Ontogeny of cholinergic amacrine cells in the opossum (Didelphis aurita) retina

Immunocytochemistry for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, was used to determine the onset and to follow the maturation of the cholinergic cells in the retina of a marsupial, the South American opossum (Didelphis aurita). ChAT-immunoreactivity was first detected in amacrine cells in the ganglion cell layer by postnatal day 15 (P15) and in the inner nuclear layer by P35. Much later, at P50 a second sub-population of ChAT-immunoreactive cell bodies was evident in the inner nuclear layer. Processes from ChAT-immunoreactive amacrine cells were detected in the two bands of the inner plexiform layer before synaptogenesis. In the adult retina, these two bands correspond to sublamina 2 and 4 of the inner plexiform layer. In flat whole-mounted preparations, cholinergic cell density was 263±13 cells/mm[2] in the ganglion cell layer and it was estimated a total of 24,000 cholinergic neurons. ChAT-immunoreactive somata showed a random pattern of distribution.     

Synaptic organization of cholinergic amacrine cells in the rhesus monkey retina

In the rhesus monkey retina, choline acetyltransferase (ChAT) immunoreactivity has been used to study the localization and synaptic organization of cholinergic neurons by both light and electron microscopy with peroxidase-antiperoxidase immunohistochemistry. ChAT-containing neurons are a type of amacrine cell with 97.5% of their cell bodies localized to the ganglion cell layer and the remainder in the inner nuclear layer. Their processes arborize in a single narrow band in the inner plexiform layer in a plane dividing the outer two-thirds from the inner one-third of this synaptic region. With electron microscopy, ChAT-immunoreactive amacrine cell processes were observed to be primarily postsynaptic to the diffuse invaginating cone bipolar cells and presynaptic to ganglion cells, although they are both post- and presynaptic to immunohistochemically unlabeled amacrine cell profiles and to ChAT-containing amacrine cell processes as well.     

Laminar asymmetry in the distribution of choline acetyltransferase-immunoreactive neurons in the retina of the tree shrew (Tupaia belangeri)

Cholinergic neurons in the retina of the tree shrew were identified immunocytochemically using a monoclonal antibody directed against choline acetyltransferase (ChAT). The chief result is that roughly 4 times as many ChAT-immunoreactive neurons are found in the inner nuclear layer (INL) as in the ganglion cell layer (GCL). In the INL, two classes of cholinergic neuron can be distinguished on the basis of soma size, one large and one small. The large neurons correspond closely in size and number to the displaced cholinergic neurons in the GCL, suggesting that these are the matching populations of cholinergic amacrine cells reported in other species. The small ChAT-immunoreactive neurons, on the other hand, which make up 60% of the total number of ChAT-positive neurons in the retina, appear to have no counterpart in the GCL. Whether these small neurons are a separate class of amacrine cell or some other cell type (e.g. bipolar, interplexiform, etc.) remains to be determined.     

Co-localization of vasoactive intestinal polypeptide, γ-aminobutyric acid and choline acetyltransferase in neocortical interneurons of the adult rat

Interneurons immunoreactive for vasoactive intestinal polypeptide (VIP) are integral elements of columnar organization patterns in the rat cerebral cortex. By application of the sensitive mirror technique, the co-localization of VIP with the classical inhibitory neurotransmitter γ-aminobutyric acid (GABA) and the acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT), was investigated in neocortical neurons. Furthermore, the frequency of co-localization of ChAT with GABA was determined. In a sample of 118 VIP-immunoreactive neurons, mostly from the primary somatosensory cortex, it was demonstrated that virtually all of them reveal immunoreactivity for GABA and, therefore, are to be GABAergic. Moreover, 34% of mostly bipolar, VIP-positive neurons contained ChAT and are, thus, supposedly cholinergic as well. Co-localization of VIP and ChAT varied according to cortical laminae. Finally, 88% of a total of 60 ChAT-immunoreactive neurons were also immunostained for GABA. It is concluded that almost all VIP-immunoreactive neurons and most of the cholinergic neurons in rat neocortex represent partly overlapping subpopulations of inhibitory interneurons utilizing GABA.     

