Circuit directionality for motivation:

lateral accumbens-pallidum, but not

pallidum-accumbens, connections
regulate motivational attraction to
reward cues Elizabeth B. Smedley &
Alyssa Dileo & Kyle S. Smith
Visit to download the full and correct content document:
https://ebookmass.com/product/circuit-directionality-for-motivation-lateral-accumbens-
pallidum-but-not-pallidum-accumbens-connections-regulate-motivational-attraction-to-
reward-cues-elizabeth-b-smedley-alyssa-dileo-kyle-s/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...

Elsevier Weekblad - Week 26 - 2022 Gebruiker

https://ebookmass.com/product/elsevier-weekblad-
week-26-2022-gebruiker/

Jock Seeks Geek: The Holidates Series Book #26 Jill

Brashear

https://ebookmass.com/product/jock-seeks-geek-the-holidates-
series-book-26-jill-brashear/

Last But Not Leashed Eileen Brady

https://ebookmass.com/product/last-but-not-leashed-eileen-brady/

Last But Not Leashed Eileen Brady

https://ebookmass.com/product/last-but-not-leashed-eileen-
brady-2/
Pain-Induced Negative Affect Is Mediated via
Recruitment of The Nucleus Accumbens Kappa Opioid
System

https://ebookmass.com/product/pain-induced-negative-affect-is-
mediated-via-recruitment-of-the-nucleus-accumbens-kappa-opioid-
system/

The New York Review of Books – N. 09, May 26 2022

Various Authors

https://ebookmass.com/product/the-new-york-review-of-
books-n-09-may-26-2022-various-authors/

Calculate with Confidence, 8e (Oct 26,

2021)_(0323696953)_(Elsevier) 8th Edition Morris Rn
Bsn Ma Lnc

https://ebookmass.com/product/calculate-with-
confidence-8e-oct-26-2021_0323696953_elsevier-8th-edition-morris-
rn-bsn-ma-lnc/

What Kind of Girl Alyssa B. Sheinmel

https://ebookmass.com/product/what-kind-of-girl-alyssa-b-
sheinmel/

Taming the Corporation: How to Regulate for Success

Robert Baldwin

https://ebookmass.com/product/taming-the-corporation-how-to-
regulate-for-success-robert-baldwin/
Accepted Manuscript

Circuit directionality for motivation: lateral accumbens-pallidum, but not pal-

lidum-accumbens, connections regulate motivational attraction to reward cues

Elizabeth B. Smedley, Alyssa DiLeo, Kyle S. Smith

PII: S1074-7427(19)30089-9
DOI: https://doi.org/10.1016/j.nlm.2019.05.001
Reference: YNLME 7028

To appear in: Neurobiology of Learning and Memory

Received Date: 28 September 2018

Revised Date: 23 April 2019
Accepted Date: 10 May 2019

Please cite this article as: Smedley, E.B., DiLeo, A., Smith, K.S., Circuit directionality for motivation: lateral
accumbens-pallidum, but not pallidum-accumbens, connections regulate motivational attraction to reward cues,
Neurobiology of Learning and Memory (2019), doi: https://doi.org/10.1016/j.nlm.2019.05.001

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Circuit directionality for motivation: lateral accumbens-pallidum, but not pallidum-
accumbens, connections regulate motivational attraction to reward cues

Elizabeth B. Smedley1, Alyssa DiLeo2, Kyle S. Smith1

1Dartmouth College
Department of Psychological and Brain Sciences

2 Department of Neuroscience, Sackler School of Graduate Biomedical Sciences, Tufts

University School of Medicine
Abstract
Sign-tracking behavior, in which animals interact with a cue that predicts reward,
provides an example of how incentive salience can be attributed to cues and elicit motivation.
The nucleus accumbens (NAc) and ventral pallidum (VP) are two regions involved in cue-driven
motivation. The VP, and NAc subregions including the medial shell and core, are critical for
sign-tracking. Further, connections between the medial shell and VP are known to participate in
sign-tracking and other motivated behaviors. The NAc lateral shell (NAcLSh) is a distinct and
understudied subdivision of the NAc, and its contribution to the process by which reward cues
acquire value remains unclear. The NAcLSh has been implicated in reward-directed behavior,
and has reciprocal connections with the VP, suggesting that NAcLSh and VP interactions could
be important mechanisms for incentive salience. Here, we use DREADDs (Designer Receptors
Exclusively Activated by Designer Drugs) and an intersectional viral delivery strategy to produce
a biased inhibition of NAcLSh neurons projecting to the VP, and vice versa. We find that
disruption of connections from NAcLSh to VP reduces sign-tracking behavior while not affecting
consumption of food rewards. In contrast, VP to NAcLSh disruption affected neither sign-
tracking nor reward consumption, but did produce a greater shift in animals’ behavior more
towards the reward source when it was available. These findings indicate that the NAcLSh→VP
pathway plays an important role in guiding animals towards reward cues, while VP→NAcLSh
back-projections may not and may instead bias motivated behavior towards rewards.
Introduction
Sign-tracking, or autoshaping, includes a behavioral phenomenon where animals
interact with a conditioned stimulus (CS+) that predicts an unconditioned stimulus (US), like a
reward, even though the US delivery is not contingent on this behavior (Brown and Jenkins
1968; Flagel and Robinson 2017; Boakes 1977). Sign-tracking reflects the attribution of
incentive salience to the CS+ and can be highly sensitive to changes in motivational state and
cue-reward relationships (Jenkins and Moore 1973; Robinson and Berridge 2013; Berridge and
Robinson 2003; Berridge 2004; Chang and Smith 2016; Smedley and Smith 2018a, 2018b;
Flagel and Robinson 2017). The nucleus accumbens (NAc) and ventral pallidum (VP), two
reciprocally connected limbic regions, have long been implicated in motivated behaviors
directed towards CS+s and their paired rewards (Smith et al. 2009; Root et al. 2015; Mogenson
1980). For example, manipulations to decrease the function of the NAc can result in reduced
sign-tracking behaviors (Chang and Holland 2012; Chang and Holland 2013; Cardinal et al.
2002). Phasic activity patterns of NAc neurons, and release patterns of neurotransmitter input,
can also represent the reward-related value of CS+ cues including those that evoke sign-
tracking behavior (Day and Carelli 2007; Day et al. 2006; Batten et al. 2018; Flagel et al. 2011;
Singer et al. 2016; Wan and Peoples 2008; Ambroggi et al. 2011; Saunders and Robinson,
2012). VP neuronal activity is similarly modulated by CS+ cues that are imbued with incentive
salience (Tindell 2005; Tindell 2009; Smith et al. 2011; Richard et al. 2016; Ahrens et al. 2016;
Ahrens et al. 2018). Inhibition of the VP also disrupts sign-tracking behavior and does so in a
manner not attributable to changes in motor expression or the value of the reward (Chang,
Todd, Bucci, & Smith 2015).
The contributions of different anatomically organized projections between the specific
subregions of the NAc and VP to incentive salience remains unclear. The NAc can be divided
into core and shell regions, and the shell further subdivided into medial and lateral segments
(Heimer et al. 1997; Zahm 2000; van Dongen et al. 2005; Yang et al. 2018; Zahm and Brog
1992; Kuo and Chang 1992; Zaborsky et al. 1985). There is evidence that connections between
the medial NAc shell and VP play an important role in forms of appetitive motivation including
cue-triggered reward seeking, reward consumption, and sign-tracking (Stratford and Kelley
1997, Smith and Berridge 2007, Leung and Balleine 2015, Chang et al 2018, Smith et al. 2011).
In contrast, roles for connections between the lateral NAc shell (NAcLSh) and VP remain
highly understudied. The NAcLSh itself participates in positive motivation (Zhang and Kelley
2000; Lammel et al. 2012; Mahler and Aston-Jones 2012; Yang et al. 2018). However, it is
distinct from medial NAc shell in its anatomical connectivity (Zahm and Brog 1992, Deutch and
Cameron 1993). For example, the NAcLSh has reciprocal connections with VP in a more mid-
lateral VP zone that is partly dissociable from medial NAc shell-VP connectivity (Brog et al.
1993, Zahm 2000; Churchill and Kalivas, 1994). NAcLSh is also connected with areas linked
with appetitive behavior including the ventral tegmental area, substantia nigra pars compacta,
lateral hypothalamus, and extended amygdala (Yang et al. 2018, Heimer et al. 1991,
Groenewegen and Russchen 1984, Brog et al. 1993). Thus, anatomically, both NAcLSh and VP
structures are poised to interact with one another reciprocally and to affect a broader neural
network that includes areas implicated in motivation and behavioral control.
To begin addressing the role of this reciprocal connection in motivation, we investigated
the effect of biasing chemogenetic inhibition of VP projections to the NAcLSh (VP→NAcLSh), or
NAcLSh projections to the VP (NAcLSh→VP), in a Pavlovian conditioning procedure for food
and on the primary motivation to eat food. We found that NAcLSh→VP selectively reduced sign-
tracking, that VP→NAcLSh inhibition selectively increased goal-approach that also occurred
during the cues, and that neither pathway manipulation detectably affected free feeding
behavior. These results highlight a preferential role for the NAcLSh→VP pathway in regulating
the motivational attraction to reward-paired cues. Moreover, the functional dissociation between
the pathway manipulations indicates that the NAcLSh→VP conveys information to the VP for
the regulation of reward cue attraction that could be insensitive to the integrity of information
transferred back from the VP.

Materials and Methods

Subjects. Experimentally naïve male Long Evans rats (arrival weight 250-300g) were
obtained from Charles River (n = 52; Charles River, Indianapolis, IN, USA). Rats were single
housed in ventilated plastic cages in a climate-controlled colony room set to a 12h light/dark
cycle (lights on at 7:00 A.M.). Experiments were conducted during the light cycle. Food and
water were available ad libitum until 7 days before magazine training, at which point weight was
restricted to 85% of ad libitum weight prior to testing. Restriction was maintained throughout the
experiment. For restriction, rats were provided with 5-12g of standard chow (Harlan Teklad
2014) and free access to water after each testing session. All procedures were approved by the
Dartmouth Institutional College Animal Care and Use Committee.
Surgical Procedures. Rats were anesthetized with isoflurane gas and placed in a
stereotaxic apparatus (Stoelting, Kiel, WI, USA). Surgery was conducted under aseptic
conditions. A 5 µl, 33-gauge beveled needle-tipped syringe (World Precision Instruments,
Sarasota, FL, USA) was lowered to the bilateral target sites and allowed to rest for 3 minutes.
Viral vectors were infused at a rate of 0.15 µl/min the following targets in mm from bregma: VP
(-0.12 AP, +/- 2.4 ML, -8.2 DV), NAcLSh (+1.44 AP, +/- 2.0 ML, -8.2 DV) (Paxinos and Watson,
2007). Post-infusion, the needle was allowed to rest for 5 minutes to allow viral dispersion. Two
groups of animals were used to assess the NAcLSh→VP pathway: NAcLSh→VP Inhibition and
NAcLSh→VP Control. The inhibition group received 0.6 µl of AAV-hSyn-DIO-hM4D(Gi)-
mCherry (n = 13; AAV5, UNC Vector Core; n = 4; AAV8, Addgene) in the NAcLSh and 1.0 µl
CAV-Cre (IGMM, France) or CAV-Cre-GFP (IGMM, France) in the VP. Controls for the
NAcLSh→VP projection received an AAV vector lacking the DREADD molecule (AAV5-hSyn-
DIO-mCherry; n = 9, UNC Vector Core) in the NAcLSh and 1.0 µl CAV-Cre or CAV-Cre-GFP in
the VP. Viral injection volumes were determined through pilot surgeries prior to experimentation.
Adequate expression was seen using the reported 0.6uL volume for the DREADD and 1.0uL
volume for the CAV-Cre. The decided upon volumes were similar to what had worked previously
for other members of the lab in chemogenetic targeting of VP (Chang et al. 2015; Chang et al.
2018) and medial regions of NAc shell (Chang et al. 2018). As in those studies, we targeted
here a central region of both structures along their rostrocaudal axis; ultimate variation in
rostrocaudal placement (see histology maps) relates to surgical variables.
Likewise, two groups of animals were used to assess the VP→NAcLSh pathway:
VP→NAcLSh Inhibition and VP→NAcLSh Control. The inhibition group received 0.6 µl of AAV-
hSyn-DIO-hM4D(Gi)-mCherry (n = 12, AAV5, UNC Vector Core; n = 4, AAV8, Addgene) in the
VP and 1.0 µl CAV2-Cre or CAV2-Cre-GFP in the NAcLSh. Controls of the VP→NAcLSh
projection received the same virus structure with the omission of the receptor (AAV5-hSyn-DIO-
mCherry; n = 10, UNC Vector Core) in VP and 1.0 µl CAV2-Cre or CAV2-Cre-GFP in the
NAcLSh. Similar intersectional strategies to the one used here have shown to be effective in
previous reports (Allsop et al. 2018; Boender et al. 2014; Carter et al. 2013; Nair et al. 2013;
Senn et al. 2014). Surgical incisions were closed with surgical clips and covered with Neosporin.
Rats were given intraperitoneal (IP) injections at 3 mg/kg of Ketoprofen and 5 ml of 0.9% sterile
saline after surgery and monitored for the remainder of the experiment. Clips were removed
under isoflurane anesthetic within 2 weeks of surgery. Animals were recovered with food,
DietGel (Clear H2O, ME, USA), and water. Food restriction and behavioral procedures began a
minimum of 3 weeks post-surgery.
Test Apparatus. Conditioning procedures were conducted in standard operant chambers
(Med Associates, St. Albans, VT) that were enclosed in sound- and light-attenuating cabinets
and were outfitted with fans for ventilation and white noise. Chambers contained two retractable
levers on either side of a recessed magazine where food rewards would be delivered. Lever
depressions were recorded automatically, and magazine entries were recorded through breaks
in an infrared beam at the magazine site. Free feeding procedures were conducted in cleaned
plastic home-cages affixed with a glass petri dish to contain food.
Test Procedures. The experimental design included one day of magazine training,
twelve days of Pavlovian conditioning training, and 2 days of free-feeding testing. For magazine
training, one 30-minute session of magazine training was conducted to habituate rats to the
chamber and grain pellet reward delivery (BioServ, 45 mg Dustless Precision Pellets, Rodent
Grain-Based Diet). Pellets were delivered such that over a 30 minute period, about 60 pellets
were delivered [p(pellet per second) = 1/30]. Magazines were checked after testing to confirm
consumption of reward pellets.
Pavlovian conditioning then began for 12 consecutive daily sessions. A given session
contained 25 CS+ trials where the 10 sec insertion of a retractable lever was followed by the
delivery of 2 grain pellets into the magazine, and 25 CS- trials where the 10 sec insertion of the
other lever was followed by nothing. Trials were pseudorandomized such that no more than two
of the same trial followed in sequence with intertrial intervals of approximately 2 minutes.
Conditioning sessions were roughly 1 hour in length. Thirty minutes prior to each session,
intraperitoneal injections of clozapine-n-oxide (CNO) were given. CNO was dissolved in sterile
water to a concentration of 0.001 g/ml and was given at a relatively low 1 mg/kg dose that we
and others have found effective for behavioral studies including Pavlovian conditioning tasks
(Smith et al. 2016; Chang et al. 2015; Chang et al. 2018).
Within 5 days of the last conditioning session, rats were administered free feeding tests
in 2 consecutive sessions, 24 hours apart. Rats were given CNO injections as above, and then
30 minutes later given access to 16 grams of grain pellets in clean home-cages. Rats were
given 1 hour to consume 16 grams of grain pellets. Food weight was measured pre- and post-
feeding to calculate grams consumed.
Histology. After completion of behavioral procedures, rats were deeply anesthetized with
1 ml phenobarbital and perfused with 0.9% saline solution for approximately 6-8 minutes
followed by perfusion of 10% formalin until fixture of head and neck tissue (approximately 3-4
minutes). Brain tissue was extracted, saturated with 20% sucrose and frozen to -80°C until
sliced to 60 µm thick sections and mounted. Slides were coverslipped with Vectashield
mounting medium containing DAPI (Vector Labs). Fluorescent expression was imaged via
Olympus BX53 fluorescent microscope with DP73 camera. A small number of animals (n = 5 in
NAcLSh→VP; n = 3 in VP→NAcLSh) underwent mCherry immunofluorescence if they showed
dimmer, although still present, viral expression that could still be mapped. In these cases,
expression was atypical in the form of incomplete cell body expression that appeared “fleck-like”
and resulted as a consequence in overall dimmer levels of fluorescence (see Results, Viral
Expression and Supplemental Analysis & Figures). For this, 60 µm slices were washed in 0.1M
PBS (3x10 minutes), blocked in a 3% normal donkey serum (one hour), and incubated overnight
in primary antibody (rabbit anti-DsRed, 1:500; Clontech). The next day, slices were again rinsed
in 0.1M PBS (3x10 minutes) and then incubated for 4-5 hr in secondary antibody (donkey anti-
rabbit Alexafluor 594, 1:500; Thermo Scientific). After a last rinse in 0.1M PB (3x10 minutes),
slices were mounted and coverslipped with DAPA-containing Vectashield (Vector Labs). Per-
animal expression was manually transcribed onto printed images (Paxinos & Watson, 2007) and
then transcribed digitally via PowerPoint (Microsoft) at 90% transparency. Per-animal
expression maps were then combined into group expression maps by digitally overlaying the
expression areas. VP expression was defined by Paxinos and Watson (2009) coordinates (AP: -
0.12, ML: 2.4, DV: -8.2) with the target being ventral to anterior commissure and lateral/anterior
to the substantia innominata and lateral preoptic area, but dorsal to magnocellular preoptic
nucleus. NAcLSh expression was targeted on coordinates (AP: +1.44mm, ML: 2.0mm, DV: -
8.2mm) with expression localized to the area lateral and ventral to the accumbens core, but
dorsal to the most rostral portion of VP and medial to the endopiriform nucleus.
Our histology inclusion criteria were as follows: (1) no defects (lesions, cell death, or
infection) that would exclude the animal on the basis of health. (2) The presence of dense
clusters of labeled cells within the area of interest (i.e., the upstream area; VP in the
VP→NAcLSh animals; NAcLSh in the NAcLSh→VP animals). Dim/atypical viral expression
merited mCherry immunochemistry (this was applied for 24% of animals that were included in
analysis [n = 8/33]). In order for animals to qualify for immunochemistry protocols, they needed
to show expression (even if expression was atypical) in the regions of interest. An absence of
florescent expression merited exclusion from the study. Qualification for inclusion based on
histology was not related to overall fluorescence level or density, which would have been
problematic by having some animals undergo immunostaining. (3) Minimal (less than
approximately 20 cells) or unilateral only spread of Gi outside the area of interest. Any
fluorescent expression that was unexpected (i.e., in an area it wasn’t supposed to be) was
checked under multiple filters to determine if it was mCherry-specific. If it was generally
fluorescent (i.e., glowed regardless of filter) and not cell bound it was regarded as non-cellular,
fluorescent remnants and deemed non-problematic. (4) Acceptable expression was mCherry-
specific, cell-bound, bilateral, and greater than an approximate 40 cells in the area of interest.
Animals who met the above criteria were included in analysis.
 Fluorescent Retrograde Tracer (CAV2-zsGreen). As the GFP tag in the subset of rats
with CAV2-Cre-GFP injections was not reliably detectable, we estimated CAV2 spread in 6
separate animals (3 received a 1.0µl injection into the VP; 3 received a 1.0µl injection into the
NAcLSh) that were unilaterally injected with CAV2-zsGreen (Zweifel Laboratory, University of
Washington). Injection, histology, and imaging procedures were performed as above.
Statistical Modeling and Analysis. All statistical tests were carried out using R (R Core
Team, 2013). Categorical variables with multiple levels (e.g., Cue Block) were dummy coded to
make predetermined comparisons between levels (e.g., pre CS+ vs. CS+ block, post CS+ vs.
CS+ block). All linear mixed models are fit by maximum likelihood and t-tests use Satterthwaite
approximations of degrees of freedom (R; “lmerTest”, Kuznetsova et al. 2015). The reported
statistics will include parameter estimates (β values), confidence intervals (95% bootstrapped
confidence intervals around dependent variable), standard error of the parameter estimate (SE),
and p-values (R; “lmerTest”, Kuznetsova et al. 2015). Graphs were created through GraphPad
Prism (version 7.0a) and designed with Adobe Illustrator.
Conditioning data were analyzed initially for lever presses per minute (total lever presses
over the session / minutes of lever availability) in a linear mixed model which accounts for the
fixed effects of lever type (CS+ or CS-) by group (inhibition or control) by interaction with
session of training (sessions 1-12), and for random effects of individual rat intercepts (i.e.,
individual session one values) and slopes (i.e., individual rat learning rates). NAcLSh→VP
inhibition group and NAcLSh→VP controls were analyzed in an individual model while the
opposite projection group and its control was analyzed separately. Following the initial analysis,
additional models investigate components of the primary analysis by modeling either lever
response rate (i.e., CS+ presses per minute during conditioning, CS- presses per minute during
conditioning) and either subgroup (i.e., CS+ vs. CS- ppm during conditioning: NAcLSh→VP
inhibition group, CS+ vs. CS- ppm during conditioning: NAcLSh→VP site specific control group).
See example of the main analysis below:

