Loss Of Choline Acetyltransferase Activity Research Articles

Neurochemistry of Alzheimer's disease

Investigation of neurochemical correlates of Alzheimer's disease (AD) has led to the development of current treatment strategies. The cholinergic system has profound loss of the acetylcholine synthesis enzyme choline acetyltransferase (ChAT) and neurons in the forebrain's nucleus basalis of Meynert (origin of major cortical cholinergic projections). Loss of ChAT activity is the most consistent finding in post-mortem AD brains and the degree of cognitive impairment has been found to correspond with the extent of deficit. Reductions in acetylcholine release, muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors are other cholinergic features of AD; this accumulating evidence has led to the ‘cholinergic hypothesis’ of dementia. There are indications that loss of glutamate activity also correlates with cognitive impairment. Changes in monoamine activity are implicated in the behavioural symptoms of AD. The principal therapeutic agents in AD are cholinesterase inhibitors, which inhibit acetylcholine breakdown. Further treatment targets include mAChRs, nAChRs and glutamate receptors.

Lipid-Mediated In Vivo Gene Transfer Replaces the Loss of Choline Acetyltransferase Activity after Unilateral Fimbria-Fornix Aspiration

In Alzheimer's disease cholinergic neurons degenerate, resulting in loss of hippocampal acetylcholine. The fimbria-fornix aspiration is a well-known animal model mimicking hippocampal cholinergic deficiency. The aim of the present study was to use in vivo lipid-mediated gene transfer to introduce an expression vector coding for the acetylcholine synthesizing enzyme choline acetyltransferase into the hippocampus to replace the loss of enzyme activity after unilateral fimbria-fornix aspiration. Our data show that the lipid FuGene is useful to transfer DNA in vitro into 3T3 fibroblasts, C6 glioma cells, and primary astroglia and to express the respective enzyme. Lipid-mediated gene transfer in vivo resulted in a marked but transient expression of green fluorescent protein below the injection site peaking 5 days after the injection. Unilateral fimbria-fornix aspiration led to a marked reduction in the activity of choline acetyltransferase in the hippocampus, which was completely replaced 5 days after lipid-mediated gene transfer of the choline acetyltransferase vector. In conclusion, our data provide evidence that lipid-mediated gene transfer using FuGene is a useful tool to replace loss of choline acetyltranseferase activity in the hippocampus after fimbria-fornix aspiration; however, the lack of good gene transfer efficiency and the transient nature of expression limit its use for clinical applications.

Huperzine A attenuates cognitive dysfunction and neuronal degeneration caused by β-amyloid protein-(1–40) in rat

Huperzine A, a promising therapeutic agent for Alzheimer's disease, was examined for its potential to antagonize the deleterious neurochemical, structural, and cognitive effects of infusing β-amyloid protein-(1–40) into the cerebral ventricles of rats. Daily intraperitoneal administration of huperzine A for 12 consecutive days produced significant reversals of the β-amyloid-induced deficit in learning a water maze task. This treatment also reduced the loss of choline acetyltransferase activity in cerebral cortex, and the neuronal degeneration induced by β-amyloid protein-(1–40). In addition, huperzine A partly reversed the down-regulation of anti-apoptotic Bcl-2 and the up-regulation of pro-apoptotic Bax and P53 proteins and reduced the apoptosis that normally followed β-amyloid injection. The present findings confirm that huperzine A can alleviate the cognitive dysfunction induced by intracerebroventricular infusion of β-amyloid protein-(1–40) in rats. The beneficial effects are not confined to the cholinergic system, but also include favorable changes in the expression of apoptosis-related proteins and in the extent of apoptosis in widespread regions of the brain.

