Tomoyo Ochiishi

Cellular localization of adenosine A1 receptors in rat forebrain: immunohistochemical analysis using adenosine A1 receptor-specific monoclonal antibody.

Monoclonal antibodies were generated against the adenosine A1 receptor (A1R) purified from rat brain. In immunoblot analyses of purified or partially purified A1R preparations from rat brain, these antibodies recognized a solitary band, the size of which corresponded to that expected for A1R. These antibodies recognized not only the native form of A1R but also the deglycosylated form of A1R. Immunocytochemical analysis of Chinese hamster ovarian cells that were transfected stably with rat A1R cDNA showed that their cell bodies were stained intensely by these antibodies, whereas nontransfected Chinese hamster ovarian cells were not. These antibodies detected the A1R naturally present in the DDT1 MF‐2 smooth muscle cells. One of these antibodies (the 511CA antibody) was then used to examine the immunohistochemical distribution of A1Rs in rat forebrain. On light microscopy, A1R immunoreactivity was observed in the cerebral cortex, septum, basal ganglia, hippocampal formation, and thalamus. However, in some regions of the forebrain, regional differences in staining intensity were found as follows: In the cerebral cortex, the strongest immunoreactivity was found in the large pyramidal neurons of layer V. This immunoreactivity was detected in the pyramidal cell bodies, dendrites, and axon initial segments. In the hippocampus, A1R immunoreactivity was detected mainly in the stratum pyramidale. The pyramidal cells in fields CA2–CA3 of the hippocampus were stained more intensely or more clearly than those in field CA1 or the dentate gyrus. More intense A1R immunoreactivity of the apical dendrites was detected in field CA2 compared with other hippocampal fields and the dentate gyrus. Many interneurons of the hippocampus were stained by the 511CA antibody. The subcellular distribution of A1Rs in the forebrain was examined by using a digital deconvolution system and electron microscopy. In the cerebral cortex, the view obtained by removing the background haze by deconvolution revealed that the immunofluoresence‐labeled A1Rs were distributed on the surfaces of the cell bodies and dendrites and in the cytoplasm of layer V neurons as small spots. In field CA1, immunoreactivity was detected in the areas surrounding pyramidal cells. Electron microscopy revealed the presence of A1R‐immunoreactive products in both the presynaptic terminals and the postsynaptic structures. The specific cellular distribution of A1Rs is consistent with the physiological premise that endogeneously released adenosine exerts control over the excitability of forebrain neurons at both presynaptic and postsynaptic sites through A1Rs. J. Comp. Neurol. 411:301–316, 1999.

Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase II in the rat retina

Abstract Ca 2+ / calmodulin-dependent protein kinase II (CaM kinase II) consisting of α and β isoforms is highly expressed in the central nervous system and is implicated in the regulation of various Ca 2+ -dependent physiological processes. We investigated the immunohistochemical distribution of the α and β isoforms of this enzyme in the rat retina, using highly specific monoclonal antibodies which recognize each isoform. Immunoblotting revealed that not only the α but also the β isoform of CaM kinase II were expressed in the retina. The immunohistochemical study showed that highly α-immunoreactive products were localized in amacrine cells in the inner nuclear layer and displaced amacrine cells and ganglion cells in the ganglion cell layer. In addition, two well-defined bands within the inner plexiform layer were densely stained with the anti-α antibody. By contrast, immunoreactivity against the anti-β antibody was very weak in the same neuronal components of the retina. β-Immunoreactive products were homogeneously distributed throughout the inner plexiform layer and no well-defined bands were detected in this layer. Glial cells such as Muller cells were immunoreactive neither to α nor β antibody. A possible co-existence of choline acetyl transferase (ChAT) within CaM kinase II α-immunopositive neurons was examined by evaluating adjacent sections stained with anti-CaM kinase II α antibody and anti-ChAT antibody, respectively. The distribution of CaM kinase II α immunoreactivity in the rat retina was remarkably similar to that of ChAT immunoreactivity. About 32% of total ChAT-immunopositive neurons in the inner nuclear layer contained the CaM II α isoform, whereas only an occasional co-existence of both enzymes was observed in the ganglion cell layer. The present immunoblot and immunohistochemical study elucidated that not only α but also β isoforms of CaM kinase II β were expressed and this enzyme may participate in the cholinergic system in the rat retina.

Specific distribution of Ca2+ / calmodulin-dependent protein kinase II α and β isoforms in some structures of the rat forebrain

Abstract The immunohistochemical distribution of Ca 2+ / calmodulin-dependent protein kinase II (CaM kinase II) α and β isoforms in the rat forebrain was examined by using monoclonal antibodies specific to each isoform. The present study confirmed that α and β immunoreactives are localized only in neuronal elements. At the light microscopic level, specific distribution patterns of these isoforms and staining characteristics were recognized in some regions of the forebrain as follows. Firstly, α-immunoreactive neurons were more homogeneously distributed throughout the cellular layers of the cerebral cortex (i.e., layers II-IV) than β-immunoreactive ones. Secondly, neurons in the globus pallidus were immunostained by the anti-β antibody, but not by the anti-α antibody. Thirdly, neurons in the medial habenular nucleus, the subthalamic nucleus and the reticular thalamic nucleus were more densely stained with the anti-β antibody than with the anti-α antibody. However, marked differences were not observed in the hippocampal formation at the light microscopic level. The electron microscopic analysi of the cerebral cortex demostrated that subcellular localizations of α- and β-immunoreactive products within the cortical neurons were quite dissimilar: (i) the nucleus was stained only with the anti-α antibody, but not with the antiβ antibody, and (ii) β-immunoreactive products were more sporadically localized in the cytoplasms of the perikarya and dendrited than the α-immunoreactive ones. These rigional and subcellular differences between the distribution patterns of α and β immunoreactivities suggest the functional diversity of CaM kinase II α and β isoforms in the central system.

Musculotopic organization of the facial nucleus of the reeler mutant mouse

The migration of facial motoneurons is affected by the reeler gene, and the facial nucleus of the reeler mutant is cytoarchitecturally abnormal. The present study was undertaken to compare the musculotopic organization of the reeler facial nucleus with that of the normal mouse by the retrograde horseradish peroxidase method. In the normal mouse, motoneurons supplying the nasolabial muscle were located in the lateral and dorsolateral subnuclei, those supplying the posterior auricular muscle in the ventromedial and dorsomedial subnuclei, those supplying the mentalis/platysma muscle in the ventral intermediate subnucleus of the facial nucleus, and those supplying the posterior belly of the digastric muscle in the accessory facial nucleus. This musculotopic representation on the main facial nucleus and accessory facial nucleus also appears in the reeler mouse. The musculotopic representation of the facial nucleus of the reeler mