Loss of Developing Cholinergic Basal Forebrain Neurons Following Excitotoxic Lesions of the Hippocampus: Rescue by Neurotrophins

Previous studies have demonstrated that the viability of developing cholinergic basal forebrain neurons is dependent upon the integrity of neurotrophin-secreting target cells. In the present study, we examined whether infusions of nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) could prevent the loss of cholinergic septal/diagonal band neurons following excitotoxic lesions of their target neurons within the hippocampus. Postnatal Day 10 rat pups received unilateral intrahippocampal injections of ibotenic acid. Rats then received intracerebroventricular (icv) injections of nerve growth factor (30 μg/injection), brain-derived neurotrophic factor (60 μg/injection), or saline immediately following the lesion and continuing every third day for 27 days. Both saline- and BDNF-treated rats displayed a significant loss of septal/diagonal band neurons expressing the protein and mRNA for choline acetyltransferase (ChAT) and p75 low-affinity nerve growth factor receptor ipsilateral to the lesion. The magnitude of this loss was significantly attenuated in BDNF-treated rats. Many remaining neurons were atrophic with stunted dendritic processes. In contrast, NGF treatment completely rescued these cells and prevented the shrinkage of remaining cholinergic septal neurons. In addition, both NGF and BDNF induced a sprouting of cholinergic processes within the residual hippocampal remnant ipsilateral to the infusions. The present study demonstrates that icv injections of NGF, and to a lesser extent BDNF, prevent the loss of developing basal forebrain neurons which occurs following removal of normal target cells. Diffusion studies revealed relatively poor penetration of BDNF into brain parenchyma. Thus, it remains to be determined whether the failure of BDNF to provide optimal trophic support for these cells is biological or due to restricted bioavailability of this trophic factor.     

Plasticity of Galaninergic Fibers Following Neurotoxic Damage within the Rat Basal Forebrain: Initial Observations

Galanin immunoreactive fibers hypertrophy and hyperinnervate remaining cholinergic basal forebrain neurons within the septum–diagonal band complex in Alzheimer's disease. The present investigation determined whether a similar hyperinnervation of galanin immunoreactive fibers occurs following intraparenchymal injections of ibotenic acid within the cholinergic medial septum or diagonal band nucleus in young adult rats. Sections through the medial septum and the diagonal band were either concurrently immunostained for galanin and the low-affinity p75 neurotrophin receptor (an excellent marker of cholinergic basal forebrain neurons) or single stained for choline acetyltransferase. Following chemical lesion, an increase in the density of galanin immunoreactivity was seen within the medial septum on the lesion, as opposed to the contralateral control side. In contrast, within diagonal band-lesioned animals, the increase in galanin immunoreactivity was low to moderate. In either lesion paradigm we did not observe hyperinnervation of remaining cholinergic basal forebrain neurons. In fact, there was no correlation between the galanin hypertrophy and the amount of cholinergic cell loss. We hypothesize that galanin hyperinnervation within the cholinergic basal forebrain may provide a protective effect by down-regulating acetylcholine release following brain insult.     

Stability of septohippocampal neurons following excitotoxic lesions of the rat hippocampus.

The present study examined the effects of removing hippocampal nerve growth factor (NGF)-producing neurons upon cholinergic and noncholinergic septohippocampal projecting neurons. To deplete septal/diagonal band neurons of their intrinsic source of NGF, rats received unilateral intrahippocampal injections of ibotenic acid and were sacrificed 2-24 weeks later. Choline acetyltransferase and parvalbumin immunohistochemistry failed to reveal changes in the number of cholinergic or gamma-aminobutyric acid-containing neurons, respectively, within the septal/diagonal band region ipsilateral to the hippocampal lesion at any time point examined. Additionally, immunocytochemical localization of nonphosphorylated and phosphorylated neurofilament proteins did not reveal abnormal staining characteristics within the septal/diagonal band complex, suggesting that this lesion does not alter cytoskeletal features of neurons which project to the hippocampus. Selected rats received unilateral hippocampal lesions and 3 months later were injected with fluorogold into the remaining hippocampal remnant and with wheat germ agglutinin conjugated to horse radish peroxidase into the intact contralateral hippocampus. Both retrograde tracers were predominantly transported to their respective ipsilateral septum and vertical limb of the diagonal band. This indicates that following the lesion, septal/diagonal band neurons still project ipsilaterally and sprouting to the NGF-rich contralateral side does not occur. RNA blot analysis revealed a decrease in NGF mRNA expression within the lesioned hippocampus with a maximum reduction of approximately 70%. In contrast, no change in NGF mRNA expression was observed within the ipsilateral septum relative to the contralateral side. The present study demonstrates that removal of hippocampal target neurons does not alter the number, morphology, or projections of both cholinergic and noncholinergic septal/diagonal band neurons.     