Presses per Minute (ppm) = Lever Type x Group x Session + (1 + Session | Rat)

When trends appeared non-linear, transformations of session were tested to best fit learning
rate curves (including logarithmic, quadratic, and exponential growth) and utilized if the
transformation was of statistically better fit as determined by an analysis of variance comparing
model deviances (p < 0.05, R; “anova” {lmerTest}) for nested models and Akaike information
criterion (AIC) for non-nested model comparison (i.e., the model with the lowest AIC, ΔAIC =
Model AIC - Null Model AIC). It is worth noting that when examining logarithmic or exponential
components of session, the linear term is not included in the model. Both the logarithmic and
exponential curves fit an increasing or decreasing function that levels off by themselves. They
do not need, and should not add, the linear term as it is redundant. However, quadratic models
include the linear fit as well, as in this case it is not redundant. An equation containing  and 2
is a second order polynomial. It fits empirical curves that are partly linear but have a bend or
leveling off. The coefficient for the  term shows how much the curve increases and the 2
term shows how prominent the bend is. Parameter estimates for the transformed independent
variable (i.e. session) are presented in the units of the dependent variable (i.e. ppm) and can be
interpreted such that the dependent variable units increase for every transformed component of
the independent variable. For example, the logarithmic transformation of session might yield a
significant effect with an estimate of 1.28 ppm and this can be interpreted to mean that the
press rate increases 1.28 for every increase of one log session. We similarly analyzed the
latency to lever press upon CS+ delivery (see Supplementary Analysis and Figures).
Magazine entry data was analyzed for overall magazine entries made by each group
over each of the training sessions in linear mixed models similar to the structure for lever
presses (above). Further analysis investigates when some of those magazine entries occurred
by looking at the 10 second block leading up to the CS+, the 10 second CS+ presentation block,
and the 10 second block after the CS+ presentation (the reward period). Predetermined
contrasts of the cue blocks were constructed such that each block was compared to the reward
period.
Free feeding data was similarly analyzed for amount of food consumed (grams) in a
linear mixed model which accounts for the group (i.e., NAcLSh→VP inhibition group and
NAcLSh→VP control were analyzed in an individual model while the opposite projection group
and its control were analyzed separately), individual animal weights taken during free feeding
experiments, session (i.e., session 1 and 2 of free feeding), and random effects of rat.

Results
Viral expression.
All animals were evaluated for robust expression of hM4Di receptors in the upstream
area of interest (i.e. in VP for the VP→NAcLSh projection); those without clear expression or
expression outside the area of interest were excluded from analysis (n = 15, due mainly to a
faulty virus batch). Additionally, 3 animals were excluded during behavioral testing for health
reasons. Thus, analysis was run with group sizes as: 14 animals in NAcLSh→VP analysis
(control group = 6; inhibition group = 8), 19 animals in VP→NAcLSh analysis (control group = 9,
inhibition group = 10). Linear mixed model analysis of repeated measures data can
accommodate uneven group sizes (Gibbons et al. 2010).
The vast majority of animals in the NAcLSh→VP inhibition group exhibited hM4Di-
mCherry expression in the lateral shell area as previously defined between bregma +1.20-2.16
mm with one animal showing some expression slightly more medial (although this animal had
anterior expression more lateral; Fig.1A). Two animals had very discrete expression of hM4Di-
mCherry in anterior portions of VP, but with the vast majority of expression in NAcLSh, and
were thus included in analyses. Similarly, animals in the NAcLSh→VP site-specific control
exhibited mCherry expression in the NAcLSh area. Notedly, mCherry expression from the
control virus appeared more robust, and tended spread into a larger area than the DREADD-
containing inhibitory virus (Fig.1B). These controls were included in analysis given that they
would control for viral mediated gene delivery at a level covering the DREADD expression areas
and even beyond. Animals in the VP→NAcLSh inhibition group had robust hM4Di expression in
the defined VP region between bregma -0.36-+0.36 mm (Fig.2A). Two animals showed more
rostral VP expression up to +1.68 mm, but by far the most dense expression occurred in the VP
in these animals. Given the minor spread beyond the target area of hM4Di expression, we refer
to manipulations here as pathway-biased rather than pathway-specific. VP → NAcLSh control
group expression was similarly greatest in VP with more spread dorsally as seen in the NAcLSh
→ VP control group (Fig. 2B).
A few animals in manipulation groups (NAcLSh→VP inhibition [n= 5 of 8], VP→NAcLSh
inhibition [n = 3 of 10]), showed hM4Di-mCherry expression that could be characterized as
“fleck-like” where expression was not clearly arranged in the neuronal membranes. This
expression was only visible in the TexasRed filter, and not in other filters, nor was there any
sign of cell death as judged by DAPI expression. This provided confidence that the expression
was the mCherry tag, as did the statistically similar results on the main behavior of interest (CS+
responding) of these animals (Supplementary Fig. 1).
CAV expression estimation (CAV2-zsGreen). In the animals who received NAcLSh
injections of CAV-zsGreen, discrete localization of zsGreen was seen in lateral VP (Fig. 3A). In
animals who received VP injections, discrete localizations of zsGreen was seen in very lateral
portions of our NAcLSh target (Fig. 3B). In both of these cases, the zsGreen expression was
highly confined anatomically.

NAcLSh to VP projection inhibition analysis

NAcLSh to VP General Lever Responding during Conditioning. To generally determine if press
rates differed by lever type (CS+ vs. CS-), group (NAcLSh → VP inhibition vs. NAcLSh → VP
control), and session (1-12), a linear mixed model with random effects of session and rat was
constructed. Linear variables were scaled (standardized).

ppm = Lever Type x Group x Session + Session2 + (1 + Session + Session2 | Rat)

Results indicate that while all rats preferred the CS+ lever, the NAcLSh → VP inhibition group
sign-tracked less towards the CS+ lever than NAcLSh → VP controls. The quadratic fit of
session significantly contributed to the model (2(4) = 31.89, p < 0.001). A significant main
effect of lever type revealed the CS+ lever was generally preferred over the CS- by all animals
(Fig. 4A; est: 23.3 ppm; CI: 21.2-25.2; SE: 1.02; p < 0.001). The main effect of group was
significant (est: 6.16 ppm; CI: 0.41- 11.7; SE: 2.83; p = 0.044) and the NAcLSh → VP inhibition
animals pressed the CS+ lever less than NAcLSh → VP controls as seen in a significant
interaction of lever type by group (est: 22.8 ppm; CI: 18.3-26.3; SE: 2.05; p < 0.001). All animals
had increased press rates by the end of training as determined by a main effect of linear
session (est: 10.3 ppm; CI: 4.00-16.7; SE: 3.29; p = 0.006). The quadratic component of
session was also significant (est: -8.67; CI: -15.6-(-1.53); SE: 3.64; p = 0.031). However, the
CS+ is pressed more by all animals by the end of training as determined by a significant
interaction of lever type by linear session (est: 4.76 ppm; CI: 2.84-6.80; SE: 1.03; p < 0.001).
Press rates differed by group over sessions (est: 3.62 ppm; CI: 0.23-6.72; SE: 1.54; p = 0.033),
however the three-way interaction of lever type by linear session by group was not significant
(est: 3.91 ppm; CI: -0.40-7.84; SE: 2.05; p = 0.058).

CS+ presses per minute during conditioning. The inhibition of the NAcLSh→VP pathway
resulted in a decrease in ppm toward the CS+ lever compared to the NAcLSh → VP control
group. The NAcLSh→VP projection data of CS+ ppm over time appeared non-linear in form;
comparison of a model containing quadratic and linear components against a model containing
only linear components revealed a significant contribution of quadratic session to the model
(2(4) = 72.62, p < 0.001). Thus, a linear mixed model was constructed with CS+ ppm by main
effects of group (NAcLSh → VP inhibition vs. NAcLSh → VP control), by session (1 - 12; both
linear and quadratic fits), with random effects of animal starting level and learning rate:

CS+ ppm = Group x Session + Session2 + (1 + Session + Session2 | Rat)

This model had both significant quadratic (est:-0.39 ppm; CI: -0.68-(-0.11); SE: 0.15; p = 0.017)
and linear (est: 6.40 ppm; CI: 3.15-9.72; SE: 1.69; p = 0.002) fit components and did not show a
main effect of group (est: -4.48 ppm; CI: -17.9-9.18; SE: 6.14; p = 0.478). The group by linear fit
(est: 2.36 ppm; CI: 0.34-4.27; SE: 0.86; p = 0.015) interaction was significant. This result
indicated that animals with the NAcLSh→VP pathway inhibited showed markedly reduced sign-
tracking behavior as a function of training time compared to controls (Fig. 4A).

CS- ppm during conditioning. Both the NAcLSh→VP inhibition group and the NAcLSh → VP
control group decreased ppm toward the non-paired lever similarly. CS- ppm data appeared
linear and a linear mixed model fitting CS- press rates by main effects of group by session (1-
12; natural log transformation), with random effects of animal starting level and learning rate:

CS- ppm = Group x Session + (1 + Session | Rat)

We detected no significant difference between Gi and controls (est: 0.80 ppm; CI: -1.35-3.26;
SE: 1.12; p = 0.486) nor a significant interaction of group by session (est: 0.04 ppm; CI: -0.14-
0.21; SE: 0.09; p = 0.676), indicating that NAcLSh → VP inhibition and NAcLSh → VP control
groups did not differ overall in interaction with the non-predictive lever nor in how they
terminated pressing overtime. There was a significant main effect of session (est: -0.19 ppm; CI:
-0.28-(-0.09); SE: 0.04; p < 0.001) showing that both groups decreased CS- pressing over the
course of training (Fig. 4A).

CS+ vs. CS- ppm during conditioning. Ppm on the paired (CS+) and non-paired (CS-) lever
were compared within each group by fixed effects of time (i.e., session) and lever type (CS+ vs.
CS-; see analysis below). Both NAcLSh→VP inhibition animals and the NAcLSh → VP control
group showed a preference for the paired CS+ lever over the non-paired CS- lever. Although
the degree of CS+ sign-tracking was reduced in the inhibition group compared to controls (see
analysis above), the inhibition group still showed a preference for the CS+ compared to the CS-.
In short, sign-tracking during NAcLSh→VP inhibition was reduced but was not eliminated to the
level of CS- sign-tracking.

