Choline Acetyltransferase Activity Activity Research Articles

Exercise prevents HFD-induced insulin resistance risk: involvement of TNF-\u03b1 level regulated by vagus nerve-related anti-inflammatory pathway in the spleen

ObjectivesRegular physical exercise can improve insulin resistance in insulin target tissues. However, the mechanisms about the beneficial effect of exercise on insulin resistance are not yet fully resolved. This study was carried out to address whether insulin resistance improvement by exercise is involved in an anti-inflammatory pathway in the spleen in high-fat diet (HFD) feeding mice.MethodsMale C57Bl/6J mice with or without subdiaphragmatic vagotomy (sVNS) were subjected to medium-intensity treadmill exercise during HFD feeding. Glucose tolerance test and insulin tolerance test were detected, and spleen acetylcholine level, choline acetyltransferase activity (ChAT), protein kinase C (PKC) and tumor necrosis factor-alpha (TNF-α) were assayed.ResultsWe found that exercise significantly improves HFD-induced glucose intolerance and insulin resistance, along with an increase in acetylcholine level, ChAT activity, and PKC activity, and decrease in TNF-α level in the system and the spleen from HFD-fed mice. However, sVNS abolished the beneficial effect of exercise on glucose intolerance and insulin resistance, decreased acetylcholine level, ChAT activity, and PKC activity, and increase TNF-α level of the spleen in HFD-mice exercise intervention.ConclusionsThese data reveal that the prevention of HFD-associated insulin resistance by exercise intervention involves reducing splenic TNF-α level, which is mediated by cholinergic anti-inflammatory activity via influencing PKC activity, ChAT activity, and acetylcholine concentration in mice spleen.

Antioxidants Reverse the Changes in the Cholinergic System Caused by L-Tyrosine Administration in Rats.

Tyrosinemia type II is an inborn error of metabolism caused by a deficiency in the activity of the enzyme tyrosine aminotransferase, leading to tyrosine accumulation in the body. Although the mechanisms involved are still poorly understood, several studies have showed that higher levels of tyrosine are related to oxidative stress and therefore may affect the cholinergic system. Thus, the aim of this study was to investigate the effects of chronic administration of L-tyrosine on choline acetyltransferase activity (ChAT) and acetylcholinesterase (AChE) in the brain of rats. Moreover, we also examined the effects of one antioxidant treatment (N-acetylcysteine (NAC) + deferoxamine (DFX)) on cholinergic system. Our results showed that the chronic administration of L-tyrosine decreases the ChAT activity in the cerebral cortex, while the AChE activity was increased in the hippocampus, striatum, and cerebral cortex. Moreover, we found that the antioxidant treatment was able to prevent the decrease in the ChAT activity in the cerebral cortex. However, the increase in AChE activity induced by L-tyrosine was partially prevented the in the hippocampus and striatum, but not in the cerebral cortex. Our results also showed no differences in the aversive and spatial memory after chronic administration of L-tyrosine. In conclusion, the results of this study demonstrated an increase in AChE activity in the hippocampus, striatum, and cerebral cortex and an increase of ChAT in the cerebral cortex, without cognitive impairment. Furthermore, the alterations in the cholinergic system were partially prevented by the co-administration of NAC and DFX. Thus, the restored central cholinergic system by antioxidant treatment further supports the view that oxidative stress may be involved in the pathophysiology of tyrosinemia type II.

Effect of solifenacin plus and minus antioxidant supplements on the response to experimental outlet obstruction and overactive bladder dysfunction in rabbits—Part 2

