Effects of food deprivation,discrimination experience and physostigmine on choline acetylase and acetylcholine esterase in the dorsal hippocampus and frontal cortex of rats

The effects of food deprivation and preliminary training treatments on choline acetylase and acetylcholine esterase activity in the dorsal hippocampus and frontal cortex of rats have been investigated. In addition, blood sugar and adrenal epinephrine levels were examined. The activity of choline acetylase and the levels of blood sugar and adrenal epinephrine were significantly altered during various stages of preliminary training but all values were comparable to pre-experimental levels before discrimination commenced. In addition, rats were injected physostigmine salicylate (0.25 mg/kg) or saline i.p. each day immediately after receiving three training trials on a food-motivated black-white task. The physostigmine injected rats were significantly better than the controls in rate learning.No differences were observed in choline acetylase activity in the dorsal hippocampus and frontal cortex in discriminating rats treated with physostigmine or saline. However, choline acetylase activity is lower in treated and control rats which did not discriminate at the end of the 14th session.     

Autoimmune neuromuscular disease induced by a preparation of choline acetyltransferase

An apparent fatal autoimmune disease was induced in guinea pigs by injection of a preparation of choline acetyltransferase in complete Freund's adjuvant. The animals probably died from respiratory failure based on the evidence from clinical, histological, and histochemical examination. At autopsy the pathogenic lesions were located at the neuromuscular junctions and near the nerves where choline actyltransferase was found. It is further proposed that this is a presynaptic autoimmune neuromuscular disease based on the finding of numerous target, as well as targetoid, fibers in muscle after histochemical staining. There was also denervation shown by electromyographic measurements in the form of positive sharp waves as well as fibrillation discharges.     

The role of hippocampal cholinergic mechanisms in the aquisition of a barpress response.

The role of hippocampal cholinergic mechanisms in learning a bar-press response reinforced with food was investigated. Firstly, an interstrain comparison showed that mice having a low choline acetyltransferase activity in the dorsal hippocampus were quicker to associate the barpress with reinforcement. Secondly, when the activity of this enzyme was reduced by a subseizure electrical stimulation of the hippocampus learning was accelerated. It is suggested that acetylcholine availability at the hippocampal synapses slowed the apparition of these learned responses.     

Brain vesicular acetylcholine transporter in human users of drugs of abuse

Limited animal data suggest that the dopaminergic neurotoxin methamphetamine is not toxic to brain (striatal) cholinergic neurons. However, we previously reported that activity of choline acetyltransferase (ChAT), the cholinergic marker synthetic enzyme, can be very low in brain of some human high-dose methamphetamine users. We measured, by quantitative immunoblotting, concentrations of a second cholinergic marker, the vesicular acetylcholine transporter (VAChT), considered to be a "stable" marker of cholinergic neurons, in autopsied brain (caudate, hippocampus) of chronic users of methamphetamine and, for comparison, in brain of users of cocaine, heroin, and matched controls. Western blot analyses showed normal levels of VAChT immunoreactivity in hippocampus of all drug user groups, whereas in the dopamine-rich caudate VAChT levels were selectively elevated (+48%) in the methamphetamine group, including the three high-dose methamphetamine users who had severely reduced ChAT activity. To the extent that cholinergic neuron integrity can be inferred from VAChT concentration, our data suggest that methamphetamine does not cause loss of striatal cholinergic neurons, but might damage/downregulate brain ChAT in some high-dose users. However, the finding of increased VAChT levels suggests that brain VAChT concentration might be subject to up- and downregulation as part of a compensatory process to maintain homeostasis of neuronal cholinergic activity. This possibility should be taken into account when utilizing VAChT as a neuroimaging outcome marker for cholinergic neuron number in human studies.     