CS+ vs. CS- ppm during conditioning: NAcLSh→VP inhibition group. Data appeared linear and
a linear mixed model was used to analyze press rate by lever type and session interaction with
random slope and rat intercepts:

ppm = Lever Type x Session + (1 + Session | Rat)

There was a significant main effect of press rates comparing the CS- to CS+ levers (est: 6.69
ppm; CI: 2.74-10.8; SE: 2.08; p = 0.002) with an average of nearly 6.69 ppm greater towards
the paired lever. There was not a significant main effect of session (est: 0.20 ppm; CI: -0.18-
0.58; SE: 0.20; p = 0.346). However, a significant session by cue interaction (est: 0.81 ppm; CI:
0.24-1.36; SE: 0.28; p = 0.005) illustrated an increase in sign-tracking to the paired CS+ lever
over time (Fig. 4A).

CS+ vs. CS- ppm during conditioning: NAcLSh→VP site-specific control group. A quadratic
transformation in session significantly contributed to the model (2(4) = 22.40, p < 0.001) and
thus was included in the final model:

ppm = Lever Type x Session + Session2 + (1 + Session + Session2 | Rat)

There was a significant main effect of press rates toward the CS+ (est: 22.1 ppm; CI: 14.0-30.6;
SE: 4.20; p < 0.001). Both linear (est: 5.84 ppm; CI: 2.61-8.96; SE: 1.62; p = 0.006) and
quadratic (est: -0.39 ppm; CI: -0.67-(-0.11); SE: 0.14; p = 0.029) transformations in session
were significant. A linear session by cue interaction (est: 1.94 ppm; CI: 0.73-3.01; SE: 0.57; p <
0.001) showed a significant increase in presses toward the CS+ lever over sessions compared
to the CS- lever (Fig. 4A).

Magazine entries during conditioning. NAcLSh→VP inhibition and NAcLSh→VP control groups
did not differ in their overall magazine entries per day nor in how they entered the magazine
over sessions. Total magazine entries per day appeared linear and were analyzed by a linear
mixed model with fixed effects of group (NAcLSh→VP inhibition and NAcLSh→VP control
groups) by session with random slopes and intercepts:

Total Magazine Entries = Group x Session + (1 + Session | Rat)

The main effect of group was not significant (est: -31.4 entries; CI: -226-176; SE: 104; p =
0.767). The main effect of session was significant (est: -16.1 entries; CI: -30.5-(-1.95); SE: 7.51;
p = 0.049), indicating that all animals decreased their number of entries by the end of training.
The interaction of group by session was not significant (est: 15.8 entries; CI: -14.6-46.7; SE:
15.0; p = 0.311), and thus NAcLSh→VP inhibition and NAcLSh→VP control groups did not
differ over sessions in their magazine entry behavior (Fig. 4B).

Magazine entries by cue block during conditioning. Although the NAcLSh→VP inhibition group
and the NAcLSh→VP control group did not differ in overall magazine entries, as above, there
was a difference in how each group distributed their entries with respect to the CS+ versus post-
CS+ time blocks over sessions. The NAcLSh→VP control group displayed more traditional sign-
tracking behavior where they developed a decreased magazine entry rate during cue
presentation and increased magazine entries during the post-cue block when reward was
available. The NAcLSh→VP inhibition group did not show this shift over time, exhibiting less of
a difference in entries between the CS+ and post CS+ block.
Magazine entries recorded during ten-sec cue blocks (pre CS+, CS+, post CS+ [i.e.,
reward delivery]) each were averaged over 25 trials per day (i.e., an average entries per trial in
the pre CS+ block). A predetermined contrast analyzed the CS+ presentation block against the
reward delivery block and if more magazine entries were performed during the reward block (as
expected) compared to the cue presentation. Another contrast analyzed the 10 second block
prior to CS+ presentation against the reward delivery and if magazine entries were greater
during the reward (as expected) compared to the time prior to cue delivery. The average entries
per block appeared linear and were analyzed in a linear mixed model by fixed effects of session,
group, and cue, with random effects for individual rat intercepts (random slopes could not be
included due to failed convergence):
Daily Average Entries per Cue Block = Group x Session x Cue Block + (1 | Rat)
No significant main effect of group was found (est: -0.02 avg. entries; CI: -0.90-1.03; SE: 0.49; p
= 0.961). However, a significant session effect was found (est: 0.10 avg. entries; CI: 0.05-0.15;
SE: 0.03; p < 0.001). Regardless of group, contrasts to compare pre CS+ to post CS+ blocks
was significant with 2.85 average entries greater during the post CS+ block compared to pre
CS+ (est: -2.85 avg. entries; CI: -3.42-(-2.32); SE: 0.29; p < 0.001). CS+ presentation to post
CS+ block was also significant with 2.90 average entries greater during the post CS+ block
compared to pre CS+ (est: -2.09 avg. entries; CI: -3.56-(-2.36); SE: 0.29; p < 0.001). The trend
towards greater entries during the post CS+ block developed over time as seen in a significant
pre CS+ to post CS+ by session interaction (est: -0.16 avg. entries; CI: -0.22-(-0.08); SE: 0.04; p
< 0.001) and significant CS+ to post CS+ by session interaction (est: -0.15 avg. entries; CI: -
0.21-(-0.07); SE: 0.04; p < 0.001; Fig. 4C). The NAcLSh→VP inhibition group and the
NAcLSh→VP control group differed in how they entered the magazine over sessions, with a
significant group by session interaction (est: 0.13 avg. entries; CI: 0.02-0.22; SE: 0.05; p =
0.018). A three way interaction of group by session by CS+ vs. post CS+ blocks was significant
(est: -0.20 avg. entries; CI: -0.33-(-0.04); SE: 0.08; p = 0.009), indicating that the NAcLSh→VP
control group made slightly more magazine entries during the post-CS+ reward block, and fewer
entries during the CS+ itself, over sessions compared to the NAcLSh→VP inhibition group (Fig.
4C). Notedly, this difference was only seen over sessions and was not present overall as
determined by a non-significant CS+ versus post CS+ by group interaction (est: -0.17 avg.
entries; CI: -1.29-0.91; SE: 0.58; p = 0.767; Fig. 4D, 4E). The NAcLSh→VP inhibition group and
the NAcLSh→VP control group did not differ over sessions in how they entered the magazine
during the ten second blocks before vs. during CS+ presentation (i.e., the three way interaction
of group by session by contrast of pre CS+ to post CS+; est: -0.10 avg. entries; CI: -0.25-0.05;
SE: 0.08; p = 0.173). The overall interaction of preCS+ versus post CS+ by group interaction
was not significant (est: -0.15 avg. entries; CI: -1.16-1.03; SE: 0.58; p = 0.942; Fig. 4D, 4E).

Free feeding. The NAcLSh→VP inhibition and the NAcLSh → VP control groups did not differ in
the amount of food consumed during free feeding tests nor did their weights impact feeding
behavior. A linear mixed model of total grams of food consumed as a function of group
(NAcLSh→VP control versus NAcLSh→VP inhibition) by day (days 1 and 2) and body weight (in
grams) by day, with random effects of individual animal, was constructed:

Grams Consumed = Group x Day + Animal Weight x Group + (1 | Rat)

There was not a significant main effect of grams consumed by day of testing (est: 0.42 grams;
CI: -0.44-1.18; SE: 0.41; p = 0.319) nor main effect of grams consumed by group (est: -11.25
grams; CI: -32.8-9.79; SE: 11.1; p = 0.325). Grams consumed did not differ by body weight (est:
-0.01 grams; CI: -0.05-0.02; SE: 0.02; p = 0.410). There was not a significant group by day
interaction (est: -1.33 grams; CI: -3.23-0.26; SE: 0.82; p = 0.123) nor group by weight
interaction (est: 0.03 grams; CI: -0.03-0.10; SE: 0.03; p = 0.323; Fig. 4F).

VP→NAcLSh projection inhibition analysis

VP to NAcLSh General Lever Responding during Conditioning. To generally determine if press
rates differed by lever type (CS+ vs. CS-), group (VP à NAcLSh inhibition vs. VP à NAcLSh
control), and session (1-12) a linear mixed model with random effects of session and rat was
constructed.
ppm = Lever Type x Group x Session + (1+Session | Rat)

Rats in both the VP → NAcLSh inhibition group and the VP → NAcLSh control group showed a
preference for the CS+ lever, which developed over training. However, the VP → NAcLSh
inhibition group and the VP → NAcLSh control group did not differ in their behavior toward
either the CS+ or CS- levers. Transformations in session did not significantly contribute to the
model structure and as such only linear forms of session were included in the final model. The
CS+ was preferred by all animals as determined by a significant main effect of lever type (Fig
5A; est: 11.1 ppm; CI: 8.29-13.8; SE: 1.40; p < 0.001). A non-significant main effect of group
indicated that the VP → NAcLSh inhibition and VP → NAcLSh control groups did not generally
differ in press rates (est: -3.36 ppm; CI: -7.95-1.51; SE: 2.36; p = 0.171). The VP → NAcLSh
inhibition and VP → NAcLSh control groups had similar press rates toward both the CS+ and
CS- levers as determined by a non-significant interaction of lever type by group (est: -2.68 ppm;
CI: -7.97-2.68; SE: 2.79; p = 0.338). The CS+ lever preference also developed over sessions as
seen in a significant lever type by session interaction (est: 1.13 ppm; CI: 0.76-1.52; SE: 0.19; p
< 0.001). Press rates did not differ by group over sessions (est: 0.19 ppm; CI: -0.39-0.85; SE:
0.30; p = 0.534) nor did the three way interaction of lever type by session by group (est: -0.01
ppm; CI: -0.75-0.78; SE: 0.380; p = 0.988).

CS+ ppm during conditioning. The VP→NAcLSh inhibition group and the VP→NAcLSh controls
did not differ in sign-tracking to the CS+ lever. The VP→NAcLSh projection data of CS+
responses over time appeared non-linear in form and model fit significantly improved after
addition of a quadratic fit of session (2(4) = 50.9, p < 0.001) and thus were included in the final
model. The same model structure for CS+ ppm analysis for the opposite projection group (see
above) was used here:

CS+ ppm = Group x Session + Session2 + (1 + Session + Session2 | Rat)

Results showed an insignificant main effect of group (est: -4.57 ppm; CI: -12.5-4.02; SE: 4.16; p
= 0.285). A significant linear component of session (est: 3.16 ppm; CI: 0.95-5.45; SE: 1.13; p =
0.012) identified that all animals learned over training. The quadratic component of session was
also significant (est: -0.176 ppm; CI: -0.34-(-0.01); p = 0.037) again indicating that animals
learned to acquire CS+ responding at a similar rate. Group by linear session was not significant
in analysis (est: 0.213 ppm; CI: -0.93-1.40; SE: 0.56; p = 0.705; Fig. 5A), indicating that both the
VP→NAcLSh inhibition and VP→NAcLSh control groups learned at the same rate over
sessions. Thus, unlike the reduced sign-tracking observed with NAcLSh→VP inhibition
compared to the NAcLSh → VP controls, sign-tracking with VP→NAcLSh inhibition appeared
equivalent to the VP→NAcLSh controls. Sign-tracking levels were generally lower in the
VP→NAcLSh groups compared to those targeting the NAcLSh→VP, but still in a normal range
based on prior studies, which highlights the importance of controls that are specific to pathways
being targeted (Fig. 5A).

CS- ppm during conditioning. A significant main effect of group showed that rats with
VP→NAcLSh inhibition pressed the non-paired CS- more than the VP→NAcLSh controls.
However, over sessions both groups decreased press rates similarly toward the CS-. Data
appeared linear and the model of CS- responses by fixed effects of group by linear session was
constructed:
CS- ppm = Group x Session + (1 + Session | Rat)

There was a significant main effect of group (est: -2.02 ppm; CI: -3.79-(-0.34); SE: 0.88; p =
0.034). There was a significant main effect of linear session (est: -0.27 ppm; CI: -0.38-(-0.17);
SE: 0.05; p < 0.001), indicating that all animals decreased pressing toward the CS- lever. The
group by linear session interaction was not significant (est: 0.19 ppm; CI: 0.01-0.40; SE: 0.10; p
= 0.063; Fig. 5A), indicating that all animals showed similar decreases in CS- press rates over
sessions.

CS+ vs. CS- ppm during conditioning. Both the VP→NAcLSh inhibition group and the
VP→NAcLSh control group preferred the paired CS+ lever over the non-paired CS- lever. Press
rates on the CS+ and CS- levers were compared within each group by fixed effects of time (i.e.
session) and lever type (CS+ v. CS-). The following model structure is used in the next two
sections.
ppm = Lever Type x Session + (1 + Session | Rat)

CS+ vs. CS- ppm during conditioning: VP→NAcLSh inhibition group. The VP→NAcLSh
inhibition group preferred the CS+ lever over the CS- lever. There was a significant main effect
of cue type (est: 12.4 ppm; CI: 8.24-16.6; SE: 2.11; p < 0.001) such that the CS+ was
significantly preferred over the CS- in pressing behavior. There was not a significant main effect
of linear session (est: 0.20 ppm; CI: -0.32-0.74; SE: 0.26; p = 0.450). However, a lever cue by
session interaction was significant (est: 1.14 ppm; CI: 0.58-1.69; SE: 0.29; p < 0.001), such that
rates of preference toward the CS+ lever increased over time (Fig. 5A).

CS+ vs. CS- ppm during conditioning: VP→NAcLSh site specific control group. The
VP→NAcLSh control group also preferred the CS+ lever over the CS- lever. There was a main
effect of cue type (est: 9.73 ppm; CI: 6.10-13.4; SE: 1.78; p < 0.001) as confirmation of this.
There was a significant main effect of session (est: 0.39 ppm; CI: 0.14-0.67; SE: 0.14; p =
0.018) and session by lever type interaction (est: 1.13 ppm; CI: 0.65-1.64; SE: 0.24; p < 0.001),
indicating that control animals interacting with the CS+ lever more over time (Fig. 5A).

Magazine entries during conditioning. Total magazine entries per session appeared linear and
model fit a linear mixed model of magazine entries by group (VP→NAcLSh inhibition group and
the VP→NAcLSh control group) and day with random slopes and intercepts was constructed:

Total Magazine Entries = Group x Session + (1 + Session | Rat)

The VP→NAcLSh inhibition and the VP→NAcLSh control groups did not differ in magazine
entries as determined by a non-significant group effect (est: 122 entries; CI: -50.8-296; SE:
91.4; p = 0.198). Magazine entries did not change over sessions as seen in an insignificant
main effect of session (est: -11.7 entries; CI: -30.0-7.27; SE: 8.76; p = 0.196). The VP→NAcLSh
inhibition and the VP→NAcLSh control groups similarly maintained magazine entry rates over
time as seen in an insignificant group by session interaction (est: -19.7 entries; CI: -57.3-12.8;
SE: 17.5; p = 0.276; Fig. 5B).

Magazine entries during cue blocks. Despite the lack of effect of VP→NAcLSh inhibition on
sign-tracking behavior and overall magazine entries, it did lead to a difference in magazine entry
distribution: the VP→NAcLSh inhibition group distributed their entries such that fewer entries
were made in the CS+ presentation block and more entries in the post CS+ block compared to
the VP→NAcLSh controls over sessions. The statistical model structure is the same as the
opposite projection group above and includes the same cue block contrasts described above.

