Changes In Cholinergic Neurons Research Articles

Characteristics and Transcriptomic Analysis of Cholinergic Neurons Derived from Induced Pluripotent Stem Cells with APP Mutation in Alzheimer's Disease.

The cholinergic hypothesis is one of the main theories that describe the pathogenesis of Alzheimer's disease (AD). Cholinergic neurons degenerate early and are severely damaged in AD. Despite extensive research, the causes of cholinergic neuron damage and the underlying molecular changes remain unclear. This study aimed to explore the characteristics and transcriptomic changes in cholinergic neurons derived from human induced pluripotent stem cells (iPSCs) with APP mutation. Peripheral blood mononuclear cells from patients with AD and healthy individuals were reprogrammed into iPSCs. The iPSCs were differentiated into cholinergic neurons. Cholinergic neurons were stained, neurotoxically tested, and electrophysiologically and transcriptomically analyzed. The iPSCs-derived cholinergic neurons from a patient with AD carrying a mutation in APP displayed enhanced susceptibility to Aβ1-42-induced neurotoxicity, characterized by severe neurotoxic effects, such as cell body coagulation and neurite fragmentation. Cholinergic neurons exhibited electrophysiological impairments and neuronal death after 21 days of culture in the AD group. Transcriptome analysis disclosed 883 differentially expressed genes (DEGs, 420 upregulated and 463 downregulated) participating in several signaling pathways implicated in AD pathogenesis. To assess the reliability of RNA sequencing, the expression of 16 target DEGs was validated using qPCR. Finally, the expression of the 8 core genes in different cell types of brain was analyzed by the AlzData database. In this study, iPSCs-derived cholinergic neurons from AD patients with APP mutations exhibit characteristics reminiscent of neurodegenerative disease. Transcriptome analysis revealed the corresponding DEGs and pathways, providing potential biomarkers and therapeutic targets for advancing AD research.

Impaired physiological responses and neurotoxicity induced by a chlorpyrifos-based formulation in Caenorhabditis elegans are not solely dependent on the active ingredient

The current massive and indiscriminate agrochemicals usage, which is inexorably linked to the toxic consequences to the environment and people, represents a great concern. Our work aimed to compare the toxicity induced by chlorpyrifos in its pure form (CPF) with that of a commercial formulation containing allegedly inert ingredients (CBCF) using Caenorhabditis elegans as in vivo model. After a 48 h exposure period, CBCF was 14 times more lethal than CPF; Hatching, brood size, body length and motor-related behavioral parameters were decreased, but these effects were significantly higher in CBCF-exposed worms. Additionally, CBCF induced significant morphological changes in cholinergic neurons, which are associated with the motor-related behavioral parameters. Finally, by analyzing the CBCF, we detected the presence of potentially-toxic metals that were not specified in the label. The presented results highlight the toxicological relevance of components present in the commercial formulations of pesticides, which have been claimed as inert compounds.

Neuropeptide-S prevents 6-OHDA-induced gastric dysmotility in rats

This study aims to explore the effect of chronic central neuropeptide-S (NPS) treatment on gastrointestinal dysmotility and the changes of cholinergic neurons in the dorsal motor nucleus of the vagus (DMV) of a Parkinson’s disease (PD) rat model. The PD model was induced through a unilateral medial forebrain bundle (MFB) administration of the 6-hydroxydopamine (6-OHDA). Locomotor activity (LMA), solid gastric emptying (GE), and gastrointestinal transit (GIT) were measured 7 days after the surgery. NPS was daily administered (1 nmol, icv, 7 days). In substantia nigra (SN), dorsal motor nucleus of the vagus (DMV), and gastric whole-mount samples, changes in tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), glial fibrillary acidic protein (GFAP), NPS receptor (NPSR), and alpha-synuclein (Ser129) were examined by immunohistochemistry. Cuprolinic blue staining was used to evaluate the number of neuronal cells in myenteric ganglia. The GIT rate, the total number of myenteric neurons, and the expressions of ChAT, nNOS, TH, and GFAP in the myenteric plexus were not changed in rats that received the 6-OHDA. Chronic NPS treatment reversed 6-OHDA-induced impairment of the motor performance, and GE, while preventing the loss of dopaminergic and cholinergic neurons in SN and DMV, respectively. NPS attenuated 6-OHDA-induced α-syn (Ser129) pathology both in SN and DMV. Additionally, expression of NPSR protein was detected in gastro-projecting cells in DMV. Taken together, centrally applied NPS seems to prevent 6-OHDA-induced gastric dysmotility through a neuroprotective action on central vagal circuitry.

