Hiroshi Takagi

Region-specific expression of subunits of ionotropic glutamate receptors (AMPA-type, KA-type and NMDA receptors) in the rat spinal cord with special reference to nociception

The present study attempted to explore the gene expression of the subunits (GluR1-4) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor, subunit (GluR5) of kainic acid (KA)-type receptor, NR1 [a subunit of N-methyl-D-aspartate (NMDA) receptors] and the possible glutamate-binding subunit of an NMDA receptor complex in the dorsal horn of the rat spinal cords using in situ hybridization histochemistry. These results were compared with those of the spinal motor neurons. Expression of the subunits of the AMPA-type receptor was also examined at the protein level using immunocytochemistry, with reference to the motor neurons. Although all the four subunits of the AMPA-type receptor were expressed throughout the dorsal horn, the pattern of expression was different according to the dorsal horn region and to the subunits. GluR2 showed the strongest expression in the dorsal horn. Huge numbers of strongly labelled cells formed a dense collection in lamina II and superficial parts of lamina III. Many neurons in lamina II and superficial parts of lamina III expressed GluR1 moderately. Scattered neurons moderately expressing GluR3 were also seen in these regions, while the expression of GluR4 was very low. Labelling of the dorsal horn neurons by the GluR5 probe was low, and NR1 probe and a glutamate-binding subunit of an NMDA receptor complex probe labelled them diffusely with low to moderate intensity. These findings show a close relationship between the glutamergic nociceptive primary afferent system and AMPA-type receptors in which GluR2 is especially highly expressed. The present study further showed that the expression pattern of the glutamate receptors in the spinal sensory neurons differs considerably from that of spinal motor neurons. Motor neurons very strongly express GluR3 and 4, while the expression of GluR2 and GluR1 is moderate and low, respectively. Expression of GluR5 is also low in the motor neurons. However, expression of NR1 and the glutamate-binding subunit of an NMDA receptor complex is very strong. These findings indicate that the subunit composition of the AMPA-type receptors regulating motor neurons is different from that of the AMPA-type receptors in the spinal sensory neurons, and that there are at least two kinds of glutamergic systems which regulate motor neurons: via AMPA-type receptors and via NMDA receptors.

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.

Localization of nitric oxide synthase-immunoreactive neurons in the solitary nucleus and ventrolateral medulla oblongata of the rat : their relation to catecholaminergic neurons

The morphological relationship between nitric oxide (NO) and catecholamines in the solitary nucleus (SOL) and ventrolateral medulla oblongata (VLM) was studied by a double immunostaining method with antibodies against NO synthase (NOS), an NO-synthesizing enzyme, and tyrosine hydroxylase (TH), a catecholamine-synthesizing enzyme. Although NOS- and TH-immunoreactive neurons were widely distributed in the SOL and VLM, these immunoreactivities did not coexist in any single neurons. NOS-immunoreactive neurons formed clusters in some restricted regions, i.e. in the medial subnucleus of the SOL, where both NOS- and TH-immunoreactive neurons showed a complementary distribution. These findings suggest that NO-producing neurons constitute a subclass that is distinct from that of catecholaminergic neurons.

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.

Differentially expressed olfactomedin-related glycoproteins (Pancortins) in the brain

Messenger RNA differential display is conducted to search for genes that are expressed in a region-specific pattern in the rodent brain. Eleven novel gene fragments are isolated. One of these genes which we call pancortin, based on its predominant mRNA expression in the cerebral cortex of the adult, is studied. These pancortin cDNA clones are grouped into four different types of cDNA, designated as pancortin-1 to -4. All pancortin cDNAs share a common sequence in the middle of their structure, having two alternative sequences at both 5- and 3-ends, respectively. Deduced amino acid sequence shows that all pancortins have sequences of hydrophobic amino acids at N-terminus and no obvious membrane spanning regions. In situ hybridization histochemistry using oligonucleotide probes specific for 5- and 3-end variable parts has revealed that these four pancortin mRNAs are expressed differentially in the adult rodent brain. Robust expression of pancortin-1 and -2 mRNA is observed in the cerebral cortex (including the hippocampus and the olfactory bulb). However, little of pancortin-3 and -4 mRNA is observed there. In the cortex, some neurons are stained by an antibody raised against Pancortin. Immuno-electron microscopic study has revealed that Pancortin-like immunoreactive products are localized mainly in the endoplasmic reticulum and not in the Golgi apparatus indicating that Pancortins are the endoplasmic reticulum-anchored proteins. Our results suggest that each Pancortin is differentially regulated and may perform different functions in the brain.

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.

Identification of a novel adenylate kinase system in the brain: cloning of the fourth adenylate kinase.

We identify a novel subtype of adenylate kinase, which is the 4th adenylate kinase (AK4), in the vertebrate. AK4 mRNA is expressed in the mammalian central nervous system in a region-specific manner from the middle stage of embryogenesis to the adulthood in the rodent. The presence of three isozymes of adenylate kinase (AK1, AK2 and AK3) that maintains the homeostasis of adenine and guanine nucleotide composition has been reported in the vertebrate. Obtained mouse AK4 cDNA is 3667 bp in size. The predicted open reading frame consists of 223 amino acid residues. Rat AK4 cDNA is also obtained, and the predicted open reading frame is the same length as that of the mouse. The predicted rat AK4 molecule shows 97.8% homology with mouse AK4. Rat AK4 protein is distinct from rat AK3, 53.8% homologous with rat AK3, although the adenylate kinase signature and the mitochondrial energy transfer protein signature are found in both sequences. Interestingly, rat AK4 is 89.2% homologous with the human AK3 over 223 amino acid residues and rat AK3 is 53.7% homologous with the human AK3 indicating that the reported human AK3 actually belongs to the AK4 group (therefore, it should be referred to as human AK4). Although the sequence of AK4 is most similar to that of AK3 among the AK isozymes, its in vivo expression is completely different from AK3; AK4 mRNA is expressed in the pyramidal cells in the hippocampus (mainly in the subfield CA3), the granular cells in the cerebellum, nasal neuroepithelium and the liver while AK3 mRNA is expressed ubiquitously in the body. It is probable that AK4 acts on the specific mechanism of energy metabolism rather than control of the homeostasis of the ADP pool ubiquitously.

Single stranded DNA as an immunocytochemical marker for apoptotic change of ischemia in the gerbil hippocampus

The light and electron microscopic localizations of single stranded DNA (SSD) protein, a marker of apoptosis and programmed cell death, in the gerbil hippocampus were examined by immunocytochemistry after transient brain ischemia. SSD-immunoreactive (IR) cells appeared from post-operative day 1 (PO 1) to PO 7 after 5- or 10-min ischemia. Immunoreaction was recognized in the nucleus of the CA1 pyramidal neurons without remarkable morphological changes on PO 1. These findings suggest that SSD degradation can occur during delayed neuronal death in the CA1, preceding the appearance of double strand breaks, one of the characteristic features of apoptosis.

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.