Andrej Rotter

Glycine receptor distribution in mouse CNS: autoradiographic localization of [3H]strychnine binding sites

Biochemical and electrophysiological studies of mammalian CNS indicate that the amino-acid, glycine, is a major inhibitory neurotransmitter whose location is, for the large part, confined to the spinal cord and brain stem. In this study, autoradiographs of C57BL/6J mouse brain sections labeled with [3H] strychnine, a potent antagonist of glycine, were used to map the distribution of glycine receptors in the CNS. Autoradiographs showed highly localized areas of grain density, which confirmed the gross distributions described in homogenate binding studies and gave a more precise regional localization of glycine receptors in this animal. The highest overall labeling was observed in the spinal cord and medulla; areas of highest grain density included the dorsal horn of the spinal cord, the cranial nerve nuclei, the dorsal column nuclei and nuclei of the medullary reticular formation. A decrease in overall grain density was observed rostrally throughout the midbrain and pons; in caudal regions, however, dense labeling was observed over the trigeminal, vestibular and facial nuclei and over the major nuclei of the auditory system. In more rostral areas, the interpeduncular nucleus and the substantia nigra were also clearly delineated, as were certain thalamic nuclei. The cerebellum, cortex, hippocampus and olfactory bulbs showed only very low levels of grain density. In summary, it appears that high concentrations of glycine receptors in the brain and spinal cord may be preferentially localized to neurons involved in the processing of information originating from exteroceptive sensory mechanoreceptors.

Neuronal localization of pseudocholinesterase in the rat cerebellum: sagittal bands of Purkinje cells in the nodulus and uvula

The histochemical distribution of pseudocholinesterase was studied in the rat cerebellum using Koelles copper-thiocholine method. Throughout the cerebellum, pseudocholinesterase is uniformly localized in the endothelial cells of blood vessels and in the cell bodies and processes of the Bergmann glia. In addition, we demonstrate that exclusively in the ventral uvula and in the nodulus (lobules IXc and X of Larsell) pseudocholinesterase is localized in a small subpopulation of Purkinje cells. The cell bodies and dendrites of these labeled Purkinje cells form at least 4 distinct parallel bands extending along the sagittal plane of each of the lobules. Two broad bands on either side of the midline, approximately 800-900 microns wide and composed of 15-20 Purkinje cells have been designated as A bands. Two narrower bands, approximately 160-300 microns wide and composed of 5-7 Purkinje cells, on the lateral aspects of the lobules have been designated as B bands. The unique distribution of pseudocholinesterase in a small and anatomically restricted population of neurons suggests that in the cerebellum this enzyme may play a role in the metabolism of neuroactive substances.

Autoradiographic Localization of Receptors in the Cochlear Nucleus of the Mouse

Light microscopic autoradiography of bound radiolabeled ligands was used to describe the distribution of six receptor types in the dorsal and ventral mouse cochlear nuclei: Glycine receptor ([3H]strychnine); GABA receptor ([3H]muscimol); benzodiazepine receptor ([3H]flunitrazepam); adenosine receptor ([3H]cyclohexyladenosine); muscarinic ACh receptor ([3H]quinuclidinyl benzilate); histamine receptor ([3H]mepyramine). The most intense [3H]strychnine labeling was observed in the deep region of the dorsal cochlear nucleus (DCN), with slightly lower levels in the molecular and pyramidal layers. Highest density of [3H]muscimol binding sites was observed in the granule cell layer of the posterior ventral nucleus (PVCN) and in the pyramidal layer of the DCN. Diffuse [3H]flunitrazepam labeling was distributed over all laminar regions of the DCN; the highest grain density was observed over the granule cell layer of the PVCN. Intense [3H]cyclohexyladenosine labeling was seen over the molecular layer, possibly extending into the pyramidal layer, of the DCN. The granule cell layer of the PVCN was also densely labeled. High concentrations of [3H]quinuclidinyl benzilate sites were seen in the molecular layer, possibly extending into the pyramidal layer, of the DCN. A thin band of high grain density was also visible over the granule cell layer of the PVCN. Moderate, diffuse [3H]mepyramine labeling was visible throughout the DCN, with slightly higher grain density over the molecular, and possibly the pyramidal layers, than over the deep region of the DCN.

Neurochemical identification of cholinergic forebrain projection sites of the nucleus tegmentalis dorsalis lateralis.