ObjectiveOne of the most common forms of bladder dysfunction is related to the development of unstable bladder contractions (bladder overactivity). Solifenacin is a relatively new, selective, antimuscarinic agent that has been shown to be particularly useful in the treatment of overactive bladder (OAB) dysfunctions in both men and women. Experimentally, we have demonstrated that OAB and obstructive bladder dysfunction are associated with the generation of free radicals and oxidative damage to the bladder. The hypothesis tested was that solifenacin + coenzyme Q10 + α-lipoic acid (CoQ + LA) would be more effective in the treatment of OAB than either would be individually. Materials and methodsForty-eight male White New Zealand rabbits were separated into 8 groups of 6 rabbits each. The following oral treatments were given to each group: Groups 1 and 5, vehicle (saline); Groups 2 and 6, solifenacin; Groups 3 and 7, CoQ + LA; Groups 4 and 8, solifenacin + CoQ + LA. After three weeks of treatment (by oral gavage), the rabbits in Groups 1–4 received partial outlet obstruction as detailed above. The rabbits continued their treatments for four weeks following surgery. At the end of this four-week period, immediately following urodynamic and physiological studies, the bladder was excised and the muscle and mucosa were separated and frozen for biochemical research. ResultsThe following marker enzymes were quantitated: choline acetyltransferase, citrate synthase, catalase, superoxide dismutase, and malondialdehyde (MDA, marker for lipid peroxidation). Solifenacin had no effect on citrate synthase activity of the bladder smooth muscle. However, pretreatment with both the antioxidants or the combination of solifenacin + antioxidants protected the citrate synthase activity such that it remained at control values. Partial outlet obstruction (PBOO) resulted in a 60% decrease in choline acetyltransferase activity (ChAT) activity, while solifenacin had no effect on reduced ChAT activity. Both the antioxidant and combination therapy resulted in maintaining ChAT activity at control values. PBOO resulted in a significant increase in MDA of both the muscle and mucosa. For both tissues, all treatments resulted in a significantly lower MDA content. In general, the results demonstrate the combination of solifenacin + antioxidants was more effective than either solifenacin or antioxidants alone. ConclusionThe addition of the antioxidants CoQ + LA works synergistically with solifenacin in the treatment of obstructive bladder dysfunction and OAB.

Treatment with 7-nitroindazole enhances kainic acid induced cholinergic neurotoxicity in the rat striatum: a neuroprotective role for neuronal nitric oxide.

1. In this study we investigated the effect of 7-nitroindazole (7-NI), a preferential inhibitor of neuronal nitric oxide synthase (nNOS), on kainic acid (KA) induced neurotoxicity in rats. Choline acetyltransferase activity (CAT), a cholinergic marker, and histological changes were employed to assess neurotoxicity. 2. In control rats, the local intrastriatal injection of 0.5 microg of KA reduced CAT from 22.9 +/- 2.2 to 14.7 +/- 2.0 nmol/h/mg tissue ((38 +/- 6)% reduction) (P < 0.001). Greater reductions in CAT were observed with 1 and 2 microg of KA ((70 +/- 6)% and (80 +/- 3)%, respectively). 7-NI aggravated KA-induced cholinergic and histological damage. KA reduced CAT by (68.2 +/- 4)% in 7-NI-treated rats, by (38 +/- 6)% in saline-treated controls, and by (41 +/- 4)% in peanut-oil- (7-NI-vehicle-) treated rats (P = 0.0047). 3. After KA, CAT activity averaged 14.3 +/- 2.0 in peanut-oil-treated rats and 7.9 +/- 1.0 nmol/h/mg tissue in 7-NI- (peanut-oil-) treated rats (P = 0.015). Similarly to changes in CAT, 7-NI treatment aggravated KA-induced histological changes indicative of neuronal damage (acute ischemic neuronal changes, disorganization of myelinated fibers bundle, and vacuolation changes of the neuropil). Treatment with 7-NI was not associated with increased mortality. 4. Our findings suggest that neuronal NO plays a neuroprotective action on excitotoxicity.

Cholinergic deficits contribute to behavioral disturbance in patients with dementia.

