Cholinergic innervation of the primate hippocampal formation: II. Effects of fimbria/fornix transection

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 demonstrated remarkable sparing that contrasted with the almost complete loss of fibers in the parasubiculum. Interestingly, animals killed approximately 1.5 years after the fornix transection 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-

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 formation via four routes (Milner and Amaral, 1984; Gage et al., 1984; Gaykema et al., 1990), though the fimbriaifornix is

gic neurons in the basal forebrain, which parallels decreases 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. in the levels Of choline acetyltransferase (ChAT) in the hippocampal formation and reductions Of

active (ChAT-ir) fibers in all hippocampal fields (White- E-mail: dgamaralti ucdavis.edu

o 1996 WILEY-LISS, INC.

528 J.R. ALONSO ET AL.

by far the main route. Other septohippocampal fibers travel through the dorsal fornix and the supracallosal stria and along a ventral pathway through the external capsule. The cholinergic innervation of the rat hippocampus is somewhat more substantial to the ventral or temporal region, which is the main recipient of fibers from the so-called ventral pathway (Gage et al., 1984). In the monkey, the major septal projection to the hippocampal formation also travels via the fimbriaifornix, and fewer fibers follow the 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 arise from the basal nucleus of Meynert rather than the septal complex (Kitt et al., 1987). As in the rat, the rostral portion of the monkey hippocampal formation demon- strates a somewhat higher density of ChAT-ir fibers (Alonso and Amaral, 1995).

Transection of the rat fimbria/fornix leads to a massive depletion (85-95%) of hippocampal ChAT immunostaining (Storm-Mathisen, 1977; Fibiger, 1982; Gage et al., 1983, 1986; Dravid and Van Deusen, 1984; Gasser and Dravid, 1987; Matthews et al., 1987; Blaker et al., 1988; Yunshao et al., 1992; Lahtinen et al., 1993). AChE labeling is also depleted (to about 25% of control values) after fimbriai fornix lesion (Gage et al., 1983). Only in the most temporal levels of the rat hippocampal formation is there noticeably greater sparing of cholinergic fibers. Interestingly, there is a partial recovery of cholinergic innervation of temporal levels of the rat hippocampus with postlesion survival periods of 6 months or more (Gage et al., 1983). At 6 months after the fimbria/fornix transection, the level of ChAT immunostaining at the temporal one-fifth of the hippocampus increases threefold relative to levels observed 1 week after transection. This apparent sprouting of residual cholinergic fibers returned levels of ChAT to approximately 40% of control levels. At this time point, there was a detectable, albeit more modest, increase in the levels of ChAT throughout the remainder of the hippocampus (Gage et al., 1983).

35 36 a ah CA1-CA3 cc DG DNMS Ec Ecr. EI EL EL< E L r EntC ElJ ER f GCL I-VI Im ML

P Pas PL PrS r S WM

0

Abbreviations

area 35 (Brodmann) area 36 (Brodmann) alveus angular bundle fields of the hippocampus corpus callosum dentate gyms delayed nonmatching to sample caudal division of the entorhinal cortex caudal limiting division of the entorhinal cortex intermediate division of the entorhinal cortex lateral division of the entorhinal cortex caudal portion of the lateral division of the entorhinal cortex rostral portion of the lateral division of the entorhinal cortex entorhinal cortex olfactory division of the entorhinal cortex rostral division of the entorhinal cortex fimbria granule cell layer layers of the entorhinal cortex stratum lacunosum moleculare molecular layer stratum oriens pyramidal cell layer parasubiculum polymorphic layer of the dentate gyrus presubiculum stratum radiatum subiculum white matter

In the studies reported herein, we investigated the extent to which fimbriai fornix transection deafferents the monkey hippocampal formation of cholinergic innervation. We have also investigated whether in the monkey, as in the rat, residual cholinergic fibers are capable of sprouting to reinnervate denervated portions of the hippocampal formation. Thus far, this type of sprouting response has not been observed in primates with a postsurgical period as long as 6 months (Samson et al., 1991).

