Extensive reinnervation of the hippocampus by embryonic basal forebrain cholinergic neurons grafted...

THE .JOURNAL OF COMPARATIVE NEUROLOGY 373:355-372 (1996)

Extensive Reinnervation of the Hippocampus by Embryonic

Basal Forebrain Cholinergic Neurons Grafted Into the Septum of Neonatal Rats

With Selective Cholinergic Lesions

GIAMPIERO LEANZA, GUIDO NIKKHAH, OLA G. NILSSON,

Department of Physiology and Neuroscience-Section of Neurobiology, Wallenberg Neuroscience Center, Lund University, 223 62 Lund, Sweden (G.L., G.N., O.G.N., A.B.);

Institute of Human Physiology, University of Catania, 95125 Catania, Italy (G.L. ); Neurosurgical Clinic, Nordstadt Hospital, D30167 Hannover, Germany (G.N.); Neurology

Service (127), VAMC and Vanderbilt University, Nashville, Tennessee 37212-2637 (R.G.W.)

RONALD G. WILEY, AND ANDERS BJORKLUND

ABSTRACT Reconstruction of the septohippocampal pathways by axons extending from embryonic

cholinergic neuroblasts grafted into the neuron-depleted septum has been explored in the neonatal rat by using a novel lesioning and grafting protocol. Neonatal ablation of the basal forebrain cholinergic projection neurons, accompanied by extensive bilateral cholinergic denervation of the hippocampus and neocortex, was produced at postnatal day (PD) 4 by 192 immunoglobulin (IgG)-saporin intraventricularly. Four days later, cholinergic neuroblasts (from embryonic day 14 rats) were implanted bilaterally into the neuron-depleted septum by using a microtransplantation approach. The results show that homotopically implanted septal neurons survive and integrate well into the developing septal area, extending axons caudally along the myelinated fimbria-fornix and supracallosal pathways that are able to reach the appropriate targets in the denervated hippocampus and cingulate cortex as early as 4 weeks postgrafting. Moreover, the laminar innervation patterns established by the graft-derived axons closely resembled the normal ones and remained essentially unchanged up to at least 6 months, which was the longest postoperative time studied. The reinnervating fibers restored tissue choline acetyltransferase activity (up to 50% of normal) in the dorsal hippocampus and the parietooccipital cortex. Retrograde labeling with Fluoro-Gold from the host hippocampus combined with immunocytochemistry confirmed that most of the projecting neurons, indeed, were cholinergic. The results suggest that the graft-host interactions that are necessary for target-directed axon growth are present in the septohippocampal system during early postnatal maturation. Thus, the present approach may contribute to overcome the functional limitations inherent in the use of ectopically placed intrahippocampal transplants. I iwti WiIey-I,iss, Inc.

Indexing terms: axon growth, myelinated fiber pathways, target reinnemation, immunotoxin, neural transplantation

In adult rats, cholinergic-rich grafts of embryonic basal forebrain tissue are able to establish a new cholinergic innervation (and promote functional recovery) in the damaged septohippocampal system provided that the tissue is placed close to, or within, the denervated target territory. Under these conditions, graft-derived cholinergic axons can grow over relatively long distances and provide a new homotypic innervation of the initially denervated host hippocampus (Bjorklund and Stenevi, 1977; Lewis and Cotman, 1980; Bjorklund et al., 1983c; Ezerman and

Kromer, 1987; Nilsson et al., 1988). The ingrowing axons have been shown to establish functional synaptic connections with neuronal elements of the host (Low et al., 1982; Segal et al., 1985, 1987; Anderson et al., 1986; Clarke et al., 1986; Buzsaki et al., 1987; Shapiro et al., 1989) and to reinstate close to normal cholinergic neurotransmission

Accepted March 29. 1996. Address reprint requests to Giampiero Leanza, Wallenberg Neuroscience

Center-Section oi' Neurobiology Solvegatan 17, 223 62 Lund, Sweden.

1996 WILEY-LISS, INC.

356 G. LEANZA ET AL.

within the previously denervated target (Nilsson et al., 1990; Nilsson and Bjorklund, 1992; Leanza et al., 1993). In most cases, however, the reinnervating axons were confined within the gray matter of the target territory, with very little or no growth along principal fiber pathways. Similar findings of limited axonal regeneration along myelinated fiber tracts in the lesioned adult central nervous system (CNS) have been obtained in other transplantation models (see, e.g., Nornes et al., 1983; Foster et al., 1985; Zhou et al., 1985; Dasuzta et al., 1988). They have generally been ascribed to the inhibitory influence of membrane proteins expressed by the resident glial cell populations, particularly oligodendrocytes and astrocytes (for reviews, see Schwab et al., 1993; Johnson, 19941 and/or to the reduced or lack of expression of substrate-related adhesion and guidance molecules (for review, see Carbonetto, 1991).

The concept of the CNS as a nonpermissive tissue for growing neurites has been challenged by several recent studies reporting extensive axonal outgrowth along myelinated tracts in adult rats grafted with either rat (Tonder et al., 1990; Wictorin et al., 1990b), mouse (Wictorin et al., 1991; Davies et al., 1993, 1994; Li and Raisman, 19931, pig (Isacson et al., 1995), or human (Wictorin et al., 1990a; Stromberg et al., 1992; Wictorin and Bjorklund, 1992; Wictorin et al., 1992) neuroblasts. The capacity for target- directed fiber growth from grafts of fetal CNS tissue is particularly evident when the tissue is placed into the immature brain (see, e.g., Lewis and Cotman, 1980; Floeter and Jones, 1984; Sunde et al., 1984; Castro et al, 1985, 1987; Stanfield and O’Leary, 1985; Hankin and Lund, 1987; Herman et al., 1991). These observations suggest that, under certain conditions, the axons extending from implanted neuroblasts or young postmitotic neurons can escape or neutralize the inhibitory features of the growth trajectory, or benefit of the host’s intrinsic developmental plasticity, to reinnervate distant targets with high precision and specificity in the lesioned adult or immature CNS.

Thus far, successful reformation of the septohippocampal pathway by fibers growing from embryonic septal grafts placed homotopically into the neuron-depleted septum has not been achieved. In the only study where such an approach has been attempted (Hodges et al., 19911, no directed fiber outgrowth (towards the hippocampus or neocortex) was observed from basal forebrain grafts placed into the ibo- tenic acid lesioned septum or nucleus basalis region in adult rats. Along a similar line, grafts of fetal rat dopamine-rich tissue placed homotopically into the lesion-depleted substantia nigra of adult recipients failed to show any significant fiber outgrowth along the nigrostriatal pathway towards the denervated target (Bjorklund et al., 198313; Robertson et al., 1991; Nikkhah et al., 1994a). Recently, Nikkhah et al. ( 1994b) have introduced a microtransplantation technique that allows precise placement of cellular deposits within small target structures in the neonatal brain and better integration of the fetal cells with the surrounding host tissue. Interestingly, when this microtransplantation approach was applied to immature, dopamine-depleted rats, extensive growth of tyrosine hydroxylase-positive axons could be detected along the course of the original nigrostriatal pathway. The newly formed axonal projection from the intranigral micrografts provided a functional reinnervation of the host’s caudate-putamen (Nikkhah et al., 1995a) as well as a partial restoration of the lesion-induced behavioral deficits (Nikkhah et al., 1995b1, thus, confirming the permis-

sive nature of the developing CNS tissue environment for graft survival and target-directed fiber outgrowth.