Noncognitive behavioral changes such as depression, aggressive behavior, psychosis, and overactivity occur frequently in patients with dementia, in addition to cognitive impairment, and often determine the need for institutionalization. The biochemical basis of such changes is poorly understood. Clinical trial data indicate that cholinomimetics improve noncognitive behaviors. This study investigated the relationship between markers of the cholinergic and dopaminergic neurotransmitter systems and noncognitive behavioral symptoms assessed during the course of dementing illness. Brains from 46 patients with dementia (36 with AD and 10 with mixed or other dementias using Consortium to Establish a Registry for AD criteria) were examined together with 32 normal controls. The patients with dementia had been evaluated every 4 months, often over several years, for cognitive performance (Mini-Mental State Examination) and behavior (Present Behavioral Examination). Concentrations of dopamine (DA) and major metabolites, choline acetyltransferase activity (ChAT), and density (Bmax) of DA D1 receptors in frontal and temporal cortex were studied by radioligand binding protocols. None of the patients was receiving cholinomimetic drugs. ChAT activity, but no other neurochemical markers, was reduced in AD compared with controls. Loss of ChAT activity correlated with cognitive impairment. Lowered ChAT activity also correlated with increasing overactivity in patients with dementia in both frontal and temporal cortex whereas ChAT:DA and ChAT:D1 ratios in temporal cortex correlated negatively with aggressive behavior. Disturbance of the cholinergic system may underlie both cognitive and some noncognitive behavioral changes in dementia, providing a basis for rational therapy.-1467

Modeling adolescent nicotine exposure: effects on cholinergic systems in rat brain regions

Smoking among teenagers is increasing and the initiation of tobacco use during adolescence is associated with subsequently higher cigarette consumption and lower rates of quitting. Few animal studies have addressed whether adolescent nicotine exposure exerts unique or lasting effects on brain structure or function. Initial investigations with a rat model of adolescent nicotine exposure have demonstrated that the vulnerable developmental period for nicotine-induced brain cell damage extends into adolescence. In the current study, we examined the effect of nicotine on cholinergic systems in male and female adolescent rats with an infusion paradigm designed to match the plasma levels found in human smokers or in users of the transdermal nicotine patch. Choline acetyltransferase activity (ChAT) and [ 3H]hemicholinium-3 binding (HC-3) were monitored; ChAT is a static marker that closely reflects the density of cholinergic innervation, whereas HC-3 binding, which labels the presynaptic high-affinity choline transporter, is responsive additionally to nerve impulse activity. Measurements were carried out in the midbrain, the region most closely involved in reward and addiction pathways, as well as in the cerebral cortex and hippocampus. During nicotine treatment and for 1 month after the termination of treatment, ChAT activity was reduced significantly in the midbrain but not in the other regions. HC-3 binding showed a substantial increase during the course of nicotine treatment and again, the effect was limited to the midbrain. Midbrain values returned to normal immediately after the cessation of nicotine exposure and then showed a subsequent, transient suppression of activity. Although the cerebral cortex showed little or no change in HC-3 binding during or after nicotine administration, activity was reduced persistently in the hippocampus. The regionally-selective effects of adolescent nicotine treatment on cholinergic systems support the concept that adolescence is a vulnerable developmental period for ultimate effects on behavior.

Complement-mediated lesion of sympathetic ganglia in vitro with acetylcholinesterase antibodies

When administered to rats, antibodies against acetylcholinesterase (AChE) selectively destroy presynaptic inputs to sympathetic ganglia. To investigate the mechanism of this immunolesion, we created an in vitro system in which relevant components could be manipulated. Freshly dissected rat superior cervical ganglia (SCG) were incubated 15–20 h at 37°C in fresh human serum (a potent source of complement) with continuous oxygenation. More than 96% of neurons in six control ganglia retained synaptic inputs, as defined by action potentials or excitatory postsynaptic potentials (EPSP) upon stimulation of the preganglionic trunk. However, when anti-AChE antibodies were present (0.16 mg/ml), none of 61 neurons from six incubated ganglia showed synaptic responses although membrane potential and input resistance remained normal. Staining for AChE and synaptophysin (a synaptic vesicle marker) was also disrupted in ganglia exposed to AChE antibodies in complement-sufficient serum. When complement was eliminated by substituting serum that was heat-inactivated or deficient in C3, synaptic input was retained in 60–90% of neurons incubated with AChE antibodies. Choline acetyltransferase activity (ChAT), an enzymatic marker of cholinergic cytoplasm in sympathetic ganglia, was largely lost after incubation with AChE antibodies and serum. However, incubation with AChE antibodies in heat-inactivated serum, or serum that was deficient in C3 or C8, caused no measurable loss of ganglionic ChAT activity. These findings strongly implicate the complement cascade in the destruction of preganglionic sympathetic terminals that follows binding of AChE antibodies.

