The Drosophila NSF protein, dNSF1, plays a similar role at neuromuscular and some central synapses.

The N-ethylmaleimide sensitive fusion protein (NSF) was originally identified as a cytosolic factor required for constitutive vesicular transport and later implicated in synaptic vesicle trafficking as well. Our previous work at neuromuscular synapses in the temperature-sensitive NSF mutant, comatose (comt), has shown that the comt gene product, dNSF1, functions after synaptic vesicle docking in the priming of vesicles for fast calcium-triggered fusion. Here we investigate whether dNSF1 performs a similar function at central synapses associated with the well-characterized giant fiber neural pathway. These include a synapse within the giant fiber pathway, made by the peripherally synapsing interneuron (PSI), as well as synapses providing input to the giant fiber pathway. The latency (delay) between stimulation and a resulting muscle action potential was used to assess the function of each class of synapses. Repetitive stimulation of the giant fiber pathway in comt produced wild-type responses at both 20 and 36 degrees C, exhibiting a characteristic and constant latency between stimulation and the muscle response. In contrast, stimulation of presynaptic inputs to the giant fiber (referred to as the "long latency pathway") revealed a striking difference between wild type and comt at 36 degrees C. Repetitive stimulation of the long latency pathway led to a progressive, activity-dependent increase in the response latency in comt, but not in wild type. Thus the giant fiber pathway, including the PSI synapse, appears to function normally in comt, whereas the presynaptic inputs to the giant fiber pathway are disrupted. Several aspects of the progressive latency increase observed in the long latency pathway can be understood in the context of the activity-dependent reduction in neurotransmitter release we observed previously at neuromuscular synapses. These results suggest that repetitive stimulation causes a progressive reduction in neurotransmitter release by presynaptic inputs to the giant fiber neuron, resulting in an increased latency preceding a giant fiber action potential. Thus synapses presynaptic to the giant fiber appear to utilize dNSF1 in a manner similar to the neuromuscular synapse, whereas the PSI chemical synapse may differ with respect to the expression or activity of dNSF1.     

Cholinergic properties of embryonic chick sensory neurons

Experiments were carried out to determine the cholinergic properties of sensory neurons of the chick embryo by measuring the choline acetyltransferase activity (ChAT) and [3H]choline uptake. The choline acetyltransferase activity in the dorsal root ganglia of an 8-day-old chick embryo was 24.2 +/- 2.52, which increased to 45.4 +/- 9.69 pmol ACh/mg protein/min in the ganglia of 12-day-old embryos. Sensory neurons derived from dorsal root ganglia of 10-day-old embryos and maintained in a serum-free culture medium supplemented with insulin, transferrin and nerve growth factor (NGF) also contained significant amounts of ChAT (21.9 pmol ACh/mg protein/min). Omission of NGF resulted in neuronal death, and the enzyme activity could not be measured in these cultures. A specific inhibitor of ChAT, hydroxyethyl naphthylvinyl pyridine (NVP), when added to the assay mix produced a dose-dependent inhibition of ChAT from cultured neurons. Cultured sensory neurons incubated with [3H]choline followed by repeated washouts took up and retained [3H]choline. The uptake of [3H]choline was reduced by about 45% when NaCl, in the incubation medium, was replaced by LiCl. A specific inhibitor of choline uptake, hemicholinium-3, caused about 75% inhibition of [3H]choline uptake. It is implied that sensory neurons of the chick dorsal root ganglia express cholinergic properties during development.     

