THE JOURNAL OF COMPARATIVE NEUROLOGY 375:527-551 (1996)

Cholinergic Innervation of the Primate

Hippocampal Formation: 11. Effects of
Fimbria/Fornix Transection
JOSE R. ALONSO, HOI SANG U, AND DAVID G. AMARAL
Universidad de Salamanca, Departamento de Biologia Celular y Patologia, 37007 Salamanca,
Spain (J.R.A.); Department of Neurosurgery, University of California, San Diego, La Jolla,
California 92093 (H.S.U.);Department of Psychiatry and the Center for Neuroscience,
University of California, Davis, Davis, California 95616 (D.G.A.)

ABSTRACT
The distribution of choline acetyltransferase (ChAT)-immunoreactive and acetylcholines-
terase (AChE)-positive fibers and terminals was analyzed in the hippocampal formation of
macaque monkeys subjected to transection of the fimbriaifornix. Cases with either unilateral or
bilateral transections were prepared, with post transection survival times ranging from 2 weeks
to 1.5 years. The fimbriaifornix transection resulted in a dramatic decrease in the number of
cholinergic fibers in most regions of the hippocampal formation. Some hippocampal regions,
however, showed relatively greater sparing of C U T - or AChE-positive fibers. In practically all
regions of the hippocampal formation, residual AChE-positive fibers were more abundant than
ChAT-immunoreactive fibers. In animals with unilateral lesions, the distribution patterns and
density of AChE and ChAT staining on the side contralateral to the lesion were generally
similar to those of sections from unlesioned control brains. The largest decreases in the
densities of positive fibers were observed in the dentate gyrus, CA3 and CA2 fields of the
hippocampus, subiculum, parasubiculum, and medial and caudal parts of the entorhinal cortex.
Fibers were relatively better preserved in the rostral or uncal portion of the hippocampus and
dentate gyrus and in the rostral portion of the entorhinal cortex. The presubiculum demon-
strated remarkable sparing that contrasted with the almost complete loss of fibers in the
parasubiculum. Interestingly, animals killed approximately 1.5years after the fornix transec-
tion showed essentially the same pattern of fiber loss as the cases with shorter survival periods.
This indicates that the residual ChAT-immunoreactive fibers, many of which reach the
hippocampal formation through a ventral cholinergic pathway, are not capable of reinnervating
the denervated portions of the hippocampal formation. This appears to distinguish the monkey
from the rat, for which substantial sprouting and reinnervation of cholinergic fibers have been
reported after similar lesions. o 1996 Wiley-Liss, Inc.

Indexing terms: acetylcholinesterase, choline acetyltransferase, hippocampus,entorhinal cortex,

fornix lesion

The neuroanatomical organization of the cholinergic
septohippocampal pathway has been the subject of intense
research interest. This is due, in part, to the putative
cholinergic involvement in normal learning and memory
and to the possible involvement of cholinergic systems in
the genesis and progress of neurodegenerative diseases
such as Alzheimer’s disease (Davies and Maloney, 1976;
Whitehouse et al., 1981, 1982; Bartus et al., 1982; Coyle et
al., 1983). In Alzheimer’s disease, there is a loss of choliner-
gic neurons in the basal forebrain, which parallels decreases
in the levels Of choline acetyltransferase (ChAT) in the
hippocampal formation and reductions Of
active (ChAT-ir) fibers in all hippocampal fields (White-
house et al., 1982). Moreover, the extent of cognitive
dysfunction observed in patients with Alzheimer’s disease
is closely correlated with the extent of damage to the cells of
the basal forebrain cholinergic system (Bartus et al., 1982;
Coyle et al., 1983).
In rats, cholinergic fibers reach the hippocampal forma-
tion via four routes (Milner and Amaral, 1984; Gage et al.,
1984; Gaykema et al., 1990), though the fimbriaifornix is
Accepted May 13, 1996.
Address reprint requests to Dr. David G. Amaral, Center for Neuro-
science, University of California, Davis, 1544 Newton Ct., Davis, CA 95616.
E-mail: dgamaralti ucdavis.edu

o 1996 WILEY-LISS, INC.

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528 J.R. ALONSO ET AL.

by far the main route. Other septohippocampal fibers travel In the studies reported herein, we investigated the extent
through the dorsal fornix and the supracallosal stria and to which fimbriai fornix transection deafferents the monkey
along a ventral pathway through the external capsule. The hippocampal formation of cholinergic innervation. We have
cholinergic innervation of the rat hippocampus is some- also investigated whether in the monkey, as in the rat,
what more substantial to the ventral or temporal region, residual cholinergic fibers are capable of sprouting to
which is the main recipient of fibers from the so-called reinnervate denervated portions of the hippocampal forma-
ventral pathway (Gage et al., 1984). In the monkey, the tion. Thus far, this type of sprouting response has not been
major septal projection to the hippocampal formation also observed in primates with a postsurgical period as long as 6
travels via the fimbriaifornix, and fewer fibers follow the months (Samson et al., 1991).
supracallosal stria (Kitt et al., 1987; Alonso and Amaral,
1995). Small numbers of fibers also enter the hippocampal
formation via a ventral pathway, and at least some of these MATERIALS AND METHODS
arise from the basal nucleus of Meynert rather than the Animals and surgical procedure
septal complex (Kitt et al., 1987). As in the rat, the rostral
Ten adult Macaca fascicularis (cynomolgus) monkeys
portion of the monkey hippocampal formation demon-
with fimbria/fornix transections (FFT) were used in the
strates a somewhat higher density of ChAT-ir fibers (Alonso
present study. Four animals were subjected to unilateral
and Amaral, 1995).
FFT, and they were killed 4 weeks following the lesion. The
Transection of the rat fimbria/fornix leads to a massive
brains of these animals were previously used as control
depletion (85-95%) of hippocampal ChAT immunostaining
material in studies on the effects of nerve growth factor
(Storm-Mathisen, 1977; Fibiger, 1982; Gage et al., 1983,
infusion on the survival of septal neurons following FFT
1986; Dravid and Van Deusen, 1984; Gasser and Dravid,
(Tuszynski et al., 1990). These animals were fitted with an
1987; Matthews et al.,1987; Blaker et al., 1988; Yunshao et
indwelling ventricular cannula and received continuous
al., 1992; Lahtinen et al., 1993). AChE labeling is also
infusion of vehicle solution during the month-long survival
depleted (to about 25% of control values) after fimbriai
period (see Tuszynski et al., 1990, for details). The remain-
fornix lesion (Gage et al., 1983). Only in the most temporal
ing six animals received a bilateral FFT and were allowed to
levels of the rat hippocampal formation is there noticeably
survive for 2 weeks (n = 3) or approximately 18 months
greater sparing of cholinergic fibers. Interestingly, there is
following the lesion (n = 3). The latter three animals
a partial recovery of cholinergic innervation of temporal
participated in behavioral studies that assessed the effect of
levels of the rat hippocampus with postlesion survival
FFT on the performance of various learning and memory
periods of 6 months or more (Gage et al., 1983). At 6
tasks (Zola-Morgan et al., 1989). Material from 12 M .
months after the fimbria/fornix transection, the level of
fascicularis monkeys prepared for the analysis of the
ChAT immunostaining at the temporal one-fifth of the
normal distribution of ChAT-ir fibers (Alonso and Amaral,
hippocampus increases threefold relative to levels observed
1995) was used as a control for the normal distribution of
1 week after transection. This apparent sprouting of residual
ChAT and/or AChE labeling.
cholinergic fibers returned levels of ChAT to approximately
In preparation for FFT surgery, the animals were tran-
40% of control levels. At this time point, there was a detectable,
quilized with ketamine HC1 (7 mgikg, i.m.) and deeply
albeit more modest, increase in the levels of ChAT throughout
anesthetized with Nembutal (25-30 mgikg); heart rate,
the remainder of the hippocampus (Gageet al., 1983).
body temperature, and respiration were continuously moni-
tored. Although various surgical approaches were taken to
Abbreviations expose and transect the fimbriaifornix (see, for example,
Zola-Morgan et al., 19891, the most efficient and reliable
35 area 35 (Brodmann) method was the one reported by Tuszynski et al. (1990).
36 area 36 (Brodmann) The essential features of this procedure are the following: A
a alveus
ah angular bundle craniotomy 20 mm in diameter was performed with its
CA1-CA3 fields of the hippocampus center located 4 mm anterior to the intraural line and
cc corpus callosum biased to the right of the midline. The dura was opened and
DG dentate g y m s retracted over the superior sagittal sinus, exposing the
DNMS delayed nonmatching to sample
Ec caudal division of the entorhinal cortex interhemispheric fissure and the cingulate gyrus. The
Ecr. caudal limiting division of the entorhinal cortex ipsilateral hemisphere was then retracted laterally to ex-
EI intermediate division of the entorhinal cortex pose the underlying corpus callosum. Veins entering the
EL lateral division of the entorhinal cortex superior sagittal sinus were preserved wherever possible.
EL< caudal portion of the lateral division of the entorhinal cortex
ELr rostral portion of the lateral division of the entorhinal cortex After identification of the splenium and the anterior cere-
EntC entorhinal cortex bral arteries, a self-retaining retractor was used to facilitate
ElJ olfactory division of the entorhinal cortex exposure of the ipsilateral cingulate gyrus. A point 5 mm
ER rostral division of the entorhinal cortex
f fimbria
anterior to the tip of the splenium, on the surface of the
GCL granule cell layer cingulum, was selected for entry into the trigone of the
I-VI layers of the entorhinal cortex lateral ventricle. The corpus callosum was broached with a
Im stratum lacunosum moleculare suction pipette, and hemostasis was maintained with bipo-
ML molecular layer lar coagulation. After visual identification of its lateral and
0 stratum oriens
P pyramidal cell layer medial borders, the fornix was separated from the epen-
Pas parasubiculum dyma and transected either with microscissors or with the
PL polymorphic layer of the dentate gyrus bipolar coagulator. The dura was then sutured and the
PrS presubiculum craniotomy wound closed in three layers. Each animal
r stratum radiatum
S subiculum received postoperative ampicillin (250 mgiday, i.m.1 for 7
WM white matter days. Though the intent of this surgical procedure was to
 10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
CHOLINERGIC DENERVATION OF THE MONKEY HIPPOCAMPAL FORMATION 529