In the present study, a novel lesioning and grafting protocol for use in neonatal rats was designed to explore the growth properties and reinnervation capacity of embryonic basal forebrain neurons grafted into the developing sep tum. Selective destruction of the immature basal forebrain cholinergic neurons was obtained by bilateral intraventricular injections of the immunotoxin 192 immunoglobulin (1gG)-saporin at postnatal day (PD) 4 (Leanza et al., 1996). This approach makes it possible to achieve extensive cholinergic denervation of the hippocampus and the overlying neocortex without any damage to the fimbria-fornix and the supracallosal pathways; these white matter tracts, therefore, may serve as substrate structures along which cholinergic neuroblasts, micrografted into the medial septum, would be able to extend axons towards their targets. We report here that fetal septalidiagonal band neurons survive and integrate well after implantation into the toxin- lesioned developing septal region and that the grafted cholinergic neuroblasts are capable of extending axons caudally along their normal projection routes (the fimbria- fornix and supracallosal pathways) to reinnervate the host hippocampus and cingulate cortex with a near-normal termination pattern.

MATERIALS AND METHODS Subjects and design

A total of 45 male and female Sprague-Dawley rats (B and K Universal, Stockholm, Sweden) were used in the experiments. Litters (one per cage) were fostered by the mothers until weaning at 21 days of age. Animals were allocated into four groups housed under a 12 hour light-dark cycle with ad libitum access to food and water. These four groups in- cluded unoperated controls (n = 71, vehicle-injected animals (n = 71, lesion-only controls (n = 141, and lesioned and grafted animals (n = 17). Pilot histochemical evaluations of graft survival and graft-derived fiber outgrowth into the host hippocampus were performed at 1 month (one vehicle injected animal, one lesion-only animal, and two transplanted animals) and 2 months (two vehicle-injected animals, two lesion-only animals, and three transplanted animals) after transplantation, whereas animals in the main study were allowed to survive for 6 months. At this time point, half of the animals in the groups were injected with a retrograde fluorescent tracer, Fluoro-Gold (FG), into the hippocampus and were perfused 1 week later, and the fixed brains were processed for histochemistry and immunocytochemistry. The remaining animals were decapitated, the brains were dissected, and tissue specimens were processed for biochemistry and quantitative immunocytochemistry.

Lesion and transplantation surgery Selective lesions of the basal forebrain cholinergic system

were performed on pups at PD4 (day of birth = PDO; n = 31) under hypothermic anesthesia. The immunotoxin was prepared by disulfide conjugation of saporin to 192 IgG, as previously described (Wiley et al., 1991; Wiley and Lappi, 1993), was and stored frozen in -80°C until use. For lesioning, 0.4 pg/pup of stock 192 IgC-saporin (0.75 kg/pl) was dissolved in 5 + 5 pl of sterile Hank’s balanced saline solution (HBSS) and injected into the lateral ventricles at the followingcoordinates: Ap, -0.6; L, +0.8; V, -2.1 (rela-

LONG-DISTANCE DIRECTED GROWTH OF CHOLINERGIC AXONS 357

tive to Bregma and outer skull surface), allowing 1 minute for diffusion before the cannula was retracted. Seven additional pups received an equal volume of vehicle alone, whereas seven pups were not injected and served as intact controls.

Four days later (at PD8), cholinergic-rich cell suspensions were prepared according to a modified microtransplantation protocol (Nikkhah et al., 1994b) based on the previously established cell suspension technique (Bjorklund et al., 1983a; Herman et al., 1986). Briefly, the developing septal-diagonal band region was dissected from the basal forebrain of embryonic day (El14 donor rat fetuses [crown- rump length (CRL) 12-14 mm] of the same strain as the graft recipients. Tissues from 20-25 fetuses were collected in cold Dulbecco’s modified Eagle’s medium (DMEM), cut into pieces, and submitted to enzymatic digestion with 0.1% trypsin (Worthington) and 0.054 DNase (Sigma) in DMEM for 20 minutes at 37°C. After four rinses with 0.05% DNase in DMEM, the tissue was mechanically dissociated by using a 200 p1 micropipette fitted with a 0.5-mm-wide plastic tip. The tissue was then centrifuged at 600 rpm for 5 minutes, and the pellet was resuspended in 0.05% DNase in DMEM at a volume of 4 kliseptum. The number of cells in the suspension was about 1 x lo5 cells/$ with 95% viability, as determined by using the trypan blue exclusion method. For transplantation surgery, lesioned 8-day-old pups were re- anesthetized by cooling on ice and were fixed in a Cunning- ham hypodermic miniaturized device (Cunningham and McKay, 1993; Stoelting Co., Wood Dale, IL). By using a glass capillary (OD 50-70 pm), 1.0 kl of the septa1 cell suspension was then stereotaxically injected in each side of the neuron-depleted septum at the following coordinates: AP, +0.5; L, 20.5, V, -4.0, -3.5 (0.5 pl in each deposit), with Bregma and dura as references and setting the tooth bar at the level of the interaural line. The suspension was delivered at a rate of 1 pli2 minutes, and the capillary was held in place for additional 3 minutes. After each surgery, the pups were allowed to recover fully and to reacquire normal body temperature under a filament bulb before being returned to the mother.

Retrograde tracing from the host brain At 6 months postsurgery, about half of the animals from

the main experiment (three normal, two vehicle-injected, three lesion-only, and five grafted animals) received bilateral intrahippocampal injections of the fluorescent retrograde tracer Fluoro-Gold (Fluorochrome Inc.; 4% in saline) in order to retrogradely label cells in the graft that may have established efferent connections with the host hippocampus. The tracer was applied iontophoretically at a single site into the dentate gyrus region of both hippocampi (coordinates: AP, -4.5; L, k2.5; V, -2.5; tooth bar set at the interaural line level) by using glass capillaries with a tip diameter of 40 pm connected to a constant current genera- tor (Midgard) delivering a pulsed (7 seconds oni7 seconds off) positive current (5 FA) over 20 minutes. One week after injection, the animals were perfused, and the fixed brains were processed for histochemistry and immunocytochemistry (see below).