Intravenously injected CDP-choline increases blood pressure and reverses hypotension in haemorrhagic shock: effect is mediated by central cholinergic activation

Intravenous (i.v.) administration of cytidine-5′-diphosphate choline (CDP-choline) (100, 250 and 500 mg/kg) increased blood pressure in normal rats and reversed hypotension in haemorrhagic shock. Choline (54 mg/kg; i.v.), at the dose equimolar to 250 mg/kg CDP-choline decreased blood pressure of rats in both conditions and caused the death of all hypotensive animals within 2-5 min. Equimolar dose of cytidine (124 mg/kg; i.v.) did not change cardiovascular parameters. Choline levels in plasma, lateral cerebral ventricle and hypothalamus increased after CDP-choline administration. Intracerebroventricular (i.c.v.) hemicholinium-3 pretreatment (20 μg), greatly attenuated the pressor effect of CDP-choline in both conditions. Atropine pretreatment (10 μg; i.c.v.) did not change the pressor effect of CDP-choline while mecamylamine (50 μg; i.c.v.) abolished the pressor response to drug. Besides, acetylcholine (1 μmol; i.c.v.) produced similar increases in blood pressure in normal and hypotensive conditions to that observed in CDP-choline given rats. CDP-choline (250 mg/kg; i.v.) increased plasma catecholamines and vasopressin levels but not plasma renin activity. Pretreatment of rats with either prazosin (0.5 mg/kg; i.v.) or vasopressin V₁ receptor antagonist, [β-mercapto,β,β-cyclopentamethylenepropionyl¹,O-Me-Tyr²-Arg⁸]vasopressin (10 μg/kg; i.v.), attenuated the pressor response to CDP-choline while simultaneous administration of these antagonists before CDP-choline injection completely blocked the pressor effect. Results show that i.v. CDP-choline increases blood pressure and reverses hypotension in haemorrhagic shock. Activation of central nicotinic cholinergic mechanisms by the increases in plasma and brain choline concentrations appears to be involved in the pressor effect of this drug. Moreover, the increases in plasma catecholamines and vasopressin levels mediate these effects.     

Effects of ageing and long-term hormone replacement on cholinergic neurones in the medial septum and nucleus basalis magnocellularis of ovariectomized rats

Ovariectomized aged rats, some of which received long-term hormone replacement with oestrogen or oestrogen plus progesterone, were evaluated for the number and size of basal forebrain cholinergic neurones, as well as relative levels of choline acetyltransferase (ChAT) and trkA mRNA, in order to determine whether effects on basal forebrain cholinergic cell survival and function correspond with differences in cognitive performance previously described. The results show that ageing combined with long-term loss of ovarian function produced substantial reductions in the levels of ChAT and trkA mRNA in the medial septum and nucleus basalis magnocellularis, relative to much younger ovariectomized controls. In contrast, no significant effects on the number or size of the cholinergic cells were detected, indicating that loss of ovarian function does not cause a loss of cholinergic neurones with age. Long-term hormone replacement had no apparent effect on the number of ChAT-positive neurones detected, and did not prevent the reductions in ChAT and trkA mRNA associated with ovariectomy and ageing. Collectively, the data suggest that ageing combined with long-term loss of ovarian function has a severe negative impact on basal forebrain cholinergic function, but not on cholinergic cell survival per se.     

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.     

A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers

Central cholinergic systems have been visualized by immunohistochemistry using antibodies to choline acetyltransferase (ChAT). Peripheral cholinergic cells and fibers, however, have been hardly detectable with most of these antibodies. This phenomenon suggests that a different form of ChAT may exist in peripheral tissues. Here we report two types of mRNA for ChAT expressed by alternative splicing in rat pterygopalatine ganglion. One is exactly identical with ChAT mRNA reported in the central nervous system (ChAT of a common type; cChAT). The other lacks exons 6, 7, 8 and 9, which was detected only in the pterygopalatine ganglion (ChAT of a peripheral type; pChAT). The peculiarity of pChAT in chemical structure, possessing a splice joint of the exons 5 and 10, led us to produce rabbit antisera against a recombinant peptide of 41 amino acids which spans over the splice joint. On Western blots using a successfully obtained antiserum, an intense band of about 50 kDa, corresponding to the expected molecular weight of pChAT, was detected in the pterygopalatine ganglion but not in the brain. Immunohistochemistry using the antiserum failed to reveal positive staining of known brain cholinergic structures, while it permitted us to observe peripheral, probably cholinergic, nerve cells and fibers including those in the pterygopalatine ganglion and enteric nervous system.