Ontogeny of cholinergic amacrine cells in the oppossum (Didelphis aurita) retina.

Immunocytochemistry for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, was used to determine the onset and to follow the maturation of the cholinergic cells in the retina of a marsupial, the South American opossum (Didelphis aurita). ChAT-immunoreactivity was first detected in amacrine cells in the ganglion cell layer by postnatal day 15 (P15) and in the inner nuclear layer by P35. Much later, at P50 a second sub-population of ChAT-immunoreactive cell bodies was evident in the inner nuclear layer. Processes from ChAT-immunoreactive amacrine cells were detected in the two bands of the inner plexiform layer before synaptogenesis. In the adult retina, these two bands correspond to sublamina 2 and 4 of the inner plexiform layer. In flat whole-mounted preparations, cholinergic cell density was 263±13 cells/mm[2] in the ganglion cell layer and it was estimated a total of 24,000 cholinergic neurons. ChAT-immunoreactive somata showed a random pattern of distribution.     

Immunocytochemical analysis of cholinergic amacrine cells in the tiger salamander retina

Cholinergic amacrine cells in the tiger salamander retina were observed for the first time by using antibodies against choline acetyltransferase (ChAT). ChAT-immunoreactive cells were present in the inner nuclear layer (INL) and in the ganglion cell layer (GCL), and the somas of the former population (average diameter = 15.13 microm) were slightly smaller than those of the latter population (average diameter = 16.42 microm). The processes of these cells form two distinct narrow bands in the inner plexiform layer (IPL), one located near 0.2 inner plexiform units (IU) and the other near 0.65-0.7 IU. Soma size, cell density and spatial distribution of ChAT-positive cells were quantitatively analyzed. Our results suggest that cholinergic amacrine cells in the salamander retina are very similar to their counter parts in other species, and they can be used as a model system for studying cholinergic functions in the visual system.     

Development of the cholinergic system in the brain and retina of the zebrafish

We have analyzed the distribution pattern of choline acetyltransferase (ChAT) in the zebrafish brain and retina during ontogeny. ChAT-immunoreactive (ChAT-ir) neurons are observed in the prosencephalon from 60 h postfertilization (hpf) onwards, exclusively in the preoptic area (basal plate of p6) derived from the secondary prosencephalon. In the mesencephalon, ChAT-ir cells are observed in both the optic tectum and the tegmentum. Stained cells in the tegmentum are observed from 60 hpf onwards, while in the optic tectum they appear after hatching. In the rhombencephalon, ChAT-ir cells are first observed in the isthmic region (rh1) and in the medulla oblongata (rh5-rh7) at the end of embryonic life. The rhombencephalic cholinergic cell groups develop in a gradual caudorostral sequence. Motoneurons of the spinal cord are ChAT-ir from 48 hpf onwards. The retina displays ChAT-ir neuropil in both the inner and outer plexiform layers from embryonic life, whereas stained amacrine cells are only observed after hatching. The staining in the outer plexiform layer gradually decreases during juvenile development. The optic nerve axons show a transient expression of ChAT at the end of embryonic development. The early presence of ChAT immunolabeling suggests an important neuromodulator role for acetylcholine in the first developmental stages.     

Cholinergic innervation of hippocampal GAD- and somatostatin-immunoreactive commissural neurons