Quercetin protects against the Aβ25–35-induced amnesic injury: Involvement of inactivation of RAGE-mediated pathway and conservation of the NVU

Quercetin has demonstrated protective effects against Aβ-induced toxicity on both neurons and endothelial cells. However, whether or not quercetin has an effect on the neurovascular coupling is unclear. In the present study, we aim to investigate the anti-amnesic effects of quercetin and to explore the underlying mechanisms. Aβ25–35 (10 nmol) was administrated to mice i.c.v. Quercetin was administrated orally for 8 days after injection. Learning and memory behaviors were evaluated by measuring spontaneous alternation in Morris Water Maze test and the step-through positive avoidance test. The regional cerebral blood flow was monitored before the Aβ25–35 injection and on seven consecutive days after injection. Mice were sacrificed and cerebral cortices were isolated on the last day. The effects of quercetin on the neurovascular unit (NVU) integrity, microvascular function and cholinergic neuronal changes, and the modification of signaling pathways were tested. Our results demonstrate that quercetin treatment for Aβ25–35-induced amnesic mice improved the learning and memory capabilities and conferred robust neurovascular coupling protection, involving maintenance of the NVU integrity, reduction of neurovascular oxidation, modulation of microvascular function, improvement of cholinergic system, and regulation of neurovascular RAGE signaling pathway and ERK/CREB/BDNF pathway. In conclusion, in Aβ25–35-induced amnesic mice, optimal doses of quercetin administration were beneficial. Quercetin protected the NVU likely through reduction of oxidative damage, inactivation of RAGE-mediated pathway and preservation of cholinergic neurons, offering an alternative medication for Alzheimer's disease.

Dual Radiotracer Analysis of Cholinergic Neuronal Changes in Prediabetic Mouse Pancreas

Pancreatic neuronal changes associated with beta cell loss in type 1 diabetes mellitus are complex, involving, in part, parasympathetic mechanisms to compensate for preclinical hyperglycemia. The parasympathetic neurotransmitter acetylcholine (ACh) mediates insulin release via M3 muscarinic receptors on islet beta cells. The vesicular ACh transporter (VAChT) receptor has been shown to be a useful marker of cholinergic activity in vivo. The positron emission tomography (PET) radiotracer (+)-4-[(18)F]fluorobenzyltrozamicol ([(18)F]FBT) binds to the VAChT receptor on presynaptic cholinergic neurons and can be quantified by PET. The compound 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), available in a tritiated form, binds to M3 muscarinic receptors on beta cells and is a potential target for assessing pancreatic beta cell mass. In this study, we investigate the feasibility of dual radiotracer analysis in identifying neurofunctional changes that may signify type 1 diabetes mellitus in its early preclinical state. Ex vivo determinations of pancreatic uptake were performed in prediabetic nonobese diabetic mice and controls after intravenous injection of [(18)F]FBT or 4-[(3)H]DAMP. Beta cell loss in prediabetic mice was confirmed using immunohistochemical methods. [(18)F]FBT uptake was significantly higher in prediabetic pancreata than controls: 3.22 +/- 0.81 and 2.51 +/- 1.04, respectively (P < 0.03). 4-[(3)H]DAMP uptake was significantly lower in prediabetic pancreata than controls: 0.612 +/- 0.161 and 0.968 +/- 0.364, respectively (P = 0.01). These data suggest that a combination of radiotracer imaging agents that bind to neuronal elements intimately involved in insulin production may be an effective method of evaluating changes associated with early beta cell loss using PET.

