Delayed Death of Septal Cholinergic Neurons after Excitotoxic Ablation of Hippocampal Neurons during Early Postnatal Development in the Rat

EXPERIMENTAL NEUROLOGY ARTICLE NO.

139, 143–155 (1996)

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Delayed Death of Septal Cholinergic Neurons after Excitotoxic Ablation of Hippocampal Neurons during Early Postnatal Development in the Rat JONATHAN D. COOPER,*,† JEREMY N. SKEPPER,* MARIA DA PENHA BERZAGHI,† DAN LINDHOLM,† AND MICHAEL V. SOFRONIEW*,1 *MRC Cambridge Centre for Brain Repair and Department of Anatomy, University of Cambridge, Forvie Site, Robinson Way, Cambridge, CB2 2PY, United Kingdom; and †Department of Neurochemistry, Max-Planck-Institute for Psychiatry, Am Klopferspitz 18A, D-82152 Planegg-Martinsried, Munich, Germany

To investigate the role of neuron–target interactions in regulating the survival of developing septo-hippocampal cholinergic neurons, hippocampal neurons were excitotoxically ablated in early postnatal rats. Four weeks after hippocampal ablation, hippocampal levels of brain-derived neurotrophic factor and nerve growth factor (NGF) mRNA had fallen to 15% of control values, and ipsilateral septal levels of NGF protein had fallen to 45% of control values. Four weeks after hippocampal ablation, the number of immunoreactive septal cholinergic neurons had fallen to 30% of control values. The number of cholinergic neurons in the septum correlated significantly with the amount of hippocampal tissue present. Ultrastructural analysis of the septal region at 3 days after hippocampal ablation showed no evidence of excitotoxic damage, but at 7 or 10 days showed degenerative profiles compatible with the delayed cell death of large septal neurons. Two weeks of NGF administration, initiated at 4 weeks after hippocampal lesions, failed to increase the number of detectable cholinergic neurons in the septal region, suggesting that the loss of immunoreactive neurons seen at 4 weeks represented cell death rather than downregulation of cholinergic markers. These findings suggest that septal cholinergic neurons depend for survival during early postnatal development on interactions with hippocampal neurons and are compatible with the possibility that neurotrophins play a role in these interactions. r 1996 Academic Press, Inc.

INTRODUCTION

The development of the nervous system is characterized by the death of excess neurons and pruning of exuberant collateral connections which may be regulated by interactions between afferent neurons and target cells (14). Experimental studies show that in

1 To whom correspondence should be addressed. Fax: (44) (1223) 331174. E-mail: 72640,[email protected].

part, the number of afferent neurons is regulated in proportion to target size (14). Studies on peripheral neurons suggest that the molecular nature of developmental neuron–target interactions may involve the retrograde transfer to afferent neurons of sustaining trophic molecules from their target cells (36). Neurotrophic molecules are present in the central nervous system (CNS), but the roles of neuron–target interactions and target-derived neurotrophic molecules in the development of the CNS is far less well characterized. Basal forebrain cholinergic neurons represent a good model system for investigation in the CNS of the role of neuron–target interactions and of target-derived trophic molecules during development. In developing and adult rodents these cholinergic neurons bear high and low affinity nerve growth factor (NGF) receptors and transport radioactively labeled NGF from their target regions, where target neurons produce mRNAs encoding NGF and related neurotrophins (25, 41, 44). Development of the rodent septohippocampal system is accompanied by a series of temporally regulated changes in biochemical and histological markers of cholinergic function. Septal neurogenesis on Embryonic Days 15–17 in rats (6, 45) is followed by successive development of cholinergic phenotype as revealed by progressive incremental changes in neuronal staining for acetyl cholinesterase, choline acetyltransferase (ChAT), and low affinity neurotrophin receptor (p75NTR) during the late prenatal and early postnatal period (4, 33, 51). The development of neurons in the hippocampus follows a similar time course (7). Afferent cholinergic fibers from the septum reach the hippocampus at approximately the time of parturition (4, 18); however, synaptogenesis and the successive postnatal maturation of the projection to achieve the adult pattern of lamination, choline uptake, and ChAT activity do not occur until the second or third postnatal week (16, 46). Within the developing hippocampus, NGF levels make a sharp peak around Postnatal Days 12 to 14, which is followed by pronounced increases in ChAT levels in the hippocampus