This study describes the cholinergic innervation of chemically defined nonpyramidal neurons in the hilar region of the rat hippocampus. Cholinergic terminals were identified by immunocytochemistry employing a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, and the avidin-biotin-peroxidase (ABC) technique. Nonpyramidal neurons in the hilar region were characterized by immunostaining with antibodies against glutamate decarboxylase (GAD), the gamma aminobutyric acid (GABA)-synthesizing enzyme, and somatostatin (SS). The immunoreactivity to these antibodies was detected by using biotinylated secondary antibodies and avidinated ferritin as an electron-dense marker. This electron microscopic double immunostaining procedure enabled us to demonstrate that immunoperoxidase-labeled ChAT-immunoreactive terminals established symmetric synaptic contacts on the ferritin-labeled GAD- and SS-immunoreactive hilar cells. In additional experiments at least some of the GAD- and SS-immunoreactive hilar neurons were further characterized as commissural neurons by retrograde filling with horseradish peroxidase (HRP) following an injection of the tracer into the contralateral hilus. From these triple labeling experiments, we concluded that at least some GABAergic and somatostatin-containing neurons in the hilar region, which are postsynaptic to cholinergic terminals, project to the contralateral hippocampus. Together with previous studies on the cholinergic innervation of the hippocampus and fascia dentata, our present results thus demonstrate that different types of hippocampal cells, including GABAergic and peptidergic commissural neurons in the hilar region, receive a cholinergic input.     

Cholinergic amacrine cells are not required for the progression and atropine-mediated suppression of form-deprivation myopia

Muscarinic cholinergic pathways have been implicated in the visual control of ocular growth. However, the source(s) of acetylcholine and the tissue(s) which regulate ocular growth via muscarinic acetylcholine receptors (mAChRs) remain unknown. We sought to determine whether retinal sources of acetylcholine and mAChRs contribute to visually guided ocular growth in the chick. Cholinergic amacrine cells were ablated by intraocular injections of either ethylcholine mustard aziridinium ion (ECMA; a selective cholinotoxin) or quisqualic acid (QA; an excitotoxin that destroys many amacrine cells, including those that release acetylcholine). Disruption of cholinergic pathways was assessed immunocytochemically with antibodies to the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) and three different isoforms of mAChR, and by biochemical assay for ChAT activity. ECMA (25 nmol) destroyed two of the four subtypes of cholinergic amacrine cells and attenuated retinal ChAT activity, but left retinal mAChR-immunoreactivity intact. QA (200 nmol) destroyed the majority of all four subtypes of cholinergic amacrine cells, and ablated most mAChR-immunoreactivity and ChAT activity in the retina. ECMA and QA had no apparent effect on mAChRs or cholinergic fibres in the choroid, only marginally reduced choroidal ChAT activity, and had little effect on ChAT activity in the anterior segment. Toxin-treated eyes remained emmetropic and responded to form-deprivation by growing excessively and becoming myopic. Furthermore, daily intravitreal injection of 40 μg atropine for 6 days into form-deprived toxin-treated eyes completely prevented ocular elongation and myopia. We conclude that neither cholinergic amacrine cells nor mAChRs in the retina are required for visual regulation of ocular growth, and that atropine may exert its growth-suppressing influence by acting upon extraretinal mAChRs, possibly in the choroid, retinal pigmented epithelium, or sclera.     

Choline acetyltransferase immunocytochemistry of Edinger-Westphal and ciliary ganglion afferent neurons in the cat

The distribution of cholinergic neurons in the region of the cat Edinger-Westphal nucleus (EW) was determined by immunocytochemical localization of the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT). Neurons containing ChAT-like immunoreactivity (ChAT-LI) were densely distributed within EW, the anteromedian nucleus (AM), and the oculomotor nucleus (III), and were also present in immediately adjacent regions of the periaqueductal gray and ventral tegmental region. The majority of labelled neurons in EW and AM showed a markedly lower intensity of ChAT-LI than the labelled neurons in III and adjacent regions. To determine the relationship of cells with ChAT-LI to the distribution of ciliary ganglion afferent neurons, a double labelling immunocytochemistry/retrograde transport technique was also used. These experiments showed that many of the cells located outside of III that stained intensely for ChAT-LI project to ciliary ganglion. Very few ciliary ganglion afferent neurons were found in EW or AM itself; instead, the distribution of lightly labelled ChAT-LI-positive neurons in EW and AM more closely matched the known distribution of peptide-containing cells that have descending, central projections.     

