Central neuronal loss and behavioral impairment in mice lacking neurotrophin receptor p75

The neurotrophin receptor p75 is a low-affinity receptor that binds neurotrophins. To investigate the role of p75 in the survival and function of central neurons, p75 null-mutant and wild type litter mate mice were tested on behavioral tasks. Null mutants showed significant performance deficits on water maze, inhibitory avoidance, motor activity, and habituation tasks that may be attributed to cognitive dysfunction or may represent a global sensorimotor impairment. The p75 null-mutant and wild type litter mate mice were assessed for central cholinergic deficit by using quantitative stereology to estimate the total neuronal number in basal forebrain and striatum and for subpopulations expressing the high-affinity tyrosine receptor kinase A (trkA) neurotrophin receptor and choline acetyltransferase (ChAT). In the adult brain, cholinergic neurons of the basal forebrain receive target-derived trophic support, whereas cholinergic striatal neurons do not. Adult p75 null-mutant mice had significant reduction of basal forebrain volume by 25% and had a corresponding significant loss of 37% of total basal forebrain neurons. The basal forebrain population of ChAT-positive neurons in p75-deficient mice declined significantly by 27%, whereas the trkA-positive population did not change significantly. There was no significant change in striatal volume or in striatal neuronal number either in total or by cholinergic subpopulation. These results demonstrate vulnerability to the lack of p75 in adult central neurons that are neurotrophin dependent. In addition, the loss of noncholinergic central neurons in mice lacking p75 suggests a role for p75 in cell survival by an as yet undetermined mechanism. Possible direct and indirect effects of p75 loss on neuronal survival are discussed. J. Comp. Neurol. 404:1–20, 1999. © 1999 Wiley-Liss, Inc.     

The p75 neurotrophin receptor has nonapoptotic antineurotrophic actions in the basal forebrain

Because of controversy about the role of the p75 neurotrophin receptor (p75(NTR) ) in the cholinergic basal forebrain (CBF), we investigated this region in p75(NTR) third exon knockout mice that were congenic with 129/Sv controls. They express a shortened intracellular form of p75(NTR) , permitting detection of p75(NTR) -expressing cells. We performed separate counts of choline acetyltransferase (ChAT)-expressing and p75(NTR) -expressing neurons. In agreement with past reports, the number of ChAT-immunoreactive neurons in knockout mice was greater than in wild-type mice, and this was evident in each of the main anatomical divisions of the CBF. In contrast, the number of p75(NTR) -immunoreactive neurons did not differ between genotypes. The biggest increase in ChAT neurons (27%) was in the horizontal limb of the diagonal band of Broca (HDB), in which region the number of p75(NTR) -positive neurons was unchanged. Double staining revealed that some neurons in wild-type mice expressed p75(NTR) but not ChAT. In the knockout mice, all p75(NTR) -expressing neurons expressed ChAT. The increase in cholinergic neurons, therefore, was at least partially attributable to a higher proportion of ChAT immunoreactivity within the population of p75(NTR) -expressing neurons. Cholinergic neurons were also larger in knockout mice than in controls. In the hippocampal CA1 region, knockout mice had a greater number of cholinergic fibers. There was a 77% increase in hippocampal ChAT activity in knockout mice and a 38% increase in heterozygotes. The data do not support an apoptotic role but indicate a broad antineurotrophic role of p75(NTR) in the cholinergic basal forebrain.     

Enhanced spatial memory and hippocampal long‐term potentiation in p75 neurotrophin receptor knockout mice

