Immunocytochemical Localization of TASK-3 Channels in Rat Motor Neurons

Motor neurons are large cholinergic neurons located in the brain stem and spinal cord. In recent years, a functional role for TASK channels in cellular excitability and vulnerability to anesthetics of motor neurons has been described. Using a polyclonal monospecific antibody against the tandem pore domain K⁺ channel (K2P channel) TWIK-related acid-sensitive K⁺ channel (TASK-3), we analyzed the expression of the TASK-3 protein in motor systems of the rat CNS. Immunocytochemical staining showed strong TASK-3 expression in motor neurons of the facial, trigeminal, ambiguus, and hypoglossal nuclei. Oculomotor nuclei (including trochlear and abducens nucleus) were also strongly positive for TASK-3. The parasympathetic Edinger-Westphal nucleus and dorsal vagal nucleus showed significant, but weaker expression compared with somato- and branchiomotoric neurons. In addition, motor neurons in the anterior horn of the spinal cord were also strongly labeled for TASK-3 immunoreactivity. Based on morphological criteria, TASK-3 was found in the somatodendritic compartment of motor neurons. Cellular staining using methyl green and immunofluorescence double-labeling with anti-vesicular acetylcholine transporter (anti-vAChT) indicated ubiquitous TASK-3 expression in motor neurons, whereas in other brain regions TASK-3 showed a widespread but not ubiquitous expression. In situ hybridization using a TASK-3 specific riboprobe verified the expression of TASK-3 in motor neurons at the mRNA level.     

Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system

With the use of an antiserum generated in rabbits against synthetic human calcitonin gene-related peptide (CGRP) the distribution of CGRP-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system. A detailed stereotaxic atlas of CGRP-like neurons was prepared. CGRP-like immunoreactivity was widely distributed in the rat central nervous system. CGRP positive cell bodies were observed in the preoptic area and hypothalamus (medial preoptic, periventricular, anterior hypothalamic nuclei, perifornical area, medial forebrain bundle), premamillary nucleus, amygdala medialis, hippocampus and dentate gyrus, central gray and the ventromedial nucleus of the thalamus. In the midbrain a large cluster of cells was contained in the peripeduncular area ventral to the medial geniculate body. In the hindbrain cholinergic motor nuclei (III, IV, V, VI, VII XII) contained CGRP-immunoreactivity. Cell bodies were also observed in the ventral tegmental nucleus, the parabrachial nuclei, superior olive and nucleus ambiguus. The ventral horn cells of the spinal cord, the trigeminal and dorsal root ganglia also contained CGRP-immunoreactivity. Dense accumulations of fibers were observed in the amydala centralis, caudal portion of the caudate putamen, sensory trigeminal area, substantia gelatinosa, dorsal horn of the spinal cord (laminae I and II). Other areas containing CGRP-immunoreactive fibers are the septal area, nucleus of the stria terminalis, preoptic and hypothalamic nuclei (e.g., medial preoptic, periventricular, dorsomedial, median eminence), medial forebrain bundle, central gray, medial geniculate body, peripeduncular area, interpeduncular nucleus, cochlear nucleus, parabrachial nuclei, superior olive, nucleus tractus solitarii, and in the confines of clusters of cell bodies. Some fibers were also noted in the anterior and posterior pituitary and the sensory ganglia. As with other newly described brain neuropeptides it can only be conjectured that CGRP has a neuroregulatory action on a variety of functions throughout the brain and spinal cord.     

Characterization of the autonomic innervation of mammary gland in lactating rats studied by retrograde transynaptic virus labeling and immunohistochemistry

The aim of experiments was to characterize the neurons of the autonomic chain that innervates the nipple and the mammary gland of lactating rats using retrograde transynaptic virus labeling and neurotransmitter and neuropeptide immunohistochemistry. Two days after injection of green fluorescence protein labeled virus in two nipples and underlying mammary glands, labeling was observed in the ipsilateral paravertebral sympathetic trunk and the lateral horn. Three days after inoculation the labeling appeared in the brain stem and the hypothalamic paraventricular nucleus. Above the spinal cord the labeling was bilateral. A subpopulation of virus labeled cells in the paraventricular nuclei synthesized oxytocin. Labeled neurons in the lateral horn showed cholinergic immunoreactivity. These cholinergic neurons innervated the paravertebral ganglia where the virus labeled neurons were partially noradrenergic. The noradrenergic fibers in the mammary gland innervate the smooth muscle wall of vessels, but not the mammary gland in rats. The neurons in the lateral horn receive afferents from the brain stem, and paraventricular nucleus and these afferents are noradrenergic and oxytocinergic. New findings in our work: Some oxytocinergic fibers may descend to the neurons of the lateral horn which innervate noradrenergic neurons in the paravertebral sympathetic trunk, and in turn these noradrenergic neurons reach the vessels of the mammary gland.     

c-Fos expression in the central nervous system elicited by phrenic nerve stimulation.

