Anatomical mapping of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in outer hair cell efferents in adult rats

Summary The distribution of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in the cochleae of 15 adult Wistar white rats was investigated using the peroxidase-antiperoxidase (PAP) technique. A monoclonal antibody to ChAT and a polyclonal antiserum to GAD were used. Immunoreaction was investigated quantitatively, in the electron microscope, on tangential sections of the tunnel of Corti and the rows of outer hair cells. ChAT-like and GAD-like immunoreactivity was found in all efferent nerve fibres in the tunnel of Corti and in all efferent synapses on the outer hair cells. A coexistence of ChAT and GAD in the efferent system to the outer hair cells of the rat is therefore assumed.     

Differential Effects of Insulin on Choline Acetyltransferase and Glutamic Acid Decarboxylase Activities in Neuron-Rich Striatal Cultures

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.     

Immunolocalization of choline acetyltransferase in 2 types of efferent synapses of the organ of Corti

The efferent (olivo-cochlear) innervation of the organ of Corti was studied using a monoclonal antibody against choline acetyltransferase (ChAT). In the inner spiral bundle (ISB), below the inner hair cells (IHCs), the anti-ChAT immunoreactivity was observed within unvesiculated fibers and vesiculated varicosities. Unreactive varicosities, at least as numerous as the immunoreactive ones, were also detected. Both types of vesiculated varicosities synapsed with the dendrites of the primary auditory neurons (afferent fibers) connected to the IHCs. At the outer hair cell (OHC) level, nearly all the vesiculated terminals making axo-somatic synapses with the OHCs were anti-ChAT immunoreactive. Only few terminals synapsing with the OHCs were unreactive. These findings allowed the differentiation of at least three types of efferent synapses in the organ of Corti. In the ISB, a first population of axo-dendritic synapses seems to be cholinergic whereas a second population might use another neurotransmitter. At the OHC level, our results support the hypothesis that acetylcholine is the neurotransmitter of nearly all the large axo-somatic synapses. The rare unreactive axo-somatic synapses could constitute a fourth and minor type of efferent synapse. Thus, it would be helpful to subclassify the efferent innervations of the organ of Corti according to their neurochemical nature. A re-evaluation of the whole body of available electrophysiological data would be also necessary, as until now, acetylcholine was considered as being the only efferent cochlear neurotransmitter.     

Choline acetyltransferase, glutamic acid decarboxylase and somatostatin in the kainic acid model for chronic temporal lobe epilepsy

The aim of the study was to investigate neurochemical changes in a kainic acid (KA; 10 mg/kg, s.c.)-induced spontaneous recurrent seizure model of epilepsy, 6 months after the initial KA-induced seizures. The neuronal markers of cholinergic and gamma-aminobutyric acid (GABA)ergic systems, i.e. choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activities, and a marker for neuropeptide, i.e. level of somatostatin, have been investigated. The brain regions investigated were the hippocampus, amygdala/piriform cortex, caudate nucleus, substantia nigra and the frontal, parietal, temporal and occipital cortices. Six months after KA injection, reduced ChAT activity was observed in the amygdala/piriform cortex (47% of control; p<0.001), increased ChAT activity in the hippocampus (119% of control; p<0.01) and normal ChAT activity in the other brain regions. The activity of GAD was significantly increased in all analysed cortical regions (between 146 and 171% of control), in the caudate nucleus (144% of control; p<0.01) and in the substantia nigra (126% of control; p<0.01), whereas in the amygdala/piriform cortex, the GAD activity was moderately lowered. The somatostatin level was significantly increased in all cortical regions (between 162 and 221% of control) as well as in the hippocampus (119% of control), but reduced in the amygdala/piriform cortex (45% of control; p<0.01). Six months after KA injection, the somatostatin:GAD ratio was lowered in the amygdala/piriform cortex (49% of control) and in the caudate nucleus (41% of control), whereas it was normal in the hippocampus and moderately increased in the cortical brain regions. A positive correlation was found between seizure severity and the reduction of both ChAT activities and somatostatin levels in the amygdala/piriform cortex. The results show a specific pattern of changes for cholinergic, GABAergic and somatostatinergic activities in the chronic KA model for epilepsy. The revealed data suggest a functional role for them in the new network that follows spontaneous repetitive seizures.     