Previous reports have described increases in the size and number of cholinergic neurons in the basal forebrain in p75 neurotrophin receptor (p75^NTR) knockout mice. In an earlier study, we also found improved spatial memory in these mice, raising the possibility that p75^NTR regulates hippocampal function by its effects on the cholinergic basal forebrain. We therefore investigated hippocampal long‐term potentiation in p75^NTR knockout mice that shared the same genetic background as control 129/Sv mice. We also investigated heterozygous mice, carrying just one functional p75^NTR allele. The p75^NTR knockout mice had enhanced long‐term potentiation in the Schafer collateral fiber synapses of the hippocampus. Heterozygous mice had an intermediate level, greater than controls but less than knockout mice. Hippocampal choline acetyltransferase activity was also markedly elevated in p75^NTR knockout mice, with a smaller increase in heterozygous mice. In the Barnes maze, p75^NTR knockout mice displayed markedly superior learning to controls, and this was evident over the three age brackets tested. At each age, the performance of heterozygous mice was intermediate to the other groups. In the open field test, p75^NTR knockout mice exhibited greater stress‐related behavioral responses, including freezing, than did control animals. There were no differences between the three groups in a test of olfactory function. The dose‐dependent effects of p75^NTR gene copy number on hippocampal plasticity and spatial memory indicate that p75^NTR has profound effects on hippocampal function. Bearing in mind that p75^NTR is very sparsely expressed in the adult hippocampus and has a potent effect on hippocampal choline acetyltransferase activity, the effects of p75^NTR on hippocampal function are likely to be mediated indirectly, by its actions on basal forebrain cholinergic neurons. © 2009 Wiley‐Liss, Inc.     

Nerve growth factor response to excitotoxic lesion of the cholinergic basal forebrain is slightly impaired in aged rats

Nerve growth factor (NGF) promotes survival and function of basal forebrain cholinergic neurons. We studied NGF and choline acetyltransferase (ChAT) activity after partial quisqualic acid induced lesions of the basal forebrain in 3 and 27 months-old rats, in order to investigate whether NGF-related regeneration is disturbed in old age. 2 weeks post lesion, ChAT activity decreased by 25 to 32% in adult and old rats. 3 months post lesion, the ChAT deficit receded in adult rats, but remained unchanged in old rats. 2 weeks post lesion, NGF levels were reduced by 36 to 44%, but there was no significant difference between adult and old rats. 3 months post lesion, we found increased NGF levels by 44% in the posterior cortex of adult rats. These results indicate that the compensatory NGF increase in the posterior cortex after partial cholinergic lesion of the basal forebrain is slightly impaired in old age.     

Nerve growth factor induces galanin gene expression in the rat basal forebrain: Implications for the treatment of cholinergic dysfunction

Nerve growth factor (NGF) is a potential treatment for cholinergic dysfunction associated with Alzheimer's disease (AD). In rats, NGF activates gene expression of the acetylcholine synthetic enzyme choline acetyltransferase (ChAT) and prevents age- and lesion-induced degeneration of basal forebrain (BF) cholinergic neurons. Cholinergic neurons in the BF coexpress galanin (GAL), a neuropeptide that has been shown to impair performance on memory tasks possibly through the inhibition of cholinergic memory pathways. NGF up-regulates both ChAT and GAL gene expression in cultured pheochromocytoma cells; however, the effect of chronic in vivo NGF administration on GAL gene expression within the BF has not been studied. We used in situ hybridization and quantitative autoradiography to assess GAL and ChAT gene expression within the BF of adult male rats following chronic intracerebroventricular infusion of NGF or cytochrome c. We now report that, in addition to stimulating ChAT gene expression, NGF strongly up-regulated the GAL gene in the rat cholinergic BF. NGF had no effect on GAL gene expression in other noncholinergic forebrain regions. NGF induction of GAL gene expression in the BF was specific, because gene expression for another neuropeptide, neurotensin, present within noncholinergic BF neurons was unchanged. Our data provide the first evidence that in vivo NGF administration up-regulates GAL gene expression in the cholinergic BF. These results suggest that the concurrent induction of GAL in the BF could limit the ameliorating actions of NGF on cholinergic dysfunction. J. Comp. Neurol. 379;563–570, 1997. © 1997 Wiley-Liss, Inc.     

Choline acetyltransferase-like immunoreactivity in the forebrain of the red-eared pond turtle (Pseudemys scripta elegans)

Choline acetyltransferase (ChAT) immunohistochemistry was used to map the cholinergic neurons in the forebrain of Pseudemys turtles. Cell bodies with ChAT-like immunoreactivity were seen in the septum, the nucleus of the diagonal band, and embedded within the medial and lateral forebrain bundles. The region of the medial and lateral forebrain bundles contained the greatest concentration of ChAT-positive neurons. Virtually no ChAT-like immunoreactivity was seen in the areas composing the reptilian homologue of the mammalian striatum. It is suggested that the turtle basal forebrain cholinergic neurons may represent the evolutionary precursors to the mammalian cholinergic neurons of the basal forebrain and even the striatum.     

