronel et al, 2005). Investigation of whether IGF-II is induced in the amygdala after

training would provide further insight into whether the amygdala is involved at all in the

regulation of memory by IGF-II.

Insulin transiently enhances hippocampus-dependent memory.

The question of why insulin-mediated memory enhancement is transient is also one that

requires further investigation. Considering that IR-A and IGF-IR mediate similar responses on

growth and synaptic plasticity (Belfiore et al, 2009) and that IGF-I has no effect on memory, one

can speculate that the effect of insulin may instead be due to changes in metabolic regulation

after insulin binding to the “typical” IR-B on astrocytes. In the periphery as well as in the

hippocampus, insulin also leads to translocation of the neuronal insulin-sensitive glucose

transporter type 4 (GLUT4) to the plasma membrane (Grillo et al, 2009), which further leads to

increased glucose uptake into neurons, also suggestive of a metabolic mechanism. Glucose itself

seems to lead to a transient memory enhancement (Suzuki et al, 2011), suggesting that indeed,

insulin may enhance memory primarily via glucose regulation. Insulin-analogues that bind

preferentially to one receptor isoform over the other would be greatly beneficial to elucidating

this, although the existence of hybrid IR/IGF-IR that may also be IR-isoform specific

complicates these speculations even further.

IGF-II in the anterior cingulate enhances remote memory.

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 IGF-II modulates memory strength not only at recent timepoints, but also at remote

timepoints. IGF-II enhances IA memory retention regardless of whether it is injected into the

aCC immediately after training or 2 weeks after training. Interestingly, when injected into the

aCC immediately after training, IGF-II only enhances memory at remote timepoints, but not at

24h or 7d after training. This suggests that the aCC is actively engaged early on, but that the

effects are only seen after a certain amount of time (between 1 and 2 weeks) has passed. This is

also suggested by recent literature showing that c-fos and Arc are induced early after training

(Holloway et al, 2011; Zhang et al, 2011), even though inactivation of the aCC itself only

impairs remote memory. The mechanisms by which the aCC maintains memory, but does not

modulate it’s strength, over the initial 1-2 weeks after training is an intriguing question that

further study would elucidate, but it is possible that although the aCC is activated right away by

training (as measured by IEG induction), the timescale by which changes occur is significantly

slower than that of the hippocampus.

6.2 IGF-II rapidly enhances memories via systemic injection

IGF-II crosses the BBB and is thus well suited to be administered peripherally in a

clinical setting. I found that IGF-II injected s.c. before training led to the enhancement of both

aversive and non-aversive hippocampus-dependent tasks, but not amygdala-dependent tasks.

This confirms our previous data that IGF-II enhances hippocampal-dependent memories

specifically, but adds new information, that the fear-based component of the CFC and IA tasks

are not critical parameters for memory enhancement. I also found that IGF-II enhances memory

very rapidly, as both short-term CFC memory and spatial reference memory in the Y-maze show

enhancement within 20m to 1 hour after injection. This indicates that the mechanisms by which

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IGF-II enhances memory occur very soon after training and/or injections. Another indication that

this is the case is that systemic injections of IGF-II before training enhance both short-term

memory and long-term memory for up to 7 days. However, if injected systemically after training,

IGF-II does not enhance STM and only enhances LTM at 24h, but not 7d after training. This

suggests that there are critical mechanisms that occur immediately after training upon which

IGF-II must act, and that in the time IGF-II takes to pass the BBB when injected after training,

this critical period has passed.

To that end, I found that IGF-II injected in naïve mice leads to an increase in two IEG’s,

Arc and Zif268 in the hippocampus at 1 hour after injection. Interestingly, however, IGF-II did

not further enhance the training-induced expression of either of these IEG’s when injected before

CFC training, Similarly, in the prefrontal cortex, IGF-II injected before training leads to a

decrease in a number of plasticity makers at 1hr after training, including pCREB, pCaMKII and

pcofilin. These observations seem to be in contrast with the expected results that enhancement of

memory would be correlated with an increase in plasticity markers. These results therefore

suggest a number of possibilities. The first is that IGF-II, through a currently unknown

mechanism, enhances memory and enables the system to function at an optimal level while at the

same time utilizing less of these proteins. The second is that IGF-II alters the time-course by

which the expression of these markers increase/decrease, which leads to changes in behavior.

These two possibilities are not mutually exclusive, and examining further timepoints after IGF-II

injection and training should clarify the subject.

IGF-II does not impair memory flexibility.

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 One potential tradeoff regarding systemically delivered memory enhancers is the

potential for loss of memory flexibility. The adaptive nature of forgetting enables new learning

and/or the flexibility to alter behavior based on new, incoming information. Thus, memories that

are made artificially stronger may indeed become so strong that they are unalterable, an outcome

that would be problematic clinically (Floresco et al, 2011). This is indeed the case for

dopaminergic drugs given to PD patients – although they may ameliorate a number of learning

and memory symptoms, they can also lead to flexibility and decision-making deficits (Cools et

al, 2007; Floresco et al, 2011). I tested memory flexibility after systemic IGF-II administration

in two paradigms – extinction and spatial reference memory – and found that memory flexibility

remains intact. Extinction training of contextual fear memory is a new long-term memory

process that involves one extinction trial per day over a period of 8 days, whereas reversal of the

spatial reference memory is a working memory / long-term memory process that occurs in a

number of trials over the course of 2 days. Therefore, it is likely that on both short and long time

scales, IGF-II does not cause flexibility impairments.

Interestingly, although IGF-II did not cause an impairment in extinction when

administered before training itself, it did enhance extinction learning (and therefore, memory

flexibility) when given before extinction trials. This indicates that a memory already enhanced

by IGF-II remains flexible, yet IGF-II can also act on cortical areas to regulate memory

flexibility directly. Considering that deficits in reversal learning are both a symptom of ASD, as

well as a feature in many ASD mouse models (D'Cruz et al, 2013; Hoy et al, 2013; Karvat and

Kimchi, 2014; Memari et al, 2013; Micheau et al, 2014), IGF-II may be an effective treatment

for this disorder.

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6.3 IGF-II as a therapeutic for autism spectrum disorder.

ASD is a pervasive neurodevelopmental disorder characterized by a number of symptoms

including impairments in social interaction, communication (verbal and non-verbal) and

repetitive behaviors. In patients, these symptoms may manifest in varying degrees, from high-

functioning forms of ASD known commonly as Asperger’s to low-functioning forms, as

approximately 30% of ASD patients are also intellectually disabled.

Mouse models of ASD are, for the most part, based on genetic mutations found in small

subsets of the patient population. These include models of Fragile X, Rett’s syndrome,

Angelman’s syndrome and Tuberous Sclerosis, as well as Shank3 deletions (Crawley, 2012).