Differential sensitivity of cholinergic and GABAergic neurons in chick embryos treated intracerebrally with ethanol at 8 days of embryonic age

We have shown that in embryos treated with ethanol in ovo during days 1–3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferse (ChAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in both cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergic neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick.     

Role of Thyroid Hormone and Nerve Growth Factor in the Development of Choline Acetyltransferase and Other Cell-Specific Marker Enzymes in the Basal Forebrain of the Rat

The effects of treatment with L-thyroxine (subcutaneously 0.3 microgram/g body weight daily from birth, i.e., day 1) and 2.5S nerve growth factor (NGF; intraventricularly 2 micrograms on 1, 3, 5, 7, and 9 postnatal days), separately and together, were studied on the biochemical development of different cell types in the basal forebrain of 10-day-old rats. The development of cholinergic, gamma-aminobutyric acid-ergic (GABAergic), and glutamatergic neurons was monitored respectively in terms of choline acetyltransferase (ChAT), glutamate decarboxylase (GAD), and glutaminase activities, whereas glutamine synthetase (GS) and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase) activities were used to judge the maturation of astroglial and oligodendroglial cells. Treatment with either thyroid hormone or NGF from birth significantly increased the expression of ChAT activity in the basal forebrain of neonatal rats. When both agents were administered to the same animal, in agreement with our earlier in vitro findings, the stimulation in ChAT activity was much greater than the sum of the individual effects. In hypothyroid rats, significant effects of NGF at the low doses used were not detectable, although the increase of ChAT activity induced by thyroxine was potentiated by NGF in these animals. Under the present experimental conditions neither thyroxine nor NGF treatment had an appreciable effect on the activities of glutaminase, GS, and lactate dehydrogenase. However, the administration of thyroxine markedly increased CNPase activity in normal rats, whereas in hypothyroid rats the effect on both CNPase and GAD was also significant. Similar elevations in CNPase and GAD activities were not observed after NGF treatment, suggesting that the effect of NGF was specific to the cholinergic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired Development of Rat Cerebellum Induced by Neonatal Injection of the Glycoprotein Synthesis Inhibitor, Tunicamycin

The effects of the protein glycosylation inhibitor tunicamycin on the postnatal development of the rat cerebellum were examined in vivo. Tunicamycin (0.2 micrograms) was injected intracranially into 1-day-old rats. Inhibition of glycosylation of the macromolecules in the cerebellum by tunicamycin treatment was suggested by a reduced incorporation of [3H]glucosamine into the trichloroacetic acid (TCA)-insoluble fraction. The tunicamycin treatment did not affect gain in body weight significantly. However the cerebellar weight was significantly reduced by 30-40% compared with that of the controls. Development of GABAergic and cholinergic innervations in the hypoplastic cerebellum was examined by measuring the activities of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT). The specific activity and the total activity of GAD were significantly reduced in the tunicamycin-treated cerebellum. In contrast the specific activity of ChAT was significantly increased, whereas the total activity of ChAT per cerebellum was identical with that of the controls. These results suggest that the intracranial injection of tunicamycin affects the postnatal development of rat cerebellum, such as GABAergic and cholinergic innervations.     

Specific lysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase

An antiserum to pure glutamate decarboxylase (GAD) when incubated with rat cortical synaptosomes in the presence of complement caused release of 33-53% of lactate dehydrogenase (LDH) and 22-41% of total GAD. In addition most of the gamma-aminobutyrate (GABA) present was released. Anti-GAD antiserum alone, or complement alone, were without action. The antiserum plus complement had no effect on noradrenaline or choline uptake, and did not release choline acetylase (ChAT). Anti-ChAT serum plus complement released 30-37% of ChAT and 10-13% of LDH. It prevented choline uptake. This serum did not produce GAD release or prevent GABA, choline or noradrenaline uptake. When cortical synaptosomes were exposed to both antisera plus complement, their actions were strictly additive. The data indicate specific lysis of GABAergic and cholinergic synaptosomal sub-populations.     