Immunohistochemical localization of choline acetyltransferase in rabbit forebrain

uinea pig antiserum specific to the purified bovine choline acetyltransferase was used to demonstrate the localization of this enzyme in rabbit forebrain by the peroxidase-antiperoxidase immunohistochemical method. Choline acetyltransferase was localized in olfactory bulb, olfactory tract, olfactory tubercle, piriform cortex, septum, diagonal band, basal ganglia, thalamus, hypothalamus, subthalamus, habenula, cerebral cortex, hippocampal region, corpus callosum, internal capsule, fornix, longitudinal striae and other areas. The findings reflect the distribution of cholinergic axons and, possibly, their terminals. These observations correlate well with biochemical determinations of choline acetyltransferase and with previously proposed cholinergic pathways.     

TRK and p75 neurotrophin receptor systems in the developing human brain

The prenatal development of the neurons immunoreactive for high-affinity tropomycin-related kinase (trk) receptor (pan trk which recognizes trkA, trkB, and trkC) and low-affinity p75 neurotrophin receptor (p75^NTR) was examined in the human brain from embryonic weeks 10 to 34 of gestation. In the embryonic week 10 specimen in which only brainstem regions were available for evaluation, trk immunoreactivity (trk-ir) was observed in the ventral cochlear, solitary, raphe, spinal trigeminal, and hypoglossal nuclei, as well as the vestibular complex and medullary reticular formation. At this time point of gestation, p75^NTR-immunoreactive (p75^NTR-ir) staining was observed within these same regions plus the inferior olivary and ambiguus nuclei. At embryonic week 14, trk-ir neurons were seen within the subplate zone of the entorhinal cortex, basal forebrain, caudate nucleus, putamen, external segment of the globus pallidus, specific thalamic nuclei, lateral mammillary nucleus, habenula nucleus, select brainstem nuclei, and the dentate nucleus of cerebellum. At this gestational time point, p75^NTR-ir neurons were observed in each of these structures, with the exception of the caudate nucleus, specific thalamic nuclei, lateral mammillary nucleus, and habenula nucleus. Additionally, p75^NTR-ir neurons were observed within the corpus callosum. The staining pattern for both trk and p75^NTR remained unchanged at embryonic weeks 15 to 16 except for the addition of trk-ir and p75^NTR-ir within the cortical subplate zone, hippocampus, and subthalamic nucleus. By embryonic week 18, trk-ir neurons were widely expressed within mostly all thalamic nuclei. In contrast, trk-ir was no longer seen within the hypoglossal, cuneate, and gracile nuclei at this time point. This staining pattern for trk and p75^NTR remained virtually unchanged from embryonic weeks 19 to 20 and embryonic weeks 16 to 20, respectively. From embryonic weeks 22 to 34, the distribution of both trk-ir and p75^NTR-ir neurons changed gradually. During this period, neurons in most thalamic and some brainstem nuclei became progressively immunonegative for trk, whereas neurons in the neocortical subplate zone, corpus callosum, and hilar region of dentate gyrus gradually lost immunoreactivity for p75^NTR. These data demonstrate an important and complex role for both the high- (trk) and low-(p75) affinity neurotrophin receptors during the development of multiple neuronal systems in the human brain. © 1996 Wiley-Liss, Inc.     

Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice

The p75 low affinity neurotrophin receptor (p75) can induce apoptosis in various neuronal and glial cell types. Because p75 is expressed in the cholinergic neurons of the basal forebrain, p75 knockout mice may be expected to show an increased number of neurons in this region. Previous studies, however, have produced conflicting results, suggesting that genetic background and choice of control mice are critical. To try to clarify the conflicting results from previous reports, we undertook a further study of the basal forebrain in p75 knockout mice, paying particular attention to the use of genetically valid controls. The genetic backgrounds of p75 knockout and control mice used in this study were identical at 95% of loci. There was a small decrease in the number of cholinergic basal forebrain neurons in p75 knockout mice at four months of age compared with controls. This difference was no longer apparent at 15 months due to a reduction in numbers in control mice between the ages of 4 and 15 months. Cholinergic cell size in the basal forebrain was markedly increased in p75 knockout mice compared with controls. Spatial learning performance was consistently better in p75 knockout mice than in controls, and did not show any deterioration with age. The results indicate that p75 exerts a negative influence on the size of cholinergic forebrain neurons, but little effect on neuronal numbers. The markedly better spatial learning suggests that the function, as well as the size, of cholinergic neurons is negatively modulated by p75.     