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0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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and basal forebrain and in the size of basal forebrain cholinergic neurons (5, 33, 51). The ordering of the events just described strongly suggests that one or more target-derived neurotrophic factors may influence the maturation of the cholinergic input to the hippocampus. In agreement with this possibility, considerable evidence from in vitro studies and from pharmacological administration in vivo indicates that the neurotrophins NGF and brain-derived neurotrophic factor (BDNF) have effects on basal forebrain cholinergic neurons. In vitro studies show that exogenous NGF and BDNF substantially increase ChAT levels in basal forebrain cultures and will increase the survival of cholinergic neurons under certain culture conditions (2, 26). Withdrawal of NGF is lethal by an apoptotic mechanism (32, 54) to immature septal cholinergic neurons but has progressively less effect on cell survival as the neurons mature in vitro (54), in agreement with observations in sympathetic ganglion cultures (21). Infusions of exogenous NGF to neonatal and adult rats increase basal forebrain levels of ChAT and cause hypertrophy of basal forebrain cholinergic neurons (24, 28, 56). After transection of their axons in the fimbria–fornix, cholinergic septal neurons which would otherwise die can be rescued by administration of pharmacological doses of NGF or BDNF in adult rats (27, 57, 58). Nevertheless, in young adult rats, excitotoxic ablation of the hippocampus which abolishes hippocampal production of mRNA for neurotrophins and significantly reduces septal levels of NGF protein, does not result in the death of afferent septal cholinergic neurons even at long survival times (34, 49, 50), suggesting that target-derived NGF or BDNF do not regulate the survival of these neurons in the adult. In neonatal rats, infusions of NGF will transiently raise, while infusions of NGF-antibodies will transiently lower, septal ChAT levels (24, 56), but it is not known if these changes in ChAT levels are accompanied by changes in neuronal survival. In addition, mice generated with targeted disruptions of either the NGF or BDNF genes exhibit many basal forebrain cholinergic neurons for at least 4 weeks of postnatal life (17, 30). Thus, despite the considerable evidence that NGF and BDNF have effects on basal forebrain cholinergic neurons, it remains unclear whether target-derived neurotrophins regulate the survival of these neurons during development in vivo. In this study we investigate the importance of neuron– target interactions in regulating the survival of developing medial septal cholinergic neurons. Hippocampal target neurons were ablated in early neonatal rats using the excitotoxic amino acid NMDA as originally described for adult animals (50). Similar studies by others have shown that such lesions in neonatal rats lead to a significant loss of immunoreactive cholinergic septal neurons (8, 42) and that this loss can be pre-

vented by infusions of NGF (9). Here we examine in particular whether this loss of cholinergic neurons represents cell death or a down regulation of immunoreactive markers, by ultrastructural analysis of septal neurons and by testing the effects of delayed infusions of NGF. We also determined the effect of these lesions on hippocampal production of mRNA encoding BDNF and NGF and on septal levels of NGF protein. EXPERIMENTAL PROCEDURES

Animals and Surgical Procedures Wistar rat pups of either sex, 9–11 days of age at the time of surgery, were used in this study. Litter mates were assigned to six different experimental groups: (1 and 2) hippocampal injections of the excitotoxic amino acid, n-methyl-D-aspartic acid (NMDA) at two different doses, (3) hippocampal injections of saline vehicle, (4) sham operated, (5) transection of the fimbria–fornix, or (6) unoperated. Animals were operated under deep anesthesia with Avertin and ether. For hippocampal injections, the cerebral neocortex cortex was unilaterally aspirated to expose the hippocampus, and injections of either NMDA solution or vehicle were placed into the hippocampus using a stereotaxic device guided under direct visualization with a Zeiss operating microscope. Parameters for stereotaxic injection were previously determined by histological examination of trial injections of pontamine blue into the hippocampus of animals of the same age. In each animal, injections were made at four sites spread evenly throughout the hippocampal formation. Each injection site received 1 µl of either 9 or 3 µg/µl NMDA (Sigma), or 0.9% NaCl (vehicle) delivered over 2 min (49). Sham operations consisted of aspiration of the cerebral neocortex to expose the hippocampus without placing injections. Complete unilateral transections of the fimbria–fornix were made by aspiration under direct microscopic visualization after removal of the overlying cerebral neocortex. Following surgery, pups were returned to their mothers and after survival times of 28 days were taken either for determination of mRNA levels and ELISA levels in unfixed tissue (n 5 4 from each treatment group) or were perfusion fixed under terminal barbiturate anesthesia for histological evaluation (n 5 5 from each treatment group). Two further groups of animals received intraventricular infusions of 1 µl NGF (0.2 µg/µl of purified mouse NGF in saline) (n 5 5) or vehicle (n 5 3) for every other day for 14 days, commencing 28 days after ablation of hippocampal neurons by injections of 9 µg/µl NMDA to the hippocampal region. NGF or vehicle infusions were made via a chronic indwelling catheter sterotaxically placed into the lateral ventricle near the septal region and held in place by dental cement. These animals were perfused for histological analysis.