Daily Average Entries per Cue Block = Group x Session x Cue Block + (1 | Rat)
There was a significant main effect of group (est: 1.63 entries; CI: 0.41-2.95; SE: 0.66; p =
0.016) and linear session (est: 0.13 entries; CI: 0.06-0.20; SE: 0.03; p < 0.001). Main effects of
pre-CS+ versus post-CS+ block (est: -2.64 entries; CI: -3.37-(-1.85); SE: 0.36; p < 0.001) and
CS+ versus post-CS+ block (est: -2.27 entries; CI: -2.95-(-1.52); SE: 0.36; p < 0.001) were
significant in that magazine entries were greatest in the post-CS+ reward block compared to the
pre-CS+ and CS+ blocks (Fig. 5C, 5D). A group by linear session interaction was significant
(est: -0.21 entries; CI: -0.33-(-0.07); SE: 0.07; p = 0.002). The group by pre-CS+ versus post-
CS+ block interaction was not significant (est: -1.16 entries; CI: -2.48-0.23; SE: 0.72; p = 0.108;
Fig. 5D). The group by CS+ versus post-CS+ block interaction was not significant (est: -0.94
entries; CI: -2.29-0.63; SE: 0.72; p = 0.193). All animals tended to increase magazine entries
over sessions during the reward block compared to the pre-CS+ block (est: -0.17 entries; CI: -
0.27-(-0.07); SE: 0.05; p < 0.001) and compared to the CS+ presentation (est: -0.18; CI: -0.27-(-
0.09); SE: 0.05; p < 0.001; Fig. 5C). The three-way interaction of group by session by pre-CS+
vs. reward block was not significant (est: 0.10 entries; CI: -0.08-0.28; SE: 0.10; p = 0.307),
indicating that all animals distributed their magazine entries similarly toward the post-CS+
reward block compared to the pre-CS+ block over sessions (Fig. 5C). The three-way interaction
of group by session by CS+ presentation vs. reward block was significant (est: 0.33 entries; CI:
0.14-0.51; SE: 0.09; p < 0.001), indicating that over time, the VP→NAcLSh inhibition group
entered the magazine less during the CS+ presentation and more during the post-CS+ reward
block compared to VP→NAcLSh controls (Fig. 5C).

Free feeding. There was no difference in food consumption during free feeding tests between
animals with VP→NAcLSh inhibition and the VP→NAcLSh controls. The same model structure
used in the NAcLSh→VP feeding analysis was used here:

Grams Consumed = Group x Day + Animal Weight x Group + (1 | Rat)

There was not a main effect of group (est: -7.48 grams; CI: -21.8-7.84; SE: 7.89; p = 0.354),
meaning all animals consumed food similarly in free feeding tests. Animals remained consistent
with their food intake over the two days of testing as seen in an insignificant main effect of day
(est: 0.11 grams; CI: -1.10; SE: 0.61; p = 0.856) and insignificant main effect of group by day
(est: -0.98 grams; CI: -3.61-1.53; SE: 1.23; p = 0.432). However, intriguingly, there was a main
effect of weight (est: -0.028 grams; CI: -0.05-(-0.01); SE: 0.01; p = 0.020) whereby animals of
greater weight tended to eat slightly less. However, this effect of weight was generalized over
both the VP→NAcLSh inhibition and the VP→NAcLSh control groups as seen in an insignificant
group by weight interaction (est: 0.022 grams; CI: -0.02-0.06; SE: 0.02; p = 0.322; Fig. 5F).

Discussion
To investigate the importance of NAcLsh and VP connections in the ability of reward
cues to draw in motivated behavior, we used a pathway-biased chemogenetic manipulation
strategy to inhibit projections from the NAcLSh to the VP, and vice versa, in a Pavlovian
conditioning procedure. Inhibition of the NAcLsh→VP pathway resulted in a marked reduction of
sign-tracking behavior to a CS+ lever. In stark contrast, inhibition of the reverse VP→NAcLSh
pathway left sign-tracking behavior normal. However, it was not as though VP→NAcLSh
inhibition was unremarkable. Those animals showed a shift over time to having greater
magazine-directed behavior when reward was available after the post CS+ reward block versus
to when the CS+ was presented compared to controls. Inhibition of the NAcLSh→VP instead
had a greater tendency to exhibit magazine entries during the CS+ itself.
It is unlikely that the NAcLSh→VP inhibition results can be explained by a loss of the
CS+-reward association. These animals still retained their normal magazine directed behavior,
some of which was CS+-driven, and did not change their normally low level of CS- interaction.
An increase in entries in the post-cue block (i.e., reward delivery) is generally expected given
that this is when animals are consuming the reward. However, of interest here is the
VP→NAcLSh inhibition group showing greater entries than controls during the reward period
relative to the CS+ lever presentation period. And given the NAcLSh→VP animals are not
interacting with the CS+ lever as much when it is presented, they tend to increase their
magazine entries during the CS+ compared to controls (as control CS+ behavior is competing
with their ability to enter the magazine as much). None of the sign-tracking or magazine entry
effects we observed could be explained by a change in the motivational value of the food
reward because free feeding behavior was unaffected by any manipulation. We hesitate to
conclude that neither pathway is necessary for the motivation to eat, given that robust
manipulations of either the NAcLSh or the VP can indeed affect eating behavior (e.g., Zhang
and Kelley 2000; Cromwell and Berridge 1993). Two possibilities are either that the DREADD
manipulation was subtle enough to leave other brain areas capable of driving eating behavior
normally or that neither pathway is involved in free eating. In either case, the roles for other
areas connected with the NAc and VP that are known to regulate eating (e.g., the lateral
hypothalamus) deserve a similar circuit-based investigation. NAc projections to the lateral
hypothalamus may well be an important circuit for feeding behavior.
These results for magazine entries and sign-tracking further underscore the importance
of NAc/VP interactions in regulating motivated behaviors and give particular new importance to
the NAcLSh→VP pathway in determining how motivationally attractive a reward-paired cue is to
animals. It remains to be tested whether connections from other subregions of the NAc to the
VP play a similarly important role. The NAc core and medial shell subregions participate
importantly in sign-tracking behavior (Day and Carelli 2007; Flagel et al. 2011; Cardinal et al.
2002; Singer et al. 2016; Chang et al. 2012). Therefore, it’s possible that NAc output to the VP
contributes similarly to incentive salience processes. As a potential caveat to this notion, we
have recently found that a disconnection procedure to reduce communication between the NAc
medial shell and VP not only failed to reduce sign-tracking, but instead increased it (Chang et al.
2018). This result was curious with respect to other studies that have shown decreases in cue-
directed reward seeking and reward consumption using similar manipulations (e.g., Smith and
Berridge 2007, Leung and Balleine 2015). Such findings collectively raise the importance of
comparing NAc-VP subregional interactions across multiple motivation measures, and for
comparing disconnection vs. pathway-based manipulation procedures to fully understand how
NAc and VP subregions interact for motivated behavior.
Another intriguing notion that these results raise is that the NAcLSh neurons that project
to the VP do not appear to require a fully intact feedback signal to affect motivation. On the
conceptual side, feedback projections are a common organizational principle of the brain and it
remains unclear to what extent such feedback is necessary for the function of the principle site.
In the case here, we can speculate that the motivationally-relevant information conveyed form
the NAcLSh to the VP does not necessarily require information to be received back from the VP
to the NAcLSh. Mechanistically, there are many details yet to resolve. For instance, it is possible
that the VP projections to the NAcLSh do not actually synapse on the same NAcLSh neurons
that project to the VP, which would help explain the pathway dissociation here. More generally,
both the NAc and VP are composed of heterogeneous cell types (Smith et al. 2009; Root et al.
2015; Bobadilla et al 2017; Meredith and Totterdell 1999; Yang et al. 2018), suggesting that
feedforward or feedback signals between these structures will likely result in a complex mix of
inhibitory and excitatory neuronal effects (Hakan et al. 1992; Hakan et al. 1994; Hakan et al.
1995; Heimer & Wilson 1975; Churchill & Kalivas 1994; Napier & Mitrovic 1999). As such, the
VP→NAcLSh DREADD manipulation here might not have led to the “relevant” sort of
modulation of NAcLSh activity - whether targeting the relevant neuronal subpopulations or
engaging the related activity patterns - to effect sign-tracking behavior (Gore et al. 2015).
Nevertheless, the dissociation of NAcLSh→VP inhibition reducing sign-tracking and shifting
magazine-directed behavior to the CS+ time, and VP→NAcLSh inhibition shifting magazine-
directed behavior to the reward when it was presented, lays the groundwork for considering that
the function of these forward and back projections might well be different for directing motivated
behaviors.
Further regarding the cellular consequences of our manipulations, we cannot be certain
what changes in circuit-level activity are occurring during the DREADD manipulations as both
areas interact in larger circuits. It is known that CNO will reduce the activity of putative Gi-
DREADD-expressing neurons in awake behaving animals (Chang et al. 2015; Smith et al.
2016). However, non-Gi-expressing neurons can also be affected locally, perhaps through
interneurons or axon collaterals (Chang et al. 2015). As NAc and VP interact through
bidirectional GABAergic connections, plausibly a consequence of inhibiting a pathway would be
to disinhibit the recipient site. However, there are also neuromodulators involved in NAc-VP
interaction, such as NAc projections targeting cholinergic neurons in VP (Churchill and Kalivas
1994; Zahm 2000; Zaborszky and Cullinan 1992; Root et al. 2015; Smith et al. 2009). Thus, we
must remain agnostic as to what the circuit activity changes are that relate to the behavioral
effects here.
We note that the dual lever, CS+ and CS-, procedure used here seems to reliably
produce an abundance of sign-tracking responses (Chang et al. 2015). Just as various CRs can
occur from different forms of CSs (Holland, 1977), we speculate that the paired CS+ becomes
more salient when another, explicitly unpaired, lever is presented. However, this hypothesis has
not been directly tested. Another possibility could be a genetic predisposition for sign-tracking
responses in the animals acquired through our vendor. However, we have also seen high rates
of sign-tracking in genetically distinct, transgenic lines acquired elsewhere, on this task
(unpublished observations). The sign-tracking/goal-tracking distinction that other labs find in the
single CS+ condition (often with a paired cue light) reflects a behavioral tendency of animals
from different genetic backgrounds (Flagel et al., 2010b; Flagel et al., 2011). However,
generally, the form a conditioned response takes can vary based on task conditions. Animals
can be made to goal-track as a dominant response, such as if an auditory CS+ is used, or to
sign-track as a dominant response, such as here by using CS+/CS- lever cues (Holland, 1997).
When an animal sign-tracks - regardless of their natural tendencies - we regard them as having
attributed motivational value to the lever CS+ (Berridge, 2004). Thus, we argue that animals
here equivalently valued the CS+ as expressed through sign-tracking.
 Finally, we acknowledge limitations of this study. We note that there is evidence that
CNO metabolizes into clozapine which in turn could activate DREADD receptors or affect
behavior on its own (Gomez et al. 2017; Löffler et al. 2012; Mahler and Aston-Jones 2018). We
additionally note that, in our data, both control groups exhibit a level of sign-tracking and
magazine entry behavior that is consistent with prior lab records of behavior in animals not
treated with CNO, and moreover that the lack of CNO effect in the VP→NAcLSh DREADD
group compared to the positive CNO effect in the NAcLSh→VP DREADD group strongly favors
the interpretation of behavior being changed as a pathway-biased result of inhibition rather than
nonspecific effects of CNO/clozapine itself. Additionally, we and others have found previously
that there is no effect on behavior, sign-tracking or otherwise, when DREADD receptors are
expressed but a vehicle is given instead of CNO compared to when CNO is given to control-
virus-expressing rats; that is, behavior is comparable when CNO or vehicle is given
(Chang et al. 2015; Smith et al. 2016). Finally, we note that we did not evaluate whether
behavioral effects were the result of learning or performance, and issue that deserves future
attention. However, a prior study on DREADD inhibition of the VP found a reduction in sign-
tracking that was related to acquisition rather than performance (Chang et al. 2005).
The results presented here further support the notion that both the NAc shell and VP are
involved in cue-driven motivation. However, we show these circuits are uniquely contributing to
behavior as the DREADD inhibition of NAcLSh projection to VP resulting in a decrease in sign-
tracking behavior that was not present in the inhibition of the VP to NAcLSh projection. Instead,
the VP→NAcLSh projection inhibition seemed to bias animal behavior toward the magazine
during reward delivery. Importantly, neither projection inhibition resulted in a reduction in
primary motivation to consume food rewards in free consumption test sessions. Our results call
for more insight into the reciprocity and circuit dynamics of these, and adjacent areas
contributing to motivation and reward processes.

Funding
This work was supported by funding from The Whitehall Foundation (2014-05-77), the
NIH (R01DA044199), and the NSF (IOS1557987) to KSS. The authors have no competing
interests to declare.

Acknowledgements
We would like to thank Alex Brown for assistance with histology and the Zweifel
laboratory for CAV2-zsGreen.

References
Ahrens AM, Meyer PJ, Ferguson LM, Robinson TE, Aldridge JW. 2016. Neural activity in the
ventral pallidum encodes variation in the incentive value of a reward cue. Journal of
Neuroscience 36:7957-7970.

Ahrens AM, Ferguson LM, Robinson TE, Aldridge JW. 2018. Dynamic Encoding of Incentive
Salience in the Ventral Pallidum: Dependence on the Form of the Reward Cue. eNeuro
5:ENEURO.0328-17.2018.

Allsop SA, Wichmann R, Mills F, Burgos-Robles A, Chang CJ, Felix-Ortiz AC, Vienne A, Beyeler
A, Izadmehr EM, Glober G, Cum MI, Stergiadou J, Anandalingam KK, Farris K, Namburi
P, Leppla CA, Weddington JC, Nieh EH, Smith AC, Ba D, Brown EN, Tye KM. 2018.
Corticoamygdala Transfer of Socially Derived Information Gates Observational Learning.
Cell 173:1329–1342.e18.

Ambroggi F, Ghazizadeh A, Nicola SM, Fields HL. 2011. Roles of Nucleus Accumbens Core
and Shell in Incentive-Cue Responding and Behavioral Inhibition. Journal of
Neuroscience 31:6820–6830.

Bates D, Maechler M, Bolker B, Walker S. 2015. Fitting Linear Mixed-Effects Models Using
lme4. Journal of Statistical Software 67:1-48.

Batten SR, Pomerleau F, Quintero J, Gerhardt GA, Beckmann JS. 2018. The role of glutamate
signaling in incentive salience: second-by-second glutamate recordings in awake
Sprague-Dawley rats. Journal of Neurochemistry 145:276–286.

Berridge KC. 2004. Motivation concepts in behavioral neuroscience. Physiology & Behavior
81:179-209.

Berridge KC, Robinson TE. 2003. Parsing reward. Trends in Neurosciences 26:507-513.
Boakes R. 1977. Performance on learning to associate a stimulus with positive reinforcement. In
Davis, H. & Hurwitz, H. (Eds), Operant-Pavlovian Interactions. Lawrence Erlbaum
Associates, Hillsdale, NJ, pp. 67–97.

Bobadilla AC, Heinsbroek JA, Gipson CD, Griffin WC, Fowler CD, Kenny PJ, Kalivas PW. 2017.
Corticostriatal plasticity, neuronal ensembles, and regulation of drug-seeking behavior.
Progress in Brain Research 235:93–112.

Boender AJ, de Jong JW, Boekhoudt L, Luijendijk MCM, van der Plasse G, et al. 2014.
Combined Use of the Canine Adenovirus-2 and DREADD-Technology to Activate
Specific Neural Pathways In Vivo. PLOS ONE 9: e95392.