However, these syndromes make up a total of less than 25% patients with autism, whereas most

cases present without a known underlying genetic cause. For this reason, the BTBR mice, which

are inbred, provide a model that is analogous to the vast majority of ASD patients.

Here, I found that in a mouse model of ASD, the BTBR mouse, IGF-II rescued social

interaction deficits, repetitive behaviors and memory deficits. BTBR is an inbred strain of mice

exhibiting strong phenotypes on all the major autism symptoms. Recent studies have shown a

partial rescuing of social deficits with AMPA receptor modulators, Ampakines (Silverman

2006), a rescue of repetitive behaviors, but not social deficits, with Methyl-6-phenylethynyl-

pyridine (MPEP), an antagonist of the mGluR5 metabotropic glutamate receptor (Silverman

2010), and both repetitive behavior and social impairments with negative allosteric modulator of

the mGluR5 receptor, GRN-529 (Silverman 2012). Considering that Ampakines, which

modulate AMPARs, only provides a partial rescue in BTBR mice, and IGF-II injections lead to

an increase in synaptic GluR1 (Chen et al 2011), it is likely that IGF-II acts at least in part

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through this mechanism to reverse deficits, but must also produce independent effects that lead

to the full rescue of behaviors.

In other models of ASD, modulators of mGluR5, like CDPPB, alleviate cognitive

impairments in a mouse model of tuberous sclerosis (Auerbach et al, 2011) and rescues social

deficits in Shank2-/- mice (Won et al, 2012). Moreover, (1-3)IGF-I, an active peptide fragment

of IGF-I, was found to partially rescue lethality, hypoactivity, and respiratory problems and to

normalize impaired spine density, synaptic transmission, and cortical plasticity in a Rett

syndrome mouse model (Tropea et al, 2009).

Given that all major deficits of BTBR are rescued and these effects occur rapidly (within

20 minutes in the case of social recognition and marble burying), one likely mechanism is that

the action of IGF-II may target common substrates or regulations, such as neurotransmitter

release. In fact, two recent studies have indicated that this might be the case: BTBR mice were

recently shown to have altered GABA transmission (Han et al, 2014), and another recent study

showed that an acetycholinesterase inhibitor rescues some of the BTBR deficits (Karvat et al,

2014), indicating the involvement of the cholinergic system. Given that IGF-II is known to

regulate GABA and acetycholine release in vitro (Amritraj et al, 2010; Hawkes et al, 2006), this

is a likely mechanism by which IGF-II may rescue BTBR deficits.

IGF-II may also act by rescuing the increased reactivity to stressors in BTBR mice, a

phenotype of these mice suggested by previous reports (Silverman et al, 2010c). This increased

reactivity to stressors is distinct from a classical general anxiety response and interestingly,

BTBR mice showed different responses to the two anxiety provoking tasks tested here, that is

increased anxiety in the open field and decreased anxiety in the elevated plus maze. Thus, it is

hypothesized that general anxiety does not underlie the abnormal behaviors displayed by BTBR

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(Meyza et al, 2012) and, in agreement, I also found no difference in vehicle- or IGF-II-treated

BTBR mice in open field locomotion or the elevated plus maze, even though IGF-II rescued

memory, social recognition and repetitive behaviors. It is possible that the high reactivity to

stress in BTBR may be related to a change in the regulation of glucocorticoids (Meyza et al,

2012).

Last but not least, the effect of IGF-II as memory enhancer may target general synaptic

mechanisms like the IκB kinase (IKK)-NF-κB-dependent regulation of synapse formation. It has

been recently reported that exogenous IGF-II restores synapse density and promotes spine

maturation in IKK/NF-κB signaling-deficient neurons within 24h of treatment and that this

process requires an IGF-II receptor-mediated MEK/ERK activation (Schmeisser et al, 2012).

This hypothesis together with our data on long-term plasticity (Chen et al, 2011) suggests that

IGF-II may rescue synaptic impairments by regulating synaptic homeostasis. In agreement with

this possibility many of the genes mutated in ASD affect critical mechanisms of activity-

dependent pathways that regulate synaptic development and plasticity (Ebert and Greenberg,

2013).

6.4 Mechanisms of IGF-II mediated memory enhancement: Lysosomal degradation.

Memory enhancement mediated by IGF-II requires the IGF-IIR, both when injected intra-

hippocampally and when injected systemically. As noted previously, the IGF-IIR is identical to

the cation-independent mannose-6-phosphate receptor, and is known to be involved in lysosomal

degradation. Specifically, the 10% of the IGF-IIR that exists on the plasma membrane is known

to bind IGF-II and its other ligands, as well as mannose-6-phosphate containing lysosomal

enzymes, and via endocytosis, bring them to the lysosome for degradation.

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 The regulation of ligand binding by the IGF-IIR is complex, particularly at the plasma

membrane. Binding of some ligands alters the affinity of the receptor for others, despite having

distinct binding sites. For example, binding of certain lysosomal enzymes inhibits the ability of

IGF-II to bind the receptor and vice versa (Hawkes and Kar 2004). Additionally, IGF-IIR may be

involved in the internalization and degradation of other proteins/receptors, including epidermal

growth factor receptor. Thus, IGF-II may be well situated to alter the dynamics of lysosomal

degradation and receptor trafficking. Considering that endosomal-lysosomal degradation is the

main endpoint for membrane proteins, IGF-II is potentially capable of dramatically altering the

dynamics of receptor trafficking through the IGF-IIR. We have shown that IGF-II most likely

enhances memory through synaptic rather than cell-wide mechanism (Chen et al. 2011), and

specifically that IGF-II leads to an increase in synaptic, but not total, GluR1 (Chen et al. 2011),

indicating that indeed IGF-II may be actively involved in maintaining necessary synaptic

structures. A number of studies have examined the role of proteosomal degradation in memory

consolidation (Bingol and Sheng, 2011), yet the role of lysosomal degradation itself in memory

is an area that is not well characterized. Preliminary data that I have generated in the lab suggests

that chloroquine, an inhibitor of lysosomes, enhances memory in a similar fashion to IGF-II,

suggesting that an alteration of the lysosomal process is involved in the enhancement of memory.

6.5 Challenges remaining in the field of memory enhancement

The search for a memory enhancer that is effective in humans has remained elusive. For

disorders involving cognitive impairment, there are currently very few options available outside

of behavioral interventions. Autism is currently treated with only two drugs – Risperedone and

Abilify. Neither treats the core symptoms of ASD, but rather, mainly target irritability

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(Ghanizadeh et al, 2014). The only approved drug in the treatment of AD is Aricept, an

acetylcholinesterase inhibitor. This drug provides modest effects in patients in the early stages of

the disease, and enhances overall quality of life, but has not been shown to substantially increase

memory function or alter the course of the disease significantly (Knowles, 2006). A number of

compounds are currently being actively studied in the pursuit of cognitive enhancement in a

variety of forms. Ampakines, for example, are currently being investigated for their potential to

reverse deficits associated with mild cognitive impairment in AD, Fragile X-syndrome, Rett’s

syndrome, and others. A truncated IGF-I peptide is currently in clinical trials for treatment of

Rett syndrome (Khwaja et al, 2014). All of these studies are in early stages, but thus far, show

mild improvement at best. Thus, the search for effective cognitive enhancers is still ongoing, and

there are a number of challenges to finding effective enhancers.