MATERIALS AND METHODS Animals and surgical procedure

Ten adult Macaca fascicularis (cynomolgus) monkeys with fimbria/fornix transections (FFT) were used in the present study. Four animals were subjected to unilateral FFT, and they were killed 4 weeks following the lesion. The brains of these animals were previously used as control material in studies on the effects of nerve growth factor infusion on the survival of septal neurons following FFT (Tuszynski et al., 1990). These animals were fitted with an indwelling ventricular cannula and received continuous infusion of vehicle solution during the month-long survival period (see Tuszynski et al., 1990, for details). The remaining six animals received a bilateral FFT and were allowed to survive for 2 weeks (n = 3) or approximately 18 months following the lesion (n = 3). The latter three animals participated in behavioral studies that assessed the effect of FFT on the performance of various learning and memory tasks (Zola-Morgan et al., 1989). Material from 12 M . fascicularis monkeys prepared for the analysis of the normal distribution of ChAT-ir fibers (Alonso and Amaral, 1995) was used as a control for the normal distribution of ChAT and/or AChE labeling.

In preparation for FFT surgery, the animals were tran- quilized with ketamine HC1 (7 mgikg, i.m.) and deeply anesthetized with Nembutal (25-30 mgikg); heart rate, body temperature, and respiration were continuously moni- tored. Although various surgical approaches were taken to expose and transect the fimbriaifornix (see, for example, Zola-Morgan et al., 19891, the most efficient and reliable method was the one reported by Tuszynski et al. (1990). The essential features of this procedure are the following: A craniotomy 20 mm in diameter was performed with its center located 4 mm anterior to the intraural line and biased to the right of the midline. The dura was opened and retracted over the superior sagittal sinus, exposing the interhemispheric fissure and the cingulate gyrus. The ipsilateral hemisphere was then retracted laterally to expose the underlying corpus callosum. Veins entering the superior sagittal sinus were preserved wherever possible. After identification of the splenium and the anterior cerebral arteries, a self-retaining retractor was used to facilitate exposure of the ipsilateral cingulate gyrus. A point 5 mm anterior to the tip of the splenium, on the surface of the cingulum, was selected for entry into the trigone of the lateral ventricle. The corpus callosum was broached with a suction pipette, and hemostasis was maintained with bipolar coagulation. After visual identification of its lateral and medial borders, the fornix was separated from the epen- dyma and transected either with microscissors or with the bipolar coagulator. The dura was then sutured and the craniotomy wound closed in three layers. Each animal received postoperative ampicillin (250 mgiday, i.m.1 for 7 days. Though the intent of this surgical procedure was to

CHOLINERGIC DENERVATION OF THE MONKEY HIPPOCAMPAL FORMATION 529

transect the fimbria, it is likely that the approach taken to visualize the fimbria necessarily resulted in substantial damage of the ipsilateral supracallosal stria.

After the survival period, animals were deeply anesthetized with ketamine HCl(10 mgikg, i.m.1 and Nembutal(50 mgikg, i.p.) and perfused transcardially for 1 hour with a 4% solution of paraformaldehyde in 0.1 M phosphate buffer, followed by 5% sucrose solution in the same buffer for 20 minutes to clear the fixative. The brains were stereotaxically blocked in the coronal plane and cryopro- tected for histological processing.

ChAT immunohistochemistry Immunohistochemical analysis was performed with a

monoclonal ChAT antibody (AB8) kindly donated by Dr. Bruce Wainer. Immunolabeling was revealed with the double-bridging peroxidase-antiperoxidase (PAP) method as described elsewhere (Alonso and Amaral, 1995). Briefly, brains were cut on a sliding microtome, and the free- floating sections were incubated in primary antibody diluted 1:500 in 0.1 M Tris-buffered saline (TBS) containing 2% bovine serum albumin, 20% normal rabbit serum, and 0.5% Triton X-100 for 48 hours at 4°C. The sections were washed and incubated for 1 hour in rabbit anti-rat IgG secondary antiserum (Cappel Laboratories, Durham, NC) diluted 1 5 0 in TBS containing 10% normal monkey serum, 20% normal rabbit serum, and 0.2% Triton X-100 for 1 hour at room temperature. The sections were then incubated in rat PAP (Sternberger Monoclonals, Jarretsville, MD) that was diluted 1 5 0 in the same solution as the secondary antiserum for 2 hours at room temperature, rinsed in buffer, and then exposed to a second secondary antiserum and PAP incubation. The tissue-bound peroxidase was revealed with a 0.05% solution of 3,3'-diaminoben- zidine in phosphate buffer plus 0.015% H202 for 15 minutes. The sections were mounted onto gelatin-coated glass slides, air dried, intensified with a 0.005% osmium tetroxide solution, and coverslipped with Permount.

