Evaluation of the endogenous glucocorticoid hypothesis of denervation atrophy

The effects are studied of the oral administration of RU38486, a potent selective glucocorticoid antagonist, on muscle weight, non-collagen protein content, and selected enzyme activities (choline acetyltransferase, glucose 6-phosphate dehydrogenase, and glutamine synthetase) following denervation of rat skeletal muscle. Neither decreases in muscle weight, protein content, and choline acetyltransferase activity, nor increases in the activities of glucose 6-phosphate dehydrogernase and glutamine synthetase were affected by RU38486. These data do not support the hypothesis that denervation atrophy results from enhanced sensitivity of muscle to endogenous glucocorticoids

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the teleost cyprinus carpio

Cholinergic systems play a role in basic cerebral functions and its dysfunction is associated with deficit in neurodegenerative disease. Mechanisms involved in human brain diseases, are often approached by using fish models, especially cyprinids, given basic similarities of the fish brain to that of mammals. In the present paper, the organization of central cholinergic systems have been described in the cyprinid Cyprinus carpio, the common carp, by using specific polyclonal antibodies against ChAT, the synthetic enzyme of acetylcholine, that is currently used as a specific marker for cholinergic neurons in all vertebrates. In this work, serial transverse sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei of the forebrain, the midbrain, the hindbrain and the spinal cord. Moreover, ChAT-positive neurons were detected in the synencephalon and in the cerebellum. In addition to neuronal bodies, afferent varicose fibers were stained for ChAT in the ventral telencephalon, the preoptic area, the hypothalamus and the posterior tuberculum. No neuronal cell bodies were present in the telencephalon. The comparison of cholinergic distribution pattern in the Cyprinus carpio central nervous system has revealed similarities but also some interesting differences with other cyprinids. Our results provide additional information on the cholinergic system from a phylogenetic point of view and may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases

Kinesin-II is required for axonal transport of choline acetyltransferase in Drosophila

KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase. in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport

Cell type-specific regulation of choline acetyltransferase gene expression - Role of the neuron-restrictive silencer element and cholinergic-specific enhancer

This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pair-long region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. A 21-base pair-long neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region. This element was recognized by the neuron-restrictive silencer factor (NRSF), previously shown to regulate expression of other neuron-specific genes. The ChAT NRSE was inactive in both cholinergic and non-cholinergic neuronal cells, but repressed expression from a heterologous promoter in non-neuronal cells. Specific deletion of this element allowed ChAT gene promoter activity in non-neuronal cells, and overexpression of NRSF repressed ChAT gene promoter activity in cholinergic cells. The distal part of the ChAT gene promoter showed cholinergic-specific enhancing activity, which stimulated promoter activity in cholinergic cells, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. Thus, at least two kinds of regulatory elements cooperate to direct ChAT gene expression to cholinergic neurons, namely a neuron-restrictive silencer element and a cholinergic-specific enhancer

The influence of non-neuronal cells on catecholamine and acetylcholine synthesis and accumulation in cultures of dissociated sympathetic neurons

The effects of several non-neuronal cell types on neurotransmitter synthesis in cultures of dissociated sympathetic neurons from the new-born rat were studied. Acetylcholine synthesis from radioactive choline was increased 100- to 1000-fold in the presence of non-neuronal cells from sympathetic ganglia. This increase was roughly dependent on the number of ganglionic non-neuronal cells present. The effect did not appear to be due to an increased plating efficiency of neurons, since the non-neuronal cells were capable of increasing acetylcholine synthesis after only 48-hr contact with neurons that had been previously grown without non-neuronal cells for 2 weeks. C6 rat glioma cells were also able to stimulate acetylcholine synthesis, but 3T3 mouse fibroblast cells had little or no effect. None of the non-neuronal cell types synthesized detectable acetylcholine in the absence of the neurons. The ganglionic non-neuronal cells had no significant effect on catecholamine synthesis (which occurs in the absence of non-neuronal cells)

Brain muscarinic cholinergic receptors in Huntington's disease

Muscarinic cholinergic receptors and choline acetyltransferase (ChAT) activity were studied in postmortem brain tissue from patients with Huntington's disease and matched control subjects. In comparison with controls, reductions in ChAT activity were found in the hippocampus, but not in the temporal cortex in Huntington's disease. Patients with Huntington's disease showed reduced densities of the total number of muscarinic receptors and of M-2 receptors in the hippocampus while the density of M-1 receptors was unaltered. Muscarinic receptor binding was unchanged in the temporal cortex. These results indicate a degeneration in Huntington's disease of the septo-hippocampal cholinergic pathway, but no impairment of the innominato-cortical cholinergic system

Changes in neuromuscular structure and functions of human colon during ageing are region-dependent