One particular challenge in the field of cognitive/memory enhancement is that different

diseases likely represent deficits in different stages of memory. For example, a prominent

symptom of the devastating AD seems to be the inability to consolidate long-term memories, as a

gradient of retrograde amnesia accompanies AD progression (Carlesimo and Oscar-Berman,

1992; Simard and Reekum, 1999). However, many AD patients also have attention deficits,

which would in turn cause a problem with the initial encoding (Perry and Hodges, 1999) In this

case, a substance that enhances attention may increase the probability of the information being

encoded successfully, and therefore may enhance the memory (Lynch et al, 2011), but it is

equally plausible that a drug targeting consolidation processes specifically would be efficacious.

But even without the extremes of such a devastating pathology, in healthy individuals –

particularly with aging – a common complaint is not being able to “remember” well enough,

which commonly refers to the inability to retrieve memories (Cansino, 2009), though it is not

105
entirely clear where the exact impairment in normal aging lies (Light, 1991). Thus, in an

experimental setting, it is important to consider which stage of memory a putative memory

enhancer is affecting. This is relatively straightforward via pharmacological means, which can be

administered at any time point, before or after training or retrieval. Genetic or molecular

approaches represent other means to manipulate memories, and different behavioral tasks may be

tested to get a sense of which stages are affected by the manipulation, for example working

memory tasks vs. long-term memory tasks.

Another challenge is that for disease models, there is often a difficulty in discerning

whether a memory deficit lies in the consolidation process (ie. the memory was never formed or

was eliminated) or the retrieval process (ie. the memory is stored but cannot be retrieved, or

recalled). Furthermore, it is likely that substances that enhance retrieval might be clinically

beneficial, yet this stage of memory has received little attention thus far.

Yet another challenge in the field of cognitive enhancement is that of drug delivery to the

brain. The blood-brain barrier is designed to keep agents from attaining access to the brain.

Therefore, many drugs that are developed are not able to cross the BBB, despite the fact that they

might have therapeutic value. Thus, a great deal of effort has been exerted to manipulate drugs to

be able to cross the BBB, with varying success, or to find ways to administer drugs that will

bypass the BBB, such as intranasal administration. One advantage of IGF-II is that is crosses the

BBB naturally and thus would be straightforward to administer clinically.

In conclusion, in this thesis, I have found a potent enhancer of hippocampal aversive- and

non-aversive memories, the Insulin-Like Growth Factor II. I have shown that the effects of IGF-

II are persistent and impact remote memories. I have also shown that IGF-II is effective in

enhancing memory when injected centrally and peripherally, and that IGF-II is potentially

106
effective in the treatment of ASD, leading to potential for new treatments for ASD and other

cognitive disorders.

107
Table 2: Detailed statistics for Chapter II
Parameter Statistical Post-hoc
Test n Test Drug Mean s.e.m. p-value p-value Figure
Measured Test Test
Veh 280.5 69.1 Drug: F1,26=21.42,
Test 1 ***p<0.0001; Test: Test 1: *p<0.05;
IGF-2 393.32 69.13 Two-way
IA 7-8 Latency F1,26=3.85, p=0.0604; Bonferroni Test 2: 1A
Veh 134.7 71.9 ANOVA
Test 2 Interaction: F1,26=0.34, **p<0.01
IGF-2 200.68 53.19 p=0.5569
Veh 275.3 83.5
2.5ng: *p<0.05
2.5ng 552.4 23.3 One-way F3,18=10.5; Neuman-
IA 4-6 Latency Test 1 25ng: ***p<0.001 1B
25ng 798.4 57.55 ANOVA ***p=0.0003 Keuls
250ng: ***p<0.001
250ng 758.9 93.6
Veh 111.4 3.2
IA 5-6 Latency Test 1 t-test *p=0.0261 N/A N/A 1C
IGF-II 455.8 136.5
Veh 333.7 87.9
IGF-II 777.58 92.06
IGF- Veh vs. IGF-II:
II/Anti- *p<0.05;
225.9 86.9
IGF-IIR One-way Neuman-
IA 6-9 Latency Test 1 F5,40=3.82, **p=0.0023 1D
IGF- ANOVA Keuls IGF-II vs. IGF-
625.1 121.3
II/JB1 II/Anti-IGF-IIR:
Anti- **p<0.01
460.8 136.9
IGF-IIR
JB1 241.6 91.2
Veh 30.2 5.9
CFC 7 % Freezing Test 1 t-test *p=0.0434 N/A N/A 1E
IGF-II 49.2 5.8
Veh 79.5 4.4
AFC 7 % Freezing Test 1 t-test p=0.8115 N/A N/A 1E
IGF-II 81 4.3
Veh 52.61 10.13
AFC 9 % Freezing Test 1 t-test p=0.4648 N/A N/A 2
IGF-II 43.46 6.82
1,24
Veh 222.53 31.01 *Drug: F =5.73, p=0.249;
Test 1
Insulin 569.57 129.86 Two-way Test: F1,24=0.82, p=0.3744;
IA 7 Latency Bonferroni Test 1: *p<0.05 3A
Veh 283.01 54.65 ANOVA Interaction: F1,24=2.56,
Test 2
Insulin 351.79 96.89 p=0.1224
Veh 30.21 5.87 Drug: F1,24=0.88, p=0.3564;
Test 1
Insulin 51.98 10.63 Two-way ***Test: F1,24=19.34,
CFC 7 % Freezing Bonferroni Test 1: ***p<0.001 3B
Veh 15.13 6.21 ANOVA p=0.0002; *Interaction:
Test 2
Insulin 6.34 2.15 F1,24=4.80, p=0.0366
AFC 7 % Freezing Test 1 Veh 74.08 5.95 Two-way Drug: F1,24=2.17, p=0.1536; N/A N/A 3B