Cerebellar unipolar brush cells are targets of primary vestibular afferents: an experimental study in the gerbil

The unipolar brush cell (UBC) is an excitatory glutamatergic interneuron, situated in the cerebellar granular layer, that itself receives excitatory synaptic input on its dendritic brush from a single mossy fiber terminal in the form of a giant glutamatergic synapse. The UBC axon branches within the granular layer, giving rise to large terminals that synapse with both granule cell and UBC dendrites within glomeruli and resemble in morphological and functional terms those formed by extrinsic mossy fibers. So far, the only demonstrated extrinsic afferents to the UBC are the choline acetyltransferase (ChAT)-positive mossy fibers, some of which originate from the medial and descending vestibular nuclei. To ascertain whether UBCs are innervated by primary vestibular fibers, we performed a tract-tracing light and electron microscopic study of the vestibulocerebellum in gerbils. Macular and canal vestibular end-organs were individually labeled by injection of biotinylated dextran amine. After an appropriate survival time, gerbils were then processed for light and electron microscopic analysis of central vestibular projections. In the nodulus and uvula, labeled primary vestibular fibers formed mossy terminals synapsing with both granule cells and UBCs in all of the injected gerbils. Thus, innervation of UBCs by extrinsic mossy fibers carrying static and dynamic vestibular signals represents the first synapse of networks that contribute a powerful form of distributed excitation in the granular layer. Electronic Publication     

Changes in choline acetyltransferase immunoreactivity and the number of immunoreactive fibers remaining after lesions to the magnocellular basal nucleus of rats

Electrolytic and kainic acid lesions of the magnocellular basal nucleus of rats caused a homogeneous reduction in the density of choline acetyltransferase (ChAT)-immunoreactive fibers in the frontal and parietal cortices. ChAT immunoreactivity of the remaining fibers after unilateral lesions was increased ipsilaterally within the deafferentated areas. The number of intact immunoreactive fibers was consistently low through the period from 7 days to 6 months after the lesion. A previous finding that biochemically measured ChAT activity in the lesioned side recovered to the contralateral level should be interpreted as an increase in the content of ChAT in terminal axons rather than fiber sprouting.     

GM1 and NGF synergism on choline acetyltransferase and choline uptake in aged brain

In the brain of aged rats high affinity choline uptake (HAChU) of the striatum, hippocampus, and frontal cortex is lower than in young rats, while choline acetyltransferase (ChAT) activity is lower in striatum and frontal cortex. Infusion into the lateral cerebral ventricle with nerve growth factor (NGF) enhances the low values of these cholinergic markers in a dose- and region-dependent manner. GM 1 ganglioside infused into the lateral ventricle, at a dose that is ineffective alone, together with NGF synergistically enhances the effect of NGF on ChAT and HAChU activities in the brain of aged animals. The pharmacology of this GM 1/NGF synergism suggests potentiation of response.     

Cholinergic neurons in the rat nodose ganglia

Presence of acetylcholine (ACh) in the vagal afferent fibres of the rat was investigated. In the nodose ganglion, which contains the cell bodies of this sensitive contingent, a choline acetyltransferase (ChAT) activity, a choline (Ch) uptake and an endogenous content of acetylcholine were detected. These data were confirmed by ChAT immunohistological visualization.     

The length of hippocampal cholinergic fibers is reduced in the aging brain

Cholinergic deficits occur in the aged hippocampus and they are significant in Alzheimer's disease. Using stereological and biochemical approaches, we characterized the cholinergic septohippocampal pathway in old (24 months) and young adult (3 months) rats. The total length of choline acetyltransferase (ChAT)-positive fibers in the dorsal hippocampus was significantly decreased by 32% with aging (F((1,9))=20.94, p=0.0014), along with the levels of synaptophysin, a presynaptic marker. No significant changes were detected in ChAT activity or in the amounts of ChAT protein, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin related kinase receptor (Trk) A, TrkB, or p75 neurotrophin receptor (p75(NTR)) in the aged dorsal hippocampus. The number and size of ChAT-positive neurons and the levels of ChAT activity, NGF and BDNF were not statistically different in the septum of aged and young adult rats. This study suggests that substantial synaptic loss and cholinergic axonal degeneration occurs during aging and reinforces the importance of therapies that can protect axons and promote their growth in order to restore cholinergic neurotransmission.     