Biochemistry Animals from the main experiment that were not used

for retrograde tracing (four normal animals, two vehicle- injected animals, six lesion-only animals, and 5 grafted animals) were killed by decapitation under deep chloral

hydrate anesthesia (400 mgikg, i.p.1. The brains were removed, and the prefrontal, parietal, and occipital cortices as well as the dorsal and ventral halves of the hippocampus were quickly dissected, frozen in crushed dry ice, and assayed for determination of tissue choline acetyltransferase (ChAT) activity levels according to the micromethod of Fonnum (1975). The remaining portions of the brain, which comprised the entire basal forebrain, two striata, and the diencephalon, were immersion fixed in paraformaldehyde and processed for quantitative morphological analyses (see below).

Histochemistry and immunocytochemistry The residual parts of the brains following dissection of

tissue specimens for biochemistry (see above) were fixed by immersion in ice-cold phosphate-buffered 4% paraformaldehyde for 12-15 hours followed by 24 hours of dehydration in phosphate-buffered 20% sucrose at 4°C. From these brains, coronal sections (40 Fm thick) were cut on a freezing microtome from the level of the medial septum/ vertical limb of the diagonal band of Broca (septum/vDBB) through the nucleus basalis magnocellularis (NBM) and were collected into three series. Immunocytochemistry was performed on alternate sections by using a mouse 192 IgG monoclonal antibody (4 pgiml; courtesy of Dr. E. Johnson) for the low-affinity nerve growth factor (NGF) receptor (LNGFR) or by using a rabbit antibody for parvalbumin (1:1,000; courtesy of Dr. P. Emson). Briefly, sections were quenched for 10 minutes in 3% Hz02 and 10% methanol in 0.05 M Tris-buffered saline (TBS), pH 7.4, and then exposed for 16 hours to either primary antibody diluted in 0.25% Triton X-100 and 1% horse or to goat serum in TBS. After a 3 hour incubation in a 1:150 rat adsorbed horse anti-mouse or goat anti-rabbit biotinylated IgG (Vector), the sections were incubated in avidin-biotin complex (ABC) for 1 hour and then treated with 0.05% diaminobenzidine and 0.01% H202 in TBS. All steps were performed at room temperature.

Animals killed at 1 and 2 months postlesioning and grafting as well as those receiving bilateral injections of FG into the hippocampus (6 months postlesioning and grafting) were deeply anesthetized and perfused transcardially with ice-cold phosphate-buffered 4% paraformaldehyde. After 2 hours of immersion in the same fixative, the brains were kept at 4°C in 20% sucrose in phosphate buffer for 1-2 days until they sank. Four series of sagittal or coronal sections were then cut a t 40 pm thickness through the midline or from the prefrontal cortex level through the septumivDBB and NBM to the level of the caudal hippocampus, respectively.

One series (i.e., every fourth section) was mounted immediately onto gelatine-coated slides, coverslipped with DPX, and analyzed for FG fluorescence microscopy at 365 nm excitation. An adjacent series of sections was immunore- acted by using a fluorescein isothiocyanate (F1TC)-conju- gated secondary antibody for the simultaneous detection of fluorescent LNGFRiFITC immunoreactivity and FG labelling in the same cell bodies. The occurrence of FGiFITC double labelling within and close to the transplant site was evaluated by switching between 365 and 450-490 nm wavelength excitation filters. Another adjacent series of sections was processed free-floating for acetylcholinesterase (AChE) histochemistry following the method of He- dreen et al. (1985) in order to evaluate the placement and growth properties of the transplanted tissue as well as the


pattern of graft-derived reinnervation of the cholinergically denervated territories.

Stereological analyses and quantitative evaluation

StereoloBcal estimates were performed on LNGFR- and parvalbumin-immunostained specimen from immersion- fixed brains. Analyses were conducted as previously described (Leanza et al., 1995, 1996) in order to quantitatively assess the extent of the toxin-induced neuronal loss as well as to confirm the selectivity of the treatment, because, in the septum IvDBB, the two antigens are differentially expressed by cholinergic (Batchelor et al., 1989; Woolf et al., 1989) or y-amino butyric acid (GABA)ergic (Freund, 1989; Kiss et al., 1990) neurons, respectively. Nuclear boundaries were defined as previously reported (Fischer et al., 1989); immunoreactive neurons in the septumivDBB were measured bilaterally from the genu of the corpus callosum rostrally to the level of the crossing of the anterior commissure caudally, setting the lateral edge of vDBB at the medial border of the olfactory tubercle. The NBM neurons were measured unilaterally from the level of the caudal septum to the tail of the caudate-putamen. Unambiguously positive cells were identified at a x40 magnification by using an Olympus BH-2 microscope interfaced with a color video camera (Hitachi) and an Amiga 2000 computer connected to a color monitor (Trinitron; Sony). The G R I D software (Interactivision; Silkeborg, Denmark) commanding an X-Y step motor on the microscope stage was used to generate sampling frames in the selected regions that appeared as an image overlaid onto the microscope image displayed on the monitor. For further details on the sampling procedure and data analysis, see Gundersen et al. (1988).

RESULTS General observations

Two lesion-only and two grafted pups did not recover from anesthesia or were lost due to being neglected by the mother, thus, leaving 12 and 15 animals in the respective groups. The remaining rats grew healthy with no significant weight loss and no obvious sign of neurological deficit. No particular postoperative care was necessary. The vehicle- injected rats did not differ at any stage of the study from the intact animals in any of the biochemical or morphological parameters analyzed. Therefore, the data from these two groups were pooled and are referred to as “normal controls” throughout.

Appearance of the cholinergically depleted basal forebrain nuclei and stereological

analyses In keeping with previous observations (Leanza et al.,

1996), administration of 0.4 pg 192 IgG-saporin immunotoxin into the lateral ventricles of rats early after birth (PD4) resulted in a massive loss of LNGFR+ cholinergic neurons both in the septumivDBB (compare Fig. 1A with Fig. 1B) and in the NBM (not shown). Only a few spared immunoreactive neurons remained scattered mainly in the vertical and horizontal limbs of the DBB. The loss of LNGFR’ neurons in the two regions, as estimated by stereology, averaged -80 and -91%, respectively, and was highly significant (unpaired t test; in both cases, P < 0.001; Table 1 ). By contrast, no loss of septalivDBB parvalbumin

immunoreactive neurons was detected in any of the groups (Fig. lC,D, Table 1).