Brog JS, Salyapongse A, Deutch AY, Zahm DS. 1993. The patterns of afferent innervation of
the core and shell in the “accumbens” part of the rat ventral striatum:
Immunohistochemical detection of retrogradely transported fluoro-gold. The Journal of
Comparative Neurology 338:255-278.

Brown PL, Jenkins HM. 1968. Auto-shaping of the pigeon’s key-peck. Journal of the
Experimental Analysis of Behavior 11:1–8.

Cardinal RN, Parkinson JA, Lachenal G, Halkerston KM, Rudarakanchana N, Hall J, Morrison
CH, Howes SR, Robbins TW, Everitt BJ. 2002. Effects of selective excitotoxic lesions of
the nucleus accumbens core, anterior cingulate cortex, and central nucleus of the
amygdala on autoshaping performance in rats. Behavioral Neuroscience 116:553-67.

Carter ME, Soden ME, Zweifel LS, Palmiter RD. 2013. Genetic identification of a neural circuit
that suppresses appetite. Nature 503:111-4.

Chang SE, Wheeler DS, Holland PC. 2012. Roles of nucleus accumbens and basolateral
amygdala in autoshaped lever pressing. Neurobiology of Learning and Memory 97:444-
451.

Chang SE, Holland PC. 2013. Effects of nucleus accumbens core and shell lesions on
autoshaped lever-pressing. Behavioural Brain Research 256:36-42.
Chang SE, Smith KS. 2016. An omission procedure reorganizes the microstructure of sign-
tracking while preserving incentive salience. Learning & Memory 23:151-155.

Chang SE, Todd TP, Bucci DJ, Smith KS. 2015. Chemogenetic manipulation of ventral pallidal
neurons impairs acquisition of sign-tracking in rats. European Journal of Neuroscience
42:3105-3116.

Chang SE, Todd TP, Smith KS. 2018. Paradoxical accentuation of motivation following
accumbens-pallidum disconnection. Neurobiology of Learning and Memory 149:39–45.

Churchill L, Kalivas PW. 1994. A topographically organized gamma-aminobutyric acid projection

from the ventral pallidum to the nucleus accumbens in the rat. Journal of Comparative
Neurology 345:579-595.

Cromwell HC, Berridge KC. 1993. Where does damage lead to enhanced food aversion: the
ventral pallidum/substantia innominata or lateral hypothalamus? Brain Research 624:1–
10.

Day JJ, Carelli RM. 2007. The nucleus accumbens and Pavlovian reward learning.
Neuroscientist 13:148-159.

Day JJ, Wheeler RA, Roitman MF, Carelli RM. 2006. Nucleus accumbens neurons encode
Pavlovian approach behaviors: Evidence from an autoshaping paradigm. European
Journal of Neuroscience 23:1341–1351.

Deutch AY, Cameron DS. 1993. Pharmacological characterization of dopamine systems in the
nucleus accumbens core and shell. Neuroscience 46:49-56.

Flagel SB, Clark JJ, Robinson TE, Mayo L, Czuj A, Akers Ca, Clinton SM, Phillips PEM, Akil H.
2011. A selective role for dopamine in reward learning. Nature 469:53–57.

Flagel SB, Robinson TE. 2017. Neurobiological basis of individual variation in stimulus-reward
learning. Current Opinion in Behavioral Sciences 13:178- 185.
Flagel SB, Robinson TE, Clark JJ, Clinton SM, Watson SJ, Seeman P, Phillips PE, Akil H. 2010.
An animal model of genetic vulnerability to behavioral disinhibition and responsiveness
to reward-related cues: implications for addiction. Neuropsychopharmacology. 35:388–
400.

Gibbons RD, Hedeker D, DuToit S. 2010. Advances in analysis of longitudinal data. Annual
Reviews in Clinical Psychology 6:79-107.

Gore F, Schwartz EC, Daniel Salzman C. 2015. Manipulating neural activity in physiologically
classified neurons: Triumphs and challenges. Philosophical Transactions of the Royal
Society B 370.

Gomez JL, Bonaventura J, Lesniak W, Mathews WB, Sysa-shah P, Rodriguez LA, Ellis RJ,
Richie CT, Harvey BK, Dannals RF, Pomper MG, Bonci A, Michaelides M. 2017.
Chemogenetics Revealed: Dreadd Occupancy and Activation Via Converted Clozapine.
Science 507:503–507.

Groenewegen HJ, Russchen FT. 1984. Organization of the efferent projections of the nucleus
accumbens to pallidal, hypothalamic, and mesencephalic structures: a tracing and
immunohistochemical study in the cat. The Journal of Comparative Neurology 223:347-
367.

Hakan RL, Berg GI, Henriksen SJ. 1992. Electrophysiological evidence for reciprocal
connectivity between the nucleus accumbens septi and ventral pallidal region. Brain
Research 581:344–50.

Hakan RL, Eyl C, Henriksen SJ. 1994. Neuropharmacology of the nucleus accumbens:
systemic morphine effects on single-unit responses evoked by ventral pallidum
stimulation. Neuroscience 63:85–93.

Hakan RL, Eyl C. 1995. Neuropharmacology of the nucleus accumbens: iontophoretic

applications of morphine and nicotine have contrasting effects on single-unit responses
evoked by ventral pallidal and fimbria stimulation. Synapse 20:175–84.
Heimer L, Zahm DS, Churchill L, Kalivas PW, Wohltmann C. 1991. Specificity in the projection
patterns of accumbal core and shell in the rat. Neuroscience 41:89–125.

Heimer L, Wilson RD. 1975. The subcortical projections of allocortex: similarities in the neuronal
associations of the hippocampus, the piriform cortex and the neocortex. In Santini M
(Ed) Golgi Centennial Symposium Proceedings 173-93. Raven Press: New York.

Heimer L, Alheid GF, de Olmos JS, Groenewegen HJ, Haber SN, Harlan RE, Zahm DS. 1997.
The accumbens: Beyond the core-shell dichotomy. The Journal of Neuropsychiatry and
Clinical Neurosciences 9:354-381.

Holland P. 1977. Conditioned stimulus as a determinant of the form of the Pavlovian conditioned
response. Journal of Experimental Psychology: Animal Behavior Processes 3:77-104.

Jenkins HM, Moore BR. 1973. The form of the auto-shaped response with food or water
reinforcers. Journal of the Experimental Analysis of Behavior 20:163-181.

Kuo H, Chang HT. 1992. Ventral pallido-striatal pathway in the rat brain: a light and electron
microscopic study. Journal of Comparative Neurology 321:626-636.

Kuznetsova A, Brockhoff PB, Christensen RHB. 2016. lmerTest: Tests in Linear Mixed Effects
Models. R package version 2.0-33.

Lammel S, Lim BK, Ran C, Huang KW, Betley MJ, Tye KM, Deisseroth K, Malenka RC. 2012.
Input-specific control of reward and aversion in the ventral tegmental area. Nature
491:212–217.

Leung BK, Balleine BW. 2015. Ventral pallidal projections to mediodorsal thalamus and ventral
tegmental area play distinct roles in outcome- specific Pavlovian-instrumental transfer.
Journal of Neuroscience 35:4953- 4964.

Löffler S, Körber J, Nubbemeyer U, Fehsel K. 2012. Comment on “Impaired respiratory and

body temperature control upon acute serotonergic neuron inhibition.” Science 337:646.
Mahler SV, Aston-Jones GS. 2012. Fos Activation of Selective Afferents to Ventral Tegmental
Area during Cue-Induced Reinstatement of Cocaine Seeking in Rats. Journal of
Neuroscience 32:13309–13325.

Mahler SV, Aston-Jones GS. 2018. CNO Evil? Consideration for the Use of DREADDs in
Behavioral Neuroscience. Neuropsychopharmacology 43:934-936.

Meredith GE, Totterdell S. 1999. Microcircuits in nucleus accumbens’ shell and core involved in
cognition and reward. Psychobiology 27:165–186.

Mogenson GJ, Jones DL, Yim CY. 1980. From motivation to action: Functional interface
between the limbic system and the motor system. Progress in Neurobiology 14:69–97.

Nair SG, Strand NS, Neumaier JF. 2012. DREADDing the lateral habenula: a review of
methodological approaches for studying lateral habenula function. Brain research
1511:93-101.

Napier TC, Mitrovic I. 1999. Opioid modulation of ventral pallidal inputs. Annals of the New York
Academy of Sciences 877:176– 201.

Paxinos G, Watson C. 2009. The Rat Brain in Stereotaxic Coordinates. Academic Press, San
Diego, CA.

Richard JM, Ambroggi F, Janak PH, Fields HL, Francisco S, Sciences B. 2016. Ventral pallidum
neurons encode incentive value and promote cue-elicited instrumental actions. Neuron
90:1165–1173.

R. Core Team, 2016. R: a Language and Environment for Statistical Computing. R Foundation
for Statistical Computing, Vienna, Austria.

Robinson MJF, Berridge KC. 2013. Instant transformation of learned repulsion into motivational
‘wanting’. Current Biology 23:282-289.
Root DH, Melendez RI, Zaborszky L, Napier TC. 2015. The ventral pallidum: subregion-specific
functional anatomy and roles in motivated behaviors. Progress in Neurobiology 130:29-
70.

Saunders BT, Robinson TE. 2012. The role of dopamine in the accumbens core in the
expression of Pavlovian conditioned responses. European Journal of Neuroscience
36:2521–2532.

Senn V, Wolff SBE, Herry C, Grenier F, Ehrlich I, Gründemann J, Fadok JP, Müller C, Letzkus
JJ, Lüthi A. 2014. Long-range connectivity defines behavioral specificity of amygdala
neurons. Neuron 81:428–437.

Singer BF, Bryan MA, Popov P, Scarff R, Carter C, Wright E, Aragona BJ, Robinson TE. 2016.
The sensory features of a food cue influence its ability to act as an incentive stimulus
and evoke dopamine release in the nucleus accumbens core. Learning & Memory
23:595–606.

Smedley EB, Smith KS. 2018a. Evidence of structure and persistence in motivational attraction
to serial Pavlovian cues. Learning & Memory 25:78-89.

Smedley EB, Smith KS. 2018b. Evidence for a shared representation of sequential cues that
engage sign-tracking. Behavioral Processes 157:489-494.

Smith KS, Berridge KC, Aldridge JW. 2011. Disentangling pleasure from incentive salience and
learning signals in brain reward circuitry. Proceedings of the National Academy of
Sciences of the United States of America 108:E255-64.

Smith KS, Bucci DJ, Luikart BW, Mahler SV. 2016. DREADs: Use and application in behavioral
neuroscience. Behavioral Neuroscience 130:137-155.

Smith KS, Berridge KC. 2007. Opioid limbic circuit for reward: interaction between hedonic
hotspots of nucleus accumbens and ventral pallidum. Journal of Neuroscience 27:1594-
1605.
Smith KS, Tindell AJ, Aldridge JW, Berridge KC. 2009. Ventral pallidum roles in reward and
motivation. Behavioural Brain Research 196:155-167.

Saloman JL, Scheff NN, Snyder LM, Ross SE, Davis BM, Gold MS. 2016. Gi-DREADD
Expression in Peripheral Nerves Produces Ligand-Dependent Analgesia, as well as
Ligand-Independent Functional Changes in Sensory Neurons. The Journal of
Neuroscience 36(42):10769-10781.

Stratford TR, Kelley AE. 1997. GABA in the nucleus accumbens shell participates in the central
regulation of feeding behavior. The Journal of Neuroscience 17:4434-4440.

Tindell AJ, Berridge KC, Zhang J, Peciña S, Aldridge JW. 2005. Ventral pallidal neurons code
incentive motivation: amplification by mesolimbic sensitization and amphetamine.
European Journal of Neuroscience 22:2617–2634.

Tindell AJ, Smith KS, Berridge KC, Aldridge JW. 2009. Dynamic computation of incentive
salience: ‘wanting’ what was never ‘liked’. The Journal of Neuroscience 29(39):12220-8.

van Dongen YC, Deniau JM, Pennartz CMA, Galis-de Graaf Y, Voorn P, Thierry AM,
Groenewegen HJ. 2005. Anatomical evidence for direct connections between the shell
and core subregions of the rat nucleus accumbens. Neuroscience 136:1049-1071.

Wan X, Peoples LL. 2008. Amphetamine Exposure Enhances Accumbal Responses to Reward-
Predictive Stimuli in a Pavlovian Conditioned Approach Task. The Journal of
Neuroscience 28:7501–7512.

Yang H, de Jong JW, Tak Y, Peck J, Bateup HS, Lammel S. 2018. Nucleus accumbens
subnuclei regulate motivated behavior via direct inhibition and disinhibition of VTA
dopamine subpopulations. Neuron 97:1-16.

Záborszky L, Alheid GF, Beinfeld MC, Eiden LE, Heimer L, Palkovits M. 1985. Cholecystokinin
innervation of the ventral striatum: A morphological and radioimmunological study.
Neuroscience 14:427-453.
Záborszky L, Cullinan WE. 1992. Projections from the nucleus accumbens to cholinergic
neurons of the ventral pallidum: a correlated light and electron microscope double-
immunolableing study in rat. Brain Research 570:92-101.

Zahm DS. 1999. Functional-anatomical implications of the nucleus accumbens core and shell
subterritories. Annals of the New York Academy of Sciences 877:113-128.

Zahm DS. 2000. An integrative neuroanatomical perspective on some sub- cortical substrates of
adaptive responding with emphasis on the nucleus accumbens. Neuroscience &
Biobehavioral Reviews 24:85–105.

Zahm DS, Brog JS. 1992. On the significance of subterritories in the “accumbens” part of the rat
ventral striatum. Neuroscience 50: 751-767.

Zhang M, Kelley AE. 2000. Enhanced intake of high-fat food following striatal mu-opioid
stimulation: Microinjection mapping and Fos expression. Neuroscience 99: 267–277.

Figure Captions

Figure 1: NAcLSh→VP expression maps. Each coronal section represents a range of three
sections relative to Bregma (B, in mm). Expression for each animal is plotted at 90%
transparency with per-animal expression overlaid. Sections run anterior (top) to posterior
(bottom). A) Expression map of AAV-hSyn-DIO-hM4D(Gi)-mCherry in NAcLSh following CAV2-
Cre injections in the VP (i.e., NAcLSh→VP inhibition animals; red). B) Expression map of AAV-
hSyn-DIO-mCherry in NAcLSh (i.e., controls; black).

Figure 2: VP → NAcLSh expression maps. Each coronal section represents a range of three
sections relative to Bregma (B, in mm). Expression for each animal is plotted at 90%
transparency with per-animal expression overlaid. Sections run anterior (top) to posterior
(bottom). A) Expression map of AAV-hSyn-DIO-hM4D(Gi)-mCherry in VP following CAV2-Cre
injections in NAcLSh (i.e., VP → NAcLSh inhibition animals; red). B) Expression map of AAV-
hSyn-DIO-mCherry in VP (i.e., controls; black).
Figure 3: CAV-zsGreen histology. A) Expression of retrograde tracer CAV2-zsGreen after
injection into NAcLSh target (transparency: 70%; green). B) Expression of CAV2-zsGreen after
injection into the VP target (transparency: 70%; green). Expression will reflect retrograde
transport into neurons projecting to the injection site, including interneurons.