TOPOGRAPHICAL AND SUBCELLULAR DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND GLUTAMATE DECARBOXYLASE IN PIGEON OPTIC TECTUM

Abstract— The distribution of choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in different layers of the pigeon optic tectum and in some nuclei of the optic lobe have been investigated. About 40% of GAD and 25% of ChAT were found in the superficial part of tectum, but negligible activity was found in the stratum opticum. The highest GAD activity was found in layers 3-7 (according to the nomenclature of C ajal , 1911) with a peak in layer 4. ChAT activity peaked in layers 3, 5. 8 and 10/11. Its distribution correlated well with the staining pattern of AChE, particularly in the superficial part of the tectum. The distribution of ChAT and GAD did not change significantly 4 weeks after enucleation. ChAT and GAD activities were high in the nucleus isthmi, pars parvocellularis (Ipc). The activity of GAD was also high in the nucleus intercollicularis (ICo), the other nuclei showed less activity of both enzymes.     

Neurons with different neurotransmitters in embryonic neocortical allografts in the rat sciatic nerve

Different subsets of interneurons in the Wistar rat neocortex and in neocortical transplants developing in a damaged nerve were identified by the following immunohistochemical markers: glutamate decarboxylase (GAD 67) for GABAergic nerve cells, NO-synthase (NOS) for NO-ergic neurons, choline acetyltransferase (ChAT) for cholinergic cells, and tyrosine hydroxylase for catecholaminergic structures. Twentyeight days after surgery, individual GAD 67-ir, NO-ir, ChAT-ir, and very rarely TH-ir cells were detected in the graft. It was shown that the number of GAD 67-ir neurons per unit area in the grafts was less than in the rat neocortex P20.     

Differential susceptibilities of spinal cord neurons to retinoic acid-induced survival and differentiation

In a previous study, we demonstrated trophic effects of vitamin A and its active metabolite, retinoic acid (RA), on perinatal rat spinal cord neurons and astrocytes in vitro. We now report that RA increases the survival of cholinergic neurons without affecting that of GABAergic neurons. These results were supported by measured levels of acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and glutamic acid decarboxylase (GAD) activities, key enzymes of acetylcholine and gamma-aminobutyric acid metabolism, respectively, which showed RA-induced increases in AChE and ChAT levels but no elevations of GAD activity. In contrast to these phenotype-specific effects, most neurons showed RA-induced increases in neuritic outgrowth, density, and silver impregnation. Taken together, these results demonstrate neurotransmitter-specific and generalized effects of RA on developing CNS neurons.     

Inhibition of choline acetyltransferase as a mechanism for cholinergic dysfunction induced by amyloid-β peptide oligomers

Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-β peptide (Aβ). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aβ oligomers (AβOs). AβOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AβOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ~50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AβOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [(3)H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AβOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AβO binding sites, fully prevented AβO-induced inhibition of ChAT. Interestingly, we found that AβOs selectively bind to ~50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AβO-targeted neurons. Reduction in ChAT activity instigated by AβOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.     

Increased acetylcholine levels in skin biopsies of patients with atopic dermatitis

Recent experimental evidence indicates that non-neuronal acetylcholine is involved in the regulation of basic cell functions. Here we investigated the cholinergic system in the skin of healthy volunteers and patients with atopic dermatitis (AD). The synthesizing enzyme, choline-acetyltransferase (ChAT), was studied by anti-ChAT immunohistochemistry and enzyme assay. Skin biopsies taken from healthy volunteers and from AD patients were separated into the 2 mm superfical (epidermis and upper dermis) and 3 mm underlying portion (deeper dermis and subcutis). ChAT enzyme activity was detected in homogenized skin and subcutaneous fat (about 13 nmol/mg protein/h). ChAT immunoreactivity was expressed in keratinocytes, hair papilla, sebaceous and eccrine sweat glands, endothelial cells and mast cells. In healthy volunteers the superficial and underlying portion of skin biopsies contained 130 +/- 30 and 550 +/- 170 pmol/g acetylcholine (n = 12), respectively. In AD patients (n = 7) acetylcholine was increased 14-fold in the superficial and 3-fold in the underlying biopsy portion. The present study demonstrates the widespread expression of ChAT protein in the vast majority of human skin cells. Tissue levels of acetylcholine are greatly (14-fold) enhanced in the superficial 2 mm skin of AD patients.     

Expression of the cholinergic gene locus in the rat placenta

High amounts of acetylcholine (ACh) and its synthesising enzyme choline acetyltransferase (ChAT) have been detected in the placenta. Since the placenta is not innervated by extrinsic or intrinsic cholinergic neurons, placental ACh and ChAT originate from non-neuronal sources. In neurons, cytoplasmic ACh is imported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), and released through vesicular exocytosis. In view of the coordinate expression of VAChT and ChAT from the cholinergic gene locus in neurons, we asked whether VAChT is coexpressed with ChAT in rat placenta, and investigated this issue by means of RT-PCR, in situ hybridisation, western blot and immunohistochemistry. Messenger RNA and protein of the common type of ChAT (cChAT), its splice variant peripheral ChAT (pChAT), and VAChT were detected in rat placenta with RT-PCR and western blot. ChAT in situ hybridisation signal and immunoreactivity for cChAT and pChAT were observed in nearly all placental cell types, while VAChT mRNA and immunolabelling were detected in the trophoblast, mesenchymal cells and the visceral yolk sac epithelial cells. While ChAT is nearly ubiquitously expressed in rat placenta, VAChT immunoreactivity is localised cell type specifically, implying that both vesicular and non-vesicular ACh release machineries prevail in placental cell types.     

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.     

Developmental changes in choline acetyltransferase and glutamate decarboxylase activity in various regions of the brain of the male,female, and neonatally androgenized female rat

In an attempt to discern effects of sex hormones on the development of neurotransmitter systems in the rat brain, choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) have been measured at postnatal days 8, 12, 25, and 60 in five regions (amygdala, anterior hypothalamus, hippocampus, olfactory bulbs, and cerebral cortex) of the brains of normal male, normal female, and neonatally androgen-treated female rats. Essentially no associations between sex or of neonatal androgenization on either enzyme were found. The data, however, provide new information on the relative rates of development of ChAT and GAD in five regions of the rat brain which supplement the limited information already available in the literature. ChAT activity was highest in amygdala and hypothalamus, but developed most rapidly in hippocampus and cerebral cortex. The relative activities and patterns of development of GAD activity were similar to those of ChAT.     

Identification and Transgenic Analysis of a Murine Promoter that Targets Cholinergic Neuron Expression

Abstract : Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase ( LacZ ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system ; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.     

Cholinergic innervation of the mouse superior cervical ganglion: light-and electron-microscopic immunocytochemistry for choline acetyltransferase

Summary The cholinergic innervation of the mouse superior cervical ganglion was investigated by means of immunocytochemistry using a well-characterized monoclonal antibody against choline acetyltransferase (ChAT). Immunopositive nerve fibers entered the superior cervical ganglion from the cervical sympathetic trunk. Light-microscopically, these fibers appeared to be heterogeneously distributed among the principal ganglion cells. The rostral part of the ganglion contained more ChAT-positive fibers then the middle or the caudal one. The axons branched several times before forming numerous varicosities. Most of the ChAT-stained fibers and varicosities aggregated in glomerula-like neuropil structures that were surrounded by principal ganglion cell bodies, whereas others were isolated or formed little bundles among principal neurons. None of the neurons or other cell types in the ganglion exhibited ChAT-positivity. ChAT-immunoreactive fibers disappeared from the ganglion 5 or 13 days after transection of the cervical sympathetic trunk. At the ultrastructural level, most axon terminals and synapses showed ChAT-immunoreactivity. An ultrastructural analysis indicated that immunostained synapses occurred directly on the surface of neuronal soma (1.8%) and dendritic shafts (17.6%). Synapses were often seen on soma spines (18.4%) and on dendritic spines (62.2%). All immunoreactive synapses were of the asymmetric type. The results provide immunocytochemical evidence for a heterogeneous cholinergic innervation of the ganglion and the principal neurons.