Objective: To determine if human colonic neuromuscular functions decline with increasing age.Design: Looking for non-specific changes in neuromuscular function, a standard burst of electrical field stimulation (EFS) was used to evoke neuronally mediated (cholinergic/nitrergic) contractions/relaxations in ex vivomuscle strips of human ascending and descending colon, aged 35–91 years (macroscopically normal tissue; 239 patients undergoing cancer resection). Then, to understand mechanisms of change, numbers and phenotype of myenteric neurons (30 306 neurons stained with different markers), densities of intramuscular nerve fibres (51 patients in total) and pathways involved in functional changes were systematically investigated (by immunohistochemistry and use of pharmacological tools) in elderly (≥70 years) and adult (35–60 years) groups.Results: With increasing age, EFS was more likely to evoke muscle relaxation in ascending colon instead of contraction (linear regression: n=109, slope 0.49%±0.21%/year, 95% CI ), generally uninfluenced by comorbidity or use of medications. Similar changes were absent in descending colon. In the elderly, overall numbers of myenteric and neuronal nitric oxide synthaseimmunoreactive neurons and intramuscular nerve densities were unchanged in ascending and descending colon, compared with adults. In elderly ascending, not descending, colon numbers of cell bodies exhibiting choline acetyltransferase immunoreactivity increased compared with adults (5.0±0.6 vs 2.4±0.3 neurons/mm myenteric plexus, p=0.04). Cholinergically mediated contractions were smaller in elderly ascending colon compared with adults (2.1±0.4 and 4.1±1.1 g-tension/gtissue during EFS; n=25/14; p=0.04); there were no changes in nitrergic function or in ability of the muscle to contract/relax. Similar changes were absent in descending colon.Conclusion: In ascending not descending colon, ageing impairs cholinergic function

Altered muscarinic and nicotinic receptor densities in cortical and subcortical brain regions in Parkinson's disease

Muscarinic and nicotinic cholinergic receptors and choline acetyltransferase activity were studied in postmortem brain tissue from patients with histopathologically confirmed Parkinson's disease and matched control subjects. Using washed membrane homogenates from the frontal cortex, hippocampus, caudate nucleus, and putamen, saturation analysis of specific receptor binding was performed for the total number of muscarinic receptors with [3H]quinuclidinyl benzilate, for muscarinic M1 receptors with [3H]pirenzepine, for muscarinic M2 receptors with [3H]oxotremorine-M, and for nicotinic receptors with (-)-[3H]nicotine. In comparison with control tissues, choline acetyl-transferase activity was reduced in the frontal cortex and hippocampus and unchanged in the caudate nucleus and putamen of parkinsonian patients. In Parkinson's disease the maximal binding site density for [3H]quinuclidinyl benzilate was increased in the frontal cortex and unaltered in the hippocampus, caudate nucleus, and putamen. Specific [3H]pirenzepine binding was increased in the frontal cortex, unaltered in the hippocampus, and decreased in the caudate nucleus and putamen. In parkinsonian patients Bmax values for specific [3H]oxotremorine-M binding were reduced in the cortex and unchanged in the hippocampus and striatum compared with controls. Maximal (-)-[3H]nicotine binding was reduced in both the cortex and hippocampus and unaltered in both the caudate nucleus and putamen. Alterations of the equilibrium dissociation constant were not observed for any ligand in any of the brain areas examined. The present results suggest that both the innominatocortical and the septohippocampal cholinergic systems degenerate in Parkinson's disease.(ABSTRACT TRUNCATED AT 250 WORDS

Vascular Endothelial Growth Factor (VEGF) Prevents the Downregulation of the Cholinergic Phenotype in Axotomized Motoneurons of the Adult Rat

Vascular endothelial growth factor (VEGF) was initially characterized by its activity on the vascular system. However, there is growing evidence indicating that VEGF also acts as a neuroprotective factor, and that its administration to neurons suffering from trauma or disease is able to rescue them from cell death. We questioned whether VEGF could also maintain damaged neurons in a neurotransmissive mode by evaluating the synthesis of their neurotransmitter, and whether its action would be direct or through its well-known angiogenic activity. Adult rat extraocular motoneurons were chosen as the experimental model. Lesion was performed by monocular enucleation and immediately a gelatine sponge soaked in VEGF was implanted intraorbitally. After 7 days, abducens, trochlear, and oculomotor nuclei were examined by immunohistochemistry against choline acetyltransferase (ChAT), the biosynthetic enzyme of the motoneuronal neurotransmitter acetylcholine. Lesioned motoneurons exhibited a noticeable ChAT downregulation which was prevented by VEGF administration. To explore whether this action was mediated via an increase in blood vessels or in their permeability, we performed immunohistochemistry against laminin, glucose transporter-1 and the plasmatic protein albumin. The quantification of the immunolabeling intensity against these three proteins showed no significant differences between VEGF-treated, axotomized and control animals. Therefore, the present data indicate that VEGF is able to sustain the cholinergic phenotype in damaged motoneurons, which is a first step for adequate neuromuscular neurotransmission, and that this action seems to be mediated directly on neurons since no sign of angiogenic activity was evident. These data reinforces the therapeutical potential of VEGF in motoneuronal diseases.España, MINECO and FEDER BFU2015-64515-PJunta de Andalucía and FEDER : P10-CVI605