Figure 4: Effects of NAcLSh→VP inhibition. A) Presses per minute (ppm) on the CS+ lever
over the 12 training sessions for the NAcLSh→VP inhibition group (green) and the
NAcLSh→VP control group (black). B) Ppm on the CS- lever for both groups. C) Average
magazine entries per session during the 10 sec CS+ presentation (shaded grey background)
and the 10 sec post CS+ block (i.e., reward delivery) for both the NAcLSh→VP control (black)
and NAcLSh→VP inhibition group (green). D) Average magazine entries per 10 sec block type
(10 sec Pre CS+, 10 sec CS+ [shaded grey background], 10 sec Post CS+) in the NAcLSh→VP
inhibition group (green) and the NAcLSh → VP control group (grey). E) Average magazine
entries per block type in the NAcLSh→VP control group (left) NAcLSh → VP inhibition group
(right) with the whole bar representing the total magazine entries made during the 30 second
trial period encompassing all three blocks. F) Grams of food consumed over the two free
feeding sessions in NAcLSh→VP inhibition group (green) and the NAcLSh→VP control group
(grey). For all graphs, bars and lines show mean and errors show +/- SEM.

Figure 5: Effects of VP → NAcLSh inhibition. A) Presses per minute (ppm) on the CS+ lever
over the 12 training sessions for the VP→NAcLSh inhibition group (blue) and the VP→NAcLSh
control group (black). B) Ppm on the CS- lever for both groups. C) Average magazine entries
per session during the 10 sec CS+ presentation (shaded grey background) and the 10 sec post
CS+ block (i.e., reward delivery) for both groups. D) Average magazine entries per 10 sec block
type (10 sec Pre CS+, 10 sec CS+, 10 sec Post CS+) in the VP→NAcLSh inhibition group
(blue) and the VP → NAcLSh control group (grey). E) Average magazine entries per block type
in the VP → NAcLSh control group (left) VP→NAcLSh inhibition group (right) with the whole bar
representing the total magazine entries made during the 30 second trial period encompassing
all three blocks. F) Grams of food consumed over the two free feeding sessions in VP→NAcLSh
inhibition group (blue) and the VP→NAcLSh control group (grey). For all graphs, bars and lines
show mean and errors show +/- SEM.

Supplemental Analysis & Figures

“Fleck-like” expression analysis. Further analysis was used to determine if the animals within
the NAcLSh→VP projection inhibition group that displayed atypical mCherry expression (n = 5)
differed from the more cellular mCherry expressing animals in that group (n = 3). A linear mixed
model of CS+ response rate, the main variable of interest, by histology outcome (fleck-like or
cellular) by interaction of linear session and quadratic fit of session with random slopes and
intercepts was constructed. This model structure is the same as used for the main analyses.
CS+ ppm = Histology Outcome x Session + Session2 + (1 + Session + Session2 | Rat)
Data appeared non-linear and a quadratic fit of session significantly contributed to model fit
(2(4) = 19.83, p < 0.001). Analysis determined no significant difference between the fleck-like
animals and typically expressing animals in their CS+ ppm as seen in an insignificant main
effect of histology outcome (Supp. Fig. 1A; est: 8.78 ppm; CI: -0.60-17.2; SE: 4.34; p = 0.077).
Animals did not differ over sessions in both linear fit of session (est: 2.01 ppm; CI: -1.18-4.90;
SE: 1.54; p = 0.226) and quadratic fit of session (est: -0.12 ppm; CI: -0.28-0.08; SE: 0.09; p =
0.273). Fleck-like and cellular expression animals did not differ over time in press rates as
determined by an insignificant interaction of histology outcome and session (est: 0.06 ppm; CI: -
1.15-1.18; SE: 0.53; p = 0.910).
The same analysis as above was performed on the fleck-like (n = 2) and cellular (n = 8)
animals in the VP→NAcLSh projection inhibition group. Data appeared non-linear and model fit
significantly improved upon addition of a quadratic fit of session (2(4) = 29.17, p < 0.001). An
insignificant main effect of histology outcome (Supp. Fig. 1B; est: 0.07 ppm; CI: -13.2-15.5; SE:
7.08; p = 0.992) indicates that fleck-like animals did not differ from cellular expression animals in
CS+ ppm. All animals maintained similar rates of pressing as determined by insignificant main
effects of linear session (est: 3.07 ppm; CI: -1.07-7.30; SE: 2.03; p = 0.161) and quadratic fit of
session (est: -0.19 ppm; CI: -0.50-0.08; SE: 0.14; p = 0.203). Fleck and cellular animals did not
differ over sessions as seen by a non-significant histology outcome by session interaction (est:
0.53 ppm; CI: -1.93-2.91; SE: 1.16; p = 0.658). Examples of fleck-like and typical expression
can be seen in Supp. Fig. 1C.

Latency to respond to the CS+.

Differences in latency to respond to the CS+ might suggest something about the
eagerness or readiness to press that may be different in the inhibition groups compared to their
respective controls. Time points were taken of the first lever press during each of the 25 CS+
trials. If none existed (i.e., no lever presses were made at all) those trials were ignored in the
analysis. Latency to press was analyzed by the factors of group assignment and session.
Latency is typically a skewed right distribution (with most responses typically occurring quickly
after the lever is presented). This data was no exception and was log transformed to fit the
assumptions of the linear mixed model. The reported statistics are in log seconds.
log(Latency) = Group x Session + (1 + Session | Rat)
In the NAc → VP inhibition group and the NAcLSh→VP control group, there was a
difference at the group level in their latencies to the first press, with the NAcLSh→VP control
group being significantly faster to respond than the NAcLSh→VP inhibition group (Supp. Fig.
1D, est: -0.67 seconds; CI: -1.67-(-0.19); SE: 0.24; p = 0.021). Both groups also tended to get
faster to press as sessions went on as revealed in a significant main effect of session (Supp.
Fig. 1D; est: -0.06 seconds; CI: -0.10-(-0.02); SE: 0.02; p = 0.018). Notedly, groups got similarly
faster over time as seen in a non-significant group by session interaction (Supp. Fig. 1D; est:
0.03 seconds; CI: (-0.03-0.09); SE: 0.03; p = 0.338).
Latency analysis in the VP→NAcLSh inhibition and VP→NAcLSh control groups did not
reveal any significant difference between the VP→NAcLSh inhibition and VP→NAcLSh control
groups in their latency to press (Supp. Fig. 1E; est: 0.01 seconds; CI: -0.43-0.41; SE: 0.20; p =
0.971). Both groups got faster to respond over time as seen in a significant main effect of
session (Supp. Fig. 1E; est: -0.03 seconds; CI: -0.05-0.00; SE: 0.01; p = 0.035). Both groups
similarly got faster over time as seen in a non-significant interaction of group by session (Supp.
Fig. 1E; est: 0.02 seconds; CI: -0.02-0.06; SE: 0.02; p = 0.427).

Supplemental Figure 1: Fleck-like cellular expression and latency to respond to CS+

analysis. A) Presses per minute (ppm) on the CS+ lever over the 12 training sessions in the
conditioning paradigm in the NAcLSh→VP control group (n = 6, grey circle), inhibition group (n
= 3, black square), and the NAcLSh→VP fleck-like expression group (n = 5, red triangle). B)
Ppm on the CS+ lever over the 12 training sessions in the VP→NAcLSh inhibition group (n = 7,
black square), the VP→NAcLSh control group (n = 9, grey circle), and the VP→NAcLSh fleck-
like expression group (n = 3, red triangle). C) Example of fleck-like expression (above) and
cellular expression (below) in 10x. D) Average latency in seconds to press the CS+ over the 12
training sessions in the NAcLSh→VP control group (n = 6, black line), inhibition group (n = 8,
green line). E) Average latency in seconds to press the CS+ over the 12 training sessions in the
VP →NAcLSh control group (n = 9, black line), inhibition group (n = 10, blue line). Lines denote
mean and error bars denote +/- SEM.
Figure
A B

B + 2.16 - 1.92 B + 1.68 - 1.56

B + 1.88 - 1.56 B + 1.44 - 1.28

B + 1.44 - 1.20 B + 1.20 - 0.36

B + 0.84 - 0.60 B + 0.24 - 0.12

B + 0.48 - 0.24 B 0.00 - (- 0.12)

B + 0.12 - (-0.12) B - 0.24 - (-0.36)

A B

B + 1.92 - 1.68 B + 1.68 - 1.56

B + 1.56 - 1.28 B + 1.44 - 1.28

B + 1.20 - 0.72 B + 1.20 - 0.36

B + 0.60 - 0.36 B + 0.24 - 0.12

B + 0.24 - 0.00 B - 0.00 - (-0.12)

B - 0.12 - (- 0.36) B - 0.24 - (-0.36)

A B

B + 1.56 B + 1.56

B + 1.44
B + 1.44

B + 1.28
B + 1.28

B + 0.36
B -0.00

B + 0.24 B - 0.12

B + 0.12 B - 0.24
 A B
70 70

60 60
Presses per Minute

Presses per Minute

50 50

40 40
NAclSh --> VP Control
30 30 NAclSh --> VP Control
NAclsh--> VP Inhibition
20 20 NAclsh--> VP Inhibition

10 10

0 0
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Session Session

C D
10
8
Average Magazine Entries

Average Magazine Entries

8
CS+ NAclSh --> VP Control
6 NAclSh --> VP Control
CS+ NAclSh --> VP Inhibition
6 Post CS+ NAclSh --> VP Control NAclsh--> VP Inhibition
Post CS+ NAclSh --> VP Inhibition 4
4

2 2

0 0
1 2 3 4 5 6 7 8 9 10 11 12
Pre CS+ CS+ Post CS+
Session Trial Period

E F
8
Average Magazine Entries

12
6 10
Grams Consumed

Trial Window
NAclSh --> VP Control
Pre CS+ 8
4 NAclsh--> VP Inhibition
CS+
6
Post CS+
4
2
2

0 0
Control Gi 1 2
Group Day
 A B

40 40
VP --> NAclSh Control VP --> NAclSh Control
Presses per Minute

Presses per Minute

30 VP --> NAclSh Inhibition 30 VP --> NAclSh Inhibition

20 20

10 10

0 0
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Session Session

C D
8 8

Average Magazine Entries

Average Magazine Entries

CS+ VP --> NAclSh Control

6 6
CS+ VP --> NAclSh Inhibition
Post VP --> NAclSh Control VP --> NAclSh Control
4 Post VP --> NAclSh Inhibition 4 VP --> NAclSh Inhibition

2 2

0 0
1 2 3 4 5 6 7 8 9 10 11 12 Pre CS+ CS+ Post CS+
Session Trial Period

E F
8
Trial Window
12
Average Magazine Entries

Pre CS+ VP --> NAclSh Control

6 10
CS+
Grams Consumed

VP --> NAclSh Inhibition

Post CS+ 8
4
6

4
2
2

0
0 1 2
Control Gi
Group Day
 A B C
70 50
NAclSh --> VP Control VP --> NAclSh Control
60
Presses per Minute

Presses per Minute

NAclSh --> VP Cellular 40 VP --> NAclSh Cellular
50
NAclSh --> VP Fleck VP --> NAclSh Fleck
30
40
30 20
20
10
10
0 0 10x
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Session Session

D E
10.0 10.0
10x
Latency (seconds)

NAclSh --> VP Control

Latency (seconds)
VP --> NAclSh Control
7.5 7.5
NAclsh--> VP Inhibition VP --> NAclSh Inhibition
5.0 5.0

2.5 2.5

0.0 0.0
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Session
Session
Highlights

• Chemogenetic disruption of connections from NAcLSh à VP reduces sign-tracking

behavior but not consumption of food rewards.
• VP à NAcLSh disruption affected neither sign-tracking nor reward consumption but did
produce a greater shift in animals’ behavior more towards the reward source when it was
available.
Another random document with
no related content on Scribd:
THE 42nd (EAST LANCASHIRE)
DIVISION
 CHAPTER I
LANCASHIRE AND EGYPT
(August 1914—May 1915)

For a week or two there had been talk of war and of the likelihood
of England being involved. The prospects and possibilities formed an
interesting topic of conversation and speculation. We leant over the
sheer side of the precipice and caught glimpses of the black chasm
below, but few really doubted the soundness of the fence over which
we peered.
Warnings of disaster had been frequent—but disaster had always
been averted, and fair words had prevailed. For years we had been
living on the verge of national ruin through strikes of railway-men,
transport-workers, miners, or the spinners and weavers of
Lancashire, but at the last moment conciliation always won; there
was always room for compromise. Though civil war in Ireland
seemed imminent, it was comforting to reflect how much common
sense there was in the world. Besides, had it not been proved to
every one’s satisfaction that under modern conditions war between
great nations could not possibly last for more than a month or two,
as in that short period victor and vanquished would alike be reduced
to bankruptcy and impotence? Knowing this no Great Power would
be likely to commit suicide. We were living in the twentieth century,
and a great European war was an abstract conception, not
something that could actually occur.
In the closing days of July 1914 this complacency was giving way
to a very real dread. War might mean suicide even for the victor,
might be “unthinkable,” but it was in sight—plain, stark, menacing.
War such as other nations had known; not a war in which those who
had a taste for soldiering might take part while the rest of us could
read about it in the papers, feel proud of a success and depressed
by a disaster, and wonder whether sixpence would be added to the
income tax. The fantastic image that had thrilled us not altogether
unpleasantly—as children experience ecstatic shudders when
listening to tales of ogres and hobgoblins—was taking on an
appearance of grim reality. Could it after all be a grisly spectre and
not a mere bogey of turnip and white sheet? England began to regret
that the warnings of her greatest soldier had passed unheeded.
A day or two later Germany flung down her challenge to
Christianity and Civilization, stripped herself of the cloak of decency
and stood revealed in stark brutishness; and on Tuesday, August 4,
1914, England took up the challenge and declared war. The decision
was apparently not expected by the German Staff. To them it was
rather a matter for exasperation than for apprehension. England had
her hands full at home, and her vast possessions would prove a
source of weakness. She had a small regular army, a force with high
traditions, well trained and well equipped for service on the frontiers
of India and other outposts of the Empire, containing a larger
proportion of officers and men with experience of actual fighting than
any other army of the Great Powers, yet so small in numbers and so
scattered over the face of the globe, that one can almost sympathize
with the German belief that the few thousand men who could be
spared from the duty of policing India, Egypt, South Africa, and other
possessions, might safely be regarded as negligible. She had, too, a
small, indifferently trained and equipped, unprofessional, home-
defence force, but even the British themselves did not take the
Territorials seriously.
As to the rest of the potential fighting material of the British Isles,
had it not been proved to the satisfaction of the Germans (who had
made a special study of such matters with the typical Teutonic
thoroughness which works so efficiently when applied to material
facts, and fails so lamentably when the human factor enters) that the
young manhood of the nation was mainly decadent, of poor
physique, weak-chested, half-educated, lacking in patriotic purpose,
with no thought of the morrow and no ideals? With the exception of
the few hundred thousands who had received some training in
physical drill and discipline in the Boys’ Brigade and its daughter-
organizations which teach discipline, self-respect, and esprit de
corps, or in the School Cadet Corps, all were utterly untrained, and
they hated discipline. England had clearly shown that she was too
selfish to submit to any form of compulsory service; too wrapped up
in the love of comfort, ease and luxury to do more than bribe fools to
die for her. It was a nation that had lost its soul. The military aid she
could give to France could be contemptuously brushed aside. When
France had been paralysed and the Channel ports secured, the
British mercantile marine could be sunk or scared off the seas, and
the British Empire brought to its knees.
Teutonic reasoning was wrong. The British character is too simple
or too complex for the Hun. It may be that no other nation brings so
much froth to the top as ours; that none extends such tolerance to
cranks, nor gives so much rope to little cliques of shrieking egoists,
nor shows such stolid indifference when the few, claiming to speak
on behalf of the nation, so egregiously misrepresent her. On August
5, 1914, it was seen that practically every man, woman and child
approved what the Government had done, and felt instinctively that
their country would have been shamed had there been a day’s
hesitation. England had found her soul, not lost it. A nation supposed
to consist largely of pleasure seekers, of lovers of compromise,
conciliation and tolerance, of comfort and luxury, had decided that all
it held most dear would be as dust and ashes if it stood aside, a
passive spectator of the agony of France and Belgium. Practically
without a dissentient voice the nation prepared to sacrifice its all.
Unhappily, the politicians, unaccustomed to realities, were not ready
to make the most of this spirit of sacrifice. Unable to leave their
grooves of finesse, intrigue, and opportunism, they knew not how to
appeal simply to the noblest instincts; so they talked of “business as
usual,” and attempted to cajole the nation into giving a part when the
whole was ready to be offered.
This is the story of the part played in the most Composition of
appalling of human tragedies by the East the Division
Lancashire Territorial Division, which, on leaving
England, was composed of the following units—
 Cavalry: “A” Squadron, Duke of Lancaster’s Own Yeomanry—6
officers, 132 men.
R.F.A.: The 1st (Blackburn) and the 3rd (Bolton) East
Lancashire Brigades—55 officers, 1289 men.
R.E.: 1st and 2nd Field Companies and Signal Company—19
officers, 568 men.
Infantry: The 5th, 6th, 7th and 8th Battalions, the Lancashire
Fusiliers—120 officers, 3962 men.
The 4th and 5th Battalions, the East Lancashire Regiment
—60 officers, 1990 men.
The 5th, 6th, 7th, 8th, 9th, and 10th Battalions, the
Manchester Regiment—180 officers, 5966 men.
A.S.C.: Three Companies and the Transport and Supply Column
—16 officers, 276 men.
R.A.M.C.: Three Field Ambulances—30 officers, 665 men.
Total (including Divisional and Brigade Headquarters): 511
officers, 14,966 men.