Expression of the low affinity neurotrophin receptor, P75NGFR, in the rat forebrain, following unilateral bulbectomy

It has been hypothesized that the main olfactory bulb, with its relatively rich source of neurotrophins, may provide trophic support for neurons that project to the bulb. We monitored expression of the common, low affinity receptor for neurotrophins, p75^NGFR, in the olfactory bulb and basal forebrain of unilaterally bulbectomized and sham-treated rats, 1–16 weeks post-surgery, using the monoclonal antibody MAb192. An induction of p75^NGFR-immunoreactivity was observed in both the glomerular and olfactory nerve layers of the right, contralateral main olfactory bulb of lesioned animals. The naturally occurring regeneration taking place in the olfactory neuroepithelium is known to be altered by olfactory bulbectomy, with subsequent changes in the sensory input to the remaining bulb. These changes in expression of p75^NGFR in the olfactory bulb support the hypothesis we have developed in previous papers, that changes in the extent of the peripheral input from the olfactory neuroepithelium to the main olfactory bulb regulate p75^NGFR expression in both the glomerular and the olfactory nerve layers. Expression of p75^NGFR in the basal forebrain of bulbectomized animals was found to be no different than sham-treated controls and does not support the hypothesis that the olfactory bulb provides trophic support to this region of the central nervous system.     

Modulation of photic resetting in rats by lesions of projections to the suprachiasmatic nuclei expressing p75 neurotrophin receptor

The suprachiasmatic nuclei of the hypothalamus (SCN) are the site of the master circadian clock in mammals. The SCN clock is mainly entrained by the light–dark cycle. Light information is conveyed from the retina to the SCN through direct, retinohypothalamic fibres. The SCN also receive other projections, like cholinergic fibres from basal forebrain. To test whether cholinergic afferents are involved in photic resetting, lesions of cholinergic projections were performed in rats with intracerebroventricular (i.c.v.) injections or intra‐SCN microinjections of 192 IgG‐saporin. When injected in the SCN, this immunotoxin destroys the cholinergic projections and retinohypothalamic afferents that express p75 low‐affinity nerve growth factor (p75^NGF) receptors. The extent of lesions in the basal forebrain and SCN was assessed by acetylcholinesterase histochemistry, p75^NGF receptor, choline acetyl‐transferase, calbindin‐D28K and VIP immunocytochemistry. The intra‐SCN treatment reduced light‐induced phase advances by 30%, and induced a complete loss of forebrain and retinal afferents expressing p75^NGF receptors within the SCN and a decrease of forebrain cholinergic neurons, most likely those projecting to the SCN. The i.c.v. treatment reduced light‐induced phase advances by 40%, increased phase delays and led to extensive damage of forebrain p75^NGF‐expressing neurons, while sparing half of the fibres expressing p75^NGF receptors (retinal afferents?) in the SCN. Because the integrity of forebrain p75^NGF‐expressing neurons appears to be critical in mediating the effects on light‐induced phase advances, we therefore suggest that anterior cholinergic projections expressing p75^NGF receptors modulate the sensitivity of the SCN clock to the phase advancing effects of light.     

Co-expression of trkA and p75 neurotrophin receptor in extracranial olfactory neuroblastoma cells

Olfactory neuroblastoma (ON, esthesioneuroblastoma) is a high-grade malignant tumour of neuronal origin. Little is known about the neurobiological behaviour of this tumour. Ten cases of ON and five cases of nasopharyngeal carcinoma were examined for expression of trkA and p75 neurotrophin receptor (p75NTR) using immunohistochemistry and double labelling fluorescence. We found that all ON tissues from 10 cases expressed both trkA and p75NTR at different levels. Double staining revealed that almost all trkA-immunoreactive ON cells also contained p75NTR immunoreactivity. By contrast, no trkA or p75NTR immunoreactivity was detected in nasopharyngeal carcinoma cells from five patients. These results suggest that nerve growth factor may play a role in the generation of ON and staining of trkA and p75NTR may assist in the diagnosis of ON.     

