Rie Miyoshi

Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography.

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimers disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.

Ontogeny of muscarinic receptors in the rat brain with emphasis on the differentiation of M1- and M2-subtypes— semi-quantitative in vitro autoradiography

The ontogeny of muscarinic acetylcholine receptors (mAChR) in the rat brain was studied with emphasis on the differentiation of M1- and M2-receptor subtypes through semi-quantitative in vitro autoradiography. [3H]Quinuclidinyl benzilate (QNB) and [3H]pirenzepine (PZ) were used for labeling total mAChR and M1-receptors, respectively. In the cerebral cortex of adult rats, [3H]QNB binding sites were more richly present in the superficial and deeper layers than in the middle layer, while M1-receptors were diffusely observed in all the layers. This means that M2-receptors are highly concentrated in the superficial and deeper layers. The ontogenetical differentiation of the laminar distribution between M1- and M2-receptors first appeared at 14 days of postnatal age. In the hippocampus and striatum whose mAChR were predominantly of the M1-type in the adult rat brain, ontogenic patterns of M1-receptors were almost identical to those of total mAChR. On the other hand, mAChR in the cerebellar cortex and lower brainstem of the adult rat were mainly of the M2-subtype. In these areas, the ontogeny of total mAChR was apparently observed. However, M1-receptors were not observed at any stage of the ontogeny. The above-mentioned results indicate that M1- and M2-receptors show distinct developmental behaviors in the rat brain.

NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, increases extracellular GABA in the striatum of the freely moving rat.

We investigated the effect of an NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), on the levels of endogenous GABA in the rat striatum using in vivo microdialysis. Rats were perfused with the artificial CSF containing L-NMMA (0.1, 0.3 and 1.0 mM) or its inactive isomer D-NMMA (1.0 mM) for 1 h. Infusion of L-NMMA, but not its D-isomer, dose-dependently increased GABA concentration. Co-infusion with tetrodotoxin (1 microM) did not antagonize the increase of GABA induced by L-NMMA. These results show that decreased NO activity enhances GABA release even in the absence of depolarization of GABA neurones. We conclude that NO may be directly acting on GABA nerve terminals and tonically inhibiting GABA release or synthesis under basal conditions.

Somatostatin increases intracellular Ca2+ concentration in cultured rat hippocampal neurons

Changes of intracellular Ca2+ concentration [( Ca2+]i) in response to somatostatin were measured in cultured rat hippocampal neurons on a single cell basis by fura-2 fluorometry. Somatostatin increased [Ca2+]i dose-dependently and this effect was completely blocked in either Ca2+-depleted medium or LaCl3-containing medium. In addition, omega-conotoxin GVIA completely inhibited the effect of somatostatin. Our results indicate that somatostatin receptors couple with N-type voltage-sensitive Ca2+ channels in cultured rat hippocampal neurons.

Modulation of dopamine D1 receptor binding by neurotensin in the rat striatum

The effect of neurotensin on binding characteristics of dopamine D1 receptors was examined in the rat striatal membranes through radioreceptor assay. Neurotensin or its analogs were added to incubation medium of[(3)H]SCH 23390 saturation or dopamine/[(3)H]SCH 23390 inhibition experimental systems. Neurotensin did not modulate D1 antagonist binding but converted a part of D1 agonist high affinity binding sites to a low affinity state. Neurotensin(8-13) had the same potency as neurotensin itself, whereas neurotensin(1-8) had only weak activity in modulating D1 agonist binding. GTP and neurotensin had the same effect on D1 agonist binding. However, when both neurotensin and GTP were added, the result was the same as with either alone. These data suggest that neurotensin modulates the functional state of D1 receptors probably via a GTP binding protein in the rat striatum.

Mapping of somatostatin receptor localization in rat brain: Forebrain and diencephalon

Using quantitative in vitro receptor autoradiography, minute distributions of 125I-Tyr11-somatostatin (SS)-14 binding sites were investigated in the rat forebrain and diencephalon. In the cerebral cortex, there was a high density of receptors observed in layers V-VI and a low density in layers I-IV. The entorhinal cortex displayed the highest receptor density of the cerebral cortices. The olfactory system had a high SS receptor density. The anterior olfactory nucleus, nucleus of the lateral olfactory tract, medial habenular nucleus and the basolateral amygdaloid nucleus showed moderate densities. In the limbic system, the CA1 and subiculum regions had high receptor densities. More detailed observations revealed high receptor densities in the oriens, radiatum and lacunosum layers and a much lower density in the pyramidal cell layer. The caudate putamen and substantia nigra showed low receptor densities, while the claustrum displayed the highest density of receptors in the rat brain. These data were not consistent with those of previous studies using 125I-SS-28 and 125I-201-995, which had shown that the high receptor density area in the basolateral amygdaloid group was identified as the lateral amygdaloid nucleus, and that the pyramidal cell layer in the hippocampus showed high receptor densities.