The twelve battalions of infantry were brigaded as follows—

The Lancashire Fusiliers Brigade—the 5th, 6th, 7th, and 8th
Battalions, Lancashire Fusiliers.
The East Lancashire Brigade—the 4th and 5th Battalions, East
Lancs Regiment, and the 9th and 10th Battalions, Manchester
Regiment.
The Manchester Brigade—the 5th, 6th, 7th, and 8th Battalions,
Manchester Regiment.
The 5th L.F. was mainly composed of men from the Bury district,
the 6th from Rochdale, the 7th and 8th from Salford, the 4th E.
Lancs from Blackburn, the 5th from Burnley, the 5th Manchesters
from Wigan, the 6th and 7th from Manchester and its suburbs
(including a good proportion from the Cheshire suburbs), the 8th
from Ardwick and East Manchester, the 9th from Ashton-under-Lyne,
the 10th from Oldham.
The 1st R.F.A. Brigade (Blackburn) was composed of the following
batteries: the 4th (Blackburn), the 5th (Church), the 6th (Burnley).
The 3rd R.F.A. Brigade (Bolton) of the following: the 18th, 19th and
20th, all from Bolton and district.
Many of these units date back to 1859, when the Volunteer
movement came into being. For forty-one years the Volunteers were
an untried force, but in the year 1900 their offers of service in the
South African War were accepted, the infantry battalions providing
detachments which served with credit in that campaign. It was not
until 1907 that serious thought was given to the equipment and
organization of this fine body of patriotic men. Lord Haldane’s
Territorial Scheme, based on the model of the Regular Army, did
more than change the name from Volunteers to Territorials. Major-
General Douglas Haig, Director of Military Training at the War Office,
had charge of the scheme, and in bringing about this great change,
he gave evidence of the foresight and grasp of essentials which in
1918 enabled him to lead the armed strength of the Empire to
victory. The various batteries, battalions and corps, no longer
scattered and independent units, were organized and trained as
divisions, and the force was actually treated as a valuable and even
essential element in the system of national defence, with the result
that the Territorial Force soon reached a higher state of efficiency
than the Volunteers had ever been permitted to approach. The East
Lancashire Division had the reputation of being one of the smartest
and most efficient of the whole force.
On August 3 units in camp for their annual peace Mobilization
training were recalled, and the order to mobilize the
Division was received at 5.30 p.m. on the following day. Probably
few members of the Territorial Force had realized that their “calling-
up” notices had been ready from the day they joined; that month by
month the addresses on the envelopes were checked and altered
when necessary; that enough ball ammunition was stored at the
various headquarters to enable every man to march out with the full
complement of a fighting soldier, and that field dressings were
available for issue. It must be remembered that never before had the
Force been embodied, and that officers and men were civilians, few
of whom had ever regarded mobilization as other than a remote
possibility quite outside the range of practical politics. Yet the
absence of confusion was remarkable, as all ranks threw themselves
into their new parts with zest, making the best of unusual conditions
and treating discomfort as a jest. The men were quartered in drill-
halls or schools within easy reach of their Headquarters. Major-
General Douglas, commanding the Division, with his General Staff
Officer, Lieut.-Colonel A. W. Tufnell, was daily at the Divisional
Headquarters in Manchester, or at the Headquarters of units,
supervising the mobilization and arrangements. These have been
described as “hectic days.” Improved though the organization was as
compared with that of the Volunteers, it was anything but complete.
Animals and vehicles for transport, harness, and an infinite variety of
requisites had to be procured by free purchase or by
commandeering in great haste. This was the weakest part of the
scheme, and though much ingenuity was displayed by officers
unaccustomed to this sort of thing, the waste was great. To such
shifts were they reduced that street watering-carts were bought from
Urban District Councils, and actually taken to Egypt. Among the
weird varieties of carts thus acquired one of the best remembered is
the “Black and Green” van which did duty as a Medical Officer’s
vehicle, and was last seen bleached by the sun, but with the original
lettering still traceable under the service grey, at Gabari Docks,
Alexandria in February 1917. Nothing could better illustrate the
inability of a large and patriotic section of the public to grasp the
significance of events than the expectation freely expressed by
vendors that their horses or carts would be returned to them in the
course of a few weeks. In some cases the drivers of requisitioned
animals joined the Territorial unit in order to remain with their horses.
On August 10 Lord Kitchener invited the Territorial Force to
volunteer for foreign service. By August 12 the three Infantry
Brigades had accepted the invitation, and within a few days ninety
per cent. of the East Lancashires eagerly seized the opportunity.
England saw with pride the keenness of the Territorials to meet the
enemy, and knew that her sons were true to the breed. For more
than fifty years she had treated her citizen-army as something that
must not be taken too seriously, but that is England’s way. The
response was magnificent, but it was expected, for the nation never
doubted that they would answer the call. It was no small gift they
offered. The most powerful army the world had ever seen was
moving forward victoriously towards Paris, remorseless and
apparently irresistible; and the Territorials offered their bodies for
death and mutilation, and gave up parents, wives, children, homes,
prospects, and all they held most dear. At a later period such
sacrifice was demanded as the nation’s right, but in August 1914 a
lead had to be given, an example set. Old volunteers were not
content to look on while the younger generation fought and suffered.
Daily a stream of old members, N.C.O.s and men, many in the
autumn of their lives, besieged the Headquarters of their old corps
and clamoured to be allowed to join up, lying cheerfully and brazenly
in respect of their age. Many gained their object; more were almost
broken-hearted by their rejection. The example set in those dark
days was a stimulus and incentive to recruiting all over the country,
and especially so in the great towns of Lancashire, where the “Pals”
battalions were soon to be raised. It was the vindication of the
Volunteer. “Defence, not Defiance,” had been their motto, but who
among the prescient founders and bulwarks of the movement could
ever have conceived the idea of the glorious rôle to be filled by the
corps they had helped to raise—of the old Volunteer units from every
county and city, at full strength, fighting and laying down their lives in
Belgium, France, Egypt, Gallipoli, Palestine and Greece, and taking
an inspiring part in the greatest of all crusades against the Turk?
Until August 20 officers and men remained within easy reach of
their Headquarters. The Division was then moved into camps in the
neighbourhoods of Bolton, Bury and Rochdale, and anxiously it
awaited the summons. Another fortnight passed, and the rumour that
Egypt was to be the destination began to gain ground. Rumour was,
however, at this time a discredited jade, fit subject for scorn, for was
she not responsible for the passage through England and Scotland
of myriads of Russian soldiers, the ice and snow of Archangel still
clinging to their boots and beards, who had been seen by thousands
of British optimists at every railway junction in the land? But in the
training camps of East Lancashire she was restored to public favour
when a telegram, dated September 5, was received by the Divisional
Commander from Lord Kitchener—

“Inform the Division from me that I hope that they will push
on hard with their training in Egypt, as, before they are ready,
there will be plenty of troops from India to garrison Egypt, and
I hope they will be one of the first of the Territorial Forces to
join our Army on the Continent. All will depend on their fitness
for service against the enemy in the Field.—Kitchener.”