TrkA and mitogen-activated protein kinase phosphorylation are enhanced in sympathetic neurons lacking functional p75 neurotrophin receptor expression

This study examined the effects of hypomorphic p75 neurotrophin receptor (p75NTR) expression and high levels of nerve growth factor (NGF) on trkA phosphorylation and downstream activation of p44/42 mitogen-activated protein kinase (MAPK). Post-ganglionic sympathetic neurons from postnatal day 1 p75NTR exon III null mutant (p75(-/-)) and 129/SvJ mice were cultured in the presence of 50 ng/mL NGF and analysed by Western blotting. Levels of phosphorylated trkA are increased in p75(-/-) neurons compared with 129/SvJ neurons, and these higher levels are maintained with continuous exposure to NGF. MAPK is also phosphorylated to a greater extent in p75(-/-) neurons than in 129/SvJ neurons, both within 10 min of exposure to NGF, and with continuous NGF treatment for 5 days. These data provide new insight into the mechanism underlying enhanced neurite outgrowth in p75(-/-) neurons, demonstrating that trkA and MAPK signalling in sympathetic neurons are increased when p75NTR function is disrupted.     

Effects of ganglioside treatment in rats with a lesion of the cholinergic forebrain nuclei

The effects of GM1 ganglioside (30 mg/kg i.p.) administration for 22 days on choline acetyltransferase (ChAT) activity and noradrenaline (NA) levels in the cerebral cortex and on the acquisition of active and passive avoidance-conditioned responses were investigated in both sham-operated rats and in rats with a unilateral electrolytic lesion of the magnocellular forebrain nuclei (MFN). A statistically significant ChAT decrease in cortical areas ipsilateral to the lesion was found in saline-treated lesioned rats. In the lesioned GM1-treated rats, ChAT activity was only reduced in the frontoparietal areas and was significantly increased in the ipsilateral parietooccipital areas as well as in both contralateral regions. NA levels in the cortex were neither significantly affected by the lesion nor by GM1 treatment. The lesion impaired the acquisition of active and passive conditioned avoidance responses. GM1 treatment improved acquisition of the active avoidance response in the lesioned rats as indicated by a larger number of avoidances and a smaller number of escape failures during training in comparison with saline treatment. Ganglioside had no effect on the passive avoidance responses. These results demonstrate that GM1 administration facilitates the recovery of the cortical cholinergic system and of behavioral responses impaired by an electrolytic lesion of the cholinergic forebrain nuclei.     

In vivo knockdown of basal forebrain p75 neurotrophin receptor stimulates choline acetyltransferase activity in the mature hippocampus

This study seeks to determine whether knockdown of basal forebrain p75 neurotrophin receptor (p75^NTR) expression elicits increased hippocampal choline acetyltransferase (ChAT) activity in mature animals. Antisense (AS) oligonucleotides (oligos) targeting p75^NTR were infused into the medial septal area of mature rats continuously for 4 weeks. In all rats, the cannula outlet was placed equidistant between the left and the right sides of the vertical diagonal band of Broca. We tested phosphorothioate (PS), morpholino (Mo), and gapmer (mixed PS/RNA) oligos. Gapmer AS infusions of 7.5 and 22 μg/day decreased septal p75^NTR mRNA by 34% and 48%, respectively. The same infusions increased hippocampal ChAT activity by 41% and 55%. Increased hippocampal ChAT activity correlated strongly with septal p75^NTR downregulation in individual rats. Infusions of PS and Mo AS oligos did not downregulate p75^NTR mRNA or stimulate ChAT activity. These results demonstrate that p75^NTR can dynamically regulate hippocampal ChAT activity in the mature CNS. They also reveal the different efficacies of three diverse AS oligo chemistries when infused intracerebrally. Among the three types, gapmer oligos worked best. © 2016 Wiley Periodicals, Inc.     