AChE histochemistry A series of sections adjacent to those prepared immuno-

histochemically for ChAT was processed for the demonstration of AChE using a modification (Hedreen et al., 1985) of the Koelle method (Geneser-Jensen and Blackstad, 1971) as described previously (Bakst and Amaral, 1984; Amaral and Basset, 1989; Alonso and Amaral, 1995). A complete third series of sections from each brain was stained with 0.25% thionin for the demonstration of cell bodies.

Analysis Sections were analyzed using brightfield- and darkfield-

equipped microscopes. Low-power photomicrographs were taken from the cases with unilateral FFT. Representative adjacent sections from the side ipsilateral to the FFT stained for C U T , AChE, and thionin and from the contralateral side stained for ChAT were photographed. These panoramic views were obtained using a 4 x 5 inch photographic system (Nikon multiphot) and a darkfield base (Nikon BD-2). Higher magnification photomicrographs were taken with a Leitz Dialux 20 microscope equipped with a Wild MPS55 35 mm camera system.

RESULTS Nomenclature for the monkey hippocampal

formation The nomenclature and definition of cytoarchitectonic

divisions used herein are as previously described (Alonso and Amaral, 1995; see Figs. 1-6,9-14). Briefly, the dentate gyrus is divided into three layers, the molecular layer, the granule cell layer, and the polymorphic cell layer. The hippocampus proper is divided into three distinct fields- 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 layer, stratum lucidum, stratum radiatum, and stratum lacunosum moleculare. Following distally from the CA1 field are the subiculum, presubiculum, and parasubiculum. The presubiculum and parasubiculum have a cell-free layer I and a thicker layer I1 with densely packed neurons. Layer I1 of the presubiculum has thinner superficial and thicker deep sublaminae. The entorhinal cortex is divided into seven fields: olfactory (Eo), rostral (ER), lateral (EL) [with rostral (ELr) and caudal parts EL^)], intermediate (EI), caudal (Ec), and caudal limiting field (EcL; Amaral et al., 1987). Six layers (I-VI) can be distinguished in the entorhinal cortex (Fig. 11A).

We use the term superficial to designate those layers closer to the pia or to the hippocampal fissure and the term deep to indicate the opposite direction. The long axis of the primate hippocampus is referred to as the rostrocaudal axis (equivalent to the temporoseptal axis in rodents), whereas the axis orthogonal to the rostrocaudal axis is called the transverse mi s . The term proximal is used to refer to the portion of a particular field that is located closer to the dentate gyrus, and distal refers to the portion closer to the rhinal sulcus.

General characteristics of ChAT and AChE labeling

The FFT resulted in clear reductions in both ChAT and AChE fiber labeling in the hippocampal formation ipsilat- era1 to the lesion (Figs. 1-6, 9-14). By contrast, there was no change in the number of AChE-positive cells distributed throughout the hippocampal formation; they were perhaps even more evident because of the depletion of obscuring fiber plexuses. As in control animals, no ChAT-immunoposi- tive neurons were observed in the lesioned hippocampi. In the hemisphere contralateral to the FFT, the distribution and density of ChAT and AChE labeling resembled that in control animals.

Among the different hippocampal fields, the effects of the FFT varied from almost complete loss of cholinergic fibers to almost complete preservation. In some regions, such as the dentate gyrus, the hilar portion of CA3, and the superficial layers of the caudal entorhinal cortex, there was a massive depletion of labeled fibers (Figs. 2A, 4A, 6A, lOA, 12A, 14A). In the presubiculum, in contrast, the cholinergic innervation was substantially preserved (Fig. 8). These results were generally observed in both ChAT and AChE preparations. However, hippocampal fields that in the ChAT preparations were almost totally devoid of labeled fibers, such as the hilar portion of CA3, CA1, and dentate gyrus, demonstrated a markedly reduced yet clearly detectable plexus of AChE-labeled fibers (Figs. lB, 3B, 5B).