108
 Veh 74.08 5.95
Test 1 Test: F1,24=1.49, p=0.2334;
Insulin 82.63 3.89
AFC 7 % Freezing ANOVA Interaction: F1,24=0.02, N/A N/A 3B
Veh 65.44 10
Test 2 p=0.8955
Insulin 75.69 3.49
Veh 34.72 2.73 **Drug: F1,28=9,97,
Test 1
Insulin 63.93 7.55 p=0.0038; *Test:
Two-way Test 1: **p<0.01,
CFC 8 % Freezing Veh 31.46 4.43 F1,28=4.59, p=0.0410; Bonferonni 3C
ANOVA Insulin: *p<0.05
Test 2 Interaction: F1,28=2.59,
Insulin 40.94 8.13 p=0.1187
Veh 52.98 6.31 F1,18=0.04, p=0.8527; Test:
Test 1
Insulin 57.34 10.51 Two-way F1,18=1.86, p=0.1897;
AFC 5-6 % Freezing N/A N/A 3D
Veh 45.67 3.29 ANOVA Interaction: F1,18=0.15,
Test 2
Insulin 44.14 6.4 p=0.6998
Veh 18.4 3.07
Acq
Insulin 17.42 3.16 t-test
IA 9 Latency p=0.7593 N/A N/A 3E
Veh 199.32 47.46 (Test 1)
Test 1
Insulin 230.32 87.42
Veh 531.86 127.88 Drug: F1,30=1.49, p=0.2317;
Test 1
Insulin 741.22 133.9 Two-way Test: F1,30=0.43, p=0.5186;
IA 8-9 Latency (s) N/A N/A 4A
Veh 475.7 157.97 ANOVA Interaction: F1,30=0.07,
Test 2
Insulin 612.28 141.46 p=0.7990
Veh 51.12 14.78 Drug: F1,30=0.64, p=0.4345;
Test 1
Insulin 62.74 14.72 Two-way Test: F1,30=0.05, p=0.8250;
AFC 5-8 % Freezing N/A N/A 4B
Veh 45.42 13.02 ANOVA Interaction: F1,30=0.01,
Test 2
Insulin 60.85 18.89 p=0.9119
Veh 53.49 11.2 Drug: F1,21=0.01, p=0.9246;
AFC Test 1
Insulin 54.11 12.57 Two-way Test: F1,21=0.11, p=0.7419;
(high 5-8 % Freezing N/A N/A 4C
Veh 57.23 3.67 ANOVA Interaction: F1,21=0.00,
dose) Test 2
Insulin 59.10 14.98 p=0.9623
Veh 30.21 5.87 Drug: F1,24=0.52, p=0.4776;
Test 1
IGF-I 37.03 6.12 Two-way **Test: F1,24=8.62,
CFC 7 % Freezing N/A N/A 5A
Veh 15.13 6.21 ANOVA p=0.0072; Interaction:
Test 2
IGF-I 16.94 5.74 F1,30=0.18, p=0.6793
Veh 73.68 5.82 Drug: F1,22=1.36, p=0.2568;
Test 1
IGF-I 79.99 5.16 Two-way **Test: F1,22=0,95,
AFC 6-7 % Freezing N/A N/A 5A
Veh 65.44 10 ANOVA p=0.3408; Interaction:
Test 2
IGF-I 74.98 7 F1,22=0.06, p=0.8153
CFC 6 % Freezing Veh 36.16 3.7 Two-way Drug: F1,20=0.12, p=0.7305; N/A N/A 5B
Test 1
IGF-I 37.96 3.01 ANOVA Test: F1,20=2.81, p=0.1093;

109
 Veh 32.68 5.95 Interaction: F1,20=1.10,
Test 2
IGF-I 27.12 2.27 p=0.3072
Veh 52.98 6.31 Drug: F1,18=1.73, p=0.2045;
Test 1
IGF-I 62.76 9.11 Two-way Test: F1,18=0.99, p=0.3338;
AFC 5-6 % Freezing N/A N/A 5C
Veh 45.67 3.29 ANOVA Interaction: F1,18=0.00,
Test 2
IGF-I 55.37 7.9 p=0.9955
Veh 51.12 14.75 Drug: F1,17=0.02, p=0.8997;
Test 1
IGF-I 39.98 9/06 Two-way Test: F1,17=0.05, p=0.8293;
AFC 5-8 % Freezing N/A N/A 6A
Veh 45.42 13.02 ANOVA Interaction: F1,17=0.02,
Test 2
IGF-I 41.58 8.69 p=0.8849
Veh 527.34 147.48 Drug: F1,19=0.30, p=0.5911;
Test 1
IGF-I 482.79 157.38 Two-way Test: F1,19=0.02, p=0.8827;
IA 5-6 Latency (s) N/A N/A 6B
Veh 538.99 154.62 ANOVA Interaction: F1,19=0.07,
Test 2
IGF-I 542.08 164.73 p=0.7930

110
Table 3: Detailed statistics for Chapter III.
Parameter Statistical Post-hoc
Test n Test Drug Mean s.e.m. p-value p-value Figure
Measured Test Test
1,38
Veh 412.73 95.71 Drug: F =0.55,
Test 1
IGF-II 387.95 89.57 p=0.4621; Test:
Two-way
IA 10-11 Latency (s) Veh 401.21 100.58 F1,38=0.35, p=0.5556; N/A N/A 7
ANOVA
Test 2 Interaction: F1,38=0.13,
IGF-II 302.14 61.48 p=0.7171
Veh 421.73 95.71 Drug: F1,36=1.67,
Test 1 p=0.2045; Test:
Insulin 320 85.87 Two-way
IA 9-11 Latency (s) F1,36=0.18, p=0.6746; N/A N/A 8
Veh 401.21 100.58 ANOVA
Test 2 Interaction: F1,36=0.04,
Insulin 241.41 79.97 p=0.8397
Veh 433.58 83.29 Drug: F1,48=1.29,
Test 1
IGF-I 589.22 98.57 p=0.2610; Test:
Two-way
IA 12-14 Latency (s) Veh 516.11 92.15 F1,48=0.11, p=0.7406; N/A N/A 9
ANOVA
Test 2 Interaction: F1,48=0.33,
IGF-I 567.2 88.38 p=0.5678
Veh 330.59 165.29 Drug: F1,12=0.02,
Test 1
IGF-II 252.8 126.4 p=0.9043; Test:
Two-way
IA 4 Latency (s) Veh 331.6 165.8 F1,12=0.33, p=0.5765; N/A N/A 10A
ANOVA
Test 2 Interaction: F1,12=0.32,
IGF-II 452.26 226.13 p=0.5803
Veh 207.66 72.54 Drug: F1,20=15.35,
Test 1
IGF-II 731.98 100.88 ***p=0.0009; Test:
Two-way
IA 5-7 Latency (s) Veh 288.66 156.12 F1,20=0.01, p=0.9401; Bonferonni Test 1: **p<0.01 10B
ANOVA
Test 2 Interaction: F1,20=0.39,
IGF-II 668.55 112.7 p=0.5385
Veh 348.13 82.36 Drug: F1,20=58.49,
Test 1 Test 1:
IGF-II 860.69 39.21 ***p<0.0001; Test:
Two-way ***p<0.001
IA 6 Latency (s) Veh 213.05 13.85 F1,20=3.2, p=0.0887; Bonferonni 11A
ANOVA Test 2:
Test 2 Interaction: F1,20=0.03,
IGF-II 750.36 101.45 ***p<0.001
p=0.8586
Veh 448.51 116.17 Drug: F1,24=0.02,
Test 1
IGF-II 448.68 72.51 p=0.9011; Test:
Two-way
IA 7 Latency (s) Veh 490.15 12.42 F2,30=5.16, p=0.0118; N/A N/A 11B
ANOVA
Test 2 Interaction: F2,30=2.50,
IGF-II 512.14 115.55 p=0.0990
IA 6 Latency (s) Veh 138.38 27.44 Two-way Drug: F1,30=18.23, Bonferonni Test 1: **p<0.01 11C
Reactivation
IGF-II 214.26 40.26 ANOVA ***p=0.0002; Test: Test 2: *p<0.05