TARGET DEPENDENCE OF DEVELOPING CHOLINERGIC NEURONS

mRNA Determinations Under terminal ether anesthesia, rats were decapitated and using sterile conditions the brains were removed and the hippocampus (or its remnant) carefully dissected bilaterally under magnification with an operating microscope. RNA was extracted using the guanidium thiocyanate method (12) and a shortened cRNA standard was added to the samples before extraction to assess recovery (37). RNA was glyoxylated, run through a 1.3% agarose gel, and blotted onto Hybond N filters. The filters were consecutively hybridized with 32P-labeled cRNA probes specific for mouse BDNF, mouse NGF, and b-actin using a 50% formamide solution (60). The probes were produced by run-off transcription using the P-Gemini system. Autoradiography was conducted at 270°C for various periods of time depending on the intensity of the signals. NGF Protein Determinations NGF concentrations were determined in the ipsilateral medial septal nucleus by two-site ELISA (53). Unfixed tissue was collected for ELISA measurements as described above for the mRNA samples. The medial septum was dissected as previously described (49). NGF protein determinations were carried out using the mouse monoclonal antibody 27/21 and the values obtained were corrected for recovery of added mouse 2.5S NGF. Fixation and Processing of Tissue for Light Microscopy Rats were fixed under terminal barbiturate anesthesia and continuous positive pressure ventilation, by transcardiac perfusion with a vascular rinse followed by 150 ml of a buffered (pH 7.4) solution of 4% paraformaldehyde/0.1% glutaraldehyde (49, 50). Perfused brains were removed, fixed a further 2–3 h in buffered 4% paraformaldehyde only, and placed in 30% sucrose buffer at 4°C for at least 24 h prior to sectioning on a Leitz freezing microtome at 40 µm. Sections were collected in buffer and stored at 4°C until staining, which normally occurred within 48 h of sectioning. Immunohistochemical and Other Staining Procedures Alternate sections through the septal region of perfusion-fixed brains were immunohistochemically stained free-floating for either ChAT or low affinity NGFreceptor (p75NTR) using well characterized primary monoclonal antibodies and either the peroxidase– antiperoxidase (PAP) procedure or the biotin–avidin– peroxidase procedure, with diaminobenzidine as a chromogen (50). No difference in the quality of staining was noted between these two procedures. The primary antibodies used were a monoclonal anti-ChAT antibody (20) kindly provided by F. Eckenstein and monoclonal

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anti-p75NGFR antibody MC-19210 kindly provided by E. M. Johnson. A 1:6 series of sections through the hippocampal region of each animal was stained immunohistochemically for GFAP using a polyclonal antiserum (DACO) and the procedures described above. All immunohistochemically stained sections were mounted, dried, exposed briefly to dilute osmium tetroxide, and coverslipped from xylene. A further 1:6 series of frozen sections through the hippocampal region of each animal was stained for histochemical detection of AChE (49) and a 1:6 series of sections throughout both the septal region and hippocampal lesion site were stained with cresyl violet as a Nissl stain. Morphometric and Statistical Analysis The cross-sectional surface area of viable neuronal tissue in the hippocampal formation was determined by interactive image analysis using digitized images (captured via a black and white video camera attached to a Zeiss microscope using a 31 objective) and a Seescan image analyzer (Cambridge, England). Four levels were measured throughout the hippocampus on Nisslstained sections for every animal, and values were expressed as the total area measured on the left divided by the total area measured on the right side 3100 (i.e., L/R 3 100) for each animal. These values were used in correlations between amount of hippocampal tissue present and morphometric values of septal cholinergic neurons. Cell counts and cell size of septal cholinergic neurons were made either by interactive image analysis using digitized images (captured via a black and white video camera attached to a Zeiss microscope using a 310 planapo objective) and a Seescan image analyzer or using a drawing tube. Septal neurons immunohistochemically stained for either ChAT or p75NTR were included in the analysis which had a cross-sectional area of greater than 26 µm2, a feret diameter greater than 7 µm, and a roundness factor (KA/P 2, where K is 1.257, A is the cross-sectional area, and P is the perimeter) greater than 0.35 (to eliminate large elongated fibers). Because bilateral changes were noted in the levels NGF protein in the septal region after unilateral hippocampal ablation, we felt it necessary to compare absolute counts obtained on each side of the medial septal nucleus across all experimental groups, rather than comparing ratios of the left (treated) versus right (untreated) sides as done in previous studies. This required precise matching of septal levels for morphometric determinations. Cell counts were performed on three pairs of sections stained for ChAT or p75NTR taken from a standardized portion of the medial septum a fixed distance rostral to the decussation of the anterior commisure (48). Values for cell counts were expressed for individual animals as the mean number of ChAT- or p75NTR-stained neurons per section in either the left or

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right sides of the medial septal nucleus. The left side was always ipsilateral to the treated hippocampus. All cell counts were Abercrombie corrected (1). Values for cell size were expressed for individual animals as the mean cross-sectional area of ChAT- or p75NGFR-stained neurons in either the left or right sides of the medial septal nucleus. Morphometric values were statistically compared by ANOVA with post-hoc Neuman–Keuls pairwise analysis. Electron Microscopy Ultrastructural analysis of the medial septum was performed 3, 7, and 10 days after hippocampal ablation with multiple injections of 9 µg/µl NMDA or complete transection of the fimbria–fornix by aspiration (n 5 2 at each survival time). Unoperated control litter mates

were evaluated at the same survival times. Rats were perfused under terminal barbiturate anesthesia with Pipes-buffered saline (10 mM Pipes, 137 mM NaCl, 7 mM KCl, 1 mM CaCl2 · 3H2O) at pH 7.4 including 2500 IU heparin/dm3, followed by 40 ml of a fixative containing 3% glutaraldehyde, 0.5% paraformaldehyde in 0.1 M Pipes buffer containing 2.5% polyvinypyrollidine with a relative molecular mass of 40 kDa (PVP-40) and 1 mM CaCl2, at pH 7.2. Following perfusion, the brain was removed and the rostral forebrain sectioned on a vibratome to form specimens 250 µm thick from which the septal region was trimmed. These specimens were rinsed in 0.1 M Pipes buffer made isotonic with sucrose and containing 2 mM calcium chloride at pH 7.4 overnight at 4°C and subsequently postfixed in 1% osmium tetroxide in the same buffer. Specimens were