Fibre‐free diet leads to impairment of neuronally mediated muscle contractile response in rat distal colon

Dietary fibre consumption is known to be beneficial to increase stool bulk and frequency. In contrast, it is unclear whether chronic dietary fibre deficiency affects colonic motor functions, especially neuronally mediated muscle contractions. In this study, rats were fed a fibre-free diet or diet containing dietary fibre (cellulose or guar gum) for 27 days. Furthermore, neurogenic and myogenic contractions were evaluated in circular and longitudinal muscle strips of the distal colon. Additionally, the number of enterochromaffin (EC) cells, which play important roles in the initiation of the peristaltic reflex, was also examined by immunohistochemistry for serotonin. Myogenic contractions induced by carbachol or substance P were examined in the presence of tetrodotoxin. Circular muscle was hyposensitive to carbachol, but longitudinal muscle was hypersensitive to substance P in the fibre-free group. Nerve-mediated circular (5-20 Hz) and longitudinal (1-2 Hz) muscle contractions evoked by electrical field stimulation were attenuated in the fibre-free group and the latter response was almost abolished by atropine, suggesting functional changes of cholinergic neurons. EC cell number was decreased in the fibre-free group. In conclusion, changes in neurogenic and myogenic contractions and a decrease in EC cell number observed may affect colonic motility of the fibre-free group.

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.

Age-related changes in cholinergic neurons in the laterodorsal and the pedunculo-pontine tegmental nuclei of cats: A combined light and electron microscopic study

In aged cats, light microscopic studies revealed significant decrease in the soma size of choline acetyltransferase (ChAT)-positive neurons in the laterodorsal and pedunculo-pontine tegmental nuclei (LDT and PPT), compared with adult control animals. In addition, a significant reduction of the total dendritic length and total dendritic segment number of ChAT-positive neurons was detected in both the LDT and PPT of aged cats. However, in contrast to the changes of soma and dendrites, no significant changes in the number of ChAT-positive neurons in aged were found comparing to that in the control cats in both the LDT and PPT; nor were there differences in the staining intensity of the somata of neurons in the adult and aged cats. Electron microscopic analysis highlighted degenerative changes in cholinergic neurons in the LDT and PPT of aged cats which included somata with intracytoplasmic vacuoles, darkened mitochondria, depletion of dendritic microtubules and severe demyelination of axons. These data indicate that profound atrophic changes occur in cholinergic systems of the LDT and PPT as a consequence of the aging process. These alterations likely reflect the cellular bases for the age-related changes in REM sleep that occur in old animals.

Involvement of protein kinase C in the adaptive changes of cholinergic neurons to sympathetic denervation in the guinea pig myenteric plexus

Supersensitivity to muscarinic, κ- and μ-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (κ-opioid receptor agonist) and DAMGO (μ-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, κ- and μ-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas κ- and μ-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed.

Association of learning and memory impairments with changes in the septohippocampal cholinergic system in rats with kaolin-induced hydrocephalus.