Effect of partial outlet obstruction on choline acetyltransferase activity in the rat and rabbit.

Partial outlet obstruction of the rabbit bladder induces a rapid and significant increase in bladder mass. This increase in mass is associated with a variety of specific contractile dysfunctions, characterized by a marked decrease in the response to field stimulation (acting through the release of neurogenic transmitters). There is histological evidence indicating that the decrease in the contractile response of isolated strips of rabbit urinary bladder to field stimulation is associated with a degeneration of synaptic membranes within the bladder detrusor (neuropathy). In the current experiments, the effect of partial outlet obstruction in rabbit and rat urinary bladders on choline acetyltransferase activity (ChAT) were determined and correlated with both the level of bladder hypertrophy (increase in mass) and the contractile response to field stimulation. The results can be summarized as follows: In the rabbit, partial outlet obstruction induced a rapid 5-fold increase in bladder mass over the 7 day period of study. This increase in mass was associated with a decrease in the contractile response of isolated strips of bladder body and base to field stimulation and a decrease in ChAT activity. Interestingly, the rabbit bladder base showed a significantly higher ChAT activity than the bladder body, although the contractile response to muscarinic stimulation was significantly greater in the bladder body than in the base. In the rat, partial outlet obstruction induced a mild 2-fold increase in bladder mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial learning impairments in rats with selective immunolesion of the forebrain cholinergic system.

A monoclonal antibody to the low-affinity NGF receptor, 192 IgG, coupled to a cytotoxin, saporin, was recently introduced as an efficient selective neurotoxin for the NGFr-bearing cholinergic neurones in the rat basal forebrain. In the present study we report that an intracerebroventricular injection of this 192 IgG-saporin conjugate induces a severe, long-lasting spatial learning impairment, as assessed in the Morris water-maze task. This behavioural impairment was associated with 65-90% depletion of choline acetyltransferase activity (ChAT) in the hippocampus and cortex. ChAT activity associated with other cholinergic neurone systems in the brain (striatum, mesencephalon, spinal cord), was left virtually unaffected. This new immunotoxin holds great promise as a tool for selective and efficient lesions of the forebrain cholinergic system in functional and behavioural studies.

Modulation of tyrosine hydroxylase gene expression in the rat adrenal gland by exercise: effects of age

Both aging and exercise are associated with alterations in circulating levels of catecholamines. To determine the interactions of age and exercise on tyrosine hydroxylase (TH) activity and TH mRNA, Fischer-344 female rats aged 5 months (young) and 25 months (old) were trained by treadmill running for 10 weeks. The elevation in maximum oxygen consumption in both groups was equivalent following exercise, indicating that training had occurred. In control rats, both TH activity and TH mRNA were greater in the older groups when compared with the younger animals. In young rats, exercise decreased TH activity by 25% and TH mRNA by 27%. In older rats, exercise was not associated with a decrease in TH activity and TH mRNA. Choline acetyltransferase activity (ChAT) was decreased and glutamic acid decarboxylase activity (GAD) was increased by exercise in young rats. The decrease in ChAT activity and increase in GAD activity suggest that transynaptic mechanisms play a role in the exercise-induced alteration of TH gene expression. Neither ChAT nor GAD was altered by exercise in older groups. Our data suggest that the previously reported diminution in catecholamines associated with exercise may be due to a decrease in TH mRNA and a resulting decrease in TH activity. There was no effect of exercise in the old rats, supporting previous observations that the plasticity of the sympathoadrenal system diminishes with age.