With available material, we were able to evaluate whether the fibers of the ventral cholinergic pathway that were not

Fig. 1. Rostra1 coronal sections of the monkey hippocampal formation. 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

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 = 1 mm.

Fig. 2. Photomicrographs of coronal sections of the monkey hippocampal formation. A Section adjacent to those shown in Figure 1 stained for the demonstration of ChAT immunostaining. B: Section at about the same rostrocaudal level from the contralateral side, stained

immunohistochemically for the demonstration of C U T . Comparison of A and B illustrates the substantial loss of ChAT fibers following FFT. These low-power micrographs were taken from animals with unilateral fimbriai fornix transedion and the 1 month survival period. Scale bar = 1 mm.

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 1 month survival period. Scale bar = 1 mm.

Fig. 4. Photomicrographs of coronal sections of the monkey hippocampal 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-

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 1 month survival period. Scale bar = 1 mm.

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 1 month survival period. Scale bar = 1 mm.

Fig. 6. Photomicrographs of coronal sections of the monkey hippocampal formation. A Section adjacent to those shown in Figure 5 stained for the demonstration of ChAT immunostaining. B: Section at about the same rostrocaudal level from the contralateral side, stained

immunohistochemically for the demonstration of C U T . These low- power micrographs were taken from animals with unilateral fimbriai fornix transection and the 1 month survival period. Scale bar = 1 mm.


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 essentially the same as that in animals with 2 weeks of postsurgical 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 cholinergic markers in the principal divisions of the fornix transected 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 hippocampus (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 moleculare 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 stratum 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

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 presubiculum, 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 density 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 midrostrocaudal levels, cholinergic fiber loss was much more evident in ELc than 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

Fig. 7. Photomicrographs displaying the patterns of staining for ChAT (A,B) and AChE (C,D) in the ipsilateral (A,C) and contralateral (B,D) molecular layers of the dentate gyrus. Note the decrease in the density of labeled fibers in the molecular layer ipsilateral to the lesion

(Avs. B); only a few remaining ChAT-ir fibers (arrows) are apparent on the side ipsilateral to the lesion. Note also the persistence of the supragranular band of diffuse AChE staining (asterisks; C vs. D). Eighteen months survival time. Scale bar = 100 bm.

Fig. 8. Darkfield photomicrographs of the presubiculum and parasubiculum of the ipsilateral (A,B) and contralateral (C,Dj hemispheres, stained for ChAT (A,Cj and AChE (B,Dj. Note the persistence of labeled fibers in the presubiculum and the severe depletion in the parasubiculum. One month survival time. Scale bar = 500 um.


Fig. 9. Representative rostra1 coronal sections of the monkey entorhinal cortex. A Section from the entorhinal cortex ipsilateral to the lesion stained with thionin. B: Darkfield photomicrograph of an adjacent section stained for the demonstration of AChE. The main

divisions of the entorhinal cortex shown in 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. Scale bar = 1 mm.


Fig. 10. Photomicrographs of coronal sections of the monkey entorhinal cortex. A Section adjacent to those shown in Figure 9, stained for the demonstration of C U T . B: Section at about the same rostrocaudal level from the contralateral side, stained immunohisto-

chemically for the demonstration of ChAT. These low-power micrographs were taken from animals with unilateral fimbriaifornix transection and the 1 month survival period. Scale bar = 1 mm.

Fig. 11. Representative midlevel coronal sections of the monkey entorhinal cortex. A: Section from the entorhinal cortex ipsilateral to the lesion stained with thionin. B: Darkfield photomicrograph of an adjacent section stained for the demonstration of AChE. The main

divisions of the entorhinal cortex shown in 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. Scale bar = 1 mm.

Fig. 12. Photomicrographs of coronal sections of the monkey entorhinal cortex. A Section adjacent to those shown in Figure 11, stained for the demonstration of ChAT. B: Section at about the same rostrocaudal level from the contralateral side, stained immunohisto-

chemically for the demonstration of ChAT. These low-power micrographs were taken from animals with unilateral fimbriaifornix transection and the 1 month survival period. Scale bar = 1 mm.