Role of nitric oxide in the ethylcholine aziridinium model of delayed apoptotic neurodegeneration in vivo and in vitro

The involvement of nitric oxide in neurodegenerative processes still remains incompletely characterized. Although nitric oxide has been reported to be an important mediator in neuronal degeneration in different models of cell death involving NMDA-receptor activation, increasing evidence for protective mechanisms has been obtained. In this study the role of nitric oxide was investigated in a model of NMDA-independent, delayed apoptotic cell death, induced by the neurotoxin ethylcholine aziridinium ethylcholine aziridinium both in vivo and in vitro. For the in vivo evaluation rats received bilateral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ventricle) or vehicle. In the hippocampus a transient decrease in nitric oxide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7±9.8% of controls). The decrease coincided with the maximal reduction in choline acetyltransferase activity as marker for the extent of cholinergic lesion. The effect of pharmacological inhibition of nitric oxide synthase was tested by application of various nitric oxide synthase inhibitors with different selectivity for the nitric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition resulted in a significant potentiation of the loss of choline acetyltransferase activity in the hippocampus measured seven days after ethylcholine aziridinium application, whereas the specific inhibition of neuronal or inducible nitric oxide synthase was ineffective. These pharmacological data are suggestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-free primary neuronal cell cultures from hippocampus, cortex and septum of E15–17 Wistar rat embryos. Ethylcholine aziridinium-application in a range of 5–80 μM resulted in delayed apoptotic neurodegeneration with a maximum after three days as confirmed by morphological criteria, life–death assays and DNA laddering. Nitric oxide synthase activity in harvested cells decreased in a dose- and time-dependent manner. Nitric oxide production as determined by measurement of the accumulated metabolite nitrite in the medium was equally low in controls and in ethylcholine aziridinium treated cells (range 0.77–1.86 μM nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13 h after ethylcholine aziridinium application. The present data indicate that in a model of delayed apoptotic neurodegeneration as induced by ethylcholine aziridinium neuronal cell death in vitro and in vivo is independent of the cytotoxic potential of nitric oxide. This is confirmed by a decrease in nitric oxide synthase activity, absence of nitric oxide production and absence of inducible nitric oxide synthase expression. In contrast, evidence for a neuroprotective role of nitric oxide was obtained in vivo as indicated by the exaggeration of the cholinergic lesion after unspecific nitric oxide synthase inhibition by N-nitro- l-arginine methylester.

Effects of NMDA and its antagonists on ventral horn cholinergic neurons in organotypic roller tube spinal cord cultures

Neurotoxic effects of excitatory amino acid (EAA) receptor agonist N-methyl-D-aspartic acid (NMDA) and its antagonists on ventral horn cholinergic neurons were studied in organotypic rollertube cultures of spinal cord (OTC-SCs) using biochemical assays of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and AChE histochemistry. NMDA exposure decreased ChAT and AChE activity by 83% and 66%, respectively. Cultures treated with NMDA also showed a marked loss of AChE staining in both dorsal and ventral horns and a significant, dose-dependent decrease in the number of ventral horn AChE-positive neurons (VHANs). NMDA treatment primarily resulted in the loss of small VHANs (< 300 microns2). VHANs with a size and distribution typical of alpha-motoneurons were relatively well preserved. The effects of NMDA on OTC-SCs appeared to be independent of the age of the cultures. The NMDA antagonist DL-AP5 completely prevented the NMDA-induced loss of ChAT activity, but only attenuated the effect of NMDA on ChE activity. The antagonists DL-AP5, D-AP5 and MK-801, used alone, caused significant loss and/or shrinkage of VHANs. These effects appeared to be distinct from the NMDA mediated toxicity. The results indicate that NMDA and its antagonists exert powerful toxic effects on ventral horn cholinergic neurons. The large cholinergic alpha-motoneurons, however, appear to be relatively immune to these toxic effects.

Molecular biology of APO E alleles in Alzheimer's and non-Alzheimer's dementias.