Morphological features of intraseptal grafts AChE histochemistry at 1, 2, and 6 months after trans-

plantation surgery (performed on PD8) revealed that all transplanted animals exhibited surviving grafts that, in most cases, were well integrated into the host parenchyma. Graft tissue occurred in both gray and white matter either as small clumps of dispersed neurons or as distinct masses of heavily stained tissue. At the site of graft injection in the septum, the two deposits often fused together to form a single, strongly AChE+ tissue aggregate just below the corpus callosum and extending across the midline. In general, the grafts did not disrupt the structural integrity of the host septal parenchyma (Fig. 2A). However, in one case, the graft had grown to such an extent that it com- pressed the host septum in the mediolateral dimension.

Dispersed graft tissue, occurring either as individual AChE+ cells or as small masses of AChE+ tissue, was often found far from the injection site, sometimes as much as 3 mm along the rostrocaudal axis, at various locations outside the septum. The first such location was within the corpus callosum. Here, the graft tissue (probably deposited along the track of the injection capillary) occurred either as a compact mass of strongly AChE+ tissue (Fig. 2C) or as small clusters of cells that had migrated mainly in the caudorostral, but apparently not in the mediolateral, direction. These AChE”. neurons were scattered in the rostral part of the corpus callosum overlying the septum, and they exhibited rich AChE+ processes that coursed through the surrounding white matter and invaded the overlying cortical parenchyma (Fig. 2D,E).

Another frequent location was the fimbria-fornix pathway, where the grafted tissue was seen as clusters of AChE+ cells embedded within the fascicles of graft-derived fibers running along the myelinated fimbrial tract and the dorsal fornix pathway to reinnervate the hippocampus (Fig. 2B). In one specimen, part of the grafted tissue appeared as a small, strongly AChE+ protuberance attached to the fimbria and protruding into the adjacent lateral ventricle (not shown).

In two cases, some grafted cells were seen at the rostral tip of the hippocampus (Fig. 2F). In sagittal sections, these small aggregates appeared to derive from cells that had migrated or had been pushed caudally underneath the corpus callosum and along or within the myelinated fimbria- fornix tract towards the septal pole of the hippocampal formation. No obvious trace of grafted neurons was detected in the most ventral portion of the septum or in the vDBB.

Extent and pattern of AChE+ fiber outgrowth Consistent with previous observations (Leanza et al.,

1996), bilateral intraventricular injections of 192 IgG- saporin immunotoxin to neonatal (PD4) rats produced an almost complete disappearance of the AChE+ fibers in the dorsal hippocampus as well as in the cingulate, frontal, parietal, and occipital cortices (compare Fig. 3A,B with Fig. 4A,B). In these regions, only sparse fibers could be detected that were of similar density at all postoperative times (i.e., at 1, 2, or 6 months). In the hippocampus, the residual AChE+ fibers were distributed mainly in the part of the subiculum immediately underlying the corpus callosum (Fig. 4B) as well as in the suprapyramidalistratum oriens

Fig. 1. Representative examples of low-affinity nerve growth factor (NGFi receptor (LNGFR; A,B) and parvalbumin (C,D) immunostain- ing in the septumivertical limb of the diagonal band of Broca (septum/ vDBBi region 6 months following the intraventricular injection of 0.4 kg 192 immunoglobulin (1gG)-saporin performed at postnatal day (PDi 4. In B. note the marked loss of LNGFR-immunoreactive neurons in

the septum-vDBB and the modest neuronal sparing in the vertical and horlzontal limbs of the diagonal band compared with the normal pattern (A). In contrast, no loss of parvalbumin-immunoreactive neurons is detectable in lesioned animals (D) compared with normal controls (0. Arrows indicate the midline of the septum. Scale bar = 300 km in A.


TABLE 1. Stereological Analyses'

Region (staining) ~ ~ ~~

SeptumivDBB NBM SeptumivDBB Group t N ) (LNGFR) (LNGFR) (Parvalbumin)

Normal 15, 5929 t 83 2947 t 62 5969 i 46 Lesioned !6 i 1242 t 86' 278 i 47" 6288 i 160

'Stereologically estimated numbers of LNGFR-immunoreactive neurons irs.e.m.1 in the septum vertiral limb of the diagonal band of Broca (septumivDBBi and nucleus basalis magnocellularis i NBMiorparvdbumin-immunoreactiveneurons(zs.e.m ) i n theseptumi vDBB, months after bilateral intraventricular administration of 192 I&-saponn to neonatal !PD 4 ) rats. Imrnunoreactive neurons in the septum/vDBB were counted bilaterally, whereas estimates in the NBM refer to one side only. Asterisks indicate difference from the Normal group a t P < 0.001.

region of CA1 (Fig. 5B) and CA3 (Fig. 6B). AChE+ fibers along the so-called ventral septohippocampal pathway that passed through the amygdala-piriform region and the ventral part of the hippocampal formation appeared to be partially spared. Likewise, more substantial sparing of AChE+ fibers was detected in the more ventral cortical territories (not shown).

This pattern of cholinergic fiber depletion was confirmed upon analysis of specimens that were cut in the sagittal plane. In normal animals, the white matter encompassing the fimbria, the dorsal fornix, and the supracallosal striae (i.e., the normal routes for the so-called dorsal pathway of septohippocampal cholinergic axons; cf. Gage et al., 1983a) was richly supplied with AChE+ fibers, which terminated in the cingulate cortex and in the various fields of the dorsal hippocampal formation with a characteristic laminar pattern (Fig. 8A). By contrast, in the 192 IgG-treated animals, the same structures remained virtually devoid of cholinergic fibers, and only a few spared AChE+ processes were found scattered mainly in the supra- and infrapyramidal zones of the CA2-CA3 fields (Fig. 8B).

In the transplanted animals, the cholinergic-rich graft tissue was associated with a remarkable outgrowth of AChE+ fibers. These fibers, as early as 1-2 months postgrafting, were seen to reinnervate bilaterally the appropriate terminal areas of the host hippocampus (above all, its dorsal part) as well as the cingulate and medial parietal cortices, with a close-to-normal lamination pattern (Fig. 3C) that remained virtually unchanged at later time points. Consistent with previous findings in fimbria-fornix-lesioned adult rats (Bjorklund and Stenevi, 1977; Gage et al., 1983b; Nilsson et al., 19881, outgrowing AChE+ fibers appeared to be fine and varicose and were found consistently in areas normally receiving a dense AChE+ innervation. Thus, dense fiber bands were seen in the infra- and suprapyramidal zones of CAI (Fig. 5C) and CA3 (Fig. 6C), whereas fiber density was lower than normal in the stratum radiatum and in the stratum lacunosum-moleculare of both fields. In the dentate gyrus, AChE+ fibers were distributed densely in the hilar zone (Fig. 5C). Fiber density in this region appeared to be higher than normal in the inner molecular layer but had a clear AChE-poor band similar to that observed in the normally innervated dentate gyrus (compare Fig. 5C with Fig. 5A). Dense AChE' fibers or fiber fascicles were observed consistently in the fimbria, the dorsal fornix, the corpus callosum, and the supracallosal striae (Fig. 7E,F), displaying a pattern very similar to normal (Fig. 7A,B). No or very few such preterminal fibers were detected in the lesion-only animals (Fig. 7C,D). In sagittal sections, these AChE+ fiber bundles were observed to extend caudally from the septa1 graft deposits along the