Effect of age on α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites in the rat brain studied by in vitro autoradiography

Receptors for excitatory amino acid,L-glutamate, have been classified into three subtypes named as N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate receptors. In the present study, an effect of age on binding sites of [3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA), a QA agonist, was studied in the rat brain through quantitative in vitro autoradiography.3H-AMPA binding sites were most concentrated in the hippocampus and cerebral cortex where glutamate receptors have been demonstrated to play a role in synaptic transmission. In aged rats,3H-AMPA binding sites in the hippocampus and cerebral cortex were not significantly changed. In our previous studies, it was noticed that strychnine-insensitive glycine receptors, which functionally coupled with NMDA receptors, showed marked age-dependent decreases in telencephalic regions. It has been shown that the glutamatergic neuronal system is involved in learning and memory. Nevertheless, it is considered that AMPA binding sites are not involved in the decline of neuronal functions, especially impairment of learning and memory, accompanying with aging process.

Effects of calcium antagonists on muscarinic receptor subtypes in the rat brain

The effects of several calcium antagonists, including nicardipine, nifedipine, verapamil, and diltiazem, on muscarinic acetylcholine antagonist binding were studied in the P2 fraction of the rat cerebral cortex using either [3H]quinuclidinyl benzilate ([3H]QNB) or [3H]pirenzipine as the radioactive ligand. (1) The potency of [3H]QNB binding inhibition was in the order nicardipine greater than verapamil greater than diltiazem greater than nifedipine. The IC50 values of nicardipine, verapamil, and diltiazem were 2.56 X 10(-6) M, 1.28 X 10(-5) M, and 6.00 X 10(-5) M, respectively. (2) The inhibition of [3H]QNB binding by nicardipine was significantly decreased in the presence of Ca ions. (3) In saturation experiments of [3H]QNB binding in the presence of nicardipine, the Kd value appeared to be significantly affected, but the Bmax value was unchanged. This indicated that nicardipine probably inhibited [3H]QNB binding allosterically. On the other hand, (4) nicardipine inhibited [3H]pirenzipine binding completely with an IC50 value of 7.87 X 10(-7) M. It was concluded that nicardipine had an inhibitory effect on M1-receptor binding.

Immunohistochemical localization of neurotensin and β-endorphin in the rat anterior pituitary gland

Nakanes enzyme-labeled antibody technique revealed that cells containing neurotensin-like immunoreactivity were widely distributed in the anterior lobe of the pituitary body. Immunohistochemical studies on serial sections showed that a part of neurotensin positive anterior lobe cells contained beta-endorphin-like peptide simultaneously. The results show that beta-endorphin and neurotensin occur together in certain pituitary cells and this is an evidence of coexistence of more than one peptide within one anterior pituitary cell.

Ontogeny of phorbol ester receptors in rat brain studied by in vitro autoradiography

The ontogeny of phorbol ester receptors, which have been considered to correspond to protein kinase C, in the rat brain was studied through in vitro autoradiography with3H-phorbol 12,13-dibutyrate (3H-PDBu). The distribution of3H-PDBu binding sites in the adult rat brain was similar to the previous reports by other researchers. The developmental pattern of3H-PDBu binding sites varied with brain region.3H-PDBu binding sites in the amygdala, thalamus, stratum pyramidale of CA 1 of the hippocampus, dentate gyrus, superior colliculus, substantia nigra, interpeduncular nucleus and cerebellar molecular layer were postnatally increased to adult levels and after that they remained constant. On the other hand, in the stratum oriens and stratum radiatum of CA 1 of the hippocampus, and in the lateral and medial geniculate bodies,3H-PDBu binding sites reached peaks at 21 or 28 days of postnatal age and after that they declined to adult levels. The cerebellar granular layer showed a low level of3H-PDBu binding sites throughout all the ontogenetic stages. A distinct ontogenetic pattern of phorbol ester receptors in various regions of the brain may reflect a role of protein kinase C in the neural development of each discrete area.