Current research into the aetiology of the dementias is focused upon genetic factors which give rise to the disease process. Recently the Apolipoprotein E gene (APO E) and in particular the epsilon 4 allele has been shown to be a risk factor for late onset Alzheimer's disease (AD) where there is an increased frequency of the epsilon 4 allele. The epsilon 4 allele has also been shown to reduce the age at onset of dementia in AD in a dose dependent manner, with the epsilon 2 allele having an opposing effect. We have genotyped a large series of clinically and neuropathologically confirmed cases of AD and found the expected increase in the Apolipoprotein epsilon 4 allele frequency when compared to a control population. Similarly, in Lewy Body Dementia (LBD) an increased epsilon 4 frequency is also found though a normal epsilon 2 frequency exists, unlike in AD where the epsilon 2 frequency is reduced. No changes in APO E allele frequencies were found in presenile AD, Parkinson's disease with or without dementia, or in Down's syndrome. No association was found between any of the APO E alleles and the histopathological indices of AD, cortical senile plaques and neurofibrillary tangles, in any disease category. Neurochemical indicators of AD, loss of choline acetyltransferase activity was also unaffected by APO E genotype. Whilst their appears to be a strong association between the APO E allele and AD and also in LBD, other related neurodegenerative disorders associated with dementia do not show such a linkage. Changes in the epsilon 2 allele frequency may indicate a genetic difference between AD and LBD. The epsilon 4 allele does not appear to influence the burden of AD type pathology and this is particularly relevant given the relative lack of NFT in LBD indicating that factors other than SP or NFT may govern the onset of dementia.

Insulin-like Growth Factor-I: Potential for Treatment of Motor Neuronal Disorders

Motor neuronal disorders, such as the loss of spinal cord motor neurons in amyotrophic lateral sclerosis or the degeneration of spinal cord motor neuron axons in certain peripheral neuropathies, present a unique opportunity for therapeutic intervention with neurotrophic proteins. Normally, such proteins do not cross the blood-brain barrier, but spinal cord motor neuron axons and nerve terminals lie outside the barrier and thus may be targeted by systemic administration of protein growth factors. Insulin-like growth factor-I (IGFI) receptors are present in the spinal cord, and, like members of the neurotrophin receptor family, IGF-I receptors mediate signal transduction via a tyrosine kinase domain. IGF-I was found to prevent the loss of choline acetyltransferase activity in embryonic spinal cord cultures, as well as to reduce the programmed cell death of motor neurons in vivo during normal development or following axotomy or spinal transection. Consistent with earlier reports that IGF-I enhances motor neuronal sprouting in vivo, subcutaneous administration of IGF-I increases muscle endplate size in rats. Subcutaneous injections of IGF-I also accelerate functional recovery following sciatic nerve crush in mice, as well as attenuate the peripheral motor neuropathy induced by chronic administration of the cancer chemotherapeutic agent vincristine in mice. Doses of IGF-I that accelerate recovery from sciatic nerve crush in mice result in elevated serum levels of IGF-I which are similar to those obtained following subcutaneous injections of formulated recombinant human IGF-I (Myotrophin) in normal human subjects. Based on these findings, together with evidence of safety in animals and man, clinical trials of recombinant human IGF-I have been initiated in patients with amyotrophic lateral sclerosis and are planned to begin soon in patients with chemotherapy-induced peripheral neuropathies.

Differential alterations in adenosine A 1 and κ 1 opioid receptors in the striatum in Alzheimer's disease

The alterations in Alzheimer's disease (AD) of two binding sites in the striatum suggested to have a presynaptic localisation have been investigated by quantitative ligand binding autoradiography. Adenosine A 1 binding sites labelled with [ 3H]cyclohexyladenosine (CHA) and κ 1 opioid binding sites labelled with [ 3H]U-69593 were studied in adjacentsections of the striatum obtained postmortem from 10 patients with AD and 9 matched controls. In AD, there was a significant reduction of [ 3H]CHA binding sites in the caudate nucleus (control= 88±4;AD= 56±6 pmol/g tissue;mean ± S.E.M.) and putamen (control= 83±4;AD= 58±7pmol/g). In control subjects, highest levels of [ 3H]U-69593 binding were localised to patches within the caudate nucleus (9.66±0.58pmol/g) with lower levels in the matrix (5.54±0.48pmol/g). There was no alteration in [ 3H]U-69593 binding sites in either the caudate nucleus (patches and matrix) or putamen of AD patients. The activity of choline acetyltransferase (ChAT), determined in the same tissue samples used for autoradiographic analysis, was significantly reduced in AD (control= 124±11;AD= 64±14nmol/h/mg protein). There was a positive correlation between ChAT activity and [ 3H]CHA binding (r = 0.769), but not [ 3H]U-69593 binding (r = 0.197). The results indicate that a marked loss of adenosine A 1 receptors occurs in the striatum of AD with no loss of κ 1 opioid receptors, and that the loss of A 1 receptors parallels the loss of choline acetyltransferase activity.

