Shinobu Inagaki

localizations of α1 and β1 subunits of soluble guanylate cyclase in the rat brain

Abstract We studied the localizations of α1 and β1 subunits of soluble guanylate cyclase using in situ hybridization. The β subunit was widely distributed in most neurons throughout the brain, with different levels of expression. The α1 subunit was also distributed throughout the brain; however, it was located in more limited regions. Both subunits were expressed markedly in the glomerular layer of the olfactory bulb, dorsal and ventral striatum, and several regions in the brainstem. Regions with little or no α1 subunit expression, but with marked expression of the β1 subunit included the olfactory bulb except for the glomerular layer, pyramidal cell layer in CA1 and granular cell layer in the dentate gyrus of the hippocampus, and many brainstem nuclei. The above regions expressing both subunits are suggested to contain active soluble guanylate cyclase as a target for nitric oxide, and thus may be involved in cellular signal transduction.

Autoradiographic localization of calcitonin gene-related peptide binding sites in human and rat brains

125I-calcitonin gene-related peptide (CGRP) binding sites were mapped in the human brain and rat brains by in vitro macroautoradiography, and compared to each other. Binding experiments were made to characterize 125I-CGRP binding on the human and rat brains. Scatchard analysis of saturation experiments from slide-mounted sections of the human and rat cerebellum displayed 125I-CGRP binding sites with a dissociation constant (Kd) of 0.17 nM and 0.11 nM, respectively, and a maximal number of binding sites (Bmax) of 96.8 fmol/mg and 23.0 fmol/mg protein. 125I-CGRP binding was time-dependent, reversible and saturable with high affinity in the brains. Autoradiograms showed a discrete distribution of 125I-CGRP binding sites throughout the brains of human and rat with patterns similar to each other. In the human brain, the highest binding was seen in the cerebellum, inferior olivary nuclear complex, certain parts of the central gray matter, arcuate nuclei of the medulla oblongata and dorsal motor nucleus of the vagus, and densities of CGRP-binding sites were high in the nucleus accumbens, amygdala, tail of the nucleus caudatus, substantia nigra, ventral tegmental area, medial portion of the inferior colliculus, medial pontine nuclei, locus coeruleus, inferior vestibular nucleus, substantia gelatinosa of the spinal trigeminal nucleus, nucleus of the solitary tract and nucleus cuneatus lateralis. In the rat, high densities were found in the hippocampus pars anterior, nucleus accumbens, ventral and caudal portions of the nucleus caudatus-putamen, central and basolateral nuclei of the amygdala, caudal portion of the insular cortex, medial geniculate body, superior and inferior colliculi, certain portions of the central gray matter, locus coeruleus, inferior olivary nuclei, vagal complex, nucleus cuneatus lateralis and cerebellum. In contrast, in both species, most of the cortical areas including the hippocampus, most of the thalamus, and hypothalamus exhibited few binding sites. In addition, high quantities of the binding sites were seen on the pia mater and on walls of blood vessels in the brain and subarachnoidea. These results revealed essentially homologous locations of CGRP binding sites in the human and rat central nervous systems and well corresponding distributions of binding sites and endogenous CGRP-like immunoreactivity.

Coexistence of peptides (corticotropin releasing factor/neurotensin and substance P/somatostatin) in the bed nucleus of the stria terminalis and central amygdaloid nucleus of the rat

Coexistence of corticotropin releasing factor and neurotensin and also of substance P and somatostatin was demonstrated in the lateral bed nucleus of the stria terminalis and the central amygdaloid nucleus of the rat, by means of a light microscopic mirror method or immunofluorescent double staining. Using the former technique, a major proportion of corticotropin releasing factor-like immunoreactive cells were found to display neurotensin-like immunoreactivity in the dorsal subdivision of the lateral bed nucleus of the stria terminalis and the lateral subdivision of the central amygdaloid nucleus. On the other hand, the immunofluorescent method showed that a significant number of neurons with both substance P- and somatostatin-like immunoreactivity were located in the ventral subdivision of the lateral bed nucleus of the stria terminalis and the medial subdivision of the central amygdaloid nucleus. Distribution patterns of such co-localized peptides may indicate that there are morphological and biochemical similarities between the dorsal subdivision of the lateral bed nucleus of the stria terminalis and the lateral subdivision of the central amygdaloid nucleus, as well as between the ventral subdivision of the lateral bed nucleus of the stria terminalis and the medial subdivision of the central amygdaloid nucleus. Previous studies have demonstrated that peptide-containing neurons in the lateral bed nucleus of the stria terminalis and central amygdaloid nucleus, such as corticotropin releasing factor-, neurotensin-, substance P- and somatostatin-like immunoreactive cells, project to the lower brainstem. The results of the present study suggest that corticotropin releasing factor/neurotensin and substance P/somatostatin neurons may be part of the lateral bed nucleus of the stria terminalis/central amygdaloid nucleus-lower brainstem pathways.

Neuropeptide Y-immunoreactive neurons receive synaptic inputs from dopaminergic axon terminals in the rat neostriatum.