FIG. 1. Nissl (A–D) or AChE (H–J) stained coronal sections through the rat hippocampal formation and midbrain after unilateral aspiration of overlying neocortex and multiple stereotaxic injections of saline (A, B, E, H) or 9 µg/µl NMDA (C, D, F, G, I–K) into the hippocampus. Note that the neocortex has been removed by aspiration and that the injected hippocampus is slightly reduced in size after injections of saline and has virtually disappeared after injections of NMDA. E–G are details, respectively, of A, C, and D. Hippocampal (Hp) cytoarchitecture is moderately disturbed after saline injections (E). After NMDA injections little neuron-containing tissue remains in the hippocampus, but a remnant of the fimbria (f) persists at both rostral (F) and caudal (G) levels. AChE-stained cholinergic fibers (arrows) are present along the entire rostro–caudal extent of the fimbria after NMDA lesions (H–J). H and I are taken from sections neighboring those shown, respectively, in F and G. J is a detail of I. GFAP-positive astrocytes are present in the tissue remnant after NMDA injections (K). K is taken from a section neighboring that shown in J. Scale bars, 1150 µm for A–D; 213 µm for E, F; 52 µm for G; 72 µm for H, I; 24 µm for J, K.

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TARGET DEPENDENCE OF DEVELOPING CHOLINERGIC NEURONS

then rinsed twice in maleate buffer at pH 5.2 and bulk stained in 2% uranyl acetate in the same buffer for 2 h at 4°C. Specimens were dehydrated through a graded series of alcohols and infiltrated with and embedded in araldite epoxy resin. One-micrometer sections were cut with glass knives and stained with 1% methylene blue. Thin sections (approximately 50 nm) were cut using a diamond knife and double stained with uranyl acetate and lead citrate and examined in a Philips 300 electron microscope operated at 80 KV. RESULTS

All experimental treatments (i.e., cortical aspirations alone or followed by multiple hippocampal injections of saline or NMDA or FF transection) were well tolerated by animals who continued to feed, grow, groom, and behave normally without any obvious deficits. Lesion-Induced Changes in Hippocampal Area and Neurotrophin mRNA Levels Bilateral surface area measurements were made in Nissl-stained sections at four rostro–caudal levels of the hippocampus at 28 days after cortical aspirations and hippocampal infusions. Sham-operated animals with unilateral removal of the cerebral neocortex overlying the hippocampus showed a shrinkage of hippocampal surface area to 67.8 6 1.9% of that on the contralateral side. Multiple hippocampal injections of saline resulted in a further shrinkage of the hippocampal surface area to 53.5 6 2.0% of that on the contralateral side (Figs. 1A, 1B, and 1E). Injections of NMDA resulted in a dose-dependent ablation of the injected

TABLE 1 ELISA of NGF in the Medial Septum

Unoperated Fimbria fornix lesion NMDA

n

Ipsilateral septal NGF protein (pg/mg tissue)

4 3 4

1889 6 209 1754 6 137 845 6 83

Note. NGF protein levels from the left and right septum of unoperated animals did not differ significantly and were pooled to derive a single mean. Compared with this mean, NGF protein levels did not change significantly in the ipsilateral septal region after complete transection of the fimbria fornix, but fell significantly on the ipsilateral side after unilateral hippocampal injections of NMDA (P , 0.001, ANOVA with post hoc Neuman–Keuls pairwise analysis).

hippocampus, such that injections of 3 or 9 µg/µl of NMDA resulted, respectively, in a shrinkage of the hippocampal formation to 22.3 6 1.0% or 7.3 6 1.1% of that on the contralateral side. Injections of 9 µg/µl of NMDA caused the death of virtually all neurons within the ipsilateral hippocampal formation, including the CA subfields, dentate gyrus, most of the subiculum, and parts of the entorhinal cortex, and in these animals only a thin band of white matter persisted as the remnant of the fimbria, and virtually no neuroncontaining hippocampal tissue remained (Fig. 1). In all cases, the fimbrial remnant contained intact AChEstained fibers along its entire rostro–caudal extent (Figs. 1H–1J), and hippocampal remnants exhibited moderate gliosis, with some reactive GFAP-positive astrocytes (Fig. 1I). Relative levels of mRNA encoding NGF and BDNF were determined by quantitative Northern blot analysis of hippocampal tissue, 28 days after treatments (Fig. 2). Injection of saline (vehicle) resulted in a significant decline in hippocampal BDNF mRNA levels to 62.2 6 10.4% of control values (P , 0.01). Injection of 3 or 9 µg/µl of NMDA, caused dose-dependent declines in relative levels of hippocampal BDNF mRNA, respectively, to 20.3 6 3.0% or 11.5 6 2.3% of control values (P , 0.01; ANOVA with post hoc Neuman–Keuls pairwise analysis). The levels of mRNA encoding NGF showed similar changes across all treatment groups. Medial Septal NGF Protein Levels

FIG. 2. Northern blot analysis of hippocampal mRNA encoding BDNF after injection of 3 or 9 µg/µl NMDA or vehicle (V) into the hippocampus unilaterally on the left side (L) or in animals 28 days after sham (Sh) lesions compared with unoperated litter mates (Un). R, right (unlesioned) side.

Twenty-eight days after ablation of hippocampal neurons with 9 µg/µl NMDA, NGF protein levels in the septal region measured by ELISA had fallen significantly on the ipsilateral side to about 45% of control (i.e., untreated) values (Table 1). In FF-lesioned animals, NGF protein levels were not significantly different from control values in the ipsilateral septum (Table 1).

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Changes in Immunohistochemically Identified Septal Cholinergic Neurons Cholinergic neurons were identified by immunohistochemical detection of two markers, ChAT and p75NTR (Fig. 3). In unoperated animals approximately equal numbers of ChAT- and p75NGFR-stained neurons were present within the septal nucleus of either side (Table 2). Transection of the fimbria–fornix reduced the number of ipsilateral cholinergic septal neurons to less than 30% of unoperated control values (Figs. 3A and 3E, Table 2). In sham-operated animals with aspiration of overlying cortex to reveal the hippocampus, the number of ipsilateral septal cholinergic neurons was reduced to about 75% of unoperated control values, and multiple hippocampal injections of saline reduced this number further to about 65% (Figs. 3B and 3F, Table 2). Hippocampal ablation with 9 µg/µl NMDA resulted in massive loss of ipsilateral septal cholinergic neurons to about 30% of unoperated control values (Figs. 3C and 3G, Table 2). This loss of neurons was not significantly different from that seen after fimbria–fornix transection (Table 2). A substantial, but significantly smaller decline occurred after hippocampal injections of 3 µg/µl NMDA, to about 55% of unoperated control values. Regression analysis revealed a significant correlation between the number of neurons stained for either ChAT or p75NTR within the septal nucleus ipsilateral to the lesion and the cross-sectional area of persisting hippocampal tissue when individual animals were com-

pared across all the groups (except those with fimbria transections) (Figs. 4A and 4B). Surviving cholinergic neurons in the ipsilateral medial septal nucleus showed a significant reduction in their cross-sectional area vs unoperated control animals of similar severity after axotomy or target ablation (not shown). No significant changes occurred in the number or size of neurons stained for either immunohistochemical marker contralateral to lesions in all treatment groups. In adjacent sections stained with cresyl violet, there was no evidence of excitotoxic damage to the septal region. Effects of Delayed Infusion of NGF To test whether loss of cholinergic staining was due to down-regulation of the cholinergic marker proteins ChAT or p75NGFR in the absence of trophic support, a group of animals injected with 9 µg/µl NMDA was allowed to survive for 28 days and then infused intracerebroventricularly with exogenous NGF for 14 days. These animals showed a massive loss of ipsilateral septal neurons stained for ChAT or p75NGFR. The number of cholinergic neurons present was less than 30% of unoperated control values and was not significantly different from the number seen in animals injected with 9 µg/µl NMDA (Table 2) and having no subsequent treatment or those injected with 9 µg/µl NMDA followed by infusions of saline.

FIG. 3. Immunoreactive medial septal cholinergic neurons. (A–H) Adjacent sections through the bilateral septal region were immunohistochemically stained for ChAT (A–D) or p75NTR (E–H). Axotomy of the fimbria fornix (ff) (A, E) causes a massive loss of ipsilateral (asterix) septal cholinergic neurons. Hippocampal injections of vehicle (veh) slightly reduces the number of ipsilateral (asterix) septal cholinergic neurons (B, F), whereas hippocampal injections of 9 µg/µl NMDA (nmda) causes a massive loss of these neurons (C, G). Delayed administration of pharmacological doses of murine NGF (d-ngf) commencing 28 days after hippocampal NMDA injections fails to increase the number of ipsilateral (asterix) septal cholinergic neurons (D, H) over the number seen after NMDA injections alone. Scale bar, 105 µm.

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Ultrastructural Changes in Septal Cholinergic Neurons Electron microscopic examination of the medial septal nucleus of unoperated neonatal control animals revealed numerous large bipolar or multipolar neurons in which the nucleus was surrounded by large amounts of perinuclear cytoplasm containing dense aggregations of endoplasmic reticulum and numerous mitochondria (Fig. 5A). In unoperated animals between Postnatal Days 12 and 21, a small number of such neurons exhibited presumptive degenerative profiles consisting of uniform darkening of the cytoplasm and nucleus (Fig. 5B). No obvious increase in the number of degenerating neurons or signs of excitotoxic damage were seen at 3 days after hippocampal ablation. However, at 7 and 10 days after target ablation at Postnatal Days 9–11, many septal neurons exhibited signs of degeneration similar to those seen in a small number of neurons in unoperated animals. These signs of degeneration included prominent vacuolization of the endoplasmic reticulum without other changes at early stages (Fig. 5D), followed by darkening of both the cytoplasm and nucleus, shrinkage of the cell body, and dissolution of the nuclear membrane at later stages (Figs. 5E and 5F). Three and 7 days after axotomy of the fimbria–fornix at Postnatal Days 9–11, the ipsilateral medial septum contained many degenerating neurons which exhibited darkening of the cytoplasm but not of the nucleus, as well as early dissolution of cytoplasmic organelles (Fig. 5C). TABLE 2 Cell Counts of Septal Cholinergic Neurons Mean ipsilateral neuronal number per section Treatment group

n

ChAT

p75

Unoperated Fimbria fornix lesion Sham operated Vehicle infusion 3 µg NMDA infusion 9 µg NMDA infusion 9 µg NMDA 28 d 1 NGF 14 d