fimbria and the dorsal fornix for several millimeters and terminated in the dorsal part of the host hippocampal formation (compare Fig. 8A with Fig. 8C). In the most extensively reinnervated specimens, AChE + fibers were seen to course above and below the corpus callosum all the way back to the splenum of the corpus callosum (see Fig. 8C,D). The AChE+ fibers running along the supracallosal striae appeared to give off fibers into the medial aspects of cingulate and parietal cortices, where they distributed with a near-normal laminar pattern. In the cingulate cortex, the AChE+ innervation was densest close to the corpus callosum (compare Fig. 4A with Fig. 4 0 . Again, such patterns of fiber outgrowth were well established already at 1-2 months postlesioning and grafting and did not change in the specimens taken at the 6 month time point.

Retrograde labelling of grafted neurons projecting to the host hippocampus

FG was infused iontophoretically into the dorsal hippocampal formation on both sides in order to visualize cells in the grafts that may have established efferent connections with the host hippocampus. In all specimens, the injections were well centered in the dentate gyrus and consisted of a small necrotic core surrounded by a brightly fluorescent halo (1.0-1.3 mm wide) of cellular and neuropil labelling, with no leakage of the tracer along the capillary tract (not shown). In normal control animals (Fig. 9A), the FG injections resulted in the retrograde labelling of neurons in the medial septum and the vDBB, whereas, in the lesion- only animals, FG-labelled neurons were greatly reduced in number and were distributed mainly close to the midline (Fig. 9B). In grafted animals, substantial numbers of FG-labelled cells were observed within the transplant, where they appeared either as densely packed clusters of fluorescent cells or scattered along the myelinated fiber tracts (Fig. 10A).

Simultaneous visualization of FG and LNGFRiFITC within the same somata was performed in the fluorescence microscope by switching between excitation filters. Al- though a large proportion of FG-labelled grafted neurons was clearly LNGFR+ (Fig. 10B,C), some LNGFR'IFG- and LNGFR-/FG+ somata were observed as well (not shown). The distribution pattern of grafted LNGFR/FJTC-immunoreactive cells was similar to that seen in adjacent sections that were processed for AChE histochemistry.

Biochemistry Table 2 shows that, at 6 months postlesion, ChAT

activity levels were significantly reduced in the prefrontal (-45%) and the parietooccipital (-75%) cortices as well as in both thedorsal (-85%) and theventral (-63%) hippocampus (one-way ANOVA; in all cases, P < 0.001 vs. normal values). In the animals with neonatal intraseptal grafts, the reduction in ChAT levels remained unchanged in the prefrontal cortex ( - 45%) as well as in the ventral hippocampus cortex (-60%). The 1.6-fold increase measured in the parietooccipital cortex, compared with the levels in lesion- only control animals, failed to reach significance in the one-way ANOVA, also including the normal group (P > 0.05), but it was statistically significant when the two groups were compared directly (unpaired t-test; P < 0.05). ChAT levels were significantly increased (by 2.3-fold; P < 0.05 vs. lesion-only control values) in the dorsal half of the hippocampus he . , in the region where the densest graft- derived reinnervation was detected). In the dorsal hippocam-

Fig. 2. Photomicrographs of coronal (A$) and saGttal (B,D-F) sections stained for acetylcholinesterase (AChE) illustrating the morphology and the distribution of the cholinergic-rich grafts after bilateral placement into the neuron-depleted septum performed a t PD8. In coronal sections, graft tissue was observed as single AChE+ aggregates across the midline of the septum (A) or within the corpus callosum (C). In sagittal sect.ions (D), grafts appeared either as dispersed AChE' cells within the most rostra1 aspect of the corpus callosum (arrowheads in El or as small aggregates placed in close contact with the septa1 pole of the

hippocampus (F). Dispersed AChE+ grafted neurons were also seen embedded within the fimbria-fornix pathway (B). E and F are higher magnifications of the boxed areas in D. In D, note the dense bundle of AChE+ fibers growing caudally from the graft along the fimbria and dorsal fornix towards the hippocampus. Survival times: A, 2 months; B, 6 months; C-F, 1 month. A, alveus; CC corpus callosum: gCC, genu of the corpus callosum; DF, dorsal fornix; H, hippocampus: S, septum; T, transplant; Th, thalamus; VHC, ventral hippocampal commissure. Scale bars = 300 pm (A also applies to C, B also applies to El.


Fig. 3. Photomicrographs of coronal sections through the dorsal hippocampus stained for AChE. A Normal hippocampus showing the characteristic laminated AChE' innervation pattern. B: Dorsal hippocampus 6 months after the intraventricular injection of 0.4 yg 192 IgG-saporin performed a t PD4; note the virtually complete loss of intrinsic AChE' fibers in this region. C: Specimen with micrografts of

cholinergic-rich tissue placed bilaterally into the neuron-depleted septum at PD8 (2 months survival); note the near normal and organotypic innervation in all hippocampal subfields on both sides. Similar patterns ofAChE ' reinnervation in the same regions were observed also at 1 and 6 months postgrafting. Scale bar = 1 mm.


Fig. 4 . Photomicrographs of AChE-stained coronal sections through the cingulate cortex illustrating the normal distribution of AChE+ fibers (A) and the effects of the bilateral intraventricular administration of 192 IgC-saporin a t PD4 (B). In B, note the complete loss of AChE- innervation in the cingulate cortex as well as the lack of AChE' fibers in the dorsal fornix and supracallosal striae, with a modest sparing in the region corresponding to the fasciola cinerea. C is taken from a transplanted animal 2 months after bilateral placement of septal-diagonal band tissue into the neuron-depleted septum (performed a t PD8). Note the near normal pattern of AChE' innervation in

pus, the ChAT level averaged 35% of normal (compared with 15% of normal in the lesion-only group), reaching, in the most successful cases, as much as 47-50% of the normal ChAT content.

DISCUSSION The present study was based on the use of the 192

IgG-saporin toxin in neonatal rats to produce extensive and selective lesions of the developing cholinergic basal forebrain projection system without any disruption of the structural integrity of the axonal growth trajectory (Leanza et al., 1996) in combination with the microtransplantation approach, which allows precise and atraumatic placement of small graft deposits into small target structures of immature hosts (Nikkhah et al., 199413,1995a). The results show that embryonic basal forebrain cholinergic neurons micrografted homotopically into the septa1 region of immunotoxin-depleted neonatal rats can 1) survive and integrate into the developing host parenchyma, 2) form prominent bundles of AChE+ axons that extend for several millimeters along the original trajectories of the septohippocampal and supracallosal pathways, 3 ) innervate with remarkable accu- racy appropriate terminal fields in the host dorsal hippocampus and overlying cingulate and parietal cortices as early as 1 month postgrafting, and 4) induce long-lasting recovery of ChAT activity levels in the reinnervated regions.