Graft-derived cholinergic reinnervation of the hippocampus prevents a lasting increase of hippocampal noradrenaline concentration induced by septohippocampal damage in rats

Long-Evans female rats sustained aspirative lesions of the septohippocampal pathways and, 2 weeks later, received into the dorsal hippocampus grafts prepared from the septal area (rich in cholinergic neurons: Group Sep) or from the mesencephalic raphe (poor in cholinergic neurons; Group Rap) of rat fetuses. Lesion-only (Group Les) and virtually intact (Group Sham) rats served as controls. Between 9.5 and 10.5 months after grafting surgery, we found the lesions to decrease choline acetyltransferase activity (ChAT), high affinity synaptosomal uptake of [ 3H]choline (HACU) and serotonin concentration ([5-HT]), as well as to increase the noradrenaline concentration ([NA]) in the dorsal hippocampus. Raphe grafts increased [5-HT] to 456% of normal, but had only weak or no effects on the other lesion-induced modifications in brain neurochemistry. Septal grafts dramatically increased ChAT activity and HACU, enhanced [5-HT], and reduced [NA] to near-normal levels. We also found a significant negative correlation between HACU and [NA] in rats with lesions, whether grafted or not. These data show that grafts providing the denervated hippocampus with a new cholinergic innervation might be able to exert inhibitory effects on the lesion-induced increase of [NA]. Since such an increase is indicative of sympathetic sprouting, the finding of reduced [NA] in rats with graft-derived cholinergic reinnervation of the hippocampus is in line with the hypothesis that hippocampal cholinergic denervation plays a crucial role in the induction of sympathetic sprouting. However, our data do not allow to distinguish whether grafts rich in cholinergic neurons inhibited the sympathetic sprouting itself, or rather reduced the NA content of sprouted fibers.

Slowing of the dominant occipital rhythm in electroencephalogram is associated with low concentration of noradrenaline in the thalamus in patients with Alzheimer's disease

The thalamus is involved in the regulation of the neocortical rhythmicity reflected in EEG as the alpha rhythm. Recent evidence suggests that the thalamus is affected in Alzheimer's disease (AD). We studied the relationship between the dominant occipital rhythm of the lifetime EEG and the choline acetyltransferase activity (ChAT) and monoamine concentrations in the postmortem thalamus of 20 histologically verified AD patients. The AD patients were divided into 3 groups (5–6 Hz, 7 Hz and 8–9 Hz) according to the frequency of the dominant occipital rhythm (FOC). Noradrenaline (NA) concentrations were significantly lower for the 5–6 Hz and 7 Hz subgroups as compared to the 8–9 Hz subgroup. The NA content of the thalamus correlated significantly with the FOC ( r=0.46, P=0.04). The ChAT activity or concentra-tions of dopamine or serotonin did not correlate with the FOC or differ across the AD subgroups. The result suggests that the noradrenergic deficit of the thalamus may contribute to slowing of the dominant occipital rhythm in AD.

Lesions of nucleus basalis alter ChAT activity and EEG in rat frontal neocortex

The EEG was recorded from frontal, parietal and visual cortices of sham-operated control rats and rats having ibotenic acid lesions of the nucleus basalis. Recordings were made during a period of rest and during stimulus-evoked desynchronization. Spectral power was determined using a Fast Fourier Transform routine; 3 artifact-free 4 sec epochs of resting activity and two 4 sec epochs of activated EEG were analyzed. Choline acetyltransferase activity (ChAT) was measured in each cortical area and was reduced in lesioned animals an average of 25% in frontal cortex, 19% in the parietal region and 10% in visual cortex. The percent of low frequency activity (1–12 Hz) in the frontal EEG was significantly greater in lesioned animals than in the control group during quiet rest; a significant correlation was found between ChAT activity and power in this band. Desynchronized activity was largely unaffected except for a reduction in 25–31 Hz activity in the frontal cortex of lesioned animals. EEG activity in both the parietal and visual areas was unchanged from control values.

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.

Altered sympathetic-salivary gland development: delayed response to postnatal castration.