Fig. 13. Representative caudal coronal sections of the monkey entorhind cortex. A: Section from the entorhinal cortex ipsilateral to the lesion stained with thionin. B: Darkfield photomicrograph of an adjacent section stained for the demonstration of AChE. The main

divisions of the entorhinal cortex shown in each photograph are indicated in the Nissl-stained section. These low-power micrographs were taken from animals with unilateral fimbriaifornix transection and the I month survival period. Scale bar = 1 mm.

Fig. 14. Photomicrographs of coronal sections of the monkey chemically for the demonstration of ChAT. These low-power micrographs were taken from animals with unilateral fimbriaifornix transection and the 1 month survival period. Scale bar = 1 mm.

entorhinal cortex. A Section adjacent to those shown in Figure 13, stained for the demonstration of ChAT. B: Section at about the same rostrocaudal level from the contralateral side, stained immunohisto-


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

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.

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

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


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

substantial depletion of AChE-labeled fibers, particularly in the superficial layers of the entorhinal cortex, with some savings in the deep layers. Eighteen months survival time. Scale bar = 250 bm.

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-

cially into layer I11 of EI, however, were no longer apparent. In the mid rostrocaudal and caudal portions of the entorhinal cortex, the “rhinal sulcus bundle” of CUT-ir 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


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 intrafimbrial 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 hippocampal 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, hippocampus, 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. I t is likely that this and the rostral hippocampal 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 parasubiculum and subiculum. Although this suggests that the superficial 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 transection 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

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 innervation 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 formation (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 substantial depletions of fiber labeling in the hippocampus and dentate gyrus. Typically, however, the rostrocaudal differences in denervation were not described. In fact, Koliatsos

Fig. 17. Darkfield photomicrographs of coronal sections of the fornix stained for ChAT (A) and AChE (B). Solid arrows indicate dense groups of labeled fibers located in dorsal and ventral positions of the fornix. Note the smaller volume of the ipsilateral fornix (the right side)

and the presence of a lateral patch of labeling (open arrows). These patches contain small clusters of intrafornical neurons that appear to be innervated by the cholinergic fibers. One month survival time. Scale bar = 1 mm.


et al. (1990) reported a profound reduction in levels of ChAT immunocytochemistry in all hippocampal sectors ipsilateral to the lesion throughout the rostrocaudal axis of the hippocampal formation. Kordower and Fiandaca (1990) did not observe demonstrable changes in the AChE fiber staining within the subicular complex, whereas we observed marked depletions in both AChE and ChAT fibers in the subiculum and, especially, in the parasubiculum.

Functional considerations There are two interrelated areas of functional interest

that we wish to address. The first deals with the role of the fornix and of the cholinergic innervation of the hippocampal formation in memory function. The second is the potential for morphological reorganization of the primate hippocampal formation following fornix transection.

I t has long been claimed that blocking cholinergic hippocampal transmission, either by lesions of the septohippocampal system or by pharmacological manipulation, produces substantial learning and memory impairments (Beatty and Carbone, 1980; Bartus et al., 1982; Coyle et al., 1983; Brito et al., 1983; Kelsey and Landry, 1988; McLamb et al., 1988; Sunderland et al., 1988). In the rat (Bjorklund and Stenevi, 1977; Bjorklund et al., 1983a,b; Nilsson et al., 1988) and more recently in the marmoset monkey (Ridley et al., 1991; Ridley and Baker, 19931, these impairments have been markedly ameliorated by transplantation of cholinergic neurons into the denervated hippocampus. There is circum- stantial evidence that damage of the fornix in humans also causes a severe anterograde amnesic syndrome (Gaffan and Gaffan, 1991; Hodges and Carpenter, 1991; von Cramon and Schuri, 1992).