transect the fimbria, it is likely that the approach taken to RESULTS

visualize the fimbria necessarily resulted in substantial Nomenclature for the monkey hippocampal
damage of the ipsilateral supracallosal stria. formation
After the survival period, animals were deeply anesthe-
tized with ketamine HCl(10 mgikg, i.m.1 and Nembutal(50 The nomenclature and definition of cytoarchitectonic
mgikg, i.p.) and perfused transcardially for 1 hour with a divisions used herein are as previously described (Alonso
4% solution of paraformaldehyde in 0.1 M phosphate and Amaral, 1995; see Figs. 1-6,9-14). Briefly, the dentate
buffer, followed by 5% sucrose solution in the same buffer gyrus is divided into three layers, the molecular layer, the
for 20 minutes to clear the fixative. The brains were granule cell layer, and the polymorphic cell layer. The
stereotaxically blocked in the coronal plane and cryopro- hippocampus proper is divided into three distinct fields-
tected for histological processing. CA3, CA2, and CAl-and into five layers that are oriented
parallel to the pyramidal cell layer. The layers are (arranged
from deep to superficial): stratum oriens, pyramidal cell
ChAT immunohistochemistry layer, stratum lucidum, stratum radiatum, and stratum
Immunohistochemical analysis was performed with a lacunosum moleculare. Following distally from the CA1
monoclonal ChAT antibody (AB8) kindly donated by Dr. field are the subiculum, presubiculum, and parasubiculum.
Bruce Wainer. Immunolabeling was revealed with the The presubiculum and parasubiculum have a cell-free layer
I and a thicker layer I1 with densely packed neurons. Layer
double-bridging peroxidase-antiperoxidase (PAP) method
I1 of the presubiculum has thinner superficial and thicker
as described elsewhere (Alonso and Amaral, 1995). Briefly, deep sublaminae. The entorhinal cortex is divided into
brains were cut on a sliding microtome, and the free- seven fields: olfactory (Eo), rostral (ER),lateral (EL)[with
floating sections were incubated in primary antibody di- rostral (ELr) and caudal parts EL^)], intermediate (EI),
luted 1:500 in 0.1 M Tris-buffered saline (TBS) containing caudal (Ec), and caudal limiting field (EcL; Amaral et al.,
2% bovine serum albumin, 20% normal rabbit serum, and 1987). Six layers (I-VI) can be distinguished in the entorhi-
0.5% Triton X-100 for 48 hours at 4°C. The sections were nal cortex (Fig. 11A).
washed and incubated for 1 hour in rabbit anti-rat IgG We use the term superficial to designate those layers
secondary antiserum (Cappel Laboratories, Durham, NC) closer to the pia or to the hippocampal fissure and the term
diluted 1 5 0 in TBS containing 10%normal monkey serum, deep to indicate the opposite direction. The long axis of the
20% normal rabbit serum, and 0.2% Triton X-100 for 1 primate hippocampus is referred to as the rostrocaudal axis
hour at room temperature. The sections were then incu- (equivalent to the temporoseptal axis in rodents), whereas
bated in rat PAP (Sternberger Monoclonals, Jarretsville, the axis orthogonal to the rostrocaudal axis is called the
MD) that was diluted 1 5 0 in the same solution as the transverse m i s . The term proximal is used to refer to the
secondary antiserum for 2 hours at room temperature, portion of a particular field that is located closer to
rinsed in buffer, and then exposed to a second secondary the dentate gyrus, and distal refers to the portion closer to
antiserum and PAP incubation. The tissue-bound peroxi- the rhinal sulcus.
dase was revealed with a 0.05% solution of 3,3'-diaminoben-
zidine in phosphate buffer plus 0.015% H202 for 15 min- General characteristics of ChAT
utes. The sections were mounted onto gelatin-coated glass and AChE labeling
slides, air dried, intensified with a 0.005% osmium tetroxide The FFT resulted in clear reductions in both ChAT and
solution, and coverslipped with Permount. AChE fiber labeling in the hippocampal formation ipsilat-
era1 to the lesion (Figs. 1-6, 9-14). By contrast, there was
AChE histochemistry no change in the number of AChE-positive cells distributed
throughout the hippocampal formation; they were perhaps
A series of sections adjacent to those prepared immuno- even more evident because of the depletion of obscuring
histochemically for ChAT was processed for the demonstra- fiber plexuses. As in control animals, no ChAT-immunoposi-
tion of AChE using a modification (Hedreen et al., 1985) of tive neurons were observed in the lesioned hippocampi. In
the Koelle method (Geneser-Jensen and Blackstad, 1971) the hemisphere contralateral to the FFT, the distribution
as described previously (Bakst and Amaral, 1984; Amaral and density of ChAT and AChE labeling resembled that in
and Basset, 1989; Alonso and Amaral, 1995). A complete control animals.
third series of sections from each brain was stained with Among the different hippocampal fields, the effects of the
0.25% thionin for the demonstration of cell bodies. FFT varied from almost complete loss of cholinergic fibers
to almost complete preservation. In some regions, such as
Analysis the dentate gyrus, the hilar portion of CA3, and the
superficial layers of the caudal entorhinal cortex, there was
Sections were analyzed using brightfield- and darkfield- a massive depletion of labeled fibers (Figs. 2A, 4A, 6A, lOA,
equipped microscopes. Low-power photomicrographs were 12A, 14A). In the presubiculum, in contrast, the cholinergic
taken from the cases with unilateral FFT. Representative innervation was substantially preserved (Fig. 8). These
adjacent sections from the side ipsilateral to the FFT results were generally observed in both ChAT and AChE
stained for C U T , AChE, and thionin and from the contra- preparations. However, hippocampal fields that in the
lateral side stained for ChAT were photographed. These ChAT preparations were almost totally devoid of labeled
panoramic views were obtained using a 4 x 5 inch photo- fibers, such as the hilar portion of CA3, CA1, and dentate
graphic system (Nikon multiphot) and a darkfield base gyrus, demonstrated a markedly reduced yet clearly detect-
(Nikon BD-2). Higher magnification photomicrographs were able plexus of AChE-labeled fibers (Figs. lB, 3B, 5B).
taken with a Leitz Dialux 20 microscope equipped with a With available material, we were able to evaluate whether
Wild MPS55 35 mm camera system. the fibers of the ventral cholinergic pathway that were not

Fig. 1. Rostra1 coronal sections of the monkey hippocampal forma-
tion. A Section from the hippocampal formation ipsilateral to the
lesion stained with thionin. B: Darkfield photomicrograph of an
adjacent section stained for the demonstration of acetylcholinesterase
(AChE). The main divisions of the hippocampal formation shown in
10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
each photograph are indicated in the Nissl-stained section. These
low-power micrographs were taken from animals with unilateral
fimbriaifornix transection and the 1 month survival period. Compa-
rable sections stained for AChE from unlesioned animals are illustrated
in Alonso and Amaral(1995). Scale bar = 1mm.
 10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Fig. 2. Photomicrographs of coronal sections of the monkey hippo- immunohistochemically for the demonstration of C U T . Comparison of
campal formation. A Section adjacent to those shown in Figure 1 A and B illustrates the substantial loss of ChAT fibers following FFT. These
stained for the demonstration of ChAT immunostaining. B: Section at low-power micrographs were taken from animals with unilateral fimbriai
about the same rostrocaudal level from the contralateral side, stained fornix transedion and the 1month survivalperiod. Scale bar = 1mm.
 10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Fig. 3. Representative coronal sections of the monkey hippocampal divisions of the hippocampal formation shown in each photograph are
formation. A Section from the hippocampal formation ipsilateral to the indicated in the Nissl-stained section. These low-power micrographs
lesion stained with thionin. B: Darkfield photomicrograph of an were taken from animals with unilateral fimbriaifornix transection and
adjacent section stained for the demonstration of AChE. The main the 1month survival period. Scale bar = 1mm.

Fig. 4. Photomicrographs of coronal sections of the monkey hippo-
campal formation. A: Section adjacent to those shown in Figure 3
stained for the demonstration of C U T immunostaining. B: Section at
about the same rostrocaudal level from the contralateral side, stained
immunohistochemically for the demonstration of ChAT. Note preserva-
10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
tion of staining in the presubiculum and complete loss in the parasibicu-
lum. These low-power micrographs were taken from animals with
unilateral fimbriaifornix transection and the 1month survival period.
Scale bar = 1mm.
 10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Fig. 5. Representative coronal sections of the monkey hippocampal divisions of the hippocampal formation shown in each photograph are
formation. A: Section from the hippocampal formation ipsilateral to the indicated in the Nissl-stained section. These low-power micrographs
lesion stained with thionin. B: Darkfield photomicrograph of an were taken from animals with unilateral fimbriaifornix transection and
adjacent section stained for the demonstration of AChE. The main the 1month survival period. Scale bar = 1mm.
 10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Fig. 6. Photomicrographs of coronal sections of the monkey hippo- immunohistochemically for the demonstration of C U T . These low-
campal formation. A Section adjacent to those shown in Figure 5 power micrographs were taken from animals with unilateral fimbriai
stained for the demonstration of ChAT immunostaining. B: Section at fornix transection and the 1month survival period. Scale bar = 1mm.
about the same rostrocaudal level from the contralateral side, stained

536
involved in the fornix transection were capable of sprouting
to reinnervate deafferented regions of the hippocampal
formation. It was quite clear that the distribution of
ChAT-ir fibers in animals with bilateral FFT that had
survived approximately 18 months after lesion was essen-
tially the same as that in animals with 2 weeks of postsurgi-
cal survival; i.e., there was little or no lesion-induced
sprouting of residual cholinergic fibers. Below we describe
the main characteristics of the distribution of both choliner-
gic markers in the principal divisions of the fornix transec-
ted hippocampal formation.
Dentate gyrus
The density of ChAT- and AChE-labeled fibers was
substantially reduced in all layers of the ipsilateral dentate
gyms (Figs. 1-7). There were greater numbers of fibers
remaining medially than laterally in the dentate gyrus. In
the molecular layer, the ChAT-and AChE-labeled fibers
were slightly more abundant in the inner half, but they did
not form a distinct supragranular plexus (Fig. 7A,C). The
supragranular band of diffuse AChE labeling seen in the
unlesioned molecular layer appeared to be relatively intact,
although perhaps with a slightly reduced staining intensity
(Fig. 7C). Granule cell bodies, which in control sections are
surrounded by ChAT- and AChE-positive fibers and varicosi-
ties, were devoid of cholinergic innervation (Fig. 7A). There
were a few scattered fibers in the polymorphic cell layer, but
these did not form a distinct infragranular plexus.
Hippocampus
Transection of the fimbria/fornix resulted in a marked
depletion of cholinergic fibers. The magnitude of differentia-
tion, however, varied along the rostrocaudal and transverse
axes. As was noted above, there was substantially greater
residual AChE staining than ChAT staining in the hippo-
campus (compare Figs. lB, 3B, and 5B with Figs. 2A, 4A,
and 6A). Residual staining for both ChAT and AChE was
most prominent in the uncal region (rostromedial portion)
of the hippocampus (Figs. lB, 2A). The pattern of staining
here, however, was not normal. Whereas in control material
the cholinergic fibers of the uncal region typically extended
laterally into stratum oriens and stratum lacunosum mo-
leculare of CA3, the ChAT-ir fibers in the FFT animals were
much more restricted to the most medial portion of the
uncal region.
In control preparations, the highest density of ChAT
labeling was observed in the portion of CA3 that is enclosed
within the hilar region of the dentate gyrus, and the density
of fibers decreased towards and into CA2. The opposite
pattern was observed in the FFT material; the lowest
staining for both ChAT and AChE was observed in the hilar
and compact regions of CA3, and the density of fibers
actually increased slightly along the transverse axis (Figs.
3B, 4A). The highest density of fiber and terminal labeling
was observed at the distal border of CA1. In this region, the
oblique band of increased fiber and terminal labeling that
separated CA1 from the subiculum in normal material was
also distinguishable in the lesion material.
In the hippocampus, the highest density of residual
ChAT-ir and AChE-labeled fibers was located in the stra-
tum lacunosum-moleculare (Figs. 4A, 6A). A noticeable,
albeit decreased, density plexus of fibers extended horizon-
tally along this layer and into the superficial portion of the
stratum radiatum. Although there were some ChAT-ir
fibers in the stratum oriens close to the alveus, the number
10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
J.R. ALONSO ET AL.
was very low in the rest of the layer. There were almost no
ChAT-ir fibers in the pyramidal cell layer or stratum
lucidum. The alveus was almost completely negative for
ChAT and AChE fibers.
Subiculum, presubiculum, and parasubiculum
The effects of the FFT on the cholinergic innervation of
the hippocampal formation were especially varied in the
subiculum, presubiculum, and parasubiculum. Distal to the
slightly increased density of ChAT fiber labeling at the
CAlisubiculum border, there was a general decrease in
fiber labeling in all layers of the subiculum (Fig. 4A, 6A).
Unlike the case in most other areas of the hippocampal
formation, the density of ChAT- and AChE-positive fibers
was high in both layers of the presubiculum (Figs. 4A, 6A,
8).Though it is difficult to state confidently that there was
no decrease in fiber and terminal labeling in the presubicu-
lum, the visual impression of this region in the FFT
material was that it had a normal or even slightly increased
density of labeling.
The alterations observed in the parasubiculum differed
markedly from those observed in the presubiculum. In the
parasubiculum, there was almost complete loss of fiber and
terminal labeling (Figs. 4A, 6A, 8). Layer I maintained a low
density of labeled fibers that coursed parallel to the pial
surface, but layer I1 was virtually devoid of its cholinergic
innervation (Fig. 8). Contrary to what is observed in these
principal layers of the parasubiculum, the deep cellular
layers that surround the angular bundle demonstrated
almost normal levels of ChAT and AChE labeling (Figs. 13,
14). As will be seen below, the staining pattern observed in
these deep layers was similar to that observed in the deep
layers of the entorhinal cortex.
Entorhinal cortex
The cholinergic innervation of the entorhinal cortex was
also substantially decreased by the FFT. The overall den-
sity of ChAT-and AChE-labeled fibers in the entorhinal
cortex was much lower than in the adjacent perirhinal
cortex, whereas in normal animals the entorhinal cortex
had a much denser cholinergic innervation. In fact, the FFT
produced a rather striking border between the relatively
heavily labeled areas 35 and 36 of the perirhinal cortex and
the lateral portion of the entorhinal cortex, particularly in
the superficial layers (Figs. 12A, 14A, 15A).As in the other
hippocampal fields, there were more residual AChE-stained
fibers than ChAT-ir fibers in the entorhinal cortex (Figs.
15A, 16A).
Substantial variations were observed in the effect of the
FFT on the entorhinal cortex along the rostrocaudal and
radial axes. First, rostra1 levels of the entorhinal cortex
(divisions Eo and ER)demonstrated relatively little change
in their patterns of stained fibers (Figs. 9B, 10A). The
extent of cholinergic fiber loss became progressively greater
at increasingly more caudal levels of the entorhinal cortex.
At all levels, the amount of fiber loss was greater laterally
than medially in the entorhinal cortex. Thus, at midrostro-
caudal levels, cholinergic fiber loss was much more evident
in ELcthan in Er (Fig. 12A).
The decrease in the density of labeled fibers in these two
latter fields was first evident in layer I and extended at more
caudal levels into the deeper layers. At midrostrocaudal
levels of the entorhinal cortex, where most of the transverse
extent is occupied by EI, more dramatic changes were
observed. The fiber-rich superficial layers (1-111) were
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Fig. 7. Photomicrographs displaying the patterns of staining for (Avs. B); only a few remaining ChAT-ir fibers (arrows) are apparent on
ChAT (A,B) and AChE (C,D) in the ipsilateral (A,C) and contralateral the side ipsilateral to the lesion. Note also the persistence of the
(B,D) molecular layers of the dentate gyrus. Note the decrease in the supragranular band of diffuse AChE staining (asterisks; C vs. D).
density of labeled fibers in the molecular layer ipsilateral to the lesion Eighteen months survival time. Scale bar = 100 bm.
10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