Experimental immune-mediated damage of septal cholinergic neurons

Degeneration of cholinergic neurons in the medial septum and the diagonal band of Broca is a frequent neuropathological feature of Alzheimer's disease. To determine whether an immune process can injure these basal forebrain cholinergic neurons, we serially immunized guinea pigs with septal cholinergic hybrid cells (SN-56). Following immunization, a relatively selective damage of septal cholinergic neurons, reduction in septal choline acetyltransferase (ChAT) activity and decrease in acetylcholine release in hippocampus were detected. Serum IgG from guinea pigs immunized with SN-56 cells and stereotactically injected into the medial septal region of rats produced a loss of ChAT activity in the medial septum, frontal cortex and hippocampus, together with impairment of learning and long term spatial memory. These data suggest that relatively selective damage to septal cholinergic neurons can be caused by an immune-mediated process in experimental animals.     

Environment-spatial conditional learning in rats with selective lesions of medial septal cholinergic neurons

Cholinergic medial septal neurons may regulate several aspects of hippocampal function, including place field stability and spatial working memory. Monkeys with damage to septal cholinergic neurons are impaired in visual-spatial conditional learning tasks; however, this candidate function of septal cholinergic neurons has not been studied extensively in the rat. In the present study, rats with selective lesions of cholinergic neurons in the medial septum and vertical limb of the diagonal band of Broca (MS/VDB), made with 192 IgG-saporin, were tested on a conditional associative learning task. In this task, which we term "environment-spatial" conditional learning, the correct location of a spatial response depended on the array of local environmental cues. MS/VDB-lesioned rats were impaired when the two parts of the conditional problem were presented concurrently, but not when one environment had been learned before the full conditional problem was presented. Our findings suggest that cholinergic MS/VDB neurons participate in some aspects of conditional associative learning in rats. They may also shed light on the involvement of cholinergic projections to the hippocampus in modulating and remodeling hippocampal spatial representations.     

Galanin-like immunoreactivity in cholinergic neurons of the septum-basal forebrain complex projecting to the hippocampus of the rat

It is now wel recognized that there are several groups of cholinergic neurons in the basal forebrain with direct projections to various cortical regions. Immunohistochemical investigations of the distribution of the neuropeptide galanin (GAL) have shown that two of these brain areas, the medial septum and diagonal band, contained large numbers of GAL-immunoreactive neurons. In the present study, double staining techniques using antibodies raised against choline acetyltransferase (ChAT) revealed that GAL- and ChAT- like immunoreactivities are colocalized within a subpopulation of the cholinergic neurons within the medial septum and diagonal band. This colocalization of GAL- and ChAT-immunoreactivities was not seen to occur within other groups of forebrain cholinergic neurons. Immunohistochemistry carried out subsequent to injections of fluorescent retrograde tracers into the hippocampal formation revealed that both ChAT/GAL- and ChAT-containing neurons project to the hippocampal formation. The question of GAL as a modulator of cholinergic transmission in this projection is discussed.     

Basal forebrain neurons suppress amygdala kindling via cortical but not hippocampal cholinergic projections in rats

Intraventricular administration of the immunotoxin 192 IgG-saporin in rats has been shown to cause a selective loss of cholinergic afferents to the hippocampus and cortical areas, and to facilitate seizure development in hippocampal kindling. Here we demonstrate that this lesion also accelerates seizure progression when kindling is induced by electrical stimulations in the amygdala. However, whereas intraventricular 192 IgG-saporin facilitated the development of the initial stages of hippocampal kindling, the same lesion promoted the late stages of amygdala kindling. To explore the role of various parts of the basal forebrain cholinergic system in amygdala kindling, selective lesions of the cholinergic projections to either hippocampus or cortex were produced by intraparenchymal injections of 192 IgG-saporin into medial septum/vertical limb of the diagonal band or nucleus basalis, respectively. Cholinergic denervation of the cortical regions caused acceleration of amygdala kindling closely resembling that observed after the more widespread lesion induced by intraventricular 192 IgG-saporin. In contrast, removal of the cholinergic input to the hippocampus had no effect on the development of amygdala kindling. These data indicate that basal forebrain cholinergic neurons suppress kindling elicited from amygdala, and that this dampening effect is mediated via cortical but not hippocampal projections.     