The septohippocampal cholinergic (SHC) system plays an important role in the maintenance of normal memory and learning. However, the fact that memory and learning impairments under hydrocephalic conditions are directly related to the SHC system is less well known. We investigated the relationships between pathological changes in SHC neurons and impairments in memory and learning among hydrocephalic rats. Rats with kaolin-induced hydrocephalus were prepared with injections of kaolin suspension into the cisterna magna. Learning and memory performance was assessed with the passive avoidance and Morris water maze tests. Ventricular sizes were measured for the lateral and third ventricles. Acetylcholinesterase and choline acetyltransferase immunostaining was performed to investigate degenerative changes in cholinergic neurons in the medial septum and hippocampus. Hydrocephalic rats demonstrated significant learning and memory impairments in the passive avoidance and Morris water maze tests. Decreased hesitation times in the passive avoidance test and markedly increased acquisition times and decreased retention times in the Morris water maze test indicated learning and memory dysfunction among the hydrocephalic rats. The numbers of cholinergic neurons in the medial septum and hippocampus were decreased in the hydrocephalic rats. The decreases in choline acetyltransferase and acetylcholinesterase immunoreactivity were significantly correlated with enlargement of the ventricles. Impairment of spatial memory and learning may be attributable to degeneration of SHC neurons. These results suggest that learning and memory impairments in rats with kaolin-induced hydrocephalus are associated with the dysfunction of the SHC system induced by ventricular dilation.

Muscarinic1 and 2 receptor mRNA in the human caudate-putamen: no change in m1 mRNA in schizophrenia.

Studies using tissue obtained at autopsy suggest that changes in cholinergic neurons could be important in the pathology of schizophrenia.1-4 We have previously reported a decrease in [3H]pirenzepine binding5 and [3H]AF-DX 384 binding6 to caudate-putamen (CP) from subjects who had schizophrenia. Under the conditions chosen, [3H]pirenzepine would predominately bind to muscarinic1 (M1) and muscarinic4 (M4) receptors,7whereas [3H]AF-DX 384 would mainly bind to muscarinic2 (M2) and M4 receptors.8 Given the relative concentrations of M1, M2 and M4 receptors in the human CP and the magnitude of the decreases in radioligand binding in schizophrenia, our results most likely reflected a change in the density of M1 and M2 receptors in the CP from the schizophrenic subjects. In situ hybridisation has now been used to determine levels of m1 and m2 mRNA in CP from 14 schizophrenic and 16 control subjects previously used for radioligand binding. m2 mRNA in the CP from the schizophrenic and control subjects was below the sensitivity of in situhybridisation. There was no difference in the levels of m1 mRNA in CP from schizophrenic and control subjects (mean +/- SEM: 103 +/- 16 vs106 +/- 17 fmol [35S]oligonucleotide probe g-1estimated tissue equivalents, P = 0.91). In conclusion, data from our radioligand binding studies show decreases in [3H]pirenzepine binding that are likely to reflect a decrease in the density of M1 receptors in CP from schizophrenic subjects. Our data in this study show the absence of a concomitant change in mRNA coding for that receptor.

Changes in Cholinergic Neurons and Failure in Learning Function After Microsphere Embolism-Induced Cerebral Ischemia

Central cholinergic neurons play an important role in learning and memory functions. The present study was undertaken to elucidate the pathological changes in learning function and acetylcholine metabolism of the cerebral cortex and hippocampus, following microsphere embolism in rats. Microspheres (48 μm) were injected into the right internal carotid artery of the rats. Learning function was determined using a passive avoidance task on the seventh day after the embolism. In the biochemical study, acetylcholine and choline contents, and choline acetyltransferase activity were measured in the cerebral cortex and hippocampus. Cortical acetylcholinesterase-containing fibers were quantitatively estimated in the embolized rat. Passive avoidance was impaired in the microsphere-embolized rat. Microsphere embolism decreased the acetylcholine concentration and choline acetyltransferase activity in the cerebral cortex and hippocampus. In the histochemical study, the length of cortical acetylcholinesterase-containing fibers was decreased, but cell density was unchanged in the ipsilateral hemisphere of the microsphere-embolized rat. The results suggest that microsphere embolism induces severe damage to cholinergic neurons, which may be related to the impairment of learning function in the ischemic brain.

Serotonergic modulation of cholinergic function in the central nervous system: cognitive implications.