Electroacupuncture in rats normalizes the diabetes‐induced alterations in the septo‐hippocampal cholinergic system

Diabetes induces early sufferance in the cholinergic septo‐hippocampal system, characterized by deficits in learning and memory, reduced hippocampal plasticity and abnormal pro‐nerve growth factor (proNGF) release from hippocampal cells, all linked to dysfunctions in the muscarinic cholinergic modulation of hippocampal physiology. These alterations are associated with dysregulation of several cholinergic markers, such as the NGF receptor system and the acetylcholine biosynthetic enzyme choline‐acetyl transferase (ChAT), in the medial septum and its target, the hippocampus. Controlled and repeated sensory stimulation by electroacupuncture has been proven effective in counteracting the consequences of diabetes on cholinergic system physiology in the brain. Here, we used a well‐established Type 1 diabetes model, obtained by injecting young adult male rats with streptozotocin, to induce sufferance in the septo‐hippocampal system. We then evaluated the effects of a 3‐week treatment with low‐frequency electroacupuncture on: (a) the expression and protein distribution of proNGF in the hippocampus, (b) the tissue distribution and content of NGF receptors in the medial septum, (c) the neuronal cholinergic and glial phenotype in the septo‐hippocampal circuitry. Twice‐a‐week treatment with low‐frequency electroacupuncture normalized, in both hippocampus and medial septum, the ratio between the neurotrophic NGF and its neurotoxic counterpart, the precursor proNGF. Electroacupuncture regulated the balance between the two major proNGF variants (proNGF‐A and proNGF‐B) at both gene expression and protein synthesis levels. In addition, electroacupuncture recovered to basal level the pro‐neurotrophic NGF receptor tropomyosin receptor kinase‐A content, down‐regulated in medial septum cholinergic neurons by diabetes. Electroacupuncture also regulated ChAT content in medial septum neurons and its anterograde transport toward the hippocampus. Our data indicate that repeated sensory stimulation can positively affect brain circuits involved in learning and memory, reverting early impairment induced by diabetes development. Electroacupuncture could exert its effects on the septo‐hippocampal cholinergic neurotransmission in diabetic rats, not only by rescuing the hippocampal muscarinic responsivity, as previously described, but also normalizing acetylcholine biosynthesis and NGF metabolism in the hippocampus.     

Ultrastructural characterization of choline acetyltransferase-containing neurons in the basal forebrain of rat: evidence for a cholinergic innervation of intracerebral blood vessels

The ultrastructural morphology and vascular associations of cholinergic neurons in the horizontal limb of the nucleus of the diagonal band of Broca (nDBBhl) and amygdala of rat were determined by the immunocytochemical localization of choline acetyltransferase (ChAT), the acetylcholine biosynthetic enzyme. Within the nDBBhl peroxidase reaction product was distributed throughout the cytoplasm of selectively labeled neuronal perikarya and dendrites. Labeled perikarya were characterized by an oval cell body (7-10 microns X 17-26 microns in diameter) in which was located a large nucleus and often a prominent nucleolus. Dendrites were by far the most numerous immuno-labeled profiles in the nDBBhl. The labeled dendrites had a cross-sectional diameter of 0.4-4.6 microns and contained numerous mitochondria and microtubules. Approximately 10% of all immunolabeled dendrites received synaptic contacts from unlabeled presynaptic boutons. In contrast to the relatively large number of ChAT-labeled dendrites within the nDBBhl, ChAT-positive axons were less frequently observed and immunolabeled axon terminals were never detected. The labeled axons had an outside diameter of 0.4-1.4 micron and were myelinated. The absence or relative paucity of immunolabeled terminals in the nDBBhl indicates that most if not all of the cholinergic perikarya within this nucleus are efferent projection neurons. The nDBB is known to have widespread projections to many areas of the neocortex, hippocampus, and amygdala. In the present study we examined the amygdala and observed many ChAT-labeled axon boutons. The immunolabeled varicosities contained numerous agranular vesicles and although ChAT-positive terminals were in direct contact with unlabeled neuronal elements within the amygdala, few if any synaptic densities were detected in a single plane of section. With respect to the vasculature, immunolabeled perikarya and dendrites within the nDBBhl and axon terminals in the amygdala were often in direct apposition to blood vessels. In many instances the labeled profile was observed lying directly on the basal lamina of a capillary endothelial cell. In no instance, however, were membrane densities observed. The presence of cholinergic neuronal elements contacting the vessel wall provides morphologic evidence suggesting that the neurogenic control of cerebral vasculature is in part mediated via a cholinergic mechanism.     

Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions

Nerve growth factor (NGF) was injected intraventricularly during 4 weeks into adult rats with unilateral partial lesions of the cholinergic septo-hippocampal pathway. On the lesioned side, NGF treatment elevated choline acetyltransferase (ChAT) activity up to 60% above the activity measured on the lesioned side of cytochrome c-treated controls. On the unlesioned side, NGF treatment increased ChAT activity only to an insignificant degree. ChAT activity in the septum of NGF-treated animals was increased by 60% as compared to controls. The NGF-induced increases on the lesioned side and in the septum were not accompanied by elevations in acetylcholinesterase (AChE) activity. Furthermore, histochemical analysis revealed no difference in AChE staining pattern or intensity between NGF-treated and control animals. The lack of effect on AChE strongly suggests that the increases in ChAT activity in hippocampus and septum are due to an elevation of ChAT activity within cholinergic neurons surviving the lesion rather than to a promotion of sprouting of cholinergic fibers.     

Basic fibroblast growth factor (bFGF) increases the survival of embryonic and postnatal basal forebrain cholinergic neurons in primary culture

Basic fibroblast growth factor (bFGF) is found in high concentrations in the mammalian central nervous system. It is a mitogen for glia and it influences the development and survival of specific populations of neurons. In this study, we investigated the effect of various concentrations of bFGF on the survival of embryonic and postnatal cholinergic basal forebrain neurons plated at low and high density in the presence and absence of glia. We observed that 50 and 100 ng/ml of bFGF increased the survival of embryonic cholinergic neurons plated at high density. This effect was observed only in the presence of glia. Lower concentrations of 10 and 20 ng/ml had no effect on cholinergic neuronal survival. The number of GFAP (glial fibrillary acidic protein)-positive cells in high-density embryonic cultures was increased by all concentrations of bFGF. In low-density embryonic cultures, an increase in cholinergic neuron survival was observed at concentrations ranging from 20 to 100 ng/ml. The number of GFAP-positive cells in low-density cultures was also increased by all concentrations of bFGF. Similar to low-density embryonic cultures, the survival of cholinergic neurons from postnatal day 2 cultures was significantly increased in the presence of glia at concentrations of 20, 50 and 100 ng/ml of bFGF. Postnatal glia was affected by all concentrations of bFGF, as was observed in embryonic cultures. This study indicates that high concentrations of bFGF can influence cholinergic neuronal survival by stimulating and increasing glia, which may produce factor(s) that are necessary for cholinergic neuron survival.     

Retinoic acid and nerve growth factor induce differential regulation of nicotinic acetylcholine receptor subunit expression in SN56 cells

Retinoic acid (RA) and nerve growth factor (NGF) have multiple functions in the regulation of neuronal development. In the present study, we characterized the expression of different nicotinic acetylcholine receptor (nAChR) subtypes in the cholinergic SN56 cell line and investigated the roles of RA and NGF in the expression of choline acetyltransferase (ChAT) and different nAChR subtypes. The nAChR agonist [(3)H]epibatidine was bound to two sites, with apparent affinities of 13 and 380 pM. RT-PCR analysis revealed expression of alpha3, alpha4, alpha5, alpha7, beta2, and beta4 nAChR subunits. RA treatment induced morphological changes, and the mRNA level of ChAT was maximally elevated after 4 days of exposure. The density of [(3)H]epibatidine binding sites and the mRNA and protein level of the alpha3 and beta2 nAChR subunits were also increased by RA-induced differentiation. RA down-regulated the mRNA and protein level of the alpha4 nAChR subunit, whereas no significant change was observed in the mRNA and protein level of the alpha7 nAChR subunit. NGF treatment increased the mRNA and protein level of the alpha3 and beta2 nAChR subunits. No morphological effects of NGF were observed, and the mRNA level of ChAT and mRNA and protein level of the alpha4 and alpha7 nAChR subunits were not significantly altered. Validation was performed with real-time RT-PCR. The present results show that RA and NGF have different effects on the expression of ChAT and the morphology and the expression pattern of different nAChR subunits in cholinergic SN56 cells.