Phrenic nerve afferents (PNa) have been shown to activate neurons in the spinal cord, brain stem, and forebrain regions. The c-Fos technique has been widely used as a method to identify neuronal regions activated by afferent stimulation. This technique was used to identify central neural areas activated by PNa. The right phrenic nerve of urethane-anesthetized rats was stimulated in the thorax. The spinal cord and brain were sectioned and stained for c-Fos expression. Labeled neurons were found in the dorsal horn laminae I and II of the C3-C5 spinal cord ipsilateral to the site of PNa stimulation. c-Fos-labeled neurons were found bilaterally in the medial subnuclei of the nucleus of the solitary tract, rostral ventral respiratory group, and ventrolateral medullary reticular formation. c-Fos-labeled neurons were found bilaterally in the paraventricular and supraoptic hypothalamic nuclei, in the paraventricular thalamic nucleus, and in the central nucleus of the amygdala. The presence of c-Fos suggests that these neurons are involved in PNa information processing and a component of the central mechanisms regulating respiratory function.     

Disease-related regressive alterations of forebrain cholinergic system in SOD1 mutant transgenic mice

Transgenic mice carrying the human mutated SOD1 gene with a glycine/alanine substitution at codon 93 (G93A) are a widely used model for the fatal human disease amyotrophic lateral sclerosis (ALS). In these transgenic mice, we carried out a neurochemical study not only restricted to the primarily affected regions, the cervical and lumbar segments of the spinal cord, but also to several other brain regions. At symptomatic (110 and 125 days of age), but not at pre-symptomatic (55 days of age) stages, we found significant decreases in catalytic activity of the cholinergic enzyme, choline acetyltransferase (ChAT) in the hippocampus, olfactory cortex and fronto-parietal cortex. In parallel, we observed a decreased number of basal forebrain cholinergic neurons projecting to these areas. No alterations of the cholinergic markers were noticed in the striatum and the cerebellum. A widespread marker for GABAergic neurons, glutamate decarboxylase (GAD), was unaffected in all the areas examined. Alteration of cholinergic markers in forebrain areas was paralleled by concomitant alterations in the spinal cord and brainstem, as a consequence of progressive apoptotic elimination of cholinergic motor neuron. Gestational supplementation of choline, while able to result in long-term enhancement of cholinergic activity, did not improve transgenic mice lifespan nor counteracted cholinergic impairment in brain regions and spinal cord.     

Organization of central cholinergic neurons revealed by combined in situ hybridization histochemistry and choline-O-acetyltransferase immunocytochemistry.

Digoxigenin-labeled riboprobes and in situ hybridization of choline-O-acetyltransferase mRNA, both alone and in combination with immunohistochemical procedures for the synthetic enzyme of acetylcholine, were used to map the topography of putative cholinergic neurons in the rat central nervous system. Only the anti-sense riboprobe yielded specific labeling, which was absent in brain sections processed with sense riboprobe. Telencephalic neurons demonstrating the mRNA for choline-O-acetyltransferase and choline-O-acetyltransferase-like immunoreactivity were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, Islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis, as well as occasionally in the amygdala. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridization signal, even though some cells in those areas were observed to exhibit choline-O-acetyltransferase-like immunopositivity. Thalamic cells were devoid of hybrido- and immunoreactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few cell bodies labeled with riboprobe and co-localizing choline-O-acetyltransferase-like immunopositivity were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei evinced moderate hybridization signal, whereas cells of the parabigeminal nucleus were very weakly reactive. In contrast, motor neurons of the cranial nerve nuclei demonstrated high levels of choline-O-acetyltransferase mRNA and choline-O-acetyltransferase-like immunoreactivity. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord and around the central canal were also labeled with riboprobe. It is concluded that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons in most of the neural regions believed to contain them on the basis of acetylcholinesterase pharmacohistochemistry and choline-O-acetyltransferase immunocytochemistry, with the prominent exceptions of the cerebral cortex, hippocampus, olfactory bulb, anterior olfactory nucleus, and caudal raphe nuclei, which apparently do not possess neurons expressing detectable levels of the mRNA for the synthetic enzyme of acetylcholine.     