Accumulating evidence suggests that serotonin may modulate cholinergic function in several regions of the mammalian brain and that these serotonergic/cholinergic interactions influence cognition. The first part of this review is an overview of histological, electrophysiological and pharmacological (in vitro, in vivo) data indicating that, in several brain regions (e.g., hippocampus, cortex and striatum), there are neuroanatomical substrates for a serotonergic/cholinergic interaction, and that alterations in serotonergic activity may induce functional changes in cholinergic neurons. In the second part, the review focuses on experimental approaches showing or suggesting that central cholinergic and serotonergic mechanisms are cooperating in the regulation of cognitive functions. These arguments are based on lesion, intracerebral grafting and pharmacological techniques. It is concluded that not all mnesic perturbations induced by concurrent manipulations of the serotonergic and cholinergic systems can be attributed to a serotonergic modification of the cholinergic system. The cognitive faculties of an organism arise from interactions among several neurotransmitter systems within brain structures such as, for instance, the hippocampus or the cortex, but also from influences on memory of other general functions that may involve cerebral substrates different from those classically related to mnesic functions (e.g., attention, arousal, sensory accuracy, etc.).

Nerve growth factor prevents apoptotic cell death in injured central cholinergic neurons.

Experimental lesions have been widely used to induce neuronal degeneration and to test the ability to trophic molecules to prevent lesion-induced alterations, but these studies have not demonstrated unequivocally that afflicted neurons die as a result of these manipulations. The documentation of neuronal death in the above-described models and the time when it occurs after injury are crucial for the interpretation of trophic effects. In the present study, we combined multiple approaches to investigate the nature of retrograde neuronal changes in cholinergic neurons of the medial septal nucleus (MSN) after complete, unilateral transection of the fimbria-fornix (F-F). Projections neurons of the MSN were prelabeled with the fluorescent tracer Fluoro-gold (FG) 1 week prior to lesion. By counting both FG-labeled and choline acetyltransferase (ChAT)-immunoreactive neurons in the MSN at multiple time points postaxotomy, we differentiated the phenotypic response to injury from the degenerative process and established a critical time between the third and fourth weeks postaxotomy, during which approximately 50% of fluorescent perikarya disappear. Working in the previous time window, we identified dying cells by electron microscopy (EM) and terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) and showed that MSN neurons die via apoptosis, beginning at 16 days postaxotomy. An additional group of animals was allowed to survive for 1 month (i.e., 10 days after cell death has been completed); during this period, animals were treated with intraventricular nerve growth factor (NGF). Quantitative analysis of surviving cholinergic perikarya showed that NGF prevented degeneration of the majority of neurons. In concert, the results of the present study establish that NGF does not merely protect the phenotype but also prevents cell death in lesioned central cholinergic neurons.

Encapsulated genetically engineered fibroblasts: Release of nerve growth factor and effects in vivo on recovery of cholinergic markers after devascularizing cortical lesions

Genetically engineered rat fibroblasts producing nerve growth factor (NGF) were encapsulated in alginate-polylysine-alginate gels with the objective to produce viable “minifactories” continuously producing and secreting NGF into the rat brain. Microencapsulated fibroblasts (NGF secretors and NGF non-secretors) were placed onto the surface of the lesioned rat cortex (unilateral devascularizing lesion) and animals were sacrificed 30 days after surgery. Fibroblasts NGF-non secreters normally produce tumors after implantation, therefore, they were irradiated prior to encapsulation. Three other experimental groups were studied in parallel: non-lesioned (controls), lesioned rats receiving “empty” alginate spheres and lesioned animals without treatment and microspheres. Biochemical analysis of microdissected brain tissues of lesioned animals treated with encapsulated NGF-secretor fibroblasts showed a significant increase in choline acetyltransferase (ChAT) activity in cortices adjacent to the lesion but not far from it (entorhinal cortex). This may indicate a gradient of concentration of the released NGF and/or differential responsivity of lesioned vs nonlesioned target tissue. ChAT enzymatic activity in the microdissected nucleus basalis magnocellularis (NBM) was significantly decreased ( P < 0.05) in all lesioned animals treated with spheres without fibroblasts and those with fibroblasts not secreting NGF. Morphometric analysis of ChAT-IR and low affinity NGF-receptor IR cholinergic neurons in the middle portion of the NBM shows a prevention of neuronal shrinkage and extensive neuropil in animals treated with microencapsulated NGF-secretor fibroblasts. The results of this study demonstrate that NGF from encapsulated genetically engineered fibroblasts can be secreted for at least long enough to prevent degenerative changes of cholinergic neurons in the NBM.