Organizational changes in cholinergic activity and enhanced visuospatial memory as a function of choline administered prenatally or postnatally or both

This experiment was an examination of the effects of supplemental dietary choline chloride given prenatally (to the diet of pregnant rats) and postnatally (intubed directly into the stomachs of rat pups) on memory function and neurochemical measures of brain cholinergic activity of male albino rats when they became adults. The data demonstrate that perinatal choline supplementation causes (a) long-term facilitative effects on working and reference memory components of a 12-arm radial maze task, and (b) alternations of muscarinic receptor density as indexed by [3H]quinuclidinyl benzilate (QNB) binding and choline acetyltransferase (ChAT) levels in the hippocampus and frontal cortex of adult rats. An analysis of the relationship between these organizational changes in brain and memory function indicated that the ChAT-to-QNB ratio in the hippocampus is highly correlated with working memory errors, and this ratio in the frontal cortex is highly correlated with reference memory errors.     

Excitotoxicity of NMDA and kainic acid is modulated by nigrostriatal dopaminergic fibres

The excitotoxic action of N-methyl-D-aspartate (NMDA) and of kinase (KA) has been assessed by measuring glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) activity in the rat striatum 5 days after focal microinjections of NMDA or KA. Prior unilateral lesioning of the nigrostriatal dopaminergic pathway by focal injection of 6-hydroxydopamine decreases the excitotoxic effect of NMDA and of KA.     

Membrane-bound choline acetyltransferase in Torpedo electric organ: A marker for synaptosomal plasma membranes?

The enzyme choline-O-acetyltransferase catalyses the biosynthesis of acetylcholine from acetyl coenzyme A and choline and is considered as one of the best markers for cholinergic nerve endings. The distribution of this enzymatic activity was analysed during the purification of plasma membranes of purely cholinergic nerve endings isolated from the electric organ of the fish Torpedo marmorata. This tissue, which receives a profuse and purely cholinergic innervation, can be considered as being a "giant" neuromuscular synapse. The isolated nerve endings (synaptosomes) were first osmotically disrupted and their plasma membranes isolated by equilibrium density centrifugation (discontinuous followed by continuous sucrose gradients). Choline acetyltransferase activity was found to exist in three forms: (1) a soluble form (the major one) present in the cytoplasm of the nerve endings, (2) a form which is ionically associated with membranes and which can be solubilized by washing exhaustively the membrane fraction with solutions of high ionic strength (0.5 M NaCl) and (iii) a form which is non-ionically bound to membranes and cannot be solubilized with high salt solution. The soluble and the non-ionically bound activities exhibited very similar affinities for choline (1.34 and 1.64 mM, respectively). The non-ionically membrane-associated form of choline acetyltransferase was found to "copurify" with the cholinergic synaptosomal plasma membranes of Torpedo, its specific activity being increased from 122 (crude fraction) to 475 (purified membrane fraction) nmol/h/mg protein. An enrichment was also observed for another cholinergic marker, the enzyme acetylcholinesterase, but not for the nicotinic receptor to acetylcholine, a marker for postsynaptic membranes. No choline acetyltransferase activity could be detected in preparations of synaptic vesicles that were highly purified from the electric organ. Also, the non-ionically associated form of choline acetyltransferase activity was hardly detectable (2.4 nmol/h/mg protein) in fractions enriched in axonal membranes prepared from the cholinergic electric nerves innervating the electric organ. The partition into soluble and membrane-bound activity was also analysed for choline acetyltransferase present in human placenta, a rich source for the enzyme but a non-innervated tissue. In this case the great majority of the enzyme appeared as soluble activity. Very low levels of non-ionically membrane-bound activity were found to be present in a crude membrane fraction from human placenta (2.8 nmol/h/mg protein).(ABSTRACT TRUNCATED AT 400 WORDS)     

Testosterone locally increases vasopressin content but fails to restore choline acetyltransferase activity in other regions in the senescent male rat brain

Age-related decreases have been reported in both vasopressinergic and cholinergic innervation in the rat brain. Since both systems are also sensitive to sex steroids, the effect of testosterone supplementation on vasopressin (AVP) levels and on choline acetyltransferase (ChAT) activity was investigated in the brains of young, middle-aged and aged male rats. Although no age-related changes in AVP levels were observed in the lateral septum or the medial amygdala (MA), peripheral testosterone administration raised AVP levels in the MA in all age groups. ChAT activity decreased with age in the medial preoptic area and was not restored by testosterone.     

Reduced amounts of immunoreactive somatostatin in the temporal cortex in senile dementia of Alzheimer type

Post-mortem brain tissue from 15 patients dying with a diagnosis of senile dementia of Alzheimer type (SDAT) was compared with tissue obtained from 16 control patients at routine post-mortem. A significant fall in choline acetyltransferase (ChAT) activity was observed in the cortex, hippocampus and amygdala of the SDAT cases and was maximal in the temporal cortex. The fall in ChAT activity observed in the temporal cortex was accompanied by a significant reduction (47%) in immunoreactive somatostatin.     

Drosophila CAKI/CMG protein, a homolog of human CASK, is essential for regulation of neurotransmitter vesicle release

Vertebrate CASK is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. CASK is present in the nervous system where it binds to neurexin, a transmembrane protein localized in the presynaptic membrane. The Drosophila homologue of CASK is CAKI or CAMGUK. CAKI is expressed in the nervous system of larvae and adult flies. In adult flies, the expression of caki is particularly evident in the visual brain regions. To elucidate the functional role of CASK, we employed a caki null mutant in the model organism Drosophila melanogaster. By means of electrophysiological methods, we analyzed, in adult flies, the spontaneous and evoked neurotransmitter release at the neuromuscular junction (NMJ) as well as the functional status of the giant fiber pathway and of the visual system. We found that in caki mutants, when synaptic activity is modified, the spontaneous neurotransmitter release of the indirect flight muscle NMJ was increased, the response of the giant fiber pathway to continuous stimulation was impaired, and electroretinographic responses to single and continuous repetitive stimuli were altered and optomotor behavior was abnormal. These results support the involvement of CAKI in neurotransmitter release and nervous system function.     

Behavioral evaluation of the anti-excitotoxic properties of MK-801: comparison with neurochemical measurements

The ability of MK-801 to protect striatal neurons from the excitotoxic action of quinolinic acid was evaluated by means of apomorphine-induced rotational behavior and by measurement of striatal choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activity, neurochemical markers for cholinergic and GABAergic neurons, respectively. Animals with a unilateral quinolinic acid lesion of the striatum exhibited a vigorous rotational response when challenged with apomorphine (0.5 mg/kg, s.c.) 6 days later and were found to have an 88 90% depletion of striatal ChAT and GAD activity. Treatment with a high dose of MK-801 (10 mg/kg, i.p.) prior to intrastriatal injection of quinolinic acid eliminated the subsequent rotational response to apomorphine and resulted in complete protection of striatal ChAT and GAD activity. Lower doses of MK-801 (1, 3 and 5 mg/kg, i.p.) failed to significantly reduce the rotational response to apomorphine but provided partial, dose-dependent protection of both ChAT and GAD activity. The rotational response to apomorphine correlated with the percent reduction in both ChAT activity (r = 0.57, P less than 0.0005) and GAD activity (r = 0.49, P less than 0.0005). Rotational behavior may thus provide a means to evaluate the functional integrity of the striatum.     

Relationship between the cortical choline acetyltransferase content and EEG delta-power

Destruction of the nucleus basalis abolished normal spectral power only in the frontal cortex, where choline acetyltransferase (ChAT) activity was lower. In lesioned animals, delta-power was increased and alpha-power decreased. The increase in delta-power during immobility correlated with a reduction in ChAT activity. High-voltage spindles occurred more frequently after cholinergic denervation. The results suggest that spectral power and high-voltage spindle analysis might serve as a useful tool for evaluating the efficacy of pharmacological strategies aimed at alleviating the cholinergic deficit.     