Vasopressinergic and oxytocinergic pathways in the central nervous system

Recent data obtained by immunohistochemical and other anatomical tracing methods indicate that oxytocin and vasopressin pathways are much more complex and extensive than previously recognized. In addition to the classic magnocellular neurons that project from the supraoptic and paraventricular (PVN) nuclei to the posterior pituitary gland, generally smaller neurons in various parts of the PVN send vasopressin fibers to the portal capillary bed in the median eminence, or send oxytocin or vasopressin projections to other brain and spinal cord sites. In addition, vasopressin neurons are also found in the suprachiasmatic nucleus and may contribute to extrahypothalamic projection areas. Many of these axonal projections appear to form synapses with other neurons in forebrain, hindbrain, and spinal cord regions, which suggests roles for these peptides in neuronal communication. In brain stem and spinal cord, terminal fields include both parasympathetic and sympathetic regulatory centers. Oxytocin terminals are also found on large intracerebral arteries where the peptide may regulate cerebral blood flow.     

Neurochemical Organization and Connections of the Cerebral Preglomerular Complex of the Masu Salmon

Using immunohistochemical labeling of the cells containing neuronal NO synthase (nNOS), tyrosine hydroxylase (TH), GABA, and parvalbumin (PA), as well as histochemical marking of choline acetyltransferase-containing neurons, we examined the neurochemical organization of the glomerular nuclei and preglomerular complex in the brain of the masu salmon (Oncorhynchus masou). Injections of the carbocyanine dye DiI allowed us to examine projections of neurons of the preglomerular and mammillary nuclei in the salmon brain. We showed that cholinergic, GABA-, PA-, TH-, and nNOS-immunopositive neurons belonging to different morphological types are present in the glomerular and medial preglomerular nuclei. The analysis of correlations between morphometric characteristics of the cells belonging to different neurochemical types and densitometric estimates of amounts of neurochemical agents present in these cells allowed us to hypothesize that there are close morphofunctional interrelations in cell populations possessing different neurochemical and morphometric characteristics. These interrelations of the cells belonging to different chemotypes are, probably, realized as mediatory/modulatory ones. The presence of a great number of small slightly differentiated cells in the preglomerular and glomerular nuclei allows us to suppose that the growth of the greatest sensory center of the salmon brain is provided by neuroblasts that migrate from the proliferative zones in the course of postembryonal neurogenesis. It is also hypothesized that NO, TH, and GABA are involved in paracrine control of the postnatal morphogenesis of the salmon preglomerular complex. The data obtained by hodological analysis indicate that the nuclei of the preglomerular complex obtain afferent projections from the dorsomedial and ventroventral telencephalic regions, preoptic nucleus, periventricular layer of the tectum, and posterior central thalamic nucleus. Our study demonstrated the existence of reciprocal functional connections between the preglomerular complex (most important diencephalic center for transmission of sensory information) and dorsomedial and ventral regions of the telencephalon in the masu salmon.     

THE NUCLEAR DNA CONTENT OF LARGE VENTRAL SPINAL NEURONS

Abstract— A nuclear fraction has been obtained from large spinal neurons previously isolated from bovine ventral spinal cord in bulk suspensions. The fraction contained an average of 5.3 ± 0.9 pg DNA/nucleus, indicating a high incidence of diploid nuclei. This conclusion was confirmed by distribution analysis of DNA in propidium iodide-stained nuclei examined by flow microfluorometry. That technique showed that at least 90% of the nuclei from large spinal neurons are diploid. Mixed, mostly non-neuronal nuclei derived from many types of cells in the ventral spinal cord contained an average of 5.9 ± 0.6 pg DNA/nucleus, 19% of which possibly possess more than diploid amounts of DNA. The uniform DNA content in nuclei of large spinal neurons and most other types of cells in the ventral spinal cord contrasts sharply with a wide variation (av 26-fold) in the nuclear volumes of the same cells.     