Long-term protective effects of human recombinant nerve growth factor and monosialoganglioside GM1 treatment on primate nucleus basalis cholinergic neurons after neocortical infarction

Infarction induces biochemical and morphological retrograde degenerative changes in cholinergic neurons of the rat nucleus basalis magnocellularis [Sofroniew et al. (1983) Brain Res. 289, 370–374]. In the present study, this lesion model has been reproduced in the non-human primate ( Cercopithecus aethiops) to investigate whether degenerative changes affecting the cortex surrounding the lesioned area and the ipsilateral basal forebrain are prevented by the early administration of recombinant human nerve growth factor alone or in combination with the monosialoganglioside GM1. Six months after surgery and treatment, the monkeys were processed either for biochemistry (choline acetyltransferase assay) or immunocytochemistry. In lesioned vehicle-treated animals, choline acetyltransferase activity significantly decreased by 28% in the cortex surrounding the injured area and by 31% in the ipsilateral nucleus basalis of Meynert when compared with values of sham-operated monkeys. These biochemical changes were fully prevented with the administration of nerve growth factor alone or in combination with the monosialoganglioside GM1. The morphometrical analysis revealed a significant shrinkage of cholinergic neurons (61 ± 1.4% of sham-operated cell size) and loss of neuritic processes (59 ± 10% of sham-operated values) within the intermediate nucleus basalis region of lesioned vehicle-treated animals. Although a protection of the cholinergic cell bodies within the nucleus basalis was found with both treatments, a significant recovery of the neuritic processes (84 ± 7.2% of sham-operated values) was assessed only in the double-treated monkeys. These results indicate that the early administration of nerve growth factor alone or in combination with the monosialoganglioside GM1 induces a long-term protective effect on the nucleus basalis cholinergic neurons in cortical injured non-human primates.

Pharmacology of Nerve Growth Factor in the Brain

This chapter discusses the pharmacology of nerve growth factor (NGF) in the brain. Neurotrophic factors regulate survival and growth of neurons, suggesting that these molecules may become useful in the treatment of neurodegeneration. NGF is the best-characterized neurotrophic factor and serves as paradigmatic example for other proteins. In the brain, NGF selectively acts on forebrain cholinergic neurons, which are involved in cognitive functions and degenerate in Alzheimer's disease. Detailed information is available on the pharmacologic effects of NGF in the brain of animals with experimental lesions of cholinergic systems. These studies indicate that NGF administration is able to counteract degenerative changes of cholinergic neurons and to stimulate presynaptic function of surviving cholinergic axons. These changes translate into functional changes at the postsynaptic level and associated behavioral functions.

Recombinant human nerve growth factor prevents retrograde degeneration of axotomized basal forebrain cholinergic neurons in the rat