5 5 5 5 5 5 5

35.3 6 1.2 10.6 6 2.1 25.8 6 2.2 20.9 6 1.8 19.3 6 0.5 12.3 6 0.9 8.1 6 0.7

33.3 6 2.1 9.2 6 1.8 27.3 6 2.4 23.9 6 2.4 18.4 6 1.0 12.1 6 0.8 9.7 6 0.9

Note. Mean number of neurons (Abercrombie corrected (1)) stained for either ChAT or p75NTR per tissue section in a standardized region of the ipsilateral septum in treatment groups described in the text. Two-factor analysis showed that fimbria–fornix transections and hippocampal NMDA lesions of either dose resulted in significant declines in the number of ipsilateral cholinergic neurons as compared with unoperated sham- or vehicle-injected animals (P , 0.01). Cell counts after injections of 9 µg/µl were significantly lower than after injections of 3 µg/µl NMDA (P , 0.01), but did not differ significantly from those following fimbria–fornix transections. The number of septal cholinergic neurons present after delayed administration of NGF did not differ significantly from the number present after infusions of NMDA alone. ANOVA with post hoc Neuman–Keuls pairwise analysis.

FIG. 4. Correlation analyses of hippocampal area [expressed as the left (i.e., treated) area divided by the right area 3100] versus the mean number of neurons stained for either ChAT (A) or p75NGFR (B) in the septum ipsilateral to the treated hippocampus in the same animals. For these analyses, all unoperated, sham-operated, vehicle, and NMDA-lesioned animals were evaluated. Significant correlations were shown by regression analysis between the number of septal ChAT (A) or p75NTR (B) stained neurons and the amount of ipsilateral hippocampal area present across treatment groups.

DISCUSSION

Many Developing Septal Cholinergic Neurons Undergo Delayed Death after Ablation of Neurotrophin-Producing Target Neurons In agreement with our previous findings in adult rats (49, 50), our findings here in neonatal rats show that excitotoxic lesions which ablate hippocampal neurons during early postnatal development also essentially abolish hippocampal production of mRNA encoding BDNF and NGF and lower septal NGF protein levels. However, in contrast to our previous findings in adult rats where essentially all septal cholinergic neurons survived such lesions (49, 50), in this study we found that ablation of hippocampal neurons resulted in the

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FIG. 5. Ultrastructural profiles of septal neurons. (A–F) Electron microscopic appearance of large (18–30 µm diameter) septal neurons in rat littermates perfused on postnatal days 18 (A–D) or 21 (E, F). (A) Typical appearance of most large septal neurons in unlesioned (unles) animals. (B) Large septal neuron in an unlesioned animal showing uniform darkening of the cytoplasm and nucleus, with beginning dissolution of the nuclear membrane. (C) Large septal neuron 7 days after fimbria fornix transection (ff les), showing darkening of the

TARGET DEPENDENCE OF DEVELOPING CHOLINERGIC NEURONS

loss of most immunohistochemically stained developing septal cholinergic neurons. The loss of immunohistochemical staining for the cholinergic markers, ChAT and p75NTR, strongly suggests, but does not prove, that septal cholinergic neurons have died. Both ChAT and p75NGFR are dynamically regulated in septal neurons (22, 28) and can be down-regulated in other CNS neurons to the complete loss of detectable immunoreactivity in the absence of cell death (35, 59). For these reasons we conducted two additional studies to investigate whether cell death or down-regulation of marker-protein expression had occurred. NGF is able to up-regulate cholinergic markers in developing and adult septal neurons (22, 24, 28, 56). We therefore investigated whether 14 days of intraventricular administration of exogenous NGF starting 28 days after lesion was able to restore immunohistochemical staining of septal neurons which might have remained alive in the absence of a detectable cholinergic phenotype. This delayed NGF treatment did not increase the number of cholinergic neurons detected with immunohistochemical staining, suggesting that these neurons had died. Degenerating neurons exhibit a characteristic range of morphological changes at the ultrastructural level (13). We therefore ultrastructurally analyzed the septal region and found widespread evidence for the death of septal neurons after target ablation. The changes appeared to be progressive over time and lead ultimately to neuronal death. Based on comparisons with previous ultrastructural studies of adult septal cholinergic neurons identified by retrograde transport from the hippocampus (38) or by immunohistochemical staining for ChAT or p75NTR (31, 38) we concluded that the degenerating profiles in our material were neuronal. It was not possible to immunohistochemically identify degenerating cholinergic neurons at the ultrastructural level since the immunoreactive marker proteins disappear from the cells very early in the degenerative process. Nevertheless, these ultrastructural findings are compatible with our other experimental findings, suggesting that the loss of immunohistochemically detectable cholinergic septal neurons after target ablation in the neonatal rat represents the death of these neurons. It also is highly unlikely that the cholinergic cell loss observed in the medial septum is due to excitotoxic changes caused by the spread of NMDA injected into