Effects of the lesion

the more ventral aspect of the cingulate cortex and the subiculum as well as the presence of AChE' fibers in the dorsal fornix and the supracallosal striae and single neurons and fibers in the corpus callosum (arrowheads). Displaced graft tissue is evident as a small aggregate or as individual cells underneath or within the corpus callosum with neurons within (arrowheads). CC, corpus callosum; CCx, cingulate cortex; DF, dorsal fornix; DHC, dorsal hippocampal commissure; FC, fasciola cinerea; S, subiculum; SCS, supracallosal striae; T, transplant. Scale bar = 300 km.

to neonatal (PD4) rats produced a substantial loss of LNGFR+ cholinergic neurons in the septumivDBB and NBM nuclei (80-90%), whereas the noncholinergic, parvalbumin-immunoreactive neuronal population in the septum/vDBB was unaffected. Cholinergic neuronal loss in the basal forebrain nuclei was paralleled by a marked reduction in ChAT activity levels in the neocortex (45-75%) and in the dorsal (-85%) and ventral (-63%) hippocampus as well as a loss of AChE' terminal innervation in the same areas. The extent and specificity of the cholinergic neuron depletion induced by the neonatal toxin treatment, as observed in our previous study (Leanza et al., 19961, greatly exceed those reported previously following electrolytic (Ben-Barak and Dudai, 1980; Hohmann et al., 1988) or excitotoxic (Seng- stock et al., 1992; Zupan et al., 1994) lesions of the developing basal forebrain. The effects obtained after neonatal treatment, however, are somewhat milder than the > 90% ChAT depletions observed in hippocampus and cortex after optimal doses of intraventricular 192 IgG-saporin in adult animals (Nilsson et al., 1992; Leanza et al., 1995; Waite et al., 1995), suggesting a slightly lower efficiency of the immunotoxin administered during the early postnatal period (for discussion, see Leanza et al., 1996). Neonatally lesioned rats consistently displayed some sparing of LNGFR- immunoreactive cholinergic neurons in the basal forebrain nuclei, particularly in the vertical and horizontal limbs of the DBB. Residual AChE+ fibers were detected in the most ventral Dortions of neocortex. the ventral Darts of the

In keeping with our previous observations (Leanza et al., 1996), bilateral intraventricular injections of 192 Is-saporin

hippocampal formation, including the ento;hinal cortex and the amygdala-piriform lobe. On the other hand, little


fiber sparing was seen in the most dorsal territories, including the dorsal hippocampus. Likewise, the fimbria- fornix and supracallosal pathways were permanently devoid of AChE+ cholinergic fibers, and no signs of fiber sprouting were detected in these areas at 1, 2, or 6 months postlesion in the lesion-only animals.

Growth properties of the intraseptal grafts Surviving grafts were observed in all transplanted ani-

mals. This suggests that the immunotoxin (injected at PD4) does not interfere with the survival of the embryonic cells subsequently grafted at PD8. The grafts were generally well placed within the septal area and displayed a high degree of anatomical integration into the host septum or associated white matter bundles without any obvious signs of scarring at the implantation site. Thus, microtransplantation proved to be a useful strategy for homotopic implantation of cell suspensions of fetal cholinergic-rich neurons into the neuron-depleted septum in immature rats. Although most of the grafted cholinergic neurons did not position themselves appropriately within the medial septal nucleus or the DBB, they were nevertheless adequately placed to extend axons along the normal trajectories of the septohippocampal projection (i.e., the fimbria-fornix and the supracallosaI striae). Whereas most of the grafted neurons occurred in the host septal parenchyma, others were found at a distance up to several millimeters from the graft site. Whether this was due to active migration of the implanted fetal cells or to passive displacement, e.g., within the lateral ventricle of the growing brain, is unclear. Displacement or migration of grafted cholinergic neurons has been reported so far only in neonatal hosts (Lewis and Cotman, 1980) but not in adult hosts (see, e.g., Bjorklund and Stenevi, 1977; Bjorklund et al., 1983c; Nilsson et al., 1988; Hodges et al., 1991). The capacity of immature fetal neurons and neuroblasts to migrate over limited distances within the neonatal brain has been observed previously after transplantation into the developing neocortex (McConnell, 1985, 1988; McConnell and Kaznowski, 1991). Similarly, dopaminergic neuroblasts exhibit some migratory properties after implantation into the striatum or the substantia nigra region of neonatal recipients (Snyder-Keller et al., 1989; Herman et al., 1991; Abrous et al., 1993; Nikkhah et al., 1995a). Against this background, it seems likely that the dispersion of AChE+ neurons along white matter tracts, such as the dorsal fornix, the fimbria, and the corpus callosum, indeed reflects active migration of grafted cholinergic neuroblasts or young neurons away from the site of injection. By contrast, theoccurrence of cellular aggregates attached to the ven-

Fig. 5. Vertical stripes from coronal sections through the dorsal hippocampus showing the distribution of AChE+ fibers in the different layers of the CA1 and dentate gyrus regions of representative animals. A Normal hippocampus. B: Six months after bilateral intraventricular administration of 0.4 pg 192 I@-saporin (performed at PD4); note the virtually complete loss of AChE+ staining patterns and the sparse residual fibers in the suprapyramidal/stratum oriens region. C: AChE' innervation produced by grafts of embryonic septal-DBB tissue 2 months after bilateral implantation into the neuron-depleted septum (performed a t PD8). Note the near normal pattern of laminar AChE' innervation in the various fields. Similar patterns of AChE' fiber distribution were observed also at 1 and 6 months postgrafting. CC, corpus callosum; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum-moleculare; sm, stratum moleculare; sg, stratum granulosum; h, hilus; Scale bar = 300 km.

Fig. 6. Photomicrographs of AChE-stained coronal sections through the CA3 subregion ofthe hippocampal formation. A Normal hippocampus. B: Six months after the bilateral intraventricular administration of 0.4 kg 192 I&-saporin performed a t PD4; note the virtually complete disappearance ofAChE' fibers in the various layer of CA3 and the sparse fiber sparing in the suprapyramidaleistratum oriens region. C: Appearance of the CA3 subregion 2 months after the bilateral implantation of embryonic septal-DBB tissue into the neuron-depleted septum performed at PD8. Note t,he near normal or supranormal laminar distribution of AChE+ fibers in the various fields. Similar patterns of AChE' innervation were observed at all time points postgrafting (i.e. 1, 2, or 6 months). sm, stratum moleculare; sr, stratum radiatum: sp, stratum pyramidale; so, stratum oriens. Scale bar = 300 um.