Ascending and descending projections of the lateral vestibular nucleus in the rat

The tracer neurobiotin was injected into the lateral vestibular nucleus in rat and the efferent fiber connections of the nucleus were studied. The labeled fibers reached the diencephalon rostrally and the sacral segments of the spinal cord caudally. In the diencephalon, the ventral posteromedial and the gustatory nuclei received the most numerous labeled fibers. In the mesencephalon, the inferior colliculus, the interstitial nucleus of Cajal, the nucleus of Darkschewitch, the periaqueductal gray matter and the red nucleus received large numbers of labeled fibers. In the rhombencephalon, commissural and internuclear connections originated from the lateral vestibular nucleus to all other vestibular nuclei. The medioventral (motor) part of the reticular formation was richly supplied, whereas fewer fibers were seen in the lateral (vegetative) part. In the spinal cord, the descending fibers were densely packed in the anterior funiculus and in the ventral part of the lateral funiculus. Collaterals invaded the entire gray matter from lamina IX up to lamina III; the fibers and terminals were most numerous in laminae VII and VIII. Collateral projections were rich in the cervical and lumbosacral segments, whereas they were relatively poor in the thoracic segments of the spinal cord. It was concluded that the fiber projection in the rostral direction was primarily aimed at sensory-motor centers; in the rhombencephalon and spinal cord, fibers projected onto structures subserving various motor functions.     

Partial cloning and expression of mRNA coding choline acetyltransferase in the spinal cord of the goldfish, Carassius auratus

Choline acetyltransferase (ChAT, EC 2.3.1.6) synthesizes a neurotransmitter, acetylcholine in cholinergic neurons. ChAT is considered to be the most specific marker for cholinergic neurons. To obtain a better marker of the neurons, as the first step, we isolated a partial ChAT cDNA from the goldfish (Carassius auratus) brain by RT-PCR methods. The partial cDNA of the goldfish ChAT was composed of 718 nucleotides. The amino acid sequence of the goldfish ChAT is approximately 70% identical to those of mammalian and chicken ChAT. Northern blot analysis demonstrated that ChAT mRNA was expressed in the brain and the spinal cord of the goldfish, and much abundant in the spinal cord. In the spinal cord of the goldfish, ChAT-positive neurons were detected mainly in the ventral horn by in situ hybridization. In addition, fluorescence in situ hybridization combined with a retrograde labeling by using True Blue demonstrated ChAT mRNA positive neurons were exactly motoneurons. In the cord, putative presynaptic sympathetic neurons were also labeled.     

Gaseous transmitters in the brain of the masu salmon, <Emphasis Type="Italic">Oncorhynchus masou</Emphasis> (Salmoniformes,Salmonidae)

Distribution of nitroxidergic and H₂S-producing neurons in the brain of the masu salmon Oncorhynchus masou was studied by methods of histochemical labeling of NADPH-diaphorase and by immunohistochemical labeling of the neuronal nitric oxide synthase and cystathionine β-synthase (CBS). The established distribution of CBS and nNOS/NADPH-d of neurons and fibers in the masu salmon telencephalon, optic tectum, and cerebellum allows suggesting that the NO- and H₂S-producing systems represent individual, non-overlapping neuronal complexes performing specialized functions in the activity of local neuronal networks. In the medullar part, the nNOS-ir and NADPH-d-positive neurons were detected in the composition of viscerosensory (V, VII, and IX–X) and visceromotor (III, IV, and VI) nuclei of craniocerebral nerves, octavolateral afferent complex, reticulospinal neurons, and medial reticular formation. CBS in the masu salmon medulla was revealed in neurons of the nerve X nucleus, reticulospinal neurons, and ventrolateral reticular formation. Distribution of NO-ergic and H₂S-producing neurons in the masu salmon medullar nuclei indicates that NO in masu salmon is the predominant neuromodulator of the medullar viscerosensory systems, while H₂S seems to modulate only the descending motor systems. The results of the performed study allow suggesting that NO in the masu salmon medulla periventricular area can act as a regulator of postnatal ontogenesis.     

Neurons of the medulla oblongata and hypothalamus projecting on the sympathetic neurons of the ventral horn of the spinal cord]

Connections of the neurons of the spinal cord ventral horn with the structures, situating above have been investigated. After injection of uranyl acetate into the TIII segment of the spinal cord, labelled neurons are found in various reticular nuclei of the medulla oblongata. At the level of the roots of the XII pair of the cranial nerves they are revealed in the reticular paramedian, ventral, parvocellular and lateral nuclei. The formations mentioned participate in regulation of the cardio-vascular system. More rostral (2 and 4 mm relatively to the roots of the XII pair of the cranial nerves) the neurons are observed in the reticular giant cellular nucleus, in nuclei of the raphe and in the group of the P-substance reactive neurons. Besides, labelled neurons are revealed in the posterior, lateral fields and in the dorso- and ventromedial nuclei of the hypothalamus.     