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the hippocampus. First, in adult animals given similar or higher doses of NMDA, no cholinergic cell loss is observed in the septal region (49, 50). Second, no evidence of excitotoxic cell damage was seen in Nisslstained sections through the septal region. Third, no degenerative changes were observed at the light- or electron-microscopic levels at 3 days after the NMDA injections, the first changes appeared only at 7 days. Changes induced by NMDA injections are acute and are apparent within 24 h after injection at both lightand electron-microscopic levels (15). These findings all argue in favor of the delayed death of many developing septal cholinergic neurons after ablation of their target neurons. Our findings are broadly in agreement with several recent studies showing that the number of immunoreactive septal cholinergic neurons is significantly reduced after excitotoxic lesions of the hippocampus in neonatal animals (8, 9, 42). The differences in the number of cholinergic neurons persisting after such lesions in the different studies may be related to the extent of the lesions or some other technical aspect; for example, in our study hippocampal surface area and neurotrophin mRNA level and number of septal cholinergic neurons were all significantly correlated to amount of NMDA infused into the hippocampus. Burke et al. (9) also report that infusion of either NGF, or to a lesser extent BDNF, is able to prevent this loss of septal cholinergic neurons, suggesting that both growth factors might be able to regulate the number of cholinergic neurons present. Our present study adds to these observations experimental evidence that the loss of staining of cholinergic neurons represents cell death, rather than down-regulation of immunoreactive markers in surviving neurons. In addition, our findings indicate that partial lesions of the Hp results in partial declines in the numbers of septal neurons such that the number of septal neurons correlates significantly with the amount of hippocampal tissue present. Comparison of Effects of Target Neuron Ablation or Axotomy Since axotomy by transection of the fimbria–fornix is lethal to many adult septal cholinergic neurons (39, 55), it is important to consider whether the experimental procedure for target ablation is not merely causing an axotomizing lesion in neonatal animals which leads

cytoplasm but not of the nucleus, and beginning dissolution of cytoplasmic organelles. (D) Large septal neuron 7 days after hippocampal ablation with NMDA (nmda) showing prominent vaculoization of the endoplasmic reticulum (arrows) but no changes in the nucleus or cytoplasmic organelles. (E) Large septal neuron 10 days after hippocampal ablation with NMDA (nmda) showing vaculoization (arrows) and darkening of the cytoplasm, darkening of the nucleus, and dissolution of the nuclear membrane (arrowheads) but intact cytoplasmic organelles. (E) Presumptive large septal neuron (compare with other septal neurons sectioned through the nucleolus) 10 days after hippocampal ablation with NMDA (nmda) showing cell shrinkage, darkening of the cytoplasm, darkening of the nucleus, and disolution of the nuclear membrane (arrowheads). Scale bar, 2.0 µm.

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to retrograde cell death. Several lines of evidence indicate that this is not the case. Similar excitotoxic lesions in adult animals do not cause axotomizing lesions, and ultrastructural evidence suggests that excitotoxic lesions do not cause extensive damage to axons (15, 49, 50). In addition, the fimbria–fornix was intact and contained numerous AChE-positive cholinergic fibers along its entire rostro–caudal extent after excitotoxic ablation of the hippocampus in all cases examined for this study. This observation strongly suggests that axotomizing lesions had not been created, since the disappearance of AChE fibers distal to the lesions is characteristic of fimbria–fornix transections. In animals with hippocampal ablation, these persisting AChE fibers probably derive from surviving cholinergic neurons in the diagonal band region which also project to the hippocampus via the fornix. In addition, septal levels of NGF protein declined significantly after target ablation but not after axotomy, and different types of degenerative profiles of septal neurons were seen at the ultrastructural level in response to either target ablation or axotomy, further suggesting that target ablation and axotomy are distinct lesions. Thus it is highly unlikely that excitotoxic target ablation caused axotomy which lead to the retrograde death of septal cholinergic neurons observed in our studies. Developing Septal Cholinergic Neurons Survive in Proportion to Target Size We also found that the number of cholinergic neurons present in the medial septum correlated significantly across all experimental groups with the cross-sectional area of the ipsilateral hippocampus. This correlation remained significant when untreated animals were removed from the analysis. Since the cross-sectional area of cortical tissue is proportional to the number of viable neurons which persist long term after experimental manipulations such as excitotoxic lesions (29), these findings suggest that during postnatal development the survival of developing afferent cholinergic neurons may be regulated in proportion to the number of hippocampal target neurons. In the context of the neurotrophic hypothesis, these findings further suggest that one or more factors derived from hippocampal neurons may be involved in this regulation. In agreement with this possibility, we found that ablation of hippocampal neurons in neonatal rats essentially abolished hippocampal production of mRNA encoding BDNF and NGF and lowered septal levels of NGF protein to approximately half the levels present in control animals, presumably by reducing the amount of NGF retrogradely transported to the septum from the hippocampus by septal cholinergic neurons. These findings are compatible with a role for these neurotrophins in regulating the survival of developing septal cholinergic neurons, but do not exclude a role for other factors