fibers in the presubiculum and the severe depletion in the parasubiculum. One month survival time. Scale
contralateral (C,Dj hemispheres, stained for ChAT (A,Cj and AChE (B,Dj. Note the persistence of labeled
Fig. 8. Darkfield photomicrographs of the presubiculum and parasubiculum of the ipsilateral (A,B)and

bar = 500 um.

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CHOLINERGIC DENERVATION OF THE MONKEY HIPPOCAMPAL FORMATION 539

Fig. 9. Representative rostra1 coronal sections of the monkey divisions of the entorhinal cortex shown in each photograph are
entorhinal cortex. A Section from the entorhinal cortex ipsilateral to indicated in the Nissl-stained section. These low-power micrographs
the lesion stained with thionin. B: Darkfield photomicrograph of an were taken from animals with unilateral fimbriaifornix transection and
adjacent section stained for the demonstration of AChE. The main the 1month survival period. Scale bar = 1mm.
 10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
540 J.R. ALONSO ET AL.

Fig. 10. Photomicrographs of coronal sections of the monkey chemically for the demonstration of ChAT. These low-power micro-
entorhinal cortex. A Section adjacent to those shown in Figure 9, graphs were taken from animals with unilateral fimbriaifornix transec-
stained for the demonstration of C U T . B: Section at about the same tion and the 1month survival period. Scale bar = 1mm.
rostrocaudal level from the contralateral side, stained immunohisto-
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Fig. 11. Representative midlevel coronal sections of the monkey divisions of the entorhinal cortex shown in each photograph are
entorhinal cortex. A: Section from the entorhinal cortex ipsilateral to indicated in the Nissl-stained section. These low-power micrographs
the lesion stained with thionin. B: Darkfield photomicrograph of an were taken from animals with unilateral fimbriaifornix transection and
adjacent section stained for the demonstration of AChE. The main the 1month survival period. Scale bar = 1mm.
 10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Fig. 12. Photomicrographs of coronal sections of the monkey chemically for the demonstration of ChAT. These low-power micro-
entorhinal cortex. A Section adjacent to those shown in Figure 11, graphs were taken from animals with unilateral fimbriaifornix transec-
stained for the demonstration of ChAT. B: Section at about the same tion and the 1 month survival period. Scale bar = 1mm.
rostrocaudal level from the contralateral side, stained immunohisto-
 10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Fig. 13. Representative caudal coronal sections of the monkey divisions of the entorhinal cortex shown in each photograph are
entorhind cortex. A: Section from the entorhinal cortex ipsilateral to indicated in the Nissl-stained section. These low-power micrographs
the lesion stained with thionin. B: Darkfield photomicrograph of an were taken from animals with unilateral fimbriaifornix transection and
adjacent section stained for the demonstration of AChE. The main the I month survival period. Scale bar = 1mm.
 10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Fig. 14. Photomicrographs of coronal sections of the monkey chemically for the demonstration of ChAT. These low-power micro-
entorhinal cortex. A Section adjacent to those shown in Figure 13, graphs were taken from animals with unilateral fimbriaifornix transec-
stained for the demonstration of ChAT. B: Section at about the same tion and the 1month survival period. Scale bar = 1mm.
rostrocaudal level from the contralateral side, stained immunohisto-

CHOLINERGIC DENERVATION OF THE MONKEY HIPPOCAMPAL FORMATION
Fig. 15. Darkfield photomicrographs of sections of the entorhinal
cortex at the level of the caudal division of the entorhinal cortex
ipsilateral (A) and contralateral (B) to the lesion, stained for the
immunohistochemical demonstration of C U T . Arrows in B demarcate
nearly devoid of ChAT-ir fibers (Fig. 12A); there was also a
strong reduction in the number of AChE-positive fibers
(Fig. 11B).At yet more caudal levels, the loss of staining
was even more pronounced in the superficial layers of the
entorhinal cortex. Even here, the deep layers of Ec and ECL
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545
the different layers of the entorhinal cortex. Note the substantial loss of
fibers throughout all layers of the entorhinal cortex, with some sparing
in the deep layers and subcortical white matter. Eighteen months
survival time. Scale bar = 250 pm.
and the white matter surrounding the angular bundle
retained a substantial cholinergic innervation (Fig. 14A).
The variable changes in the general distribution of ChAT
and AChE labeling in the entorhinal cortex apparently
reflect the fact that this region is innervated by two or more