Genetic evidence that chloroadenosine increases the specific activity of choline acetyltransferase in PC12 cells via modulation of an adenosine-dependent adenylate cyclase

Both chloroadenosine (EC50 = 3 X 10(-7) M) and cholera toxin, like nerve growth factor, increase the specific activity of choline acetyltransferase in PC12 cells over a period of several days. The increase in choline acetyltransferase activity in response to chloroadenosine appears to be caused by the ability of chloroadenosine to increase adenosine 3':5'-phosphate synthesis by binding to an adenosine receptor that activates adenylate cyclase. To test this hypothesis we determined if chloroadenosine can cause an increase in choline acetyltransferase activity in adenosine kinase-deficient PC12 cells. We have previously shown that adenosine analogues are significantly less effective at regulating adenosine 3':5'-phosphate in adenosine kinase-deficient PC12 cells than in wild type cells [Erny and Wagner (1984) Proc. natn. Acad. Sci. U.S.A. 81, 4974-4978]. Adenosine kinase-deficient PC12 cells are resistant to the induction of choline acetyltransferase in response to chloroadenosine, but not cholera toxin, supporting the role of adenosine 3':5'-phosphate in mediating the effects of chloroadenosine. The increase in choline acetyltransferase activity in wild type cells was accompanied by an increase in acetylcholine levels, demonstrating that chloroadenosine also regulates storage of acetylcholine. Acetylcholine levels were quantitated using an assay based on the ability of acetylcholine to compete with [125I]bungarotoxin for binding to the acetylcholine receptor.     

Cortical somatostatin-like immunoreactivity in cases of Alzheimer's disease and senile Dementia of the Alzheimer type

The activity of choline acetyltransferase (ChAT) and the concentration of somatostatin-like immunoreactive material (SLI) have been measured in 8 brain regions from 12 normal individuals and 12 cases of presenile and senile dementia of the Alzheimer type. ChAT activity was significantly lower in all 8 brain regions of demented patients and the SLI concentration was significantly reduced in 7 of the 8. There were correlations between the extent of the reductions of ChAT activity and SLI concentration in four brain regions, and a greater reduction of parietal cortex SLI in younger patients than in the more elderly.     

Evidence for the existence of a population of arcuate neurons costoring choline acetyltransferase and tyrosine hydroxylase immunoreactivities in the male rat

By combined immunoperoxidase and immunofluorescence histochemistry we have analyzed the distribution of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) immunoreactive (IR) perikarya within the same sections of the mediobasal hypothalamus of the male rat. Evidence was obtained for the existence of perikarya costoring TH and ChAT immunoreactivities in both the dorsomedial and ventrolateral part of the arcuate nucleus and in the adjacent periarcuate nucleus at all rostrocaudal levels. The results strongly implicate interactions between dopamine and acetylcholine as well as acetylcholine and growth hormone releasing factor in dorsomedially and ventrolaterally located TH/ChAT costoring tuberoinfundibular neurons, respectively.     

Reactive microglia in patients with senile dementia of the Alzheimer type are positive for the histocompatibility glycoprotein HLA-DR

HLA-DR is a class II cell surface glycoprotein of the human histocompatibility complex usually expressed on the surface of cells that are simultaneously presenting foreign antigen to T-lymphocytes. Using immunohistochemical procedures with two specific monoclonal antibodies to HLA-DR, HLA-DR-positive reactive microglia were found in gray matter throughout the cortex of postmortem brains of patients with senile dementia of the Alzheimer type (SDAT) and were particularly concentrated in the areas of senile plaque formation. Double immunostaining with antibodies to glial fibrillary acidic protein (GFAP) showed that the HLA-DR-positive cells were different from the reactive astrocytes although the occasional positively staining giant astrocyte was also seen. Small numbers of resting microglia were HLA-DR-positive in white matter of both normal and SDAT brains. The SDAT cases also had reduced cortical choline acetyltransferase (ChAT) levels. In the 11 brains studied, the number of hippocampal HLA-DR-positive cells was positively correlated with the numbers of plaques and negatively correlated with average cortical ChAT.