111
 Veh 183.44 34.87 F1,20=3.2, p=0.0887;
Test 1
IGF-II 593.87 87.77 Interaction: F1,20=0.03,
Veh 233.1 133.51 p=0.8586
Test 2
IGF-II 565.01 87.2
Veh 211.93 52.8
Reactivation **Drug: F1,36=8.76,
IGF-II 208.69 29.76
p=0.0054; Test:
Veh 269.58 31.99 Two-way Test 1: *p<0.05
IA 7 Latency (s) Test 1 **F2,36=6.08, p=0.0053; Bonferonni 11D
IGF-II 590.7 112.76 ANOVA Test 2: *p<0.05
Interaction: F2,36=2.26,
Veh 344.18 123.36 p=0.1188
Test 2
IGF-II 650.25 108.8
Veh 203.08 56.96 Drug: F1,40=0.89,
Test 1
IGF-II 301.91 102.25 p=0.3508; Test:
Two-way
IA 10-12 Latency (s) Veh 175.2 38.11 F1,40=6.30, p=0.0163; N/A N/A 11E
ANOVA
Test 2 Interaction: F1,40=1.66,
IGF-II 482.65 104.28 p=0.2050
Veh 171.89 36.86
Reactivation IGF-I 133.89 19.26
Insulin 193.64 37.85
Veh-No
358.67 175.02
RA
IGF-I-No
308.67 149.07
RA
Test 1 Insulin-
311.99 57.71 Drug: F5,60=0.11,
No RA
Veh 292.65 108.08 p=0.9896; Test:
Two-way
IA 6 Latency (s) IGF-I 282.15 75.81 F1,60=0.05, p=0.8292; N/A N/A 12
ANOVA
Insulin 358.29 138.24 Interaction: F5,60=0.15,
Veh-No p=0.9788
311.92 131.33
RA
IGF-I-No
298.25 152.61
RA
Test 2 Insulin-
387.68 127.49
No RA
Veh 245.8 79.45
IGF-I 325.57 115.58
Insulin 253.21 57.43

112
Table 4. Detailed statistics for Chapter IV.
Parameter Statistica Post-hoc
Test n Test Drug Mean s.e.m. p-value p-value Figure
Measured l Test Test
Freezing (%) Veh 41.87 3.07
5 ug/kg 46.30 5.49
Contextual
15 ug/kg 58.73 4.67 15 ug/kg: *<0.05;
Fear One- Dunnett's
F6,51=6.819; 30 ug/kg:
Conditioning 6-9 30 ug/kg 68.08 3.91 Way (compared 1a
***P<0.0001 ***<0.001; 45
Dose 45 ug/kg 60.32 3.07 ANOVA to vehicle)
ug/kg: **<0.01
Response
60 ug/kg 43.52 3.91
100 ug/kg 45.24 4.27
Veh 29.17 2.72 Drug: F1,33=7.09,
Contextual Test 1 *P=0.0119; Test 1: *P=0.011
IGF-II 46.03 5.24 Two-
Fear Time: F1,33=0.43,
8 to 11 Way t-test 1b
Conditioning P=0.5172;
Veh 34.34 5.07 ANOVA
(15ug/kg) Test 2 Interaction: Test 2: P=0.1211
IGF-II 48.48 7.11 F1,33=0.05, P=0.8165
Veh 28.47 3.55 Drug:F1,33=21.87,
Contextual Test 1 Test 1: **P<0.01
Two- ***P<0.0001;
Fear IGF-II 55.56 4.58
8 to 11 Way Time:F1,33=1.24, Bonferroni 1c
Conditioning Veh 38.88 4.43
Test 2 ANOVA P=0.274; Interaction: Test 2: *P<0.05
(30ug/kg) IGF-II 55.55 5.14 F1,33=1.24, P=0.274
Contextual
Fear Veh 33.33 4.20 not
Conditioning 7 t-test *P=0.047 applicable N/A 1d
(Short-term IGF-II 48.41 5.38 (N/A)
Memory)
Veh 48.82 3.61 Drug:F1,26=6.19, Test 1 (Veh vs.
Contextual Test 1 *P=0.0196; IGF-II):
Fear IGF-II 73.81 4.15 Two-
Time:F1,26=5.17, **P<0.01
Conditioning 7 to 8 way Bonferroni 1e
Veh 49.21 7.42 *P=0.0314;
(After- Test 2 ANOVA
Interaction:F1,26=7.49 IGF-II (Test 1. vs.
Training) IGF-II 47.92 4.92 , *P=0.011 Test 2): **P<0.01
Contextual
Fear Veh 37.47 4.15
Conditioning 6 t-test P=0.6039 N/A N/A 1f
(STM-After IGF-II 34.10 4.74
Training)