These studies defined the normal and hormonally altered development of activity for tyrosine hydroxylase (T-OH), the rate-limiting enzyme in catecholamine biosynthesis (Levitt et al., 1965), and choline acetyltransferase activity (CAT) in the male rat superior cervical ganglion (SCG). Additionally, salivary gland weight was monitored. Two distinct developmental plateaus for postsynaptic T-OH activity exist. The first plateau represents the prepubertal level, which is significantly lower than the second postpubertal plateau. In contrast, presynaptic CAT activity displayed only a single plateau, commencing at approximately 45 days of age. The effects of postnatal castration (at 10 or 11 days of age) on the submandibular gland and T-OH activity were delayed until after puberty. No change in T-OH activity was seen at two and four post-operative weeks between control and castrated animals; however, T-OH activity was significantly less in castrated animals at 12 and 16 post-operative weeks. Testosterone replacement reversed the effect of castration on T-OH activity. Conversely, CAT activity in the SCG was unchanged by postnatal castration for at least 16 post-operative weeks, the longest time point studied. The failure of castrated animals to display the normal developmental increase in T-OH activity following puberty was comparable with the effect of castration on the development of submandibular salivary gland weight. These results suggest that in postpubertal male rats, development of T-OH activity in the superior cervical ganglion is influenced by testosterone. The parallel effects of castration on submandibular gland weight imply that testosterone regulates T-OH activity via an indirect mechanism. In contrast to noradrenergic enzyme activity and target tissue size, the ontogeny of presynaptic CAT activity appears to be insensitive to testosterone levels.

Effects of opiates on the growth of neuron-enriched cultures from chick embryonic brain

Neuron-enriched cultures derived from 6-day-old chick embryo cerebral hemispheres were treated with morphine or methadone, 10−5 M or 10−6 M, on days 4–6 or 6–8 in culture and were evaluated morphologically and biochemically at day 9 using phase contrast microscopy and choline acetyltransferase activity (ChAT) as a cholinergic marker. The treatment of the cultures with morphine markedly affected their growth pattern; specifically, we observed an increased number of flat cells presumptively glia, and aggregates sided by flat cells and devoid of thick bundles of neuritic processes that normally characterize neuron-enriched cultures. These morphologic changes were reflected in a drastic decrease of ChAT activity in cultures treated from day 4 to day 6 but not from 6 to 8. In contrast to morphine, exposure to 10−6 M methadone from day 4 to day 6 resulted in reduced ChAT activity but the growth pattern of the cultures remained morphologically intact. We suggest that morphine exerts a general neurotoxic effect whereas methadone may affect some specific cholinergic function.

Thyrotropin-releasing hormone, luteinizing hormone-releasing hormone and substance P immuno-reactivity in post-mortem brain from cases of alzheimer-type dementia and Down's syndrome

Thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LH-RH), substance P immuno-reactivity and choline acetyltransferase activity (CAT) were measured in post-mortem brains from 6 cases of Alzheimer-type dementia (ATD), 3 cases of Down's syndrome and 6 controls and in spinal cords from two cases of ATD and two controls. The activity of CAT was lower in posterior and anterior grey matter of lumbar spinal cord from two cases of ATD than from two controls. No significant differences were observed in the levels of the 3 peptides in the ATD, Down's and control brains and spinal cords, even in areas of ATD and Down's brain in which there were significant reductions in the activity of CAT or concentration of noradrenaline. These results show that TRH, LH-RH and substance P are not affected in ATD and Down's syndrome either as a consequence of a direct effect on the peptidergic neurone or of a secondary effect related to the loss of CAT activity and noradrenaline.

Choline high-affinity uptake and metabolism and choline acetyltransferase activity in the striatum of rats chronically treated with neuroleptics.