Fig. 7. Photomicrographs ofAChE-stainedcoronal sections through the dorsal hippocampus illustratingthe pattern ofAChE+ fiber distribution in the fimbria (A,C,E) and the corpus calIosum overIying the CAI region (B,D,F). A,B: Normal hippocampus. C,D: Six months after the bilateral intraventricular injection of 0.4 pg 192 IgG-saporin performed at PD4; note the lack of AChE' fibers in the fimbria (C) and the complete disappearance of AChE' fibers in the corpus callosum and the adjacent hippocampal and cortical territories (D). Similar patterns of

AChE-fiber loss were observed a t all time points postlesion (1, 2 or 6 months). E,F: Two months after bilateral placement of embryonic cholinergic septal-DBB tissue into the neuron-depleted septum; note the presence of dense AChE* fibers in the fimbria (E) and the corpus callosum (F). Similar patterns of AChE' fiber growth in these regions were observed also at 1 and 6 months. CA, cornu ammonis; CC, corpus callosum; Cx, cortex; F, fimbria, H, hippocampus. Scale bar = 300 pm.


tricular surface of the fimbria and the alveus is most probably the result of more passive displacement of the cells within the ventricular system.

The migratory properties of grafted cells are possibly related to the extensive developmental changes that occur in the immature CNS, including differences in the composi- tion of the extracellular matrix (McKeon et al., 1991; Sanes, 1989) or cell adhesion molecules (Rutishauser and Jessel, 1988), supportive influence of immature astrocytes (Smith et al., 1986), and/or reduced expression of inhibiting factors in the neonatal brain (Savio and Schwab, 1989). On the other hand, the fact that migration of grafted cells has also

been reported in some cases in adult hosts (see, e.g., Sotelo and Alvarado-Mallart, 1986, 1987; Demierre et al., 1990) suggests that some intrinsic migratory programs may be retained in the implanted embryonic neurons during a limited period of their ontogenetic development, possibly coinciding with a short time span following their birth (which, for cholinergic neurons in the basal forebrain, lies between E l 3 and E15; see Semba, 1992).

Characteristics of the cholinergic reinnervation of the hippocampus

The speed, magnitude, and specificity of the graft-derived fiber outgrowth and reinnervation appear to be similar to those reported previously in adult fimbria-fornix lesioned rats implanted with either solid (Bjorklund and Stenevi, 1977; Ezerman and Kromer, 1987) or cell suspension (Bjorklund et al., 1983c; Nilsson et al., 1988) grafts of embryonic septal tissue. Consistent with these earlier studies, we observed a fairly normal AChE+ innervation in all subfields of the host dorsal hippocampus as early as 1-2 months postgrafting. In addition, the medial portions of the cingulate and parietal cortices displayed a rich AChE+ reinnervation that was provided by fibers coursing through the corpus callosum and the supracallosal striae. The laminated fiber patterns apparently did not change over time and resulted, 6 months postoperatively, in a significant recovery of ChAT levels in the dorsal hippocampus, which, on average, was 35% but reached values up to 47-50% of normal in the most successful cases (compare, e.g., with ChAT levels averaged from hippocampal slices 1-111 in the study of Bjorklund and Stenevi, 1977). In- creased levels of C U T activity were similarly detected in the parietooccipital cortex. This increase was of borderline significance, probably because the dissected cortical tissue comprised areas with extensive reinnervation ke . , the medial cingulate and parietal cortices) as well as nonreinner- vated or weakly reinnervated lateral cortical regions.

Although factors related, for example, to variability in the completeness of the immunotoxin-induced denervation or to sprouting of residual fibers spared by the lesion may be argued to participate in the observed changes, several lines of evidence indicate that these possibilities are unlikely: 1) All specimens taken from the lesion-only control animals displayed a near complete loss of cholinergic neurons in the medial septal nucleus and of cholinergic fibers in the

Fig. 8. Photomicrographs of AChE-stained sections illustrating on the sagittal plane the morphology of septohippocampal cholinergic fibers coursing caudally along the fimbria, dorsal fornix, and supracallosal striae and their distribution in the various fields of the hippocampal formation and in the overlying cortical territories. A Normal hippocampus. B: Six months after the intraventricular injection of 0.4 pg 192 I&-saporin performed at PD4; note the complete disappearance of AChE' innervation in the hippocampus with some fiber sparing in the CA2 field and the lack ofAChE+ fibers in the supracallosal striae, dorsal fornix, and ventral hippocampal commissure. C,D: Six months after bilateral placement of embryonic cholinergic septal-DBB tissue into the neuron-depleted septum performed at PD4; note the rich bundles of AChE' fibers growing caudally from the intraseptal grafts along the dorsal fornix and terminating in the host hippocampus. where they distribute with a normal laminar pattern. Other fibers course along the supracallosal striae to innervate the ventromedial portions of cingulate and parietal cortices. CC, corpus callosum; Cx, cortex; DF, dorsal fornix; H, hippocampus; S, septum; SCS, supracallosal striae; T, transplant; Th, thalamus; VHC, ventral hippocampal commissure. Scale bar = 1 mm


Fig. 9. Fluorescent photomicrographs of coronal sections through the septum-vDBB areas showing the distribution of retrogradely labelled neurons 1 week following bilateral iontophoretic injections of the fluorescent tracer Fluoro-Gold into the dentate gyms of the dorsal hippocampus. A Normal septum-vDBB. B: The same region in a

lesion-only control animal 6 months after bilateral intraventricular injections of 192 I&-saporin performed at PD4; note the almost complete disappearance of retrograde neuronal labelling and the few fluorescent cells scattered mainly close to the midline. Arrows indicate the midline of the septum. Scale bar = 300 km.

fimhria, dorsal fornix, supracallosal striae, dorsal hippocampus, and cingulate cortex, which remained the same at all time points postoperatively; 2 ) in the lesion-only animals, ChAT levels in the dorsal hippocampus were still markedly reduced (by about 85%) hy 6 months postlesion, which is consistent with a persistent cholinergic denervation; and 3) by using injections of FG into the host hippocampus, the cells of origin of the reinnervating fibers could be traced hack to the implant.

An interesting feature of the observed graft-derived fiber outgrowth is its remarkable directional specificity. From implants placed into or close to their original sites in the septum, a vigorous growth of AChE+ fibers occurred pre- dominantly in the caudal direction, i.e., along the fimbria- fornix pathways towards the denervated hippocampus and along the supracallosal striae into the cingulate and medial

parietal cortical areas. By contrast, a rostrally directed elongation (i.e., towards the frontal cortex) was less extensive and was seen to arise only from neurons scattered at the very rostra1 aspect of the grafts, in close proximity to, or within, the genu of the corpus callosum.