113
Auditory Fear Pre- Veh 13.89 2.78
6 t-test P=1.0 N/A N/A 1g
Conditioning tone IGF-II 13.89 5.12
Veh 38.14 0.11 Drug: F1,18=1.561,
30m
IGF-II 38.23 0.12 P=0.243; Time:
Two-
Veh 38.32 0.1 F2,18=0.1462,
Temp 5-6 Degrees (c) 24 way N/A N/A T1
IGF-II 37.88 0.33 P=0.8650;
ANOVA
Veh 38.36 0.15 Interaction:
7d F2,18=1.342, P=-.2863
IGF-II 38.0 0.21
Veh 26.0 8.34 Drug: F1,18=0.4178,
30m
IGF-II 29.83 8.66 P=0.5342; Time:
Veh 54.0 3.38 Two- F2,18=7.482,
Wire 24
5-6 Time (s) IGF-II 47.5 9.1 way **P=0.0043; N/A N/A T1
Suspension
Veh 50.6 4.28 ANOVA Interaction:
7d F2,18=1.515,
IGF-II 33.67 7.57 P+0.2466
Veh 10.6 1.12 Drug: F1,18=1.139,
30m
IGF-II 11.67 0.67 P=0.0399; Time:
Two-
Veh 10.8 1.29 F2,18=4.385,
Pole Test 5-6 Score 24 way N/A N/A T1
IGF-II 12.67 1.02 *P=0.0281;
ANOVA
Veh 13.2 0.45 Interaction:
7d F2,18=0.38, P=0.6892
IGF-II 13.67 0.61
Locomotor Veh 254.80 27.68
5-6 # Beam Breaks t-test P=0.9469 N/A N/A 1h
Activity IGF-II 252.50 20.29
Veh 20.71 7.23
# Center Entries t-test P=0.8549
IGF-II 19.19 3.65
1i
Time in Center Veh 27.78 4.25
6 24h t-test P=0.6814 N/A N/A
(s) IGF-II 24.35 6.78
Total Entries/ Veh 228.33 24.03
Locomotor t-test P=0.3718 1i
Activity IGF-II 191.83 30.76
Open Field
Veh 30.00 1.98
# Center Entries t-test P=0.8011
IGF-II 28.50 5.45
Veh 28.89 3.71
6 Time in Center 20m t-test P=0.4246 N/A N/A S1
IGF-II 23.01 6.01
Total Entries/ Veh 178.00 11.23
t-test P=0.9929
Locomotor IGF-II 177.83 14.68

114
 Activity
Veh 162.00 5.675
30m Drug:F1,30=1.78,
IGF-II 172.333 12.36
P=0.212;
Veh 175.33 10.34
12h Time:F3,30=0.92,
IGF-II 178.00 7.452 Two-
P=0.4421;
Glucose 6 mg/dl way N/A N/A 1j
Veh 151.167 4.785 Interaction:F3,30=0.23
24h ANOVA
, P=0.876
IGF-II 170.833 7.778
Matching:
Veh 170.00 14.688 F10,20=1.089, P=0.401
7d
IGF-II 178.5 15.756
Pre- Veh 22.11 0.60
Train IGF-II 22.25 0.65
Veh 22.14 0.67 Drug:F1,48=0.01,
Train
IGF-II 22.85 0.57 P=0.92;
Two-
Test 1 Veh 22.57 0.78 Time:F3,48=0.1.3,
Weight 7 Grams (g) way N/A N/A 1k
(24h) P=0.29;
IGF-II 22.00 0.48 ANOVA
Interaction:
Veh 23.39 0.89 F3,48=0.33, P=0.80
Test 2
(7d) IGF-II 23.29 0.47

115
 IgG/Veh 43.83 3.64 IgG/Veh vs.
IgG/IGF-II:
Anti- *P<0.05
IGFIIR/ 46.83 3.99
Veh One- Anti-IGFIIR/Veh
CFC – IGF- F3,30=4.274; Newman-
7-9 % Freezing Test 1 IgG/ Way vs. IgG/IGF-II: 2a
IIR 70.99 7.22 *P=0.0126 Keuls
IGF-II ANOVA *P<0.05
Anti- Anti-IGFIIR/IGF-
IGFIIR/ 50 7.47 II vs. IgG/IGF-II:
IGF-II *P<0.05
Naïve- Naïve-Veh vs.
100 5.94
Veh Trained-veh:
Trained- **P=0.0016;
Arc 169.51 17.72
Veh Naïve-Veh vs.
(Hippoc One-way F3,39=4.383;
Naïve- t-test Naïve-IGF-II: 2b
ampus) 142.36 17.68 ANOVA **P=0.0099
IGF-II *P=0.0356;
Naïve-Veh vs.
Trained- Trained-IGF-II:
145.34 9.98
IGF-II **P=0.0010
Naïve- Naïve-Veh vs.
100 7.77
Veh Trained-Veh:
Trained- **P=0.0039;
Zif268 166.62 16.45
Veh Naïve-Veh vs.
(Hippoc One-way F3,37=4.213;
Naïve- t-test Naïve-IGF-II: 2c
ampus) 142.24 11.26 ANOVA *P=0.0123
IGF-II **P=0.0098;
Naïve-Veh vs.
Trained- Trained-IGF-II
Western Blot 7-10 % of Naïve-Veh 143.54 12.83
IGF-II P=0.0151
Naïve-
100 5.99
Veh
Arc
Trained-
(Prefro 145.77 14.71 Naïve-Veh vs.
Veh One-way F3,27=3.916;
ntal t-test Trained-Veh: 2d
Naïve- ANOVA *P=0.0208
cortex) 105.29 10.34 *P=0.0138
IGF-II
Trained-
119.18 8.3
IGF-II
Naïve-
100 7.06
Veh
Zif
Trained-
(Prefro 122.71 11.6 One-way F3,27=2.511;
Veh N/A N/A 2e
ntal ANOVA P=0.0827
Naïve-
cortex) 115.61 13.6
IGF-II
Trained- 86.83 6.48

116
 IGF-II
Veh 53.74 2.8 One-sample P=0.22
Train t-test P=0.1773 S2
t-test
IGF-II 48.75 2.23 P=0.59
Veh 64.59 2.95 Drug: Test 2,
Drug:F1,34=14.41,
% Preference Bonferroni ***P<0.0001;
4h ***P=0.0006;
Two- Time: Veh,
IGF-II 66.06 2.97 Time:F1,34=1.46,
way One-sample **P<0.01 3a
P=0.24;
Veh 51.15 2.98 ANOVA t-test (24h
Interaction:F1,34=10.9
Novel Object 9 to 10 24h Vehicle) One-sample:
IGF-II 72.3 2.98 1, **P=0.0023
Recognition P=0.71
Veh 22.98 3.26
Train t-test P=0.9426
IGF-II 22.62 3.63
Total
Exploration Veh 20.43 2.65 N/A N/A S2
4h t-test P=0.4380
Time (s) IGF-II 17.89 1.88
Veh 41.31 6.65
24h t-test P=0.5890
IGF-II 36.42 5.93
Veh 45.51 5.04 One-sample P=0.41
Train t-test P=0.5931 t-test S2
IGF-II 48.63 2.56 P=0.62
Veh 63.24 3.86 Drug:F1,24=19.09,
Veh, (Test 1 vs
% Preference 4h ***P=0.0002;
IGF-II 72.29 1.81 Two- Test2): *P<0.05;
Time:F1,24=9.17,
way Bonferroni Test 2 (Veh vs. 3b
Veh 51.93 1.68 **P=0.0058;
ANOVA IGF-II):
Object 6 to 8 24h Interaction:F1,24=1.17
IGF-II 66.93 3.08 **P<0.01
Placement ;P=0.29
Veh 25.26 5.11
Train t-test P=0.95
IGF-II 25.6 2.62
Total Veh 22.85 4.53
Exploration 4h t-test P=0.37 N/A N/A S2
Time (s) IGF-II 28.61 4.31
Veh 19.36 4.09
24h t-test P=0.89
IGF-II 20.09 3.19
Social 10 % Preference Veh 69.99 3.06 Two- Drug:F1,54=8.89, Bonferroni Test 3 (Veh vs. 3c
Recognition way **P=0.0043; IGF-II):
Test 1
and Memory IGF-II 71.77 2.03 ANOVA Time:F2,54=7.28, One-sample ***P<0.001;
**P=0.0016; t-test (Test 3 Veh: (Test 2 vs.
Interaction: Veh) Test 3),
Test 2 Veh 67.5 2.64 F2,54=4.88, *P=0.011 **P<0.01; (Test 1