Cholinergic Receptor Alterations in the Brain Stem of Spinal Cord Injured Rats

Cholinergic receptors in upper motor neurons of brain stem control locomotion and coordination. Present study unravels cholinergic alterations in brain stem during spinal cord injury to understand signalling pathway changes which may be associated with spinal cord injury mediated motor deficits. We evaluated cholinergic function in brain stem by studying the expression of choline acetyl transferase and acetylcholine esterase. We quantified metabotropic muscarinic cholinergic receptors by receptor assays for total muscarinic, muscarinic M1 and M3 receptor subunits, gene expression studies using Real Time PCR and confocal imaging using FITC tagged secondary antibodies. The gene expression of ionotropic nicotinic cholinergic receptors and confocal imaging were also studied. The results from our study showed metabolic disturbance in cholinergic pathway as choline acetyl transferase is down regulated and acetylcholine esterase is up regulated in spinal cord injury group. The significant decrease in muscarinic receptors showed by decreased receptor number along with down regulated gene expression and confocal imaging accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic acetylcholine receptors and confocal imaging. The motor coordination was analysed by Grid walk test which showed an increased foot slips in spinal cord injured rats. The significant reduction in brain stem cholinergic function might have intensified the motor dysfunction and locomotor disabilities.     

Connections of brainstem respiratory nuclei studied by the retrograde horseradish peroxidase axon transport method

Intrabulbar connections of respiratory nuclei and the medullary reticular formation and also descending pathways from these structures in the spinal cord were studied by the retrograde horseradish peroxidase axonal transport method in cats. Neurons of the nucleus ambiguus and nucleus retroambigualis (ventral respiratory group) and of the ventrolateral part of the nucleus of the tractus solitarius (dorsal respiratory group) were shown to form direct two-way connections with each other and with the medial region of the medulla. Neurons of the pneumotaxic center send uncrossed axons to the nucleus ambiguus and to the medial medullary reticular formation. Neurons of the contralateral homonymous nucleus and neurons of the nucleus of the tractus solitarius are sources of projections of the locus coeruleus. A well developed system of direct connections was found between neurons of respiratory nuclei of the two halves of the brain. The possible role of these nuclear formations in genesis of the respiratory rhythm and regulation of the respiratory and other motor functions of the reticular formation is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 149–157, March–April, 1982.     

Vasoactive intestinal polypeptide immunoreactivity in the spinal cord of the guinea pig

Summary The distribution of vasoactive intestinal polypeptide-immunoreactive (VIP-IR) neurons in the lower medulla oblongata and the spinal cord has been analyzed in guinea pigs. This study includes results obtained by colchicine treatment and transection experiments. In the spinal cord, numerous VIP-IR varicosities were observed in the substantia gelatinosa of the columna dorsalis; some were also found in the substantia intermedia and the columna anterior. The spinal VIP-IR nerve fibers were mainly of intraspinal origin and oriented segmentally. VIP-IR nuclei in the spinal cord extended dorsally into corresponding regions of the caudal medulla oblongata, namely from the substantia intermedia medialis and lateralis into the vagus-solitarius complex and from the nucleus spinalis lateralis into the area of the nucleus reticularis lateralis. Additional VIP-IR perikarya were observed in the pars caudalis of the nucleus spinalis nervi trigemini. The VIP-IR nuclei within the caudal medulla oblongata probably form a continuous system with those localized within the spinal cord. They may be involved functionally in the modulation of cardiovascular and respiratory regulation in the guinea pig.Supported by the DFG, Carvas SFB 90     

Growth factor dependent cholinergic function and survival in primary mouse spinal cord cultures

In primary embryonic spinal cord cultures, synaptic transmission can be conveniently studied by monitoring radiolabeled neurotransmitter release or by recording of electrophysiological responses. However, while the mature spinal cord contains an appreciable number of cholinergic motoneurons, cultures of embryonic spinal cord have a paucity of these neurons and release little or no acetylcholine upon stimulation. To determine whether the proportion of cholinergic neurons in primary mouse spinal cord cultures can be augmented, the effects of several classes of growth factors were examined on depolarization- and Ca(2+)-evoked release of choline/acetylcholine (Ch/ACh). In the absence of growth factors, little or no evoked release of radiolabeled Ch/ACh could be demonstrated. Media supplemented with brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) were examined for their ability to preserve the population of neurons in culture. CNTF was found to increase the number of surviving neurons and to enhance the release of radiolabeled Ch/ACh; the other factors were without effect. The action of CNTF was transient, and the neuronal population decreased to levels observed in cultures lacking growth factor after 20 days in vitro. The correlation between enhanced neuron survival and increased Ch/ACh release suggests that CNTF protected cholinergic neurons, albeit transiently, from cell death.