Organization of the septal region in the rat brain: cholinergic-GABAergic interconnections and the termination of hippocampo-septal fibers

This study deals with two characteristic cell types in the rat septal complex i.e., cholinergic and GABAergic neurons, and their synaptic connections. Cholinergic elements were labeled with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. Antiserum against glutamate decarboxylase (GAD), the GABA synthesizing enzyme, was employed to identify GABAergic perikarya and terminals, by using either the peroxidase-antiperoxidase (PAP) technique or a biotinylated second antiserum and avidinated gold or ferritin. With these contrasting immunolabels we have studied the cholinergic-GABAergic interconnections in double-labeled sections of intact septal regions and the GABAergic innervation of medial septal area cholinergic neurons in sections taken from animals 1 week following lateral septal area lesion. In other electron microscopic experiments we have studied cholinergic and GABAergic neurons in the septal complex for synaptic contacts with hippocamposeptal fibers, which were identified by anterograde degeneration following fimbria-fornix transection. Our results are summarized as follows: (1) GAD-positive terminals form synaptic contacts on ChAT-immunoreactive dendrites in the medial septum/diagonal band complex (MSDB), (2) surgical lesion of the lateral septal area resulted in a dramatic decrease of the number of GABAergic boutons on MSDB cholinergic neurons, (3) cholinergic terminals establish synaptic contacts with GAD immunoreactive cell bodies and proximal dendrites in the MSDB as well as in the lateral septum (LS), (4) degenerated terminals of hippocampo-septal fibers were mainly observed in the LS, where they formed asymmetric synaptic contacts on dendrites of GABAergic neurons and on nonimmunoreactive spines. We did not observe degenerated boutons in contact with ChAT-positive dendrites or cell bodies in the MSDB. From these results and from data in the literature we conclude that excitatory hippocampo-septal fibers activate GABAergic cells, and as yet unidentified spiny neurons in the LS, which may control the discharge of medial septal cholinergic neurons known to project back to the hippocampal formation.     

Response of the monkey cholinergic septohippocampal system to fornix transection: a histochemical and cytochemical analysis

Transection of the fimbria-fornix pathway is a paradigm that has been richly exploited in rats to assess the structural and functional correlates of cognitive behavior, neural grafting, and growth factor administration. Principally, the degeneration of cholinergic neurons within the septal/diagonal band region has received detailed attention following this manipulation. In contrast, no studies have examined the response of the cholinergic septal/diagonal neurons following axotomy in nonhuman primates. This study examined the neuronal and glial responses within the septal region to selective fornix transection (without cingulate gyrus ablation) in four Cebus apella monkeys. One month following unilateral transection of the fornix by means of an open microsurgical approach, a comprehensive loss of acetylcholinesterase [AChE]-containing fibers was observed throughout the hippocampal formation and dentate gyrus ipsilateral to the lesion. Decreases in AChE fiber densities were also observed within the entorhinal cortex ipsilateral to the lesion. No such changes in AChE-fiber density were consistently observed within the subicular region. The decrease in hippocampal AChE-positive fibers was paralleled by a 49.5% reduction in cholinergic medial septal neurons as revealed by Nissl stains and immunohistochemical staining for the receptor for nerve growth factor, a marker of cholinergic basal forebrain neurons in primates. In contrast, no significant changes in the number of neurons within the vertical limb of the diagonal band were noted. Following the transection, a relatively intense reactive gliosis was observed within the dorsal half of the septal region ipsilateral to the transection and within the overlying transected corpus callosum. These data provide the foundation in nonhuman primates on which novel therapeutic factors can be evaluated in paradigms relevant to the study of Alzheimer's disease.     