Colocalization of choline acetyltransferase and gamma-aminobutyric acid in the developing and adult turtle retinas

Acetylcholine and gamma -aminobutyric acid (GABA) are putative neurotransmitters in the adult vertebrate retina. In this study, cells that coexpress choline acetyltransferase (ChAT) and GABA or glutamic acid decarboxylase (GAD) were investigated in turtle retinas from stage 14 (S14) to adulthood by using a double-labeling immunofluorescence technique. ChAT immunoreactivity was observed at S15 and included not only the presumptive starburst cholinergic amacrine cells but also a population in the ganglion cell layer (GCL) that expressed ChAT transiently during the embryonic stages (see the accompanying paper: Nguyen et al. [2000] J. Comp. Neurol. 420:512-526).     

Cholinergic and GABAergic neurons occur in both the distal and proximal turtle retina.

Turtle retinas were processed immunocytochemically and histochemically to detect the presence of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and glutamate decarboxylase (GAD). We observed cholinergic and gamma-aminobutyric acid (GABA)ergic neurons in the proximal retina, as expected, and in the distal retina as well. ChAT immunoreactivity in the distal retina was observed within the axons and pedicles of numerous cone photoreceptors, suggesting that a population of turtle cone photoreceptors uses ACh as a neurotransmitter. Type L2 horizontal cells were immunoreactive for GAD, and their dendrites invaginated into cone pedicles. AChE histochemistry revealed processes within the outer plexiform layer which formed a loosely organized lattice. In the proximal retina, labeling for ChAT and GAD was similar to that reported by previous investigators. Processes from ChAT-labeled amacrine cells in the inner nuclear layer formed a stratum within the distal inner plexiform layer (IPL) (at 16-21% relative IPL depth), and processes from ChAT-labeled amacrines in the ganglion cell layer formed a proximal ChAT stratum (at 55-58% relative IPL depth). In addition, six AChE-labeled bands and five GAD-labeled bands were observed within the IPL of stained retinas. Therefore, we determined that the two broadest AChE-labeled bands and the two broadest GAD-labeled bands overlapped the two labeled ChAT strata. The evidence for cholinergic and GABAergic processes in both the inner plexiform layer and the outer plexiform layer, combined with electrophysiological evidence from other investigators, raises the possibility that distal retinal neurons may be involved in the encoding of directional information.     

Choline acetyltransferase immunohistochemical staining in the goldfish (Carassius auratus) brain: Evidence that the Mauthner cell does not contain choline acetyltransferase

In the hatchetfish, the Mauthner cell (M-cell) is thought to be cholinergic based on electrophysiological studies using cholinergic agents and on the localization of acetylcholinesterase (AChE) and alpha-bungarotoxin to M-cell-giant fiber synapses. Immunocytochemical studies have shown that mammalian and non-mammalian cholinergic neurons stain positive for choline acetyltransferase (ChAT), the enzyme responsible for synthesizing acetylcholine. We processed tissue from the goldfish (Carassius auratus) for the immunohistochemical detection of ChAT using the monoclonal antibody AB8 and the peroxidase-antiperoxidase procedure. ChAT immunoreactivity was found in selected areas of the goldfish brain including the cranial nerve nuclei and the ventral horn motoneurons of the spinal cord. Interestingly, the M-cell soma which stains positive for AChE was ChAT negative. This immunohistochemical evidence does not support cholinergic functioning of the Mauthner cell.     

OFF-cholinergic-pathway-selective localization of P2X2 purinoceptors in the mouse retina