The FG experiments further substantiated these ohserva- tions, revealing large numbers of retrogradely labeled neurons mainly at the site of graft placement as well as within the fimhria-fornix and the overlying corpus callosum (i.e., some of the preferential locations for the displaced graft cells). Although most of these neurons appeared to he LNGFR+, the results also revealed the presence of a noncholinergic projection from the transplant, possibly from the GABAergic projection neurons that have been reported to he present in septa1 grafts (see, e.g., Cassel et al., 1991).

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TABLE 2. Choline Acetyltransferase Activity'

Area

Group N PF HD HV PO

Normal 5 266 i- 21 341 i- 19 483 i 17 259 i- 24 1,estonrd 6 148 t 19" 53 ? 5* 182 i 10" 67 i- 5* Grafted 5 144 ? 21' 120 i 20" 193 i- 38* 104 17**"

l C h o l ~ n e acetyltransferase IChAT) activity levels measured in four different cortical regions 6 months after bilateral intraventricular admtnistratiun of 0.4 wg 192 I@,- sapurin to neonatal Ipostnatal ddy IPDI 41 rats and subsequent homotopic bilateral LTafting of embryonic septal-DBB tissue performed a t PD8 Values represent t h e averages of hoth sides combined for each group and are expressed a s lo1 dpmimg tissue I+S.E.M.I. I'F, prefrontal cortex: HD, dorsal half of hippiicam~us: HV. ventral half of hippocam pus; PO. parietooccipital cortex. 'SiLmificant difference from t h e normal g m u p at P < 0.001 '"Difference [rum the lesioned proup a t P < 0.05. ***Sipxlicant difference from the Iesioned g ~ o u p a t P < 0.05 when compared directly i u n ~ a i r e d t - tes t )

Factors that may stimulate long-distance fiber outgrowth from intraseptal cholinergic-rich

grafts in neonatal hosts In previous studies of intrahippocampal implants of

embryonic basal forebrain tissue in adult hosts, graft- derived cholinergic axons have been observed to reinstate an organotypic innervation of the host hippocampus. This reinnervation has been obtained from grafts placed within or close the denervated target, generally as a result of fiber growth occurring primarily or exclusively within the gray matter of the target territory (see, e.g., Bjorklund and Stenevi, 1977; Bjorklund et al., 1 9 8 3 ~ ; Toniolo et al., 1985; Dunnett et al., 1986; Ezerman and Kromer, 1987; Nilsson et al., 1988). By contrast, in the only reported attempt at homotopical transplantation of cholinergic neuroblasts into the excitotoxin-depleted adult rat basal forebrain, no directed fiber outgrowth, even into the immediate vicinity of the grafts, was observed (Hodges et al., 1991).

The failure of grafted cholinergic neuroblasts to extend axons in large numbers along major myelinated fiber tracts in adult hosts, as opposed to the extensive axonal projections observed in the present study, as well as in other models of neonatal grafting (see, e.g., Lewis and Cotman, 1980; Floeter and Jones, 1984; Sunde et al., 1984; Castro et al., 1985, 1987; Stanfield and O'Leary, 1985; Hankin and Lund, 1987; Lund et al., 1990; Herman et al., 1991; Nikkhah et al., 1995a) is consistent with the view that axonal growth and regeneration in the CNS are regulated developmentally and that growth-promoting and/or growth-inhibiting factors along the axonal growth trajectory are expressed differentially in the neonatal or adult CNS tissue environment (see, e.g., Smith et al., 1986; Cadelli and Schwab, 1991; Carbon- etto, 1991; McKeon et al., 1991). Thus, McKeon et al. (1991) and Cadelli and Schwab (1991) have argued that the high regenerative potential seen in neonates may be linked to the low expression of growth-inhibiting molecules in the immature CNS tissue environment. Similarly, Cohen and collaborators (Cohen et al., 1989; Shewan et al., 1995) have proposed that the age-dependent decline in the capacity for axonal regeneration not only reflects a general down- regulation of growth-promoting molecules and/or their receptors but may also involve the increased expression of, and/or the acquisition of receptors for, putative inhibitory factors along the growth trajectory. In the present study, it is interesting to note that the grafted septal neurons were taken from the donor fetuses at a time point (E14) when they normally start to grow axons towards the immature

hippocampus (Koh and Loy, 1989; Semba, 1992) and that, in the host, at the time of grafting (PD8), the septohippocampal cholinergic projection is still in a phase of active growth and terminal expansion (Milner et al., 1983), suggesting that the target-directed axonal outgrowth patterns observed here are critically dependent on developmentally regulated expression of axonal guidance molecules in both the donor and the host tissue. Despite the fact that the developmental stage of the host environment was about 2 weeks ahead of that of the grafted septal neurons, the cellular interactions necessary for axonal guidance and elongation normally operating during ontogenesis of the septohippocampal projection appeared to be retained. This suggests that there is a critical time window during development when target-directed axonal outgrowth from grafted fetal neurons or neuroblasts is fully expressed. Consistent with this view, ongoing work in our laboratory has shown that fetal dopaminergic neuroblasts extend axons along the internal capsule and reinnervate the host striatum when they are grafted to the substantia nigra of neonatal rats at PD5 or PDlO but not when the same cells are grafted at PD20 (Bentlage et al., unpublished data). Similarly, intrastriatal grafts of E 14 striatal neuronal progenitors have been shown to extend axons all the way down to the substantia nigra when they are transplanted to PD1 and PD7 hosts, but not in PD21 hosts (Olsson et al., unpublished observation).

Another interesting possibility raised by the present findings is that cholinergic neuroblasts that are placed close to their normal location within the septum may be able to establish adequate functional connections with the host circuitry, thus, providing an efficient functional substitute for the lost septohippocampal cholinergic projection. Fur- ther studies will be needed to address this issue as well as to assess the capacity of homotopically placed, cholinergic-rich grafts to promote functional recovery from lesion-induced behavioral deficits in the neonatal 192 IgG-saporin lesion model.

ACKNOWLEDGMENTS We thank Christina Ciornei, Alicja Flasch, Kerstin Fogel-

strom, Birgit Haraldsson, Ulla Jarl, Sten Nilsson, Anna- Karin Olden, and Gertrude Stridsberg for expert technical assistance and Ragnar MBrtensson and Agneta Persson for the photographic work. This study was supported by grants from the Swedish MRC (04X2-3874), the National Institutes of Health (NS-06701), and the Goran Gustafsson Founda- tion.

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Extensive reinnervation of the hippocampus by embryonic basal forebrain cholinergic neurons grafted...

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