High-affinity uptake of choline and choline acetyltransferase activity (ChAT) were measured in the striatum of rats treated for 45-60 days with haloperidol (1 mg/kg per os) and pimozide (1 mg/kg per os) daily and with fluspirilene (1 mg/kg i.m.) twice a week. Haloperidol and fluspirilene caused a 20%, and pimozide a 38%, increase in high-affinity uptake of choline. They also caused a significant decrease in ChAT activity: haloperidol, 20%; pimozide, 27%; and fluspirilene, 42%. In rats treated with fluspirilene for 65-80 days the metabolism of [3H] choline taken up by striatal synaptosomes was investigated. A 33% increase in total radioactivity, a significant increase in labelled acetylcholine (ACh), a relative decrease in labelled choline, and no change in labelled phosphorylcholine and betaine were found. It is concluded that the increase in high-affinity choline uptake caused by chronic administration of neuroleptic drugs is associated with a parallel increase in choline utilization for ACh formation. On the other hand, the decrease in ChAT activity does not appear to influence ACh formation.

DEPENDENCE OF HEART RATE VARIABILITY ON INSTANTANEOUS HEART RATE IN SLEEP

This study was designed to assess: the ontogenesis of autonomic innervation in the intact embryonic and neonatal rat heart and the development of innervation of rat heart cells in primary tissue culture. Choline acetyltransferase activity (CAT), a marker for acetyl choline synthesis, was determined by radloimmunological assay in intact hearts. CAT was first detectable on the 18th gestational day (atria/ventricles = 8/2 pM/mg of protein) and reached plateau levels on the 14th postnatal day (atria/ventricles = 36/12 pM/mg of protein). Functional innervation, evidenced by cardioinhibitory vagal response, appeared at birth. Fluorescent staining demonstrated the absence of adrenergic innervation at birth. In culture preparations of newborn rats, CAT activity was not present, indicating the loss of cholinergic neurons during tissue culture preparation. Sympathetic neurons were added to rat heart cell cultures, which were free of demonstrable cholinergic and adrenergic neurons. Scanning electron microscopic, time lapse photographic, and glyoxic acid fluorescent staining indicated the development of innervation in these cultures. Conclusions: In the newborn rat heart cholinergic, but no adrenergic neuroeffector transmission is present. The trophic influence of innervation upon myocardial growth and sensitivity can be assessed with the addition of sympathetic neurons in vitro.

Choline acetyltransferase activity and mass fragmentographic measurement of acetylcholine in specific nuclei and tracts of rat brain

The mass fragmentographic assay of acetylcholine (ACh) is the only available method to measure the acetylcholine content of rat brain nuclei. The ACh concentration and the choline acetyltransferase activity (ChA) of specific rat brain nuclei and tracts are reported. The highest ACh concentration (1.2 nmol mg protein) and ChA activity (576 nmol mg protein per hr) was in the nucleus interpeduncularis, which possessed twice as much ACh and ChA as the nucleus accumbens and nucleus caudatus. The midbrain nuclei: dorsalis raphes and linearis pars caudalis contained as much ACh as the nuclei accumbens and caudatus but the ChA activity was only a fraction of that of the accumbens and caudatus. The three pontine nuclei: locus eoeruleus, tegmenti dorsalis and dorsalis vagi contained slightly less ACh than the nuclei accumbens and caudatus but the ChA varied. It was low in the locus coeruleus but 10- fold higher in the dorsalis vagi. It is proposed that the ratio of ACh content to ChA activity may have some predictive value to determine whether most of the ACh measured in various brain nuclei is located in cell bodies or axon terminals. The data presented are compared with histochemical data on the location of acetylcholinesterase ( Palkovits and Jacobowitz, 1974). This comparison suggests that when the ratio ACh/ChA × 100 is greater than 0.7 and the ACh content is 0.30 nmol mg protein or greater, the transmitter may be located in nerve terminals. When this ratio is smaller than 0.4 and the ChA activity is greater than 5.0 nmol mg protein per hr, the ACh measured may be located in cholinergic cell bodies or small cholinergic interneurones. This suggestion is supported by measurement of the ACh concentration and ChA activity in brain nuclei which are known to contain cholinergic cell bodies (e.g. motor nucleus of the vagus), small cholinergic interneurones (n. caudatus and n. accumbens) and cholinergic nerve terminals (n. locus coeruleus, dorsalis raphes).