546
Fig. 16. Darkfield photomicrographs of the entorhinal cortex at the
same level as illustrated in Figure 15 ipsilateral (A) and contralateral
(B)to the lesion, stained for the histochemical demonstration of AChE.
Arrows in B indicate the layers of the entorhinal cortex. Note the
cholinergic pathways. The C U T - and AChE-labeled fibers
observed at rostra1 levels of the entorhinal cortex, which
likely arise via a ventrally directed cholinergic bundle, were
largely intact. The transversely oriented bundles of C U T -
positive fibers that typically enter the entorhinal cortex
from the angular bundle at mid rostrocaudal levels, espe-
10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
J.R. ALONSO ET AL.
substantial depletion of AChE-labeled fibers, particularly in the superfi-
cial layers of the entorhinal cortex, with some savings in the deep
layers. Eighteen months survival time. Scale bar = 250 bm.
cially into layer I11 of EI, however, were no longer apparent.
In the mid rostrocaudal and caudal portions of the entorhi-
nal cortex, the “rhinal sulcus bundle” of C U T - i r fibers was
also eliminated. This bundle normally contributes fibers to
the lateral portions of layers I and 11, so the FFT caused a
particularly prominent loss of fibers in this region. The loss

CHOLINERGIC DENERVATION OF THE MONKEY HIPPOCAMPAL FORMATION
of staining in the rhinal sulcus bundle following FFT
indicates that the fibers constituting this bundle travel to
the hippocampal formation via the fimbria rather than the
ventral pathway.
Fimbria and other fiber bundles
In all of the animals described in this paper, the FFT
appeared to be complete. At levels caudal to the transection,
no ChAT-positive fibers could be detected and only a few
AChE-labeled fibers were present. Rostra1 to the lesion, the
ipsilateral fimbria was clearly shrunken in comparison with
the contralateral side, but abundant ChAT-ir and AChE-
positive fibers remained both ipsilateral and contralateral
to the lesion (Fig. 17). The dense clusters of fiber and
terminal labeling that surrounded groups of intrafimbrial
neurons were observed in the FFT preparations (Fig. 17).
In fact, it is likely the presence of these clusters of intrafim-
brial neurons that sustained the transected fibers within
the fimbria. Abundant AChE- and CUT-labeled axons
were also observed in the ipsilateral angular bundle, and
these presumably originated in the ventral bundle.
DISCUSSION
Alterations in the distribution of C U T - and
AChE-labeled fibers in the monkey
hippocampal formation following transection
of the fornix
Though transection of the fornix in the monkey leads to
the expected decreases in cholinergic fibers in the hippocam-
pal formation, it is interesting that the extent of deafferen-
tation is highly variable at different rostrocaudal levels and
in different hippocampal fields. The most obvious losses of
ChAT innervation were seen in the dentate gyrus, hippocam-
pus, and entorhinal cortex. Even here, however, changes
were substantially less marked at rostral levels than at
caudal levels. The enhanced fiber and terminal labeling at
the border of CA1 and the subiculum was also relatively
well preserved. It is likely that this and the rostral hippocam-
pal savings reflect the fact that cholinergic fibers of the
ventral bundle innervate these regions (Kitt et al., 1987).
Some of the variability in preserved fibers was unsuspected.
The apparently normal innervation of the presubiculum,
for example, stands in marked contrast to the almost
complete loss of cholinergic fibers in the adjacent parasubicu-
lum and subiculum. Although this suggests that the super-
ficial layers of the presubiculum receive their cholinergic
input through an entirely nonfornical route, there is no
available neural tracing evidence to corroborate this. It
should be noted that Ridley et al. (1991) have also reported
preservation of the AChE staining in the presubiculum of
the marmoset following fornix transection.
There was always substantially greater numbers of AChE-
labeled fibers than of ChAT-ir fibers in the hippocampal
formation of the fornix-transected monkeys. This is likely a
reflection, in part, of the fact that there are numerous
intrinsic AChE-positive cells in the monkey hippocampal
formation that are not ChAT positive and presumably are
not cholinergic. The dense band of AChE staining in the
inner one-third of the molecular layer of the dentate gyrus
had a near-normal appearance following the fornix transec-
tion despite the fact that CUT-immunoreactive fibers in
this area were almost entirely eliminated. The origin of this
AChE-positive and ChAT-negative staining is unclear, but
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547
it is certainly not associated with fibers entering the
hippocampal formation via the fornix and is presumably
not associated with the cholinergic system. In animals with
unilateral transections, the density of ChAT- and AChE-
labeled fibers on the side contralateral to the lesion ap-
proached normal levels. This is consistent with the finding
that the crossed septal projection is not substantial in the
monkey (Amaral and Cowan, 1980; Kitt et al., 1987),
perhaps accounting for no more than 10% of the innerva-
tion of the hippocampus.
Comparisons of the effects of fornix
transection in monkeys and in rats
In comparing our results with published findings from
similar studies of the rat, it appears that there are both
similarities and differences in the alterations in cholinergic
innervation. The hippocampus and dentate gyrus in both
rodents and primates demonstrate a marked decrease in
the density of ChAT- and AChE-positive fibers after FFT.
Our finding that there was a relative preservation of fibers
in the rostral portion of the hippocampal formation is
consistent with the finding in rodents of spared cholinergic
fibers in the temporal or ventral portion of the hippocampal
formation (Gage et al., 19831, which corresponds to the
rostral hippocampal formation in the monkey. Residual
cholinergic fibers in the rat were also typically observed in
the subiculum and in the CAl/CA2 border region (Blaker et
al., 1988). Although there were very few preserved fibers in
the monkey subiculum, there was relative sparing at the
CAl/subiculum border.
One notable difference was in the alteration found in the
presubiculum. Rats with FFT demonstrated only meager
AChE- and ChAT-positive fibers in layers I and I11 of the
presubiculum and in the molecular layers of the subiculum
and hippocampus (Mellgren and Srebro, 1973; Blaker et al.,
1988);as was emphasized above, the monkey presubiculum
demonstrated essentially completely spared ChAT- and
AChE-positive innervations throughout all layers. Also
contrary to our observations in the macaque monkey,
Matthews et al. (1987) reported a few ChAT-positive axons
in the fimbria and alveus of rats with FFT. We saw no
ChAT-ir fibers in the fornix distal to the lesion. One
possible explanation for this difference is that these ChAT-
positive axons originate from the CUT-positive intrinsic
hippocampal neurons of the rodent hippocampal formation,
which are not present in the primate hippocampal forma-
tion (monkeys: Mesulam et al., 1983, 1984; Satoh and
Fibiger, 1985; Kordower et al., 1989; Alonso and Amaral,
1995; humans: Ransmayr et al., 1989, 1992). It is not
currently known whether any of these "intrinsic" ChAT-
positive neurons give rise to fibers that enter the fimbria. It
is known, however, that the axons of these neurons do not
sprout after FFT (Blaker et al., 1988; Frotscher, 1988).
Comparison of the present observations in
Macaca fuscicularis with other studies in
primates
Our findings on the effects of FFT in the macaque are
generally consistent with previous studies carried out in the
primate. All previous studies (Rosene and Van Hoesen,
1987; Koliatsos et al., 1990; Kordower and Fiandaca, 1990;
Samson et al., 1991) using AChE staining showed substan-
tial depletions of fiber labeling in the hippocampus and
dentate gyrus. Typically, however, the rostrocaudal differ-
ences in denervation were not described. In fact, Koliatsos
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Fig. 17. Darkfield photomicrographs of coronal sections of the and the presence of a lateral patch of labeling (open arrows). These
fornix stained for ChAT (A) and AChE (B).Solid arrows indicate dense patches contain small clusters of intrafornical neurons that appear to
groups of labeled fibers located in dorsal and ventral positions of the be innervated by the cholinergic fibers. One month survival time. Scale
fornix. Note the smaller volume of the ipsilateral fornix (the right side) bar = 1mm.
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CHOLINERGIC DENERVATION OF THE MONKEY HIPPOCAMPAL FORMATION 549