It is known that, in the retina, extracellular adenosine triphosphate (ATP) inhibits acetylcholine (ACh) release from cholinergic neurons, but the types of purinoceptors on cholinergic neurons have not been examined. In the present work, we immunohistochemically examined the distribution of the purinoceptors P2X1, P2X2, P2X4, and P2X7 in relation to the cholinergic system of the retina in wild-type mice and transgenic mice expressing green fluorescent protein (GFP). Immunoreactivity for P2X2 was very strong in sublamina a of the inner plexiform layer but very weak in sublamina b of the inner plexiform layer of the retina. Immunoreactivity for P2X2 was colocalized with that for choline acetyltransferase (ChAT). When transgenic mice were treated with the immunotoxin-mediated cell-targeting technology to ablate cholinergic amacrine cells selectively, immunoreactivity for P2X2 and the signals for GFP disappeared in parallel and selectively in the OFF pathway. The distribution of immunoreactivity for P2X1, P2X4, and P2X7 differed from that of ChAT immunoreactivity. The selective distribution of P2X2 purinergic receptors in OFF-type cholinergic amacrine cells indicates that the P2X2 purinergic signaling systems in the ON and OFF pathways of the inner plexiform layer of the mouse retina are functionally different. The distribution of P2X2 purinoceptors may be responsible for the selective regulation of ACh release in the OFF pathway.     

Comparison of acetylcholinesterase and choline acetyltransferase staining patterns in the optic tectum of the goldfish Carassius auratus. A histochemical and immunocytochemical analysis

Although the optic tectum of nonmammalian vertebrates has been extensively studied anatomically, there is little information about the identification of neurotransmitters and the enzymes critical to their synthesis. Choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine synthesis, is presently regarded as the most reliable marker for cholinergic neurons, and its localization within putative cholinergic neurons has been made possible by the development of antibodies specific to ChAT. We have compared the immunocytochemical localization of ChAT to the histochemical staining of acetylcholinesterase (AChE) in the goldfish optic tectum. Goldfish brains reacted with the monoclonal antibody AB8 to ChAT have revealed that: (1) type XIV neurons are the only ChAT-positive cells in the tectum, and there are approximately 15,000 such cells per tectal hemisphere; (2) these neurons and other ChAT-containing afferent fibers form bands of label which correspond to those seen after AChE staining, and (3) many AChE-stained neurons do not contain ChAT. The immunohistochemical localization of ChAT has provided a direct method for determining the localization and organization of putative cholinergic structures in the optic tectum of goldfish. Future studies may elucidate the relationship of these cholinergic systems to the retinotectal projections, as there is close correspondence between AChE and ChAT location and the retinotectal termination patterns.     

Transient,high levels of SNAP-25 expression in cholinergic amacrine cells during postnatal development of the mammalian retina

In the present study, we have examined the development of cholinergic amacrine cells in the retina of the Brazilian opossum, Monodelphis domestica. An antibody directed against choline acetyltransferase (ChAT) revealed that ChAT-like immunoreactivity (ChAT-IR) was first observed at 15 days postnatal (15PN). By 25PN, ChAT-IR identified two matching populations of amacrine cells in the inner nuclear and ganglion cell layer. Bromodeoxyuridine birth-dating analysis coupled with immunolabeling with the anti-ChAT antibody revealed that the cholinergic amacrine cells are born postnatally, between 2PN and 15PN. In addition, we have examined the differentiation of the cholinergic amacrine cells by using an antibody directed against a presynaptic terminal-associated protein, synaptosomal-associated protein of 25 kDa (SNAP-25). Double-labeling analysis revealed that relatively high levels of SNAP-25-IR were selectively present in cholinergic amacrine cells prior to eye opening. However, in the mature retina, high levels of SNAP-25-IR were no longer observed in the ChAT-IR amacrine cells. These results reveal a distinct period in development, prior to eye opening, when high levels of SNAP-25-IR are selectively expressed in cholinergic amacrine cells. The specificity and time course of the high levels of SNAP-25 in cholinergic amacrine cells may be critical in mediating the transient properties of these cells during visual system development. J. Comp. Neurol. 394:374–385, 1998. © 1998 Wiley-Liss, Inc.     