Cholinergic neurons in the basal forebrain magnocellular complex (BFMC) respond to nerve growth factor (NGF) during development and in adult life, and it has been suggested that the administration of NGF might ameliorate some of the abnormalities that occur in neurological disorders associated with degeneration of this population of neurons. A prerequisite for the introduction of NGF in clinical trials is the availability of active recombinant human NGF (rhNGF). The present investigation was designed to test, in vivo, the efficacy of a preparation of rhNGF. Axons of cholinergic neurons of the BFMC in the rat were transected in the fimbria-fornix; this manipulation alters the phenotype and, eventually, causes retrograde degeneration of these neurons. Our investigation utilized two lesion paradigms (resection and partial transection of fibers in the fimbria-fornix), two different strains of rats, and two delivery systems. Following lesions, animals were allowed to survive for 2 weeks, during which time one group received intraventricular mouse NGF (mNGF), a second group received rhNGF, and a third group received vehicle alone. In animals receiving vehicle, there was a significant reduction in the number (resection: 70%; transection: 50%) and some reduction in size of choline acetyltransferase- or NGF receptor-immunoreactive cell bodies within the medial septal nucleus ipsilateral to the lesion. Treatment with either mNGF or rhNGF completely prevented these alterations in the number and size of cholinergic neurons. The rhNGF was shown to be equivalent in efficacy with mNGF. Thus, rhNGF is effective in preventing axotomy-induced degenerative changes in cholinergic neurons of the BFMC. Our results, taken together with the in vitro effects of rhNGF (42), indicate that an active rhNGF is now available for further in vivo studies in rodents and primates with experimentally induced or age-associated lesions of basal forebrain cholinergic neurons. These investigations provide essential information for the consideration of future utilization of rhNGF for treatment of human neurological disorders, including Alzheimer's disease.

Effects of treatment with microencapsulated monosialoganglioside GM1 on cortical and striatal acetylcholine release in rats with cortical devascularizing lesions

The present study shows a novel administration form of the monoganglioside GM1, which following microencapsulation in human serum albumin was topically applied on cortical regions damaged by devascularization in rats. The effects of microencapsulated GM1 on extracellular levels of acetylcholine, choline and dopamine in the cortex and in the striatum were analyzed using in vivo microdialysis. Cholinergic neurons in the nucleus basalis magnocellularis were studied immunohistochemically using monoclonal antibodies raised against choline acetyltransferase (ChAT). It was found that cortical devascularizing lesions produced a decrease in extracellular levels of cortical acetylcholine and choline, and retrograde morphological changes in cholinergic neurons in the nucleus basalis magnocellularis. GM1 promoted (1) recovery of the retrograde morphological changes produced by the decortication in the nucleus basalis magnocellularis and (2) a parallel increase in cortical acetylcholine release. No changes were observed in the striatum, nor on cortical or striatal dopamine levels simultaneously measured in the same perfusates.

Functional alterations in striatal cholinergic and striato-nigral gabaergic neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration

Neurochemical changes in cholinergic neurons in the striatum and ?-aminobutyric acid (GABA)ergic neurons in the midbrain, especially in the basal ganglia, have been examined following the intraperitoneal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is known to cause behavioral changes resembling those in Parkinson's disease due to the destruction of nigro-striatal dopaminergic neurons. Although the adminstration of MPTP (50 mg/kg, once a day for 5 days) to male mice of dd strain did not induce the changes in choline and acethylcholine contents in the striatum, a decrement of GABA content in the midbrain, especially in the substantia nigra, was found in addition to a significant decrease of dopamine content in the striatum. This decrement in GABA content was accompanied by a decrease of turnover rate of GABA in the substantia nigra. Furthermore, it was found that MPTP treatment induced an increase of the GABA(A) receptor-benzodiazepine receptor-chloride channel complex in the substantia nigra in addition to that of dopaminergic D(2) receptor in the striatum. Although the MPTP treatment did not induce significant changes in acetylcholinesterase and choline acetyltransferase activities as well as high affinity choline uptake, this treatment induced a decrease of [(3)H]quinuclidinyl benzilate binding to muscarinic receptor in the striatum. These results suggest that the occurrence of behavioral changes induced by MPTP administration may involve functional alterations in cholinergic interneuron in the striatum as well as those in the striato-nigral GABAergic neuron, which are associated with the destruction of nigro-striatal dopaminergic neuron with this agent.