produced by hippocampal cells or by cells locally within the septum (43). Two puzzling observations regarding septal levels of NGF were made in this study. First, hippocampal ablation which caused declines in hippocampal neurotrophin mRNA levels to about 10% of normal values resulted in a decline of septal NGF levels only to about 30% of normal. We reported similar observations previously in the adult rat after hippocampal ablation (58) and suggested as a possible explanation that the tissue blocks we dissected contained not only medial septal cholinergic neurons which project to the hippocampus, but also cholinergic neurons of the vertical and horizontal limbs of the diagonal band which retrogradely derive NGF via projections to other cortical areas (19). A reduction in the amount of NGF in the dissected septal region after lesions of the hippocampus might be masked by the continued transport of NGF from other targets by these neurons. Another possibility is that there is now clear evidence that NGF is made locally in the septal region (43), and this local production may account for the residual levels detected after hippocampal ablation. The second puzzling observation was that septal levels of NGF did not decline at all after axotomy of septal cholinergic neurons by transection of the neonatal fimbria–fornix in agreement with previous observations in adult rats (23, 49). The reason for this complete lack of decline is not clear. As just described, NGF retrogradely transported by neurons projecting to other sites and local production of NGF may account for some of the NGF detected. In addition, several lines of evidence suggest that reactive astrocytes in the forebrain can synthesize NGF in vivo (3, 49). Reactive astrocytosis in the septum may contribute to septal NGF levels after FF lesions. Target Regulation of Afferent Septal Cholinergic Neurons A large body of evidence indicates that neurotrophic factors derived from target cells regulate the survival of certain neurons during a period of naturally occurring cell death in the developing peripheral nervous system (36). It has not yet been demonstrated that septal cholinergic neurons have a period of naturally occurring developmental cell death, but our ultrastructural observation of degenerating septal neurons in untreated neonatal rats is compatible with this possibility. Moreover, our present findings and several other studies (8, 9, 42) suggest that many developing septal cholinergic neurons die in the absence of neurotrophinproducing target cells. This evidence is compatible with the possibility that target-derived neurotrophins might regulate the survival of these CNS neurons during a critical period of development. Septal cholinergic neurons exposed to NGF in vitro become dependent on NGF and die via apoptotic programmed cell death if

TARGET DEPENDENCE OF DEVELOPING CHOLINERGIC NEURONS

NGF is withdrawn within 2 weeks of plating (32, 54). Whether NGF plays such a role in vivo, and whether other factors might also be involved, needs to be determined. Alternatively, our findings are also compatible with suggestions that a contact-mediated interaction between afferent and target neurons during synaptogenesis regulates afferent neuronal survival rather than a purely neurotrophic interaction (40). Ablation of target neurons in our study would also have eliminated contact interactions of this kind. Mice which are homozygous for the targeted ablation of the genes for NGF or BDNF, or their associated Trk receptors, show some surviving septal cholinergic neurons for up to 4 weeks of postnatal life (beyond which timepoint survival of the animals appears limited), but precise counts have not yet been reported for these animals (17, 30, 47). A preliminary report suggests that the number of septal cholinergic neurons is reduced by over 25% in animals which are heterozygous for ablation of the NGF gene (11). A variety of possibilities, therefore, remain open: (1) more precise quantitative analysis may reveal more substantial losses of septal cholinergic neurons in mice homozygous for single gene ablation of NGF or BDNF; (2) loss of more than one neurotrophin may be required to show substantial cell death, and this may be revealed by cross-breeding mice with single gene knock outs for NGF or BDNF; (3) other growth factors produced by hippocampal cells may be able to sustain survival of septal cholinergic neurons in the absence of one or more neurotrophin. These possibilities are all broadly in agreement with the observations reported here that septal cholinergic neurons depend for survival during early postnatal development on target hippocampal neurons, and that one or more target-derived neurotrophin (or other trophic factor) may regulate the number of afferent septal cholinergic neurons in proportion to the number of target neurons. Available evidence suggests that septal cholinergic neurons do not remain dependent for survival on target-derived neurotrophic support after they reach a certain level of maturity. Ablation of hippocampal neurotrophin-producing target neurons in adult and aged animals results in the atrophy but not death of septal cholinergic neurons (39, 49, 50, 52). In addition, these neurons become progressively less dependent on NGF for survival as they mature in vitro, such that most neurons survive NGF withdrawal 5 weeks after plating (54). Similar observations have been reported for dorsal root ganglion neurons which are dependent on NGF for survival during early development and lose this dependence as the neurons mature in vivo or in vitro (21). Septal cholinergic neurons have receptors for, and are responsive to, different target-derived neurotrophins throughout life. Since neurotrophin levels can be dynamically regulated by factors such as synaptic input

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(37, 60), it will be interesting to elucidate how these different factors interact to regulate the survival, phenotype, and function of these neurons at different stages of development, maturity, and ageing. ACKNOWLEDGMENTS The authors thank K. J. Baker, S. J. Stevens, J. Powell, and D. Stratmann for technical assistance, J. Bashford for photography, and Dr. S. B. Dunnett for assistance with the statistical analysis. This work was supported in part by grants from the MRC, Wellcome Trust, and Merck, Sharpe & Dohme Research Laboratories.

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