Double immunocytochemistry using peroxidase-antiperoxidase and protein A-gold was performed to determine whether neuropeptide Y (NPY) immunoreactive neurons receive synaptic inputs from catecholaminergic axon terminals in the rat neostriatum. Tyrosine hydroxylase-immunoreactive axons were found to be in synaptic contact with the somas and proximal dendrites of NPY-immunoreactive neostriatal neurons. These latter neurons were medium-sized and had indented nuclei, and thus were thought to be medium aspiny interneurons. Thus nigrostriatal dopaminergic neurons may monosynaptically influence striatal NPY neurons.

Distribution of I, II and III subtypes of voltage-sensitive Na+ channel mRNA in the rat brain

We examined the expression of the I, II and III subtypes of voltage-sensitive Na+ channel mRNA in the rat brain using in situ hybridization histochemistry with oligonucleotide probes. The distribution of cells with strongly positive signals was characteristic for each subtype. Synthesis of each subtype of Na+ channel protein may be regulated by differential mRNA expression.

Localization of Mn-superoxide dismutase (Mn-SOD) in cholinergic and somatostatin-containing neurons in the rat neostriatum

We used rabbit antisera against manganese (Mn)-superoxide dismutase for immunohistochemical studies of localization in the rat neostriatum. Immunostaining was intense in large-sized neurons and several medium-sized neurons, but it was moderate to weak in other cells. Double immunostaining with monoclonal antibody to choline acetyltransferase or somatostatin demonstrated large-sized, Mn-SOD immunoreactive neurons to be cholinergic, and some medium-sized neurons which were intensely immunoreactive for Mn-SOD to contain somatostatinergic.

Immunoreactive atrial natriuretic polypeptides in the adrenal medulla and sympathetic ganglia

Atrial natriuretic polypeptide (ANP)-like immunoreactivity was found in the rat adrenal gland by using indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunostaining was present in most of chromaffin cells with varying degrees of immunoreactivity. The majority of medullary cells displayed very intense immunostaining, and several clusters revealed weaker immunostaining. No staining was found in the adrenal cortex or in the nerve fibers in this organ. In the consecutive sections treated for dopamine-beta-hydroxylase (DBH), apparently all medullary cells had intense immunofluorescence for DBH and its distribution pattern was very similar to that for ANP-like immunoreactivity. While phenylethanolamine N-methyltransferase (PNMT) immunoreactive cells largely corresponded to the intensely stained ANP-like immunoreactive cells, suggesting that adrenaline cells contained a large amount of ANP-like substance, noradrenaline cells contained a smaller amount of this substance than adrenaline cells. Ultrastructural study showed that end-products due to the immunoreaction with the ANP antiserum were primarily associating with chromaffin granules. In addition, the presence of ANP-like immunoreactivity was investigated in several sympathetic ganglia of the rat. No principal ganglion cells were ANP-positive, whereas a few small intensely fluorescent (SIF) cells were ANP-immunoreactive. The present findings suggest that catecholamines coexist with ANP which has a natriuretic and vasodilating effect, in adrenal medullary cells and SIF cells in several rat sympathetic ganglia, but not in principal ganglion cells.

Enkephalinergic projection from the ventromedial hypothalamic nucleus to the midbrain central gray matter in the rat: an immunocytochemical analysis

The projection of enkephalin-like immunoreactive (EnkI) neurons in the ventromedial hypothalamic nucleus (VMH) in the rat was investigated using immunocytochemistry. These neurons project to the dorsal portion of the midbrain central gray matter (DCG), because the destruction of the VMH resulted in a marked ipsilateral reduction of EnkI fibers there. Destruction of the dorsomedial portion of the VMH caused the appearance of fibers accumulating EnkI ventrolateral to the lesion and these fibers could be traced to the ventrolateral portion of the VMH where many EnkI neurons were concentrated. Transection of the brain at the level of the premammillary nucleus, mammillary body and superior colliculus caused the appearance of fibers accumulating EnkI in the posterior hypothalamic nucleus (PH), central gray matter medial to the fasciculus retroflexus Mynert and in the DCG, respectively. We concluded that the axons from these neurons first project dorsomedially to situate in the PH. These fibers ran caudodorsally and entered into the dorsal longitudinal fasciculus in the DCG at the midbrain.

Immunocytochemical localization of manganese superoxide dismutase (Mn-SOD) in the hippocampus of the rat

The immunocytochemical distribution of manganese superoxide dismutase (Mn-SOD) was determined in the rat hippocampus. The enzyme was localized in the mitochondria. CA1 pyramidal cells were weakly immunostained, whereas CA3 pyramidal cells were strongly reactive. These differences in the intensity of the Mn-SOD immunostaining reactions may relate to variations in the sensitivity of subfields of the hippocampus to ischemia.

Substance P afferents have synaptic contacts with dopaminergic neurons in the ventral tegmental area of the rat.

Synaptic contacts were found between dopaminergic neurons and substance P (SP)-immunoreactive axon terminals in the ventral tegmental area (VTA), by means of the immunoelectron microscopic mirror method. SP-immunoreactive terminals were found to make synaptic contact with VTA neurons exhibiting tyrosine hydroxylase immunoreactivity.