117
 vs. Test 3),
IGF-II 68.61 1.84 ***P<0.001
Veh 55.447 1.53 One-sample:
Test 3
IGF-II 69.22 2.25 **P=0.006
Veh 91.65 13.05
Test 1 t-test P=0.5061
IGF-II 82.07 5.39
Total
Veh 74.4 3.16
Exploration Test 2 t-test P=0.1840 N/A N/A S3
Time (s) IGF-II 88.1 9.4
Veh 50.88 5.08
Test 3 t-test P=0.6161
IGF-II 54.86 5.92
Contextual 9 to 10 Veh 43.89 3.36
Fear T1
IGF-II 42.22 3.63
Extinction
Veh 41.67 3.63
E1 Drug:F1,89=18.31,
IGF-II 26.67 3.68
***P<0.0001;
Veh 40.00 5.42 Two-
Time: F4,89=5.75,
% Freezing E2 way N/A N/A 3d
IGF-II 25.56 5.69 ***P=0.004;
ANOVA
Interaction:F4,89=0.86
Veh 33.49 2.06
E3 , P=0.49
IGF-II 20.72 4.70
Veh 31.27 5.19
E4
IGF-II 18.02 3.67
Veh 0.00 0.00
T1
IGF-II 0.00 0.00
Veh 4.81 8.42
E1 Drug:F1,85=11.33,
IGF-II 33.57 10.74
**P=0.0011;
Veh 9.44 13.43 Two-
Rate of Time:F4,85=6.38,
E2 way N/A N/A 3e
Extinction IGF-II 38.8 13.5 ***P=0.0002;
ANOVA
Interaction:F4,85=0.86
Veh 22.98 6.35
E3 , P=0.49;
IGF-II 39.25 11.88
Veh 33.01 9.9
E4
IGF-II 59.16 6.9
Two- Drug::F1,34=2.77, Bonferroni, E4 (Veh vs. IGF-
% Freezing of
T1 Veh 100.00 5.65 way P=0.11; II): *P<0.05; 3f
Test 1
ANOVA Time:F1,34=34.42,

118
 Veh 100.00 5.65
Veh (E1 vs. E4):
T1
*P<0.05;
Two- ***P<0.0001; Bonferroni,
% Freezing of IGF-II 100.00 8.60 way Interaction:F1,34=2.77 3f
Test 1 IGF-II: (E1 vs.
ANOVA , P=0.11
E4) ***P<0.001;
Veh 66.99 9.90
E4
IGF-II 40.84 6.55

Veh 52.60 3.197

IGF-II 46.87 1.16 One-way F2,17=4.877;
Train
IGF-II no- ANOVA P=0.0869
48.30 3.42
React
Reactiv Veh 66.15 1.89
% Preference t-test P=0.5436 3g
ate IGF-II 68.46 3.15
Veh 54.17 3.16 Veh vs. IGF-II:
***P<0.001;
IGF-II 84.35 4.6 One-way F2,17=21.44;
Test Bonferroni IGF-II vs. non-
Reactivation IGF-II no- ANOVA ***P<0.0001
59.95 2.18 React:
of Novel React ***P<0.001
6
Object Veh 17.56 2.13
Recognition
IGF-II 17.69 2.35 One-way F2,17=1.601;
Train N/A N/A
IGF-II no- ANOVA P=0.23434
13.25 1.38
React
Total Reactiv Veh 19.46 2.91
t-test P=0.4122 N/A N/A S4
Exploration (s) ate IGF-II 16.27 2.33
Veh 12.87 2.94
IGF-II 11.76 2.9 One-way F2,17=0.034;
Test N/A N/A
IGF-II no- ANOVA P=0.9094
14.6 2.44
React
Contextual 8 % Freezing Veh 40.97 4.32 2-way Drug:F1,126=20.04, N.A N/A 4a
Fear T1 ANOVA ***P<0.0001
IGF-II 55.27 6.72
Conditioning Time:F8,126=3.13,
(Flexibility) Veh 25.69 3.63 **P=0.0029;
E1
IGF-II 40.28 8.51 Interaction:F8,126=0.1
Veh 31.25 4.46 3, P=0.998
E2
IGF-II 37.5 6.94
E3 Veh 29.86 2.96

119
 IGF-II 45.42 8.6
Veh 31.95 6.53
E4
IGF-II 41.79 5.96
Veh 31.94 2.29
E5
IGF-II 43.06 6.94
Veh 23.72 4.24
E6
IGF-II 37.10 5.17
Veh 21.44 4.54
E7
IGF-II 32.4 4.61
Veh 17.19 3.31
E8
IGF-II 29.65 7.59
Veh 0 0
T1
IGF-II 0 0
Veh 28.0 14.08
E1
IGF-II 29.9 10.41
Veh 11.71 18.8
E2
IGF-II 32.38 10.35
Veh 23.22 9.32
E3
IGF-II 14.49 15.74 Drug:F1,126=0.0018,
P=0.966;
Veh 18.52 12.79 Two-
Rate of E4 Time:F8,126=2.89,
way N/A N/A 4b
Extinction IGF-II 14.59 16.19 **P=0.0054;
ANOVA
Interaction:F8,126=0.2
Veh 17.61 8.66 5, P=0.9807
E5
IGF-II 15.97 12.95
Veh 36.89 12.28
E6
IGF-II 31.87 7.81
Veh 40.51 14.52
E7
IGF-II 39.70 8.37
Veh 50.3 12.47
E8
IGF-II 45.68 13.49
% Freezing of Veh 100 10.55 Two- Drug:F1,28=0.27, Bonferroni Veh (T1 vs E8), 4c
Test 1 T1 way P=0.61; **P<0.01; IGF-II
IGF-II 100 12.16 ANOVA Time:F1,28=21.24, (T1 vs. E8),