Primate nucleus basalis of meynert p75 NGFR-containing cholinergic neurons are protected from retrograde degeneration by the ganglioside GM 1

The effects of unilateral devascularizing lesions of the neocortex in primates ( Cercopithecus aethiops) on the immunoreactivity of choline acetyltransferase and the low-affinity nerve growth factor receptor (p75 NGFR) were investigated in cell bodies of the nucleus basalis of Meynert. Choline acetyltransferase enzymatic activity was measured in the dissected ipsi- and contralateral nucleus basalis of Meynert as well as in the remaining cortex adjacent to the lesion. Cortically lesioned animals displayed a shrinkage of p75 NGFR-immunoreactive cholinergic cell bodies in only the intermediate portion of the nucleus basalis of Meynert as well as a depletion of choline acetyltransferase activity in this cellular complex. In contrast, cortically lesioned monkeys treated with monosialoganglioside did not reveal a significant loss of choline acetyltransferase activity or shrinkage of nucleus basalis of Meynert cholinergic neurons, but rather a modest hypertrophy. These results are discussed in relation to a possible use of putative trophic agents in the repair of the damaged central nervous system.

Alzheimer's disease: Is the decrease of the cholinergic innervation of the hippocampus related to intrinsic hippocampal pathology?

Consistent findings in the hippocampi of patients with Alzheimer's disease are the presence of neurofibrillary tangles in pyramidal neurons and the loss of choline acetyltransferase activity due to degeneration of hippocampal cholinergic terminals. The present study sought to clarify, in the brains of five patients with Alzheimer's disease and four controls, whether the loss of cholinergic terminals in the hippocampal stratum pyramidale in Alzheimer's disease is related to degenerative changes in hippocampal pyramidal cells. A polyclonal antibody to human choline acetyltransferase was employed to visualize immunohistochemically cholinergic terminals. Hippocampal neurons were stained with Cresyl Violet, neurofibrillary tangles with thioflavin S and a monoclonal antibody against phosphorylated neurofilament (RT97). Quantification of the stained structures was performed in CA4, CA1 and the subiculum, on five sections selected from the entire anteroposterior extent of each hippocampus. In the group of Alzheimer patients, the densities of cholinergic terminals were homogeneously diminished in the three hippocampal subregions in comparison with the controls (32–33%). In contrast, a significant loss of pyramidal neurons was found only in CA1, and the density of neurofibrillary tangles was markedly increased only in CA1 and the subiculum in Alzheimer's disease. These findings suggest that there is no relationship between the loss of cholinergic terminals and the degeneration of pyramidal cells in the hippocampus of patients with Alzheimer's disease.

Cholinergic deficit induced by ethylcholine aziridinium (AF64A) in rat hippocampus: Effect on glutamatergic systems

The reaction of the glutamatergic systems in the rat hippocampus to the withdrawal of cholinergic function after cholinergic degeneration induced by ethylcholine aziridinium (AF64A) was investigated. Furthermore, the question whether blockade of N-methyl-D-aspartate (NMDA) receptors by MK-801 has an impact on the extent of the cholinergic lesion was addressed. After bilateral intracerebroventricular injection of AF64A (2 nmol/ventricle) the activity of choline acetyltransferase (ChAT) started to decline in the hippocampus within 24 h. The reduction of ChAT activity reached its maximum within 4 days (65%) and persisted during the observation period of 65 days. The loss of ChAT activity was accompanied by a transient decline in the level of glutamate, which was most pronounced 1 to 2 days after AF64A (25% reduction). Within 65 days the glutamate level returned to normal. A detailed subdissection of the hippocampus revealed that the cholinergic system was most affected in the ventral part of the hippocampus and the CA3 subfield. On the other hand, the transient reduction in glutamate was restricted to the CA1 and CA3 area. In the dentate gyrus the marked loss of cholinergic function was not accompanied by any reduction in glutamate level. Treatment of the AF64A-injected rats with the muscarinic agonist pilocarpine prevented the decline in glutamate levels. The transient nature of the decline in glutamate as well as its reversal by treatment with pilocarpine are suggestive of an increased release of glutamate in response to the withdrawal of cholinergic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroprotectant effects of LY 274614, a structurally novel systemically active competitive NMDA receptor antagonist