The projection sites of the nucleus tegmentalis dorsalis lateralis (ntdl) were examined in rats by a biochemical technique. The ntdl was destroyed unilaterally and brains were assayed after 14 days survival for changes in choline acetyltransferase activity in discrete brain areas. A bilateral projection appears to exist to the anteroventral and the posterior nucleus of the thalamus. An ipsilateral projection to the lateral portion of the medial thalamic nucleus and the ventral geniculate was found. It is suggested that the nucleus tegmentalis dorsalis lateralis may play a role in the modulation forebrain areas.

Localization of substance P, acetylcholinesterase, muscarinic receptors and alpha-bungarotoxin binding sites in the rat interpeduncular nucleus.

On the basis of acetylcholinesterase (AChE) staining, the rat interpeduncular nucleus was subdivided into five distinct zones. Intense AChE staining was observed in the dorsal cap and the lateral zones. Moderate staining was seen in the median zone and the dorsoventral column. The perivascular zones were unlabeled. Adjacent sections were stained for AChE and for substance P immunofluorescence. Substance P like immunoreactivity was found to coincide with the localization of AChE in the dorsal cap and the lateral zones. Both muscarinic receptors and alpha-bungarotoxin binding sites had distributions resembling that of AChE. Neither unilateral nor bilateral lesions of the habenulae changed the number or distribution pattern of the receptors. It was concluded that cholinergic receptors are localized postsynaptically. Our study suggests AChE and substance P containing fibers terminate in well defined zones of the interpeduncular nucleus which also contain muscarinic and nicotinic receptors.

Regulation of glycine receptor binding in the mouse hypoglossal nucleus in response to axotomy

Recent studies have shown that muscarinic receptors in brain hypoglossal nuclei exhibit a loss of specific ligand binding in response to axotomy of the hypoglossal nerve. The mouse hypoglossal nucleus contains a high level of receptors for the inhibitory neurotransmitter, glycine; the ligand [3H]strychnine binds to the glycine receptor with high affinity. In the present study [3H]strychnine binding in mouse hypoglossal nuclei was examined at 1 to 150 days after unilateral lesions of the hypoglossal nerve. Brains were sectioned on a cryostat, thaw-mounted onto microscope slides, incubated with [3H]strychnine and processed for light microscopic autoradiography. Receptor density was assessed by counting silver grains in photomicrographs of operated and control nuclei. During the first 25 days after axotomy grain density fell to 50 percent of that of the control nucleus. After this time grain density slowly increased, returning to control levels by 150 days post lesion. These data indicate that glycine receptors on the axotomized cells of the hypoglossal nucleus are lost when connection with the target muscles of the tongue is interrupted, and that the receptors reappear when the hypoglossal nerve regenerates. It is suggested that excitatory and inhibitory neurotransmitter receptor systems may be regulated in a coordinated fashion by the functional state of the motoneuron.

Cerebellar benzodiazepine receptors: cellular localization and consequences of neurological mutations in mice

The distribution of cerebellar [3H]flunitrazepam binding sites was studied autoradiographically in Purkinje cell degeneration (pcd/pcd), weaver (wv/wv), staggerer (sg/sg) and reeler (rl/rl) mutant mice. In the normal 78-day-old C57BL/6J mouse cerebellum, the highest concentration of [3H]flunitrazepam binding sites was observed over the molecular layer. Intermediate grain density was present over the Purkinje cell layer and intermediate to high density over the deep cerebellar nuclei. Low labeling was observed over the granule cell layer. Negligible concentrations of binding sites were seen in the white matter. In 45-49-day-old Purkinje cell degeneration mutants, where essentially all Purkinje cells have disappeared by day 45, there was a small decrease in grain density over the cerebellar cortex. Concomitantly, a substantial increase in grain density was observed over the deep cerebellar nuclei of the pcd/pcd mutants when compared to littermate controls. A significant increase in [3H]flunitrazepam labeling was observed over the cerebellar cortex of 81-86-day-old wv/wv mutants; this was most pronounced in the vermis where the granule cell loss was greatest. Over the hemispheres, where fewer granule cells degenerate, a lower density of binding sites was seen. Grain density over the wv/wv deep cerebellar nuclei was comparable to that of littermates. Substantially lower [3H]flunitrazepam labeling was detected over the cerebellar cortex of 25-27-day-old sg/sg mutants in which the number of granule, Purkinje and Golgi cells is greatly reduced; the labeling over the deep nuclei, however, was significantly increased. In 27-29-day-old rl/rl mutant cerebella, where all classes of cells are malpositioned, labeling density over all areas of the cerebellar cortex, including the Purkinje cell masses, was increased. Our autoradiographic data suggest that a proportion of cerebellar cortical benzodiazepine receptors are associated with Purkinje cells; we propose that the remainder of the receptors are localized on Golgi cells, while granule cells are devoid of receptors. In the deep cerebellar nuclei, the observed increase in benzodiazepine receptors in the pcd/pcd and sg/sg mutants may be a manifestation of denervation supersensitivity subsequent to the loss of innervation by Purkinje cell axon terminals. The finding of a high receptor density in the Purkinje cell masses of the rl/rl mutant, where Purkinje cells are devoid of afferent basket cell input, suggests that benzodiazepine receptors are expressed and maintained in the absence of a full complement of GABAergic afferents.