The effects of fornix transection on the ability of macaque monkeys to perform memory tasks have been somewhat controversial. Monkeys with fornix transections can be markedly impaired in some forms of spatial memory testing and apparently normal in others (Olton and Papas, 1979; Murray et al., 1989; GafFan and Harrison, 1989). Zola- Morgan et al. (1989) demonstrated that monkeys with fornix transection are initially impaired in the delayed nonmatching to sample (DNMS) task, a test of visual recognition memory. However, when the animals were retested 1.5 years after the surgery, they performed normally on DNMS. One possible explanation for this recovery of function is that, with time, the hippocampal formation reestablishes its cholinergic innervation via sprouting by residual fibers that enter through the ventral cholinergic bundle. The cholinergic reinnervation of the fornix- transected hippocampus in the rat appears to be a robust phenomenon (Gage et al., 1983, 1984; Dravid and Van Deusen, 1984; Gasser et al., 1986; Blaker et al., 1988; Batchelor et al., 1989; He et al., 1991). In the rat, the process of sprouting is relatively fast. As early as 4 weeks after the lesion, there were significant increases in both ChAT and AChE activities relative to those observed in similar animals at 1 week of survival (Dravid and Van Deusen, 1984). Maximal recovery is seen within 8 weeks, with no further change 6 months after injury (Gasser and Dravid, 1987).

Our findings in the monkey indicate that even 1.5 years after surgery there is no significant increase in the number of ChAT-positive fibers. These data suggest that there may be a fundamental difference in the ability for morphological reorganization of the rat and monkey cholinergic innervation of the hippocampal formation. However, this lack of

sprouting may not be a general characteristic of primate hippocampal fibers; 4 weeks following FFT, substance P-containing axons are capable of sprouting in the monkey hippocampal formation (Nitsch and Leranth, 1994). Our findings are consistent with those of Samson et al. (19911, who saw no evidence of sprouting of AChE-positive fibers at 6 months after fornix transection. Thus, it is unlikely that the behavioral recovery observed by Zola-Morgan et al. (1989) can be attributed to recovery of hippocampal cholinergic innervation. It is unclear at this point why the cholinergic innervation of the monkey hippocampal formation does not demonstrate the same capacity for sprouting as is shown in rodents. Batchelor et al. (1989) have suggested that the ability of a cholinergic cell population to sprout is dependent on whether they express nerve growth factor receptor (NGFr). However, the cells of the monkey basal forebrain that innervate the hippocampal formation are positive for NGFr (Kordower et al., 1988; Kordower and Fiandaca, 1990) and, as demonstrated by the relatively well preserved fiber labeling in the rostra1 part of the hippocampal formation, do have access to denervated hippocampal fields. Further study of the plastic capacity of the monkey hippocampal formation will be necessary to determine whether the rodent model of fairly substantial reorganization can be extrapolated to the primate and human brain.

One common pathological consequence of Alzheimer’s disease is the massive loss of cholinergic innervation of the hippocampal formation. One successful strategy for restor- ing cholinergic innervation of the denervated rodent hippocampal formation has been the introduction of cholinergic cell-rich grafts (Bjorklund and Stenevi, 1977; Bjorklund et al., 1983a,b). Grafted neurons establish CUT-ir synapses with the host hippocampal formation (Clarke et al., 19861, and the release of acetylcholine by the grafted cells is under control of the host brain (Nilsson et al., 1990; Nilsson and Bjorklund, 1992). These animals improve their learning and memory abilities in comparison to lesioned animals that are devoid of transplants (Gage and Bjorklund, 1986). The use of transplanted cholinergic neurons has been touted as a possible therapy for Alzheimer’s disease. How- ever, the lack of substantial sprouting of residual cholinergic fibers as long as 1.5 years after fornix transection in our preparations raises the question of whether transplants introduced into the macaque hippocampal formation (and ultimately into the human brain) would result in an organotypic, functional distribution of cholinergic fibers. Initial successes of cholinergic graft survival and apparent host innervation in the marmoset brain (Ridley et al., 1991) raise the possibility that the lack of sprouting is not predictive of a generally less plastic morphological environ- ment in the monkey brain. Nonetheless, the specter of an inadequate reorganizational response of the primate brain to introduced grafts prods one to investigate further the potential for morphological plasticity in the nonhuman primate brain. Thus, the monkey hippocampal formation may be a valuable model system for understanding the potential for, and the mechanisms responsible for, morphological reorganization of the cholinergic system and its possible clinical significance.

ACKNOWLEDGMENTS Part of the material used in this study was prepared with

the help of Drs. Marc H. Tuszynski and Fred H. Gage; we 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).

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Cholinergic innervation of the primate hippocampal formation: II. Effects of fimbria/fornix transection