Antagonists for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor may have therapeutic potential as neuroprotectant agents in conditions of neuronal degeneration that include brain ischemia, Huntington's chorea, and Alzheimer's disease. Here we have investigated the pharmacological actions of LY274614, a structurally novel competitive NMDA receptor antagonist, for pharmacological selectivity and neuroprotectant effects following systemic administration. LY274614 potently displaced NMDA receptor ([3H]CGS19755) binding (IC50 = 58.8 +/- 10.3 nM), but had no appreciable affinity at [3H]AMPA or [3H]kainate receptor sites at up to 10,000 nM. NMDA-induced convulsions in neonatal rats or NMDA-induced lethality in mice are potently and selectively antagonized by i.p. or p.o. LY274614. Oral doses showed a delayed but prolonged duration of effect. In adult rats, the neurodegenerative effects (loss of choline acetyltransferase activity) following the intrastriatal infusions of NMDA or quinolinate, but not kainate, were prevented by LY274614 (2.5 to 20 mg/kg i.p.). LY274614 is an effective neuroprotectant agent against NMDA receptor-induced toxicity when administered systemically and is a promising therapeutic agent for conditions where glutamate plays a role in the pathology of neuronal degeneration.

Exogenous nerve growth factor stimulates choline acetyltransferase activity in aging Fischer 344 male rats

The effect of age and exogenous nerve growth factor (NGF) infusion on choline acetyltransferase (ChAT) specific activity is examined in microdissections of cerebral and hippocampal cortices, and the cholinergic nuclei of the medial septum and diagonal band of Broca (MS/DB), the nucleus basalis magnocellularis (NBM), and striatum of Fischer 344 male rats. Significant, 20% losses in ChAT activity are found in the MS/DB and striatum of 24-month-old rats (n = 21) compared to 4-month-old animals, but there is no apparent loss of enzyme activity in the NBM. Loss of ChAT activity in the MS/DB is only observed in animals older than 19 months of age, while a striatal deficit is found in animals older than 7 months. Treatment for 2 weeks with NGF at 1.2 μg/day results in significant 70% increases of ChAT activity in the MS/DB and striatum of 24-month-old rats compared to untreated and vehicle-treated 4-month-old rats, but does not stimulate activity in the NBM. Sensitivity of ChAT activity in the MS/DB and striatum to exogenous NGF increases with age. These experiments indicate that in the MS/DB, NBM, and striatum of Fischer 344 male rat there is an age-associated, differential regulation of ChAT enzyme activity and sensitivity to exogenous NGF.

Calcitonin gene-related peptide and calcitonin immunoreactivity in brain and spinal cord in Alzheimer-type dementia

Calcitonin gene-related peptide (CGRP) and calcitonin (CT) immunoreactivity were measured in hypothalamus, parahippocampal gyrus, pituitary and grey matter of the posterior and anterior spinal cord from five to six cases of Alzheimer-type dementia (ATD) and from five to six controls. CGRP was slightly increased and choline acetyltransferase decreased in the anterior grey of ATD spinal cord. No other significant differences were observed between the levels of the two peptides in the ATD and control tissues, even in the parahippocampal gyrus and posterior grey of the spinal cord which had reduced choline acetyltransferase activity in the ATD cases. These results show that CGRP and CT are not affected in ATD, either as a consequence of a direct effect on peptidergic neurons or secondary to the loss of choline acetyltransferase activity.

3H]Vesamicol binding in human brain cholinergic deficiency disorders

We measured the binding of the vesicular acetylcholine transport blocker [ 3H]vesamicol (2-[4-phenylpiperidino] cyclohexanol; AH-5183) to autopsied frontal cortex and amygdala of patients from 4 disorders having a marked brain cholinergic reduction, namely Alzheimer's disease, Parkinson's disease with dementia, dominantly inherited olivopontocerebellar atrophy and Down's syndrome. Although mean activity of the specific cholinergic marker enzyme choline acetyltransferase (ChAT) was markedly reduced by about 60% in frontal cortex in the 4 patient groups and by 80% or greater in amygdala of the Alzheimer's and Down's syndrome patients, [ 3H]vesamicol binding density was, on average, either normal or only slightly reduced as compared with the controls. This discrepancy suggests that in human brain [ 3H]vesamicol binding is either not preferentially localized to cholinergic nerve endings or, in these cholinergic deficiency syndromes, a substantial proportion of the vesamicol binding sites persist on cholinergic nerve terminals despite loss of ChAT activity.