Choline acetyltransferase-immunoreactive neurons and terminals in the rat septal complex: a combined light and electron microscopic study

A monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was used to determine the morphological characteristics of cholinergic neurons and axon terminals within the rat septum. Light microscopy revealed numerous large fusiform or multipolar ChAT-immunoreactive neurons in the medial septal nucleus/diagonal band complex (MSDB). In contrast, virtually no immunostained cells were found in the lateral septum (Nc. septalis dorsalis and Nc. septalis lateralis). Fine immunostained fibers were most abundant close to the midline in the MSDB mainly following an ascending course. A few thin ChAT-immunoreactive fibers and terminallike pericellular punctate structures were observed in the inner part of the dorsal septal nucleus. Electron microscopy of ChAT-immunoreactive neurons revealed large cell bodies rich in cytoplasmic organelles. The cell nuclei regularly exhibited multiple invaginations of the nuclear membrane. Only rarely were terminals found that established synaptic contacts on the cell bodies of immunostained neurons. In contrast, numerous terminals formed synaptic contacts on immunoreactive dendrites. ChAT-immunopositive terminals were studied in thin sections from the MSDB and from the dorsal septal nucleus. In both regions they appeared as heavily immunostained vesicle-filled boutons that established symmetric and asymmetric synaptic contacts. In the dorsal septal nucleus immunostained terminals often showed a basketlike arrangement around immunonegative cell bodies. Our fine structural study provides evidence that cholinergic neurons in the MSDB are similar to cholinergic neurons in the basal nucleus and neostriatum, which have been described by other investigators. The presence of cholinergic synapses in the septal complex indicates that this region not only contains cholinergic projection neurons, but receives a cholinergic input itself.     

IL-2 deficiency results in altered septal and hippocampal cytoarchitecture: relation to development and neurotrophins

We have found previously that brain IL-2 receptors are enriched in the hippocampal formation, and that loss of this cytokine results in cytoarchitectural alterations in the hippocampus and septum and related behavioral changes in IL-2 knockout (IL-2 KO) mice. These alterations included decreased cholinergic somata in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB) and decreased distance across the infrapyramidal (IP) granule cell layer (GCL) of the dentate gyrus (DG). To extend our previous findings, several experiments were conducted comparing IL-2 KO mice and wild-type littermates to determine (1) whether the GABAergic projection neurons of IL-2 KO mice in this region were also affected; (2) if the reduction in septal cholinergic projection neurons found in adult IL-2 KO mice is present at weaning (and prior to the development of peripheral autoimmune disease); and (3) if loss of IL-2 may result in changes in the neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), involved in maintenance of hippocampal neurons. No differences in GABAergic neurons in the MS/vDB were found in adult mice, and the reduction in cholinergic neurons seen in adult IL-2 KO mice was not found in animals at postnatal day 21. The number of neurons in the IP-GCL was also significantly reduced. Compared to wild-type mice, IL-2 KO mice had significantly reduced concentration of BDNF protein and increased concentrations of NGF. These data suggest that the septohippocampal neuronal loss in IL-2 KO mice is selective for the cholinergic neurons and appears to be due to a failure in neuronal maintenance/survival that may be, in part, associated with changes in neurotrophins.     

An immunocytochemical study of choline acetyltransferase-containing neurons and axon terminals in normal and partially deafferented hippocampal formation

Monoclonal antibodies to the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), have been used to study putative cholinergic structures in immunocytochemical preparations of normal rat hippocampal formation and of hippocampal formation deprived of its septal innervation. Small numbers of ChAT-positive (ChAT+) neuronal somata were observed scattered throughout the septotemporal extent of the normal hippocampal formation. They were most common in stratum lacunosum-moleculare of regio superior, but were also found in various layers of the dentate gyrus and occasionally in the remaining hippocampal laminae. In addition, light microscopy demonstrated that ChAT+ terminal fields in normal hippocampal formation were organized in discrete bands and laminae. Pronounced dense bands were observed: immediately superficial to stratum granulosum; deep to stratum pyramidale; and at the border between stratum radiatum and stratum lacunosum-moleculare. In the dentate gyrus, ChAT+ staining was pronounced in the hilus at temporal levels, but only moderate staining occurred in the anterior hilus and throughout the molecular layer. A close correspondence was observed in the density and distribution of ChAT+ immunoreactivity and acetylcholinesterase staining. Electrolytic lesions of the medial septal nucleus/diagonal band complex had no effect on the occurrence of ChAT+ somata, but virtually abolished the ChAT+ laminar staining pattern and eliminated all but occasional small patches of ChAT+ terminals. These results confirm that the vast majority of hippocampal cholinergic terminals originate either from neurons of the medial septum/diagonal band complex or from fibers of passage. The newly observed intrinsic hippocampal neurons can account for at least some of the ChAT activity remaining after septal lesions, and they apparently contribute to the cholinergic innervation of the hippocampal formation.     