et al. (1990) reported a profound reduction in levels of sprouting may not be a general characteristic of primate
ChAT immunocytochemistry in all hippocampal sectors hippocampal fibers; 4 weeks following FFT, substance
ipsilateral to the lesion throughout the rostrocaudal axis of P-containing axons are capable of sprouting in the monkey
the hippocampal formation. Kordower and Fiandaca (1990) hippocampal formation (Nitsch and Leranth, 1994). Our
did not observe demonstrable changes in the AChE fiber findings are consistent with those of Samson et al. (19911,
staining within the subicular complex, whereas we ob- who saw no evidence of sprouting of AChE-positive fibers at
served marked depletions in both AChE and ChAT fibers in 6 months after fornix transection. Thus, it is unlikely that
the subiculum and, especially, in the parasubiculum. the behavioral recovery observed by Zola-Morgan et al.
(1989) can be attributed to recovery of hippocampal cholin-
Functional considerations ergic innervation. It is unclear at this point why the
cholinergic innervation of the monkey hippocampal forma-
There are two interrelated areas of functional interest tion does not demonstrate the same capacity for sprouting
that we wish to address. The first deals with the role of the as is shown in rodents. Batchelor et al. (1989) have
fornix and of the cholinergic innervation of the hippocam- suggested that the ability of a cholinergic cell population to
pal formation in memory function. The second is the sprout is dependent on whether they express nerve growth
potential for morphological reorganization of the primate factor receptor (NGFr). However, the cells of the monkey
hippocampal formation following fornix transection. basal forebrain that innervate the hippocampal formation
It has long been claimed that blocking cholinergic hippo- are positive for NGFr (Kordower et al., 1988; Kordower and
campal transmission, either by lesions of the septohippocam- Fiandaca, 1990) and, as demonstrated by the relatively well
pal system or by pharmacological manipulation, produces preserved fiber labeling in the rostra1 part of the hippocam-
substantial learning and memory impairments (Beatty and pal formation, do have access to denervated hippocampal
Carbone, 1980; Bartus et al., 1982; Coyle et al., 1983; Brito fields. Further study of the plastic capacity of the monkey
et al., 1983; Kelsey and Landry, 1988; McLamb et al., 1988; hippocampal formation will be necessary to determine
Sunderland et al., 1988).In the rat (Bjorklund and Stenevi, whether the rodent model of fairly substantial reorganiza-
1977; Bjorklund et al., 1983a,b; Nilsson et al., 1988) and tion can be extrapolated to the primate and human brain.
more recently in the marmoset monkey (Ridley et al., 1991; One common pathological consequence of Alzheimer’s
Ridley and Baker, 19931, these impairments have been disease is the massive loss of cholinergic innervation of the
markedly ameliorated by transplantation of cholinergic hippocampal formation. One successful strategy for restor-
neurons into the denervated hippocampus. There is circum- ing cholinergic innervation of the denervated rodent hippo-
stantial evidence that damage of the fornix in humans also campal formation has been the introduction of cholinergic
causes a severe anterograde amnesic syndrome (Gaffan and cell-rich grafts (Bjorklund and Stenevi, 1977; Bjorklund et
Gaffan, 1991; Hodges and Carpenter, 1991; von Cramon al., 1983a,b). Grafted neurons establish C U T - i r synapses
and Schuri, 1992). with the host hippocampal formation (Clarke et al., 19861,
The effects of fornix transection on the ability of macaque and the release of acetylcholine by the grafted cells is under
monkeys to perform memory tasks have been somewhat control of the host brain (Nilsson et al., 1990; Nilsson and
controversial. Monkeys with fornix transections can be Bjorklund, 1992). These animals improve their learning
markedly impaired in some forms of spatial memory testing and memory abilities in comparison to lesioned animals
and apparently normal in others (Olton and Papas, 1979; that are devoid of transplants (Gage and Bjorklund, 1986).
Murray et al., 1989; GafFan and Harrison, 1989). Zola- The use of transplanted cholinergic neurons has been
Morgan et al. (1989) demonstrated that monkeys with touted as a possible therapy for Alzheimer’s disease. How-
fornix transection are initially impaired in the delayed ever, the lack of substantial sprouting of residual choliner-
nonmatching to sample (DNMS) task, a test of visual gic fibers as long as 1.5years after fornix transection in our
recognition memory. However, when the animals were preparations raises the question of whether transplants
retested 1.5 years after the surgery, they performed nor- introduced into the macaque hippocampal formation (and
mally on DNMS. One possible explanation for this recovery ultimately into the human brain) would result in an
of function is that, with time, the hippocampal formation organotypic, functional distribution of cholinergic fibers.
reestablishes its cholinergic innervation via sprouting by Initial successes of cholinergic graft survival and apparent
residual fibers that enter through the ventral cholinergic host innervation in the marmoset brain (Ridley et al., 1991)
bundle. The cholinergic reinnervation of the fornix- raise the possibility that the lack of sprouting is not
transected hippocampus in the rat appears to be a robust predictive of a generally less plastic morphological environ-
phenomenon (Gage et al., 1983, 1984; Dravid and Van ment in the monkey brain. Nonetheless, the specter of an
Deusen, 1984; Gasser et al., 1986; Blaker et al., 1988; inadequate reorganizational response of the primate brain
Batchelor et al., 1989; He et al., 1991). In the rat, the to introduced grafts prods one to investigate further the
process of sprouting is relatively fast. As early as 4 weeks potential for morphological plasticity in the nonhuman
after the lesion, there were significant increases in both primate brain. Thus, the monkey hippocampal formation
ChAT and AChE activities relative to those observed in may be a valuable model system for understanding the
similar animals at 1 week of survival (Dravid and Van potential for, and the mechanisms responsible for, morpho-
Deusen, 1984). Maximal recovery is seen within 8 weeks, logical reorganization of the cholinergic system and its
with no further change 6 months after injury (Gasser and possible clinical significance.
Dravid, 1987).
Our findings in the monkey indicate that even 1.5 years
after surgery there is no significant increase in the number ACKNOWLEDGMENTS
of ChAT-positive fibers. These data suggest that there may
be a fundamental difference in the ability for morphological Part of the material used in this study was prepared with
reorganization of the rat and monkey cholinergic innerva- the help of Drs. Marc H. Tuszynski and Fred H. Gage; we
tion of the hippocampal formation. However, this lack of appreciate their valuable collaboration. We also express our