Putative cholinergic projections from the nucleus isthmi and the nucleus reticularis mesencephali to the optic tectum in the goldfish (Carassius auratus)

The nucleus isthmi of fish and amphibians has reciprocal connections with the optic tectum, and biochemical studies suggested that it may provide a major cholinergic input to the tectum. In goldfish, we have combined immunohistochemical staining for choline acetyltransferase with retrograde labeling of nucleus isthmi neurons after tectal injections of horseradish peroxidase. Seven fish received tectal horseradish peroxidase injections, and brain tissue from these animals was subsequently processed for the simultaneous visualization of horseradish peroxidase and choline acetyltransferase. In many nucleus isthmi neurons the dense horseradish peroxidase label obscured the choline acetyltransferase reaction product but horseradish peroxidase and choline acetyltransferase were colocalized in 54 cells from nine nuclei isthmi. The somata of nucleus reticularis mesencephali neurons stained so intensely for choline acetyltransferase that we could not determine whether they were labelled also with horseradish peroxidase. However, the large choline acetyltransferase-immunoreactive axons of nucleus reticularis mesencephali neurons stained intensely enough for us to follow them rostrally; the axons are clustered together until the level of the rostral tectum where two groupings form: one travels into the tectum and the other travels rostroventrally to cross the midline and enter the contralateral diencephalic preoptic area. We conclude therefore that cholinergic neurons project to the optic tectum from the nucleus isthmi as well as nucleus reticularis mesencephali in goldfish.     

Cholinergic neurons in the rabbit retina: dendritic branching and ultrastructural connectivity

The structure and synaptic connectivity of rabbit retinal cholinergic neurons have been studied with an immunocytochemical technique for the localization of choline acetyltransferase (ChAT). Cholinergic processes ramified in two narrow strata within the inner plexiform layer; viewed in the plane of the retina, each immunoreactive stratum took the form of a polygonal meshwork with openings of about 20 microns. There was frequently an extensive matching of the patterns of the two strata, such that openings in one meshwork lay directly over openings of similar size in the other meshwork. It is hypothesized that the pattern of branching of these cholinergic processes is a reflection of the branching pattern of ganglion cell dendrites in the cholinergic strata. The proximal cholinergic stratum was examined ultrastructurally, and had the following characteristics: (1) ChAT-immunoreactive processes made large numbers of synaptic contacts with ganglion cell dendrites; often there were many such ganglion cell dendrites running past each other at various angles within the plane of the cholinergic stratum; (2) cholinergic processes were never observed presynaptic to any other type of inner plexiform process; (3) the principal input onto cholinergic processes was provided by bipolar cell axon terminals; (4) ganglion cell dendrites within the cholinergic stratum also received direct bipolar input; and (5) unidentified (i.e. non-cholinergic and probably non-GABAergic) amacrine cell processes were often found that were presynaptic to these same ganglion cell dendrites, and that also formed reciprocal contacts with bipolar axon terminals within these synaptic complexes.     

Localization of choline acetyltransferase in the developing and adult turtle retinas

Acetylcholine has important epigenetic roles in the developing retina. In this study, cells that expressed choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, were investigated in embryonic, postnatal, and adult turtle retinas by using immunofluorescence histochemistry. ChAT was present at stage 15 (S15) in cells near the vitreal surface. With the formation of the inner plexiform layer (IPL) at S18, ChAT-immunoreactive (-IR) cells were located in the inner nuclear layer (INL) and the ganglion cell layer (GCL). In the INL, presumed starburst amacrine cells were homogenous in appearance and formed a single row next to the IPL: This pattern was conserved until adulthood. In the GCL, however, there were multiple rows of ChAT-IR cells early in development, and this high density of labeled cells continued during the embryonic stages, until around birth. The high density of ChAT-IR cells in the GCL was due in part to a population of cells that expressed ChAT transiently. In postnatal stages and adult retinas, the presumed starburst amacrine ChAT-IR cells formed two mirror-like rows of homogenous cells on both borders of the IPL. Two cholinergic dendritic strata that were continuous with these cells were observed as early as S18, and their depths in the IPL were relatively stable throughout development. A third population of ChAT-IR cells was observed toward the middle of the INL around S25 and persisted into adulthood. Finally, cells in the outer nuclear layer (ONL) were ChAT-IR during the embryonic stages, were less immunoreactive during the postnatal stages, and were not immunoreactive in the adult retinas.     

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.