Recovery of ChAT lmmunoreactivity in Axotomized Rat Cholinergic Septal Neurons Despite Reduced NGF Receptor Expression

Previous studies have suggested that target-derived nerve growth factor (NGF) is essential for the survival of cholinergic basal forebrain neurons. Thus, axotomy of septohippocampal neurons in adult rats resulting in the withdrawal of target-derived NGF caused a dramatic loss of choline acetyltransferase (ChAT)-immunoreactive neurons in the medial septum-diagonal band complex. We have recently shown that this loss of immunolabelled neurons does not indicate cell death, since many septohippocampal cholinergic neurons recover their immunoreactivity for ChAT after a long survival time despite disconnection from target-derived neurotrophins. One possibility would be that these surviving ChAT-immunoreactive neurons have gained access to other, probably local, NGF sources. Here we provide evidence that the recovery of ChAT immunoreactivity after axotomy is not accompanied by a similar recovery of NGF receptor expression in these neurons. In situ hybridization for p75^NTR mRNA and trkA mRNA 6 months after bilateral fimbria-fornix transection revealed a substantial loss of labelled cells. In addition, there was a persisting loss of p75^NTR-immunoreactive and NGF-immunoreactive medial septal neurons. Cholinergic neurons in controls did not express NGF mRNA, but were heavily immunostained for NGF protein due to receptor-mediated uptake. These data suggest that at least some cholinergic septohippocampal neurons re-express ChAT either independently of NGF or with a reduced need for NGF.     

Direct catecholaminergic-cholinergic interactions in the basal forebrain. II. Substantia nigra-ventral tegmental area projections to cholinergic neurons

Previous observations indicate that the basal forebrain receives dopaminergic input from the ventral midbrain. The present study aimed at determining the topographic organization of these projections in the rat, and whether this input directly terminates on cholinergic neurons. Injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into discrete parts of the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNC) labeled axons and terminals in distinct parts of the basal forebrain, including medial and lateral septum, diagonal band nuclei, ventral pallidum, globus pallidus, substantia innominata, globus pallidus, and internal capsule, where PHA-L-labeled terminals abutted cholinergic (choline acetyltransferase=ChAT-containing) profiles. Three—dimensional (3-D) computerized reconstruction of immunostained sections clearly revealed distinct, albeit overlapping, subpopulations of ChAT-immunoreactive neurons apposed by PHA-L-labeled input from medial VTA (mainly in vertical and horizontal diagonal band nuclei), lateral VTA and medial SNC (ventral pallidum and anterior half of substantia innominata), and lateral SNC (caudal half of the substantia innominata and globus pallidus). At the ultrastructural level, about 40% of the selected PHA-L-labeled presynaptic terminals in the ventral pallidum and substantia innominata were found to establish synaptic specializations with ChAT-containing profiles, most of which on the cell body and proximal dendritic shafts. Convergent synaptic input of unlabeled terminals that formed asymmetric synapses with the ChAT-immunoreactive profiles were often found in close proximity to the PHA-L-labeled terminals. These observations show that the cholinergic neurons in the basal forebrain are targets of presumably dopaminergic SNC/VTA neurons, and suggest a direct modulatory role of dopamine in acetylcholine release in the cerebral cortical mantle. © 1996 Wiley-Liss, Inc.     