Effects of aging on nicotinic and muscarinic autoreceptor function in the rat brain: relationship to presynaptic cholinergic markers and binding sites

The main objective of the present work was to determine whether the regulation of ACh release by nicotinic and muscarinic autoreceptors is compromised in the aged rat brain. For this, the effects of the nicotinic agonist N-methylcarbamylcholine (MCC) and the muscarinic-M2 antagonist AF-DX 116 on ACh release from brain slices of young (3-month-old), adult (9-month-old), and aged (27-month-old) rats were tested. The ability of MCC to enhance spontaneous ACh release in hippocampal, cerebral cortical, and cerebellar slices was only modestly altered with age. In contrast, the sensitivity of muscarinic autoreceptors in the aged hippocampus and cerebral cortex, but not the striatum, to blockade by the muscarinic-M2 antagonist AF-DX 116 was severely attenuated. To assess whether the age-related changes in cholinergic autoreceptor function may be due to deficits in presynaptic cholinergic markers, we tested whether choline acetyltransferase (ChAT) activity, basal and evoked ACh release, and nicotinic and muscarinic binding sites are altered in the aged rats. ChAT activity in forebrain regions was decreased in the aged compared to the young and mature adult rats. Furthermore, the potassium-evoked, but not the spontaneous, release of ACh was markedly depressed in striatal, hippocampal, and cortical slices of aged rats. The densities of nicotinic and muscarinic-M2 binding sites, assessed using 3H-MCC and 3H-AF-DX 116 as selective ligands, respectively, were markedly reduced in homogenates of the striatum, hippocampus, cerebral cortex, and thalamus of aged rats. In contrast, muscarinic-M1 sites, selectively labeled with 3H-pirenzepine, were not affected. Therefore, it appears that age-related decrements in ChAT activity and in muscarinic-M2, but not nicotinic, binding sites in the rat brain are reflected in a decreased function of muscarinic-M2 autoreceptors. However, the positive correlation between loss of ChAT activity, decreased muscarinic-M2 binding sites, and impaired muscarinic autoreceptor function is clearly tissue dependent.

Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Ciliary neuromuscular morphology in cynomolgus monkeys after ciliary ganglionectomy

Cynomolgus monkeys underwent unilateral ciliary ganglionectomy (CG) and/or posterior ciliary neurectomy (PCN). The ciliary muscle was functionally denervated, as evidenced by loss of choline acetyltransferase activity, loss of the accommodative response to topical eserine and electrical stimulation of the Edinger-Westphal nucleus, and supersensitivity of the accommodative response to pilocarpine. Light and electron microscopy carried out 3-28 days after CG/PCN revealed degeneration of myelinated and unmyelinated nerve fibers as evidenced by axonal swelling and shrinkage, mitochondrial degeneration, axoplasmic condensation and vacuolization, and activated, phagocytosing Schwann cells. By 1-3 months after CG/PCN, few non-myelinated axons remained between the muscle bundles, larger nerve bundles were disordered and deteriorated, and Schwann cells filled with lipid and cellular debris were prominent. Beyond 6 months (6-37 months), most eyes were reinnervated by functional criteria. Concurrently, the ciliary neuromuscular morphology appeared virtually normal, exhibiting many non-myelinated axons containing numerous agranular synaptic vesicles and large mitochondria; however, remnants of degenerated axons were still present. In two eyes remaining functionally denervated, many of the unmyelinated axons between the ciliary muscle bundles were swollen or empty, with small, degenerated mitochondria and only rare synaptic vesicles, and were often ensheathed by thickened, condensed Schwann-cell cytoplasm. The muscle fibers were atrophic and separated from each other as well as from the ensheathed nerve fibers. Thus, following CG/PCN, the morphologic and functional evidence of parasympathetic denervation and reinnervation of the ciliary muscle is pathophysiologically and temporally consistent.