550
appreciation to Ms. Janet Weber, Ms. Mary Ann Lawrence,
and Ms. Barbra Mason for histological assistance; to Ms.
Belle Wamsley for secretarial assistance; to Mr. Kris Tru-
lock for photographic assistance; and to Dr. Bruce Wainer
for contributing the ChAT antibody used in this study. This
work was supported in part by NIH grant NS 16980 to
D.G.A., by the San Diego Alzheimer’s Disease Research
Center (AG051311, and by fellowships from the NATO
scientific program and the DGICyES (95-350) to J.R.A.
These studies were conducted in part, at the California
Regional Primate Research Center (RR00169).
LITERATURE CITED
Alonso, J.R., and D.G. Amaral(1995) Cholinergic innervation of the primate
hippocampal formation. I. Distribution of choline acetyltransferase
immunoreactivity in the Macaca fascicularis and Macaca mulatta
monkeys. J. Comp. Neurol. 355:135-170.
Amaral, D.G., and J.L. Bassett (1989) Cholinergic innervation of the monkey
amygdala: An immunohistochemical analysis with antisera to choline
acetyltransferase. J . Comp. Neurol. 281:337-361.
Amaral, D.G., and W.M. Cowan (1980) Subcorticalafferents to the hippocam-
pal formation in the monkey. J. Comp. Neurol. 189:573-591.
Amaral, D.G., R. Insausti, and W.M. Cowan (1987) The entorhinal cortex of
the monkey. I. Cytoarchitectonic organization. J. Comp. Neurol. 264:326-
355.
Bakst, I., and D.G. Amaral(1984)The distribution of acetylcholinesterase in
the hippocampal formation of the monkey. J. Comp. Neurol. 225344-
371.
Bartus, R.T., R.L. Dean 3d, B. Beer, and A S . Lippa (1982) The cholinergic
hypothesis of geriatric memory dysfunction. Science 21 7:408414.
Batchelor, P.E., D.M. Armstrong, S.N. Blaker, and F.H. Gage (1989) Nerve
growth factor receptor and choline acetyltransferase colocalization in
neurons within the rat forebrain: Response to fimbria-fornix transec-
tion. J . Comp. Neurol. 284:187-204.
Beatty, W.W., and C.P. Carbone (1980) Septa1 lesions, intramaze cues and
spatial behavior in rats. Physiol. Behav. 24-675-678.
Bjorklund, A., and U. Stenevi (1977) Experimental reinnervation of the rat
hippocampus by grafted sympathetic ganglia. I. Axonal regeneration
along the hippocampal fimbria. Brain Res. 138:259-270.
Bjorklund, A., F.H. Gage, R.H. Schmidt, U. Stenevi, and S.B. Dunnett
(1983a) Intracerebral grafting of neuronal cell suspensions. VII. Recov-
ery of choline acetyltransferase activity and acetylcholine synthesis in
the denervated hippocampus reinnervated by septal suspension im-
plants. Acta Physiol. Scand Suppl. 522:59-66.
Bjorklund, A,, F.H. Gage, U. Stenevi, and S.B. Dunnett (198313) Intracere-
bra1 grafting of neuronal cell suspensions. VI. Survival and growth of
intrahippocampal implants of septal cell suspensions. Acta Physiol.
Scand. Suppl. 522.49-58.
Blaker, S.N., D.M. Armstrong, and F.H. Gage (1988) Cholinergic neurons
within the rat hippocampus: Response to fimbria-fornix transection. J.
Comp. Neurol. 272-127-138.
Brito, G.N.O., B.J. Davis, L.C. Stopp, and M.E. Stanton (1983) Memoryand
the septo-hippocampal cholinergic system in the rat. Psychopharmacol-
ogy 81:315-320.
Clarke, D.J., F.H. Gage, O.G. Nilsson, and A. Bjorklund (1986) Grafted
septal neurons form cholinergic synaptic connections in the dentate
gyrus of behaviorally impaired aged rats. J. Comp. Neurol. 252:483492.
Coyle, J.T., D.L. Price, and M.R. DeLong (1983) Alzheimer’s disease: A
disorder of cortical cholinergic innervation. Science 219:1184-1190.
Davies, P., and A.J. Maloney (1976) Selective loss of central cholinergic
neurons in Alzheimer’s disease. Lancet 2:1403.
Dravid, A.R., and E.B. Van Deusen (1984) Recovery of enzyme markers for
cholinergic terminals in septo-temporal regions of the hippocampus
following selective fimbrial lesions in adult rats. Brain Res. 324:119-128.
Fibiger, H.C. (1982) The organization and somc projections of cholinergic
neurons of the mammalian forebrain. Brain Res. Rev. 4:327-388.
Frotscher, M. (1988) Cholinergic neurons in the rat hippocampus do not
compensate for the loss of septohippocampal cholinergic fibers. Neurosci.
Lett. 87:18-22.
Gaffan, D., and E.A. Gaffan (1991) Amnesia in man following transection of
the fornix. Brain 114:2611-2618.
10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
J.R. ALONSO ET AL.
Gaffan, D., and S. Harrison (1989) Place memory and scene memory: Effects
of fornix transection in the monkey. Exp. Brain Res. 74202-212.
Gage, F.H., and A. Bjorklund (1986) Cholinergic septal grafts into the
hippocampal formation improve spatial learning and memory in aged
rats by an atropine-sensitive mechanism. J. Neurosci. 62837-2847.
Gage, F.H., A. Bjorklund, and U. Stenevi (1983) Reinnervation of the
partially deafferented hippocampus by compensatory collateral sprout-
ing from spared cholinergic and noradrenergic afferents. Brain Res.
268.97-37.
Gage, F.H., A. Bjorklund, and U. Stenevi (1984) Cells of origin of the ventral
cholinergic septohippocampal pathway undergoing compensatory collat-
eral sprouting following fimbria-fornix transection. Neurosci. Lett.
44:211-216.
Gage, F.H., K. Wictorin, W. Fischer, L.R. Williams, S. Varon, and A.
Bjorklund (1986) Retrograde cell changes in medial septum and diagonal
band following fimbria-fornix transection: Quantitative temporal analy-
sis. Neuroscience 19:241-255.
Gasser, U.E., and A.R. Dravid (1987) Noradrenergic, serotonergic, and
cholinergic sprouting in the hippocampus that follows partial or com-
plete transection of the septohippocampal pathway: Contributions IX
spared inputs. Exp. Neurol. 96:352-364.
Gasser, U.E., E.B. Van Deusen, and A.R. Dravid (1986) Homologous
cholinergic efferents spared by partial fimbrial lesions contribute to the
recovery of hippocampal cholinergic enzymes in adult rats. Brain Res.
367:368-373.
Gaykema, R.P., P.G. Luiten, C. Nyakas, and J. Traber (1990) Cortical
projection patterns of the medial septum-diagonal band complex. J.
Comp. Neurol. 293:103-124.
Geneser-Jensen, F.A., and T.W. Blackstad (1971) Distribution of acetyl
cholinesterase in the hippocampal region of the guinea pig. I. Entorhinal
area, parasubiculum, and presubiculum. Z. Zellforsch. Mikrosk. Anat.
114:460481.
He, Y.S., Z.B. Yao, and Y.C. Chen (1991) Effect of nerve growth factor on the
lesioned septohippocampal cholinergic system of aged rats. Brain Res.
552159-163.
Hedreen, J.C., S.J. Bacon, and D.L. Price (1985) A modified histochemical
technique to visualize acetylcholinesterase-containing axons. J. Histo-
chem. Cytochem. 33:134-140.
Hodges, J.R., and K. Carpenter (1991) Anterograde amnesia with fornix
damage following removal of IIIrd ventricle colloid cyst. J. Neurol.
Neurosurg. Psychiatr. 54:633-638.
Kelsey, J.E., and B.A. Landry (1988) Medial septal lesions disrupt spatial
mapping ability in rats. Behav. Neurosci. 1O2:289-293.
Kitt, C.A., S.J. Mitchell, M.R. DeLong, B.H. Wainer, and D.L. Price (1987)
Fiber pathways of basal forebrain cholinergic neurons in monkeys. Brain
Res. 406:192-206.
Koliatsos, V.E., H.J. Nauta, R.E. Clatterbuck, D.M. Holtzman, W.C. Mobley,
and D.L. Price (1990) Mouse nerve growth factor prevents degeneration
of axotomized basal forebrain cholinergic neurons in the monkey. J.
Neurosci. 10:3801-3813.
Kordower, J.H., R.T. Bartus, M. Bothwell, G. Schateman, and D.M. Gash
(1988) Nerve growth factor receptor immunoreactivity in the nonhuman
primate (Cebus apella): Distribution, morphology, and colocalization
with cholinergic enzymes. J. Comp. Neurol. 277:465-486.
Kordower, J.H., and M.S. Fiandaca (1990) Response of the monkey choliner-
gic septohippocampal system to fornix transection: A histochemical and
cytochemical analysis. J. Comp. Neural. 298:443-457.
Kordower, J.H., R.T. Bartus, F.F. Marciano, and D.M. Gash (1989) Telence-
phalic cholinergic system of the New World monkey (Cebus apella):
Morphological and cytoarchitectonic assessment and analysis of the
projection to the amygdala. J. Comp. Neurol. 279:528-545.
Lahtinen, H., R. Miettinen, A. Ylinen, T. Halonen, and P.J.S. Riekkinen
(1993) Biochemical and morphological changes in the rat hippocampus
following transection of the fimbria-fornix. Brain Res. Bull. 31:311-318.
Matthews, D.A., P.M. Salvaterra, G.D. Crawford, C.R. Houser, and J.E.
Vaughn (1987) An immunocytochemical study of choline acetyltransfer-
ase-containing neurons and axon terminals in normal and partially
dederented hippocampal formation. Brain Res. 40230-43.
McLamb, R.L., W.R. Mundy, and H.A. Tilson (1988) Intradentate colchicine
impairs acquisition of a two-way active avoidance response in a Y-maze.
Neurosci. Lett. 94t338-342.
Mellgren, S.I., and B. Srebro (1973) Changes in acetylcholinesterase and
distribution of degenerating fibres in the hippocampal region after septal
lesions in the rat. Brain Res. 5219-36.
Mesulam, M.-M., E.J. Mufson, A.I. Levcy, and B.H. Wainer (1983) Choliner-
gic innervation of cortex by the basal forebrain: Cytochemistry and