Medial septal cholinergic neurons are necessary for context‐place memory but not episodic‐like memory

Loss of cholinergic cortical input is associated with diseases in which episodic memory impairment is a prominent feature, but the degree to which this neurochemical lesion can account for memory impairment in humans with neurodegenerative diseases remains unclear. Removal of cholinergic input to hippocampus impairs some of its functions in memory, perhaps by reducing the plasticity of information representation within the hippocampus, but the role of cholinergic hippocampal input in episodic‐like memories has not been investigated. To address this question, we tested rats with selective lesions of basal forebrain neurons in the medial septum and vertical limb of the diagonal band (MS/VDB), which contains hippocampal‐projecting cholinergic neurons, on a task of integrated memory for objects, places, and contexts (“what–where–which” memory). This task serves as a rodent model of human episodic memory (episodic‐like memory) and is sensitive to damage to the hippocampal system. Rats with lesions of cholinergic MS/VDB neurons performed as well on the what–where–which task as controls, but were impaired in a task that simply required them to associate places with contexts (“where–which” memory). Thus, episodic‐like memories that rely on the hippocampus do not require cholinergic neuromodulation to be formed. Nevertheless, some more specific aspects of where–which memory, which may be more dependent on the plasticity of hippocampal spatial representations, require acetylcholine. These results suggest that cholinergic projections to hippocampus are not necessary for episodic memory and, furthermore, that hippocampal spatial representations may be to some extent dissociable from episodic memory function. © 2010 Wiley‐Liss, Inc.     

Electrical activity of the cingulate cortex. II. Cholinergic modulation

The role of the cholinergic innervation in the modulation of cingulate electrical activity was studied by means of pharmacological manipulations and brain lesions. In the normal rat, an irregular slow activity (ISA) accompanied with EEG-spikes was recorded in the cingulate cortex during immobility as compared to walking. Atropine sulfate, but not atropine methyl nitrate, increased ISA and the frequency of cingulate EEG-spikes. Pilocarpine suppressed ISA and EEG-spikes during immobility, and induced a slow (4-7 Hz) theta rhythm. Unilateral or bilateral lesions of the substantia innominata and ventral globus pallidus area using kainic acid did not significantly change the cingulate EEG or its relation to behavior. Large electrolytic lesions of the medial septal nuclei and vertical limbs of the diagonal band generally decreased or abolished all theta activity in the cingulate cortex and the hippocampus. However, in 5 rats the cingulate theta rhythm increased while the hippocampal theta disappeared after a medial septal lesion. The large, postlesion cingulate theta, accompanied by sharp EEG-spikes during its negative phase, is an unequivocal demonstration of the existence of a theta rhythm in the cingulate cortex, independent of the hippocampal rhythm. Cholinergic afferents from the medial septum and diagonal band nuclei are inferred to be responsible for the behavioral suppression of cingulate EEG-spikes and ISA, and partially for the generation of a local cingulate theta rhythm. However, an atropine-resistant pathway and a theta-suppressing pathway, possibly coming from the medial septum or the hippocampus, may also be important in cingulate theta generation.     

Immunohistochemical evidence for degeneration of cholinergic neurons in the forebrain of the rat following injection of AF64A-picrylsulfonate into the dorsal hippocampus

The cholinergic neurotoxin, AF64A-picrylsulfonate, was unilaterally infused into the dorsal hippocampus of Wistar rats (2 nmol/2 μl/4 min; A 6.2, L^s 1.5, H 6.5, Paxinos and Watson). After 19 days the animals' brains were processed for immunohistochemical staining of choline acetyltransferase (ChAT). Morphometry and counting of ChAT-immunoreactive profiles revealed shrinkage and disappearance of cholinergic neurons in the medial septum and diagonal band of Broca at the lesioned brain side. These data indicate a retrograde degeneration of cholinergic neurons following injection of AF64-A-picrylsulfonate into the dorsal hippocampus of the rat.     

Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections

Several findings obtained in recent years suggest that NGF, aside from its well-established function as a neurotrophic factor for peripheral sympathetic and sensory neurons, also has trophic influence on the cholinergic neurons of the basal forebrain. The present study assessed whether NGF was able to affect survival of central cholinergic neurons after axonal transections in adult rats. The septo-hippocampal pathway was transected unilaterally by cutting the fimbria, and animals were implanted with a cannula through which NGF or control solutions were injected intraventricularly over 4 weeks. The lesions reduced the number of large cell bodies, as visualized by Nissl staining in the medial septal nucleus and in the vertical limb of the diagonal band of Broca. Furthermore, in the same nuclei, they reduced the number of cell bodies positively stained for AChE after pretreatment with diisopropylfluorophosphate (a method known to result in reliable identification of cholinergic neurons in the septal area). On lesioned sides, the number of cholinergic cells in medial septal nucleus and the vertical limb of the diagonal band was reduced by 50 +/- 4%, as compared to the number on contralateral sides. On lesioned sides of animals chronically treated with NGF, the number of AChE-positive cells in these areas was reduced only by 12 +/- 6%, as compared to control levels. These findings suggest that fimbrial transections resulted in retrograde degeneration of cholinergic septo-hippocampal neurons and that NGF treatment strongly attenuated this lesion-induced degeneration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depletion of cholinergic neurons in the nucleus of the medial septum and the vertical limb of the diagonal band in dementia with Lewy bodies

The cholinergic basal forebrain is divided into four subregions (Ch1–4), and cholinergic neuronal loss in the nucleus basalis of Meynert (Ch4) has been correlated with cognitive impairments in both Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB). However, the Ch1–2 regions, which provide the major cholinergic innervation to the hippocampus, have not been investigated in DLB. The purpose of this study was to reveal the cholinergic neuronal changes in the medial septum (Ch1) and the nucleus of the vertical limb of the diagonal band (Ch2) of DLB brains. Using choline acetyltransferase (ChAT) immunohistochemistry, we showed that the number of ChAT-immunoreactive neurons in DLB brains was significantly lower than the numbers in AD and non-demented (control) brains. No significant difference in the number of ChAT-immunoreactive neurons was found between the AD and control brains. Moreover, the size of the ChAT-immunoreactive neurons was significantly smaller in the AD and DLB brains than in the control brains. These results show that cholinergic neurons of the Ch1-2 regions are more severely affected in DLB than in AD. Our DLB cases did not fulfill the neuropathologic criteria for definite AD. Furthermore, some Lewy bodies were observed in the Ch1-2 regions. Thus, cholinergic neuronal loss in the Ch1-2 regions might be specific to the pathology of DLB. Taking the distribution of cholinergic fibers in the hippocampus into consideration, this study suggests a possibility that hippocampal cholinergic projection is involved in Lewy-related neurites in the CA2–3 regions, the origin of which remains unclear.     

Nerve growth factor mRNA-containing cells are distributed within regions of cholinergic neurons in the rat basal forebrain

It has been proposed that nerve growth factor (NGF) provides critical trophic support for the cholinergic neurons of the basal forebrain and that it becomes available to these neurons by retrograde transport from distant forebrain targets. However, neurochemical studies have detected low levels of NGF mRNA within basal forebrain areas of normal and experimental animals, thus suggesting that some NGF synthesis may actually occur within the region of the responsive cholinergic cells. In the present study with in situ hybridization and immunohistochemical techniques, the distribution of cells containing NGF mRNA within basal forebrain was compared with the distribution of cholinergic perikarya. The localization of NGF mRNA was examined by using a ³⁵S-labeled RNA probe complementary to rat preproNGF mRNA and emulsion autoradiography. Hybridization of the NGF cRNA labeled a large number of cells within the anterior olfactory nucleus and the piriform cortex as well as neurons in a continuous zone spanning the lateral aspects of both the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus. In the latter regions, large autoradiographic grain clusters labeled relatively large Nissl-pale nuclei; it did not appear that glial cells were autoradiographically labeled. Comparison of adjacent tissue sections processed for in situ hybridization to NGF mRNA and immunohistochemical localization of choline acetyltransferase (ChAT) demonstrated overlapping fields of cRNA-labeled neurons and ChAT-immunoreactive perikarya in both the horizontal limb of the diagonal band and magnocellular preoptic regions. However, no hybridization of the cRNA probe was observed in other principal cholinergic regions including the medial septum, the vertical limb of the diagonal band, or the nucleus basalis of Meynert. These results provide evidence for the synthesis of NGF mRNA by neurons within select fields of NGF-responsive cholinergic cells and suggest that the generally accepted view of “distant” target-derived neurotrophic support should be reconsidered and broadened.