General Douglas spoke for every man in the Division when he

replied: “All ranks much gratified to receive your message. They are
animated with keen desire to fit themselves to join our forces on the
Continent.”
On the 9th September the Division entrained for Southampton,
about forty trains being required to convey men, horses, guns, and
all the material of war; and on the 10th 15,500 Lancashire men, with
about a thousand officers and men of the Hertfordshire and City of
Westminster Yeomanry, embarked upon the great adventure, the first
Territorial Division to volunteer for foreign service, and the first to
leave England’s shores. It was a record of which the Lancashire men
were justly proud, and which secured to them from the start the
prestige and esprit de corps that are usually the outcome of long and
honourable tradition. The details that were not going out with the
Division became the nucleus of the Second Line, which sent drafts to
the First Line until about the end of August 1915, when the Second
Line East Lancashire (66th) Division was formed. After that date the
drafts came from the Third Line units. The Duke of Lancaster’s Own
Yeomanry had been accepted for voluntary service overseas, and it
had been hoped that the whole regiment would accompany the
Division, but finally only “A” Squadron, was ordered to embark.
It was midnight when the troopships steamed out Departure for
of the harbour, a dark night, all lights doused, no Egypt
coastwise lights to guide, the blackness stabbed by
scores of searchlights. The troops on board one of the ships began
to sing and the whole convoy took up the strains. In the morning the
first arrivals lay-to off the Eddystone, until, by late afternoon, a great
fleet had assembled. At sundown the convoy sailed in three lines
ahead, escorted by the battleship Ocean and the cruiser Minerva. It
is said that this was the first actual convoy that had left England
since the Napoleonic wars.
The rough weather of the first three days and much unavoidable
discomfort aboard ship were borne with cheerfulness. The men had
the impression that they were sadly overcrowded, but subsequent
experience of life in “cubby-holes” and “dug-outs” modified their
ideas of what lack of space really means. It was noticed that even
the worst sufferers from seasickness never lost hope, as, although
whole messes did not eat a meal during those three days, the daily
indent for one bottle of beer per man was always drawn, to be saved
for happier days. Rumours of the presence of enemy craft off
Portugal gave rise to a pleasing sense of excitement and risk, but
nothing happened. Early on September 17 the mighty rock of
Gibraltar loomed suddenly out of the mist, and soon the mystical,
sun-scorched, tawny hills of Northern Africa were sighted.
From Gibraltar the convoy was escorted by the Minerva only. As
the weather improved it was found that, in spite of the crowded life
aboard the troopships, quite a lot of theoretical training, and even a
certain amount of physical, could be given, and there was little time
for leisure in the Mediterranean. One brilliant day, after Malta had
been passed, the sight of a great fleet sailing towards the West
caused much excitement. Soon the news spread that this was the
Lahore Division bound for Marseilles—war-hardened Sikhs,
Gurkhas, Dogras, Punjabis, Pathans, and Rajputs, all eagerly
looking forward to the day when they should cross steel with the
famous soldiers of Germany, and confident of the result of such
meeting. After exchange of greetings and of escorts, the Weymouth
taking the Minerva’s place, flags were dipped and adieux waved. It
was a wondrous sight, this great gathering of mighty ships, carrying
tens of thousands of men and all their material of war from East to
West and from West to East—an object lesson of the strength and
resources of the Empire.
 On the 25th September, a misty, languorous morning, the low,
sandy coast of Egypt, fringed with surf, with here and there a clump
of palm trees, was sighted. It was the Egypt of anticipation, curiously
familiar even to men whose travels had rarely taken them beyond
the confines of Lancashire. As the troopships slowly entered
Alexandria Harbour they passed a battle-cruiser of the U.S. Navy.
Though a strict neutrality had to be maintained in respect of action,
there was no attempt to observe it in sentiment. To the delight of the
Lancashire men the Americans manned ship; their band played “God
save the King,” and generally “they did us proud.” The Division was
keen to show its appreciation. The band of the 8th Manchesters,
uncertain whether “Yankee Doodle,” “The Star-spangled Banner,” or
“My Country, ’tis of Thee,” is considered the national anthem of the
United States, and doubting their ability to render either of the first
two airs pleasingly—the third tune being that of our own national
anthem, and therefore open to misconstruction—struck up “Marching
through Georgia.” All too late came the reflection that possibly the
cruiser’s complement had been drawn from States which might not
appreciate this air. However, every one seemed delighted by the
display of cousinly emotion, and felt the better for the incident.
The disembarkation, which began the same day, Arrival in Egypt
was watched critically by regular officers who were
waiting to embark for France, and who expressed their delight at the
fine appearance and soldierly bearing of the Territorials. The first
troops to land went on by train to Cairo, where the Yeomanry, the
Lancashire Fusiliers Brigade, the Signal Company, and the Transport
and Supply Column took over Abbasia Barracks. The East
Lancashire Brigade were partly in the Citadel and Kasr-el-Nil
Barracks in Cairo and partly in camp on the Heliopolis Racecourse.
The Artillery also encamped on the racecourse, and the Engineers
occupied the Kasr-el-Nil Barracks. The 5th, 6th and 8th Battalions of
the Manchester Brigade and one company of the 7th Manchesters
remained in Alexandria as garrison, the greater part being quartered
in the Mustapha Barracks and Camp, while detachments were
accommodated in the forts of Ras-el-Tin and Kom-el-Dik. Later, half
of this company of the 7th was sent to Cyprus. The other three
companies of the battalion had been sent to Khartoum via Port
Sudan. Detachments of the R.A.M.C. accompanied the 7th
Manchesters to Cyprus and Khartoum, but the Field Ambulances as
units remained with their affiliated Brigades, the 1st at Alexandria,
the 2nd at Heliopolis, and the 3rd at Abbasia. Detachments of the T.
and S. Column took over A.S.C. duties from the Regulars at
Alexandria, Khartoum and Cyprus. Abbasia was chosen for
Divisional Headquarters.
“Khaki” helmets were quickly issued, but as the Embarkation Staff
had put the tropical clothing in bulk in one ship, some time elapsed
before the heavy serge could be discarded in favour of light drill, and
it was astonishing how well the younger lads endured the excessive
heat. They showed their grit from the start. The usual conditions of
issue were experienced. When only the smaller sizes in headgear,
boots, and clothing are available, how is it that so large a proportion
of big-footed, big-headed, big-framed men turns up? The sun-
helmets were approved. “Bill, will they let us tak’ these whoam wi’
us?” one man was heard to ask. Bill was not one of the many
optimists who took for granted that they would be home for
Christmas. “Tha’ll be lucky if tha tak’s thi yed whoam, never mind thi
⸺ ’emlet!” he replied.
Many of the horses had died on the voyage, for not only were the
ships ill-fitted for horse transport, but orders had been received from
the War Office, in spite of the Divisional Commander’s
expostulations, not to exceed one man to every six horses. It was
not possible for so small a proportion of men, most of whom were
inexperienced, to cope with so many animals. On one boat alone,
the Norseman (an Australian emigrant boat), there were more than
700 horses in charge of ninety-five men, and forty-six animals died
during the voyage. The horses at first suffered greatly from climatic
conditions, and especially from sand-colic. One apparently decided
to lay a protest before the highest quarter, so, evading the picket, it
made its way through a narrow gate into the garden of the C.R.A.
and lay down to die almost on the General’s doorstep. The
veterinary officers had a busy time, and a story concerning one of
these was told with much enjoyment by General Maxwell himself.
The “vet” was working at the highest pressure, when a gentleman in
civil garb came along and, proceeding to ask him sundry questions,
was curtly requested to seek the nether regions and mind his own
business there. His horror on discovering later that his abuse had
been levelled at the head of the Commander-in-Chief may be
imagined.
Lord Kitchener had asked the Territorials to push on with their
training, and General Douglas had assured him that all ranks were
animated by a keen desire to fit themselves for whatever service
might be required of them. Training had now begun in earnest, and
before long a new stimulus, a fresh incentive, to efficiency appeared,
and the prospect of the Division’s participation in actual warfare
became less remote. During October the suspicion that Turkey might
take the plunge and declare herself on the side of the enemy had
become a probability. An attempt to incite Egypt to revolt, and,
indeed, to persuade Mohammedans throughout the world that the
day had dawned when a swift blow should destroy British supremacy
for ever, might confidently be looked for; and an attack upon the
Suez Canal would surely follow. To Mohammedans—with the
exception of the comparatively feeble Shiah sect—the Sultan of
Turkey was the Defender of the Faith, the representative of the
Prophet. Who could say what religious fanaticism might not
accomplish if he should proclaim Jehad against the infidels? The
situation was grave; it might become critical, for the Khedive’s good
faith was distrusted, and with reason.
The Regular Army of Occupation had left Egypt for France and
Flanders, and Lancashire civilians—clerks, warehousemen, artisans,
mill-hands, merchants, professional men—were responsible for law
and order in the ancient land of the Pharaohs, for its defence and for
the safe-guarding of the Suez Canal and the communications
between West and East. They kept their charge inviolate, and during
eight months, amid strange surroundings and under trying conditions
of climate, the East Lancashire Division gained the confidence of the
Administration and the respect and esteem of British colony and
native population alike, by their efficiency, their grit, and their
exemplary conduct. As soldiers under fire they had yet to be tested;
as men they were proved and approved. Lancashire, and, indeed,
the whole Territorial Force, have reason to be proud of the first
civilian army that was placed in a position of grave responsibility.
On the 22nd of October intelligence was received of considerable
Turkish activity in the Sinai Peninsula. We were still at peace with
Turkey, but it was highly probable that a surprise attack upon the
Suez Canal might precede a formal declaration of war. The Canal
had been patrolled by a battalion of Highland Light Infantry until early
in October, when its duties had been taken over by Indian troops. To
strengthen the Canal defences the 1st and 2nd Field Companies,
R.E., were sent to Ismailia on October 26 and 27, and a few days
later a detachment of the Signal Company and the machine-gun
sections of the 5th Battalion, East Lancashires, and 10th Battalion,
Manchesters, also left Cairo for the Canal zone. The Sappers
bridged the Canal in three places, manned the searchlights, supplied
crews for steam-tugs—it was in consequence of an explosion on one
of these boats that the first casualties of the Division occurred,
Lieutenant Woods and six non-commissioned officers and men
being killed—supervised the water supply, destroyed the native
village of Kantara, and built fortifications. The Signal Company laid a
cable between Kantara, Ismailia, and Suez, and the motor-cycle
despatch riders of this company were the first ever employed on
active service outside Europe. The work of the Engineers received
much-valued commendation from the Commander-in-Chief.
A section each of the 2nd and 3rd Field Ambulances had been
sent to Ismailia and Kantara respectively to be attached to the Canal
defences, and these gave what assistance was needed to Indian
troops as well as to the men of their own Division.[1] Another section
of the 2nd Field Ambulance took charge of the Australian hospital at
Mena, at which place, and also at Meadi and Zeitoun, the East
Lancashire Transport and Supply Column had formed depots and
undertaken transport duties for the Anzacs until their own A.S.C.
units arrived.
On October 31 a force of nearly 9000 Yeomanry and Territorials
marched through the streets of Cairo. Such a display of troops had
never before been seen by the natives, and they were much
impressed at a critical time. The Commander-in-Chief, Sir John
Maxwell, expressed his appreciation of the splendid appearance of
the men and of the salutary effect of the parade on the Egyptians.
Native agitators and German and Turkish agents had for some time
been active in Egypt and the Sudan, seeking to discredit the allies,
and one of these did his best to convince spectators that what they
took to be a powerful force was in reality but a stage army—the
same unit marching in a circle. On November 1 Martial Law was
proclaimed throughout Egypt and the Sudan. On the 5th of
November war was declared against Turkey. The island of Cyprus
was formally annexed on the 7th. Two companies of the 8th
Manchesters had relieved the half-company of the 7th Manchesters
as garrison on October 20, and detachments were sent to the
various towns of the island to prevent disturbances between Greeks
and Turks when the proclamation was made. On December 20
Prince Hussein was declared Sultan of Egypt under British
Protectorate, and the 5th Lancashire Fusiliers provided a guard of
honour on the occasion of the formal entry of the new Sultan into his
capital. In Alexandria and Khartoum the Manchesters had
ceremonial marches through the towns.
For some months the Division was occupied in Work and Play
strenuous training. The men suffered from the heat,
the glare of the sun, the dust, sandstorms, flies, and other plagues of
Egypt, the remarkable drop in temperature at night, the long,
shadeless, desert marches; but they took it all cheerfully as part of
the day’s work, and by the end of the year 1914 they were fairly well
acclimatized and thoroughly fit. There was play as well as work;
cricket, rugger, soccer, lacrosse, and hockey teams were soon hard
at it; concert parties and pierrot troupes discovered unsuspected
talent. There were also boxing contests, and the race meetings on
Gezireh Island provided many dinners at the Continental and
Shepheard’s for the winners. Full advantage was taken of the
opportunities given at week-ends to visit the marvels of Egypt. The
hospitality of the British colonies at Cairo, Alexandria, and elsewhere
was warmly appreciated, and many friendships were formed
between the residents and the men from Lancashire.
 The good feeling and comradeship that prevailed throughout the
Division may be illustrated by an incident, slight but typical. On the
occasion of some special parade the 5th Manchesters had to cross
the very sandy parade ground before reaching the asphalt road that
led out of barracks. Noticing the dusty state of their boots the men of
D Company of the 6th Manchesters darted into their quarters, which
were close at hand, and, producing brushes and rags, they quickly
polished the boots of the Wigan men. It was a trifling incident, but
still a perfect expression of the spirit of good comradeship, and it had
a wonderful and lasting effect. The two battalions were knit more
closely together, and later, in Gallipoli, there was a sense of absolute
security in either battalion in the knowledge that the other was in
support.
The first Christmas on active service was celebrated in traditional
style, plum-puddings and other Christmas fare being sent out by
friends at home. The old hymns and carols at the Morning Service
stirred the emotions deeply, and thoughts of home and memories of
bygone Christmas gatherings became poignant. Dining huts and
messes, rooms and verandahs, were garlanded with palm branches,
flowers, lanterns, and chains of coloured paper. Nothing was omitted
to make the day a memorable and happy one, and one to look back
upon. Still, there were some decidedly novel features, chief of which,
perhaps, was the after-dinner swim in the sea, indulged in by a
number of Manchester men in Alexandria. On Boxing Day a great
sports meeting was held at the Gezireh Sporting Club, Cairo, and
many Lancashire men were successful competitors. They were,
however, outshone by the men of the Ceylon Planters Rifle Corps,
whose prowess was hailed with enthusiasm by their Territorial and
Yeomanry rivals.
Casual mention only has been made of the 7th Manchesters. As
the greater part of this battalion occupied a position of splendid
isolation its very interesting experiences must be referred to
separately and all too briefly. B Company had been dropped at
Alexandria to take over the duties of a company of the Suffolk
Regiment in that town and in Cyprus, and the other three companies
remained on board the troopship. Port Sudan was reached on
September 30 after a five days’ voyage, and the warm reception
given them by the Suffolks and the British officials was very welcome
after four weeks at sea. Half of C Company was left to garrison Port
Sudan and, later, among other duties, to patrol the Red Sea in
H.M.S. Enterprise, and to run an armoured train. The rest of the
battalion entrained for Khartoum, a journey of 600 kilometres, where,
on October 2, they were welcomed by the Sirdar, Sir F. R. Wingate,
and his Staff.
Khartoum and the Sudan furnished a succession of novel
experiences to the men from Burlington Street, but no surprise quite
equalled that conveyed to them by the intimation that they were at
once, without preliminary coaching, to supply a half-company to form
the British Camel Corps. The two platoons of C Company were
detailed for this duty. The departing O.C. Camel Corps had little time
in which to impart information to his successor, and there were many
documents to sign, some of these being in Arabic. He seemed
grieved that Manchester men should be ignorant of Arabic and of the
customs, habits, and requirements of camels, but volunteered as
much information as could be crammed into five minutes. Captain C.
Norbury and his subalterns gazed upon their new pets, seventy in
number, without affection, and sat down the better to enjoy the
humour of their predicament before sampling its difficulties. In
despair and not in hope, expecting rather to provoke merriment than
to elicit information, the new O.C. Camels asked his half-company if
any man among them had had experience of camels. Forth stepped
the former camel-keeper of Bostock’s menagerie. The Hour had
brought forth the Man. The 7th Manchesters now felt that they were
equal to any emergency, and soon they provided piano-tuners for the
Sirdar’s palace, trained gardeners for the barracks gardens, and
skilled artisans for every variety of job for which an expert was
demanded.
The Camel Corps was soon proficient enough to be sent by the
Sirdar on a two-weeks’ trek through villages where white men had
rarely been seen, and their presence gave the lie to reports
circulated by Turco-German emissaries that all white troops had
been recalled from Egypt and the Sudan to defend their home
country from the enemy at its gates. Incidentally they had the
opportunity of seeing the wonderful results of Manchester enterprise
in the great cotton-growing areas through which they passed. Not
only had Khartoum been transformed from a collection of mud huts
into a town of imposing buildings, shops, and public works, but the
savage Sudan of a few years ago was in process of transformation
into a land of peace and prosperity.
Training proceeded under the same trying conditions of climate
experienced by their comrades in Egypt; the same games and sports
were enjoyed with similar zest; and fitness and efficiency prevailed.
The conduct of the men received and merited high praise from the
Sirdar—who paid the battalion the distinction of becoming its
honorary Colonel—and from all with whom they came in contact.
Relations with the natives were excellent, and a firm friendship was
formed with the Egyptian regiment in Khartoum. The first of all active
service periodicals, The Manchester Sentry, was published in
Khartoum, the Sirdar and Lady Wingate contributing. The three
companies rejoined their comrades of the Manchester Brigade in
Cairo on April 23, 1915.
On January 19, 1915, the Manchester Brigade Defence of the
was transferred from Alexandria and Cyprus to Suez Canal
Cairo, and the 5th and 8th Lancashire Fusiliers
were sent to Alexandria. Reports of renewed activity on the part of
the Turks pointed to an attempt to seize the Suez Canal and invade
Egypt. On January 20 the 1st and 3rd Brigades, R.F.A., were
despatched to the Canal zone, to be followed a few days later by the
1st Field Company, R.E., which had recently been brought back to
Cairo in order to take part in divisional training. The artillery was
posted on the west bank of the Canal at El Kubri, Serapeum West,
Ferry Post (Ismailia), El Ferdan, and Kantara, most of the guns being
concealed among the pines that grow within a hundred yards of the
bank. The Field Companies assisted the garrisons of Indian troops in
the strong points of the east bank to improve their defences.
The expectations of hostile attack were realized in the early hours
of February 3, when a force of 12,000 Turks and Germans attacked,
and made an attempt to cross the Canal midway between the east
bank strong points of Serapeum and Toussoum. Heavy rifle and
machine-gun fire developed between those places about 3 a.m. The
guns of the Egyptian Mountain Battery had, by happy chance or
remarkable intuition, been mounted on the west bank within 400
yards of the main crossing-place selected by the Turks. The chanty
of the enemy, as, unaware of the battery’s proximity, he attempted to
launch the heavy steel pontoons, was much appreciated by the
gunners, who opened fire in the dark at point-blank range. When
daylight came the Battery Commander was able to congratulate
himself on some accurate “spotting,” several holed pontoons and
many dead Turks being found at the water’s edge. One of these
pontoons is now in possession of the Manchester Corporation.
Two sections of the 1st Field Company had been detailed to hold
the west bank at this point, and in the course of the fighting on
February 3 they lost one man killed and two wounded. The 19th
Battery, under Major B. P. Dobson (which had already been in action
on the previous day against the Turkish Camel Corps), played a
conspicuous part in the enemy’s defeat. They had hauled one 15-
pounder through a wood to the bank of the Canal and had fired point
blank into the Turks as they vainly attempted to launch their iron
boats. Another gun was man-hauled to a hill behind the wood, and
this found good targets as the enemy approached the Canal.
Captain P. K. Clapham did some good spotting for the battery from a
precarious and exposed position in a tall fir tree. The 18th Battery, at
Ismailia, fired on the enemy positions with good effect from 2000 to
3000 yards, and every battery of the two brigades shared in the
victory. The 20th Battery, at El Ferdan, had the distinction of being
the first Battery of the Division—and probably the first of any
Territorial unit—to open fire upon an enemy. The total casualties of
the East Lancashire Artillery were five men wounded, four of these
belonging to the 19th Battery. The men of the Signal Company, who
had done good work in this sector, also received their baptism of fire.
The attempt to invade Egypt had failed. The Punjabis (upon whom
fell the brunt of the fighting), the Rajputs and Gurkhas on the east
bank were fully prepared both to meet the attack and to assume the
offensive, inflicting a serious defeat and making important captures.
The Divisional Yeomanry arrived at Ismailia on the 4th of February in
time to co-operate with Indians, Anzacs, and the 5th Battery in
following up the enemy’s retirement. More than 1600 prisoners were
taken. On the 10th General Douglas visited the posts on the Canal,
held by units of his Division, to congratulate his men on their good
work. The 2nd Field Company reached Ismailia on the 6th, and for
the greater part of the month the sappers were kept busy
strengthening the Canal defences, making entanglements, trenches
and bomb-proof shelters, and laying mines. Another attack upon the
Canal was made on the 22nd of March, when the 5th Battery was
again in action, and the next day the Battery accompanied a column
which attacked the Turks in the Sinai Desert about nine miles N.E. of
El Kubri. While the East Lancashire Batteries remained in the Canal
zone Brigadier-General A. D’A. King, Divisional C.R.A., assumed
command of the artillery of the Canal defences.
From the beginning of the year 1915 the training Training and
had become more and more strenuous. There Fitness
were long marches in the desert—occasionally
very long ones—in full marching order, through native villages in
which the many odours were only excelled in numbers, variety, and
offensiveness by the yelping curs that were stirred into noisy activity
by the tramp of the battalions. The Khamsin wind was sickly in its
heat, the atmosphere heavy and laden with sand, the glare of the
sun pitiless, the only shade available while resting being the very
inadequate shelter provided by a blanket stretched on rifles. But the
men were physically fit—they had to be, for only fit men were
needed. The amateurs had become soldiers. The days were past
when the colour-sergeant’s whispered order to the right-flank man of
a company in extended order for “two scouts” would reach the left-
flank man in the form of “the colour-bloke wants a couple of stouts.”
In spite, however, of good physical condition and a fine spirit, it could
not truthfully be stated that the Suez road, with its five-mile towers,
and the Virgin’s Breast had endeared themselves to Lancastrians.
Familiarity had bred not contempt but a whole-hearted loathing for
that accursed highway and that distant mound. The Third Tower was
the usual goal, the advance ceasing just beyond it. How the troops
hated the sight of this detestable pile which, in the dust and glare,