CHOLINERGIC DENERVATION OF THE MONKEY HIPPOCAMPAL FORMATION
cortical connection of the septal area, diagonal band nuclei, nucleus
basalis (substantia innominata), and hypothalamus in the rhesus mon-
key. J. Comp. Neurol. 2141170-197.
Mesulam, M.-M., E.J. Mufson, A.I. Levey, and B.H. Wainer (1984) Atlas of
cholinergic neurons in the forebrain and upper brainstem of the
macaque based on monoclonal choline acetyltransferase immunohisto-
chemistry and acetylcholinesterase histochemistry. Neuroscience 12t669-
686.
Milner, T.A., and D.G. Amaral(1984) Evidence for aventral septal projection
to the hippocampal formation of the rat. Exp. Brain Res. 551579-585.
Murray, E.A., M. Davidson, D. Gaffan, D.S. Olton, and S. Suomi (1989)
Effects of fornix transection and cingulate cortical ablation on spatial
memory in rhesus monkeys. Exp. Brain Res. 74t173-186.
Nilsson, O.G., and A. Bjorklund (1992) Behaviour-dependent changes in
acetylcholine release in normal and graft-reinnervated hippocampus:
Evidence for host regulation of grafted cholinergic neurons. Neurosci-
ence 49:33-44.
Nilsson, O.G., D.J. Clarke, P. Brundin, and A. Bjorklund (1988) Comparison
of growth and reinnervation properties of cholinergic neurons from
different brain regions grafted to the hippocampus. J. Comp. Neurol.
2681204-222.
Nilsson, O.G., P. Kalen, E. Rosengren, and A. Bjorklund (1990) Acetylcho-
line release from intrahippocampal septal grafts is under control hy the
host brain: A microdialysis study. Progr. Brain Res. 82t321-328.
Nitsch, R., and C. Leranth (1994) Sprouting of remaining substance
P-immunoreactive fibers in the monkey dentate gyrus following denerva-
tion from its substance P-containing hypothalamic afferents. Exp. Brain
Res. 100:522-526.
Olton, D.S., and B.C. Papas (1979) Spatial memory and hippocampal
function. Neuropsychologia 17t669-682.
Ransmayr, G., P. Cervera, E. Hirsch, M. Ruberg, L.B. Hersch, C. Duyck-
aerts, J.-J.Hauw, C. Delumeau, and Y. Agid (1989) Choline acetyltrans-
ferase-like immunoreactivity in the hippocampal formation of control
subjects and patients with Alzheimer’s disease. Neuroscience 321701-
714.
Ransmayr, G., P. Cervera, E.C. Hirsch, W. Berger, W. Fischer, and Y. Agid
(1992) Alzheimer’s disease: Is the decrease of the cholinergic innervation
of the hippocampus related to intrinsic hippocampal pathology? Neuro-
science 47t843-85 1.
Ridley, R.M., and H.F. Baker (1993) Behavioral effects of cholinergic grafts.
Ann. N.Y. Acad. Sci 695274-277.
10969861, 1996, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-9861(19961125)375:4<527::AID-CNE1>3.0.CO;2-3 by Bcu Lausanne, Wiley Online Library on [11/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
551
Ridley, R.M., H.D. Thornley, H.F. Baker, and A. Fine (1991) Cholinergic
neural transplants into hippocampus restore learning ability in monkeys
with fornix transections. Exp. Brain Res. 83:533-538.
Rosene, D.L., and G.W. Van Hoesen (1987) The hippocampal formation of
the primate brain: A review of some comparative aspects of cytoarchitec-
ture and connections. In E.G. Jones and A. Peters (edsj: Cerebral Cortex.
New York: Plenum Press, pp. 345-456.
Samson, Y., A.H. Friedman, J.J. Wu, and J.N. Davis (1991) Loss of
hippocampal acetylcholinesterase staining after fornix lesion in the
monkey. Exp. Neurol. 114t123-131.
Satoh, K., and H.C. Fibiger (1985) Distribution of central cholinergic
neurons in the baboon (Pupzopupio). 11. A topographic atlas correlated
with catecholamine neurons. J. Comp. Neurol. 236t215-233.
Storm-Mathisen, J. (1977) Localization of putative transmitters in the
hippocampal formation: With a note on the connections to septum and
hypothalamus. Ciba Found. Symp. 58:49-86.
Sunderland, T., P.N. Tariot, and P.A. Newhouse (1988) Differential respon-
sivity of mood, behavior, and cognition to cholinergic agents in elderly
neuropsychiatric populations. Brain Res. 472t371-389.
Tuszynski, M.H., H.S. U, D.G. Amaral, and F.H. Gage (1990) Nerve growth
factor infusion in primate brain reduces lesion-induced cholinergic
neuronal degeneration. J. Neurosci. 1Ot3604-3614.
von Cramon, D.Y., and U. Schuri (1992) The septo-hippocampal pathways
and their relevance to human memory: A case report. Cortex 281411-
422.
Whitehouse, P.J., D.L. Price, A.W. Clark, J.T. Coyle, and M.R. DeLong
(1981) Alzheimer disease: Evidence for selective loss of cholinergic
neurons in the nucleus basalis. Ann. Neurol. 10:122-126.
Whitehouse, P.J., D.L. Price, R.G. Struble, A.W. Clark, J.T. Coyle, and M.R.
DeLong (1982) Alzheimer’s disease and senile dementia: Loss of neurons
in the basal forebrain. Science 215t1237-1239.
Yunshao, H., Y. Zhibin, G. Yaoming, K. Guobi, and C. Yici (1992) Nerve
growth factor promotes collateral sprouting of cholinergic fibers in the
septohippocampal cholinergic system of aged rats with fimbria transec-
tion. Brain Res. 585t27-35.
Zola-Morgan, S., L.R. Squire, and D.G. Amaral (1989) Lesions of the
hippocampal formation but not lesions of the fornix or the mammillary
nuclei produce long-lasting memory impairment in monkeys. J. Neuro-
sci. 9t898-913.