120
 Veh 100 10.55
T1 Two- ***P<0.0001;
% Freezing of IGF-II 100 12.16 way Interaction:F1,28=0.27 Bonferroni *P<0.05 4c
Test 1 Veh 41.95 8.07 ANOVA , P=0.61
E8
IGF-II 53.66 13.74
Spontaneous Veh 65.51 2.17
7 to 8 % Alternation t-test *P=0.0372 N/A N/A 4d
Alternation IGF-II 72.21 1.83
Habitua Veh 37.48 1.58
t-test P=0.1540
tion IGF-II 34.4 1.31
Veh 57.14 6.8
A1 (1)
IGF-II 77.5 7.01
Drug:F1,52=20.61,
Veh 68.57 5.95 ***P<0.0001;
A1 (2) Two-
IGF-II 80 5.35 Time:F3,52=3.3,
way
Veh 68.57 4.04 *P=0.0275;
A2 (1) ANOVA
Interaction:
IGF-II 85 3.27
Spatial F3,52=0.27, P=0.85
Veh 74.29 3.69
Reference 6 to 8 % Correct A2 (2) N/A N/A 4e
in the Y-maze IGF-II 92.5 3.66
Veh 40 5.16
R1 (1)
IGF-II 31.43 10.56
Drug:F1,44=0.64,
Veh 63.33 8.02 P=0.43;
R1 (2) Two-
IGF-II 65.71 7.19 Time:F3,44=9.86,
way
Veh 60 0 ***P<0.0001;
R2 (1) ANOVA
Interaction:F3,44=0.44
IGF-II 48.57 7.38
, P=0.73
Veh 73.33 6.67
R2 (2)
IGF-II 74.29 7.19

121
Table 5. Detailed statistics for Chapter V.
B6-Veh 69.99 3.06
BTBR- B6-Veh vs.
Test 1 50.29 1.78 BTBR-
Veh
Veh:Test1,
BTBR-
67.99 1.76 ***P<0.0001;
IGF-II
B6-Veh 67.5 2.64 Drug:F2,64=11.86,
BTBR-Veh vs.
***P<0.0001;
BTBR- Two- BTBR-IGF-II:
64.6 1.95 Time:F2,64=8.9,
% Preference Test 2 Veh way Bonferroni Test 1 1a
***P=0.0004;
ANOVA ,***P<0.001,
BTBR- Interaction:F4,64=4.59
64.75 4.27 Test 3 *P<0.05;
IGF-II , **P=0.0026
B6-Veh 55.447 1.53 Test 2 vs Test 3
BTBR- :B6-Veh,
51.61 2.52 **P<0.01;BTBR-
Test 3 Veh
BTBR- Veh,*P<0.05
Social 62.65 2.53
IGF-II
Recognition
7 to 10
and Memory
B6-Veh 74.88 4.72
(BTBR)
BTBR-
Test 1 65.88 7.35
Veh
BTBR-
64.06 5.24
IGF-II
Drug:F2,60=1.2,
B6-Veh 67.55 4.67 P=0.3070;
Total BTBR- Two-
52.27 8.28 Time:F2,60=5.33,
Exploration Veh way N/A N/A 1b
Test 2 **P=0.0070;
Time (s) ANOVA
BTBR- Interaction:F4,60=3.02
40.61 4.23 , *P=0.0245
IGF-II
B6-Veh 46.97 7.67
BTBR-
55.73 4.55
Test 3 Veh
BTBR-
61.91 7.2
IGF-II
Marble Burying Time Two- Drug:F3.42=36.72, BTBR-Veh vs
5 to 7 B6-Veh 81.13 12.49 Bonferroni 2a
Burying (s) way ***P<0.0001; B6-Veh: Test 1,
B6-IGF-II 86.02 11.2 ANOVA Time:F1,42=0.29, ***P<0.0001,Tes
Test 1 P=0.59; Interaction: t 2, ***P<0.0001;
BTBR-
277.77 26.83 F3,42=0.10, P=0.96
Veh
BTBR-Veh vs
BTBR- 150.19 23.81

122
 IGF-II 150.19 23.81 B6-IGF-II: Test
1, ***P<0.0001,
B6-Veh 96.73 9.69 Test 2,
***P<0.0001;
B6-IGF-II 88.18 14.62
BTBR-Veh vs
Test 2
BTBR- BTBR-IGF-II:
275.54 23.47 Test 1,
Veh
***P<0.001, Test
BTBR- 2, **P<0.01
166.39 21.63
IGF-II
B6-Veh 0.83 0.48
BTBR-Veh vs
B6-IGF-II 1 0.45 B6-Veh: Test 1,
Test 1 BTBR- **P<0.01, Test 2,
4.78 0.88 **P<0.01;
Veh Drug:F3,50=10.12,
BTBR- ***P<0.0001;
3.56 0.77 Two- BTBR-Veh vs.
Marbles Buried IGF-II Time:F1.50=0.78,
5 to 7 way Bonferroni B6-IGF-II: Test 2b
(#) P=0.38,
B6-Veh 1.67 0.49 ANOVA 1, **P<0.01, Test
Interaction:F3,50=0.30
B6-IGF-II 1.8 0.49 2, **P<0.01;
, P=0.82
BTBR-
Test 2 5.67 1.09 BTBR-Veh vs.
Veh
BTBR-IGF-II:
BTBR- Test 2, *P<0.05;
3.11 0.63
IGF-II

B6-Veh 42.59 4.9 BTBR-Veh vs

Contextual BTBR-IGF-II:
One-way Newman-
Fear 6 % Freezing BTBR- *P=0.0194 *P<0.05; BTBR- 3
23.15 3.01 ANOVA Keuls
Conditioning Veh Veh vs B6-Veh:
BTBR- *P<0.05
50 8.4
IGF-II
Open Field B6-Veh vs
5 to 6 B6-Veh 17.1 1.32 BTBR-Veh:
BTBR- **P<0.01;
# of Center 10.25 1.14 One-way
Veh **P=0.0029 Dunnett's 4a
Entries ANOVA
B6-Veh vs.
BTBR- BTBR-IGF-II:
9.69 1.58
IGF-II **P<0.01
Time in center One-way B6-Veh vs.
B6-Veh 28.85 3.71 *P=0.0344 Dunnett's BTBR-IGF-II: 4b
(s) ANOVA
*P<0.05
BTBR- 20.1 3.31

123
 Veh
BTBR-
16.02 2.57
IGF-II

B6-Veh 178 11.23

Total Entries/ BTBR-
248 13.93 One-way
Locomotor Veh P=0.17 N/A N/A 4c
ANOVA
Activity
BTBR-
190.67 38.12
IGF-II

B6-Veh 48.27 7.78 B6-Veh vs.

BTBR-Veh:
BTBR- **P<0.01
Elevated Plus 10 to Time in open 98.19 11.51 One-way Neuman-
Veh **P=0.0032 4d
Maze 13 arms (s) ANOVA Keuls
B6-Veh vs.
BTBR- BTBR-IGF-II:
106.58 12.87
IGF-II **P<0.01

124
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