Neurotoxicity in vitro: Model systems and practical applications. Comparative studies with the cholinergic neurotoxin in primary brain cultures and in rabbit retina in vivo

In vitro neural systems can be predictive for central nervous system neurotoxicity, except where xenobiotics primarily affect the blood-brain barrier directly. The wide range of systems now used in neurobiological studies is available for mechanistic neurotoxicological investigations although the choice of system is generally arbitrary and for this reason a more rational ‘stepwise’ approach may now be justified. There are many culture systems available, including neural cell lines, organotypic explant or reaggregation cultures, and primary monolayer cultures of individual, or mixtures of, neural cell types: neurones, astrocytes and oligodendrocytes. Of these models much success has recently been achieved using the organotypic culture. Using rat whole-brain reaggregate cultures, good in vitro/in vivo correlations for the cholinergic neurotoxicant, ethylcholine mustard aziridinium (ECMA) have been demonstrated by comparing its actions with those in the rabbit retina in vivo. Low concentrations of ECMA (12.5 μ m) cause a biphasic loss of choline acetyltransferase activity (ChAT) in foetal rat brain reaggregate cultures. Initial direct inhibition is followed by an apparently selective loss of cholinergic neurones. More widespread cytotoxicity occurs at higher ECMA concentrations. Similarly, intravitreal injection of ECMA at a final concentration of 12.5 μ m into the rabbit eye in vivo for 96 hr produced a 36% loss of retinal ChAT activity with no change in the electroretinogram (ERG), whereas 600 μ m-ECMA produced a 55% loss of ChAT with a significant loss of the ERG b-wave function, which is suggestive of a more generalized cellular toxicity. Exogenous nerve growth factor applied to the brain reaggregates 48 hr post-ECMA lesioning reverses the neurotoxin-induced loss of ChAT activity. By supplementing neurotoxicological information gained in the in vitro brain reaggregate culture system with tests using primary monolayer cultures of neurones or astrocytes a stepwise ‘screening’ system is proposed for potential neurotoxicants in vitro. In its simplest form this is: (1) screening initially by using tumour-derived neural cell line and/or primary mixed neural culture such as the ‘Micromass’ system; (2) testing of selected compounds in whole-brain reaggregates and (3) supplementing this information if necessary with effects on primary monolayer cultures of individual neural cell types.

Effect of nerve growth factor and thyrotropin releasing hormone on cholinergic neurones in developing rat brain reaggregate cultures lesioned with ethylcholine mustard aziridinium

Foetal rat whole brain reaggregate cultures were prepared in a serum-supplemented (S+) or serum-free medium (S−). Ethylcholine mustard aziridinium (ECMA) was added to the cultures at 9 days in vitro (DIV) at concentrations of 12.5, 25 or 50 μM. Choline acetyltransferase (ChAT) activity was measured at +2, +48 and +96 hr following treatment. In certain experiments the neurotrophic factors, thyrotropin releasing hormone (TRH: 50 μg/ml, daily from 9 DIV) or nerve growth factor (NGF: 7S subunit, 5 ng/ml, 0 and +48 hr following ECMA) were added during ECMA treatment. In both types of reaggregate cultured in S+ and S− media there was a 40–80% loss of ChAT activity following ECMA exposure (final concentration = 12.5 μM), presumed to reflect cholinergic cell loss. In both S+ and S− brain raggregates NGF produced increased ChAT activity with more marked effects in S+ (45–55% increase, +48–96 hr) than in S− medium (20–25% increase, 2–96 hr). No effect on cholinergic muscarinic receptors (specific 3H-QNB binding) was evident after treatment with NGF. TRH had no effect on ChAT activity in the S+ cultures but produced small increases in the S− culture condition (approx 20%, + 2–48 hr). Despite a residual “ECMA-resistant” pool of ChAT in the cultures, neither neurotrophic agent was found to cause a reversal of the lesion. In conclusion, the cholinotoxin ECMA appears to produce a cholinergic deficit in both developing S+ and S− reaggregates. This was not reversible by NGF or TRH at the concentrations and under the conditions tested. NGF had marked effects on ChAT activity without affecting muscarinic receptors in untreated developing brain reaggregates cultured in an S+ medium.