The localization of GABAA receptors in mice with mutations affecting the structure and connectivity of the cerebellum.

The distribution of cerebellar [3H]muscimol binding sites was studied autoradiographically in normal C57BL/6J mice and in the weaver, reeler, Purkinje cell degeneration and staggerer mutant mice. In the normal 79-day-old mouse cerebellum, the highest concentration of [3H]muscimol binding sites was observed in the granule cell layer. A much lower grain density was present over the Purkinje cell and molecular layers and negligible numbers of binding sites were seen over the deep cerebellar nuclei and white matter. A significant decrease in [3H]muscimol labeling was observed over the cerebellar cortex of the 81-86-day-old weaver mutant; this was most pronounced in the vermis where granule cell loss was the greatest. Over the hemispheres, where fewer granule cells degenerate, a higher density of binding sites remained. In the 27-29-old reeler cerebellum, where Purkinje cells are malpositioned, no labeling was seen over the deep Purkinje cell masses. In the quasi-normal superficial cortex, labeling density over the surviving granule cell layer was only slightly decreased. In the 54-57-day-old Purkinje cell degeneration mutant, where essentially all Purkinje cells have disappeared by day 45, a 29% decrease in grain density over the granule cell layer was observed, while labeling was still present in the molecular layer. Virtually no [3H]muscimol labeling was detected over any part of the cerebellar cortex of the 25-27-day-old staggerer mutant (which lacks parallel fiber-Purkinje cell synapses), although clusters of surviving granule cells were present in significant numbers in the lateral aspects of the cortex. Our autoradiographic data indicate that GABAA receptors are associated with granule cells in both the molecular and granule cell layers. Furthermore, our results raise the possibility that the maintenance of receptor levels may be dependent upon synaptic contacts between the granule cell and its main postsynaptic target, the Purkinje cell.

The ontogeny of [3H]muscimol binding sites in the C57BL/6J mouse cerebellum.

The characteristics of [3H]muscimol binding were investigated in cerebellar sections from 7-day-old mice. The binding sites were found to possess the kinetic properties and pharmacological specificity characteristic of high-affinity GABAA receptors. [3H]Muscimol binding sites in the developing C57BL/6J mouse cerebellum were visualized by light microscopic autoradiography. A distinct band of labeling situated over the molecular layer was apparent from day 1 to day 7. The external granule cell layer remained unlabeled throughout development. Labeling over the internal granule cell layer gradually increased from birth; it became more dense and well defined until adult levels of grain density were reached at 35-42 days of age. The deep cerebellar nuclei were moderately labeled at birth and gradually decreased in density thereafter. The observed ontogeny of granule cell [3H]muscimol binding sites suggests that the synthesis of receptors is initiated at a time immediately after cessation of cell division, coinciding with the beginning of granule cell translocation across the molecular layer. Since, at this time, granule cells have not yet formed synapses with the GABAergic Golgi II cells, nor have they, in turn, formed the vast majority of synaptic contacts with Purkinje cells, it follows that receptor appearance precedes the formation of afferent connections, and may also precede efferent synaptic contacts. The timing of the appearance of [3H]muscimol binding sites raises the possibility that their initial acquisition may be related to developmental events other than the interaction of the granule cell with its pre- or postsynaptic neuronal partners.

Cerebellar benzodiazepine receptor distribution: an autoradiographic study of the normal C57BL/6J and Purkinje cell degeneration mutant mouse.

The distribution of [3H]flunitrazepam binding sites in the cerebella of normal mice and Purkinje cell degeneration mutant mice was studied by light microscopic autoradiography. In the cerebellar cortex of normal mice, a high density of [3H]flunitrazepam binding was observed over the molecular layer, an intermediate density over the Purkinje cell layer and a low density over the granule cell layer; the white matter was devoid of labeling. The deep cerebellar nuclei were labeled to an intermediate density. In the 54-day-old Purkinje cell degeneration mutant cerebellum, which is depleted of Purkinje cells, a 36% reduction in labeling density of the cerebellar cortex was observed. The density was reduced by approximately equal amounts in both the molecular and granule cell layers; labeling in the deep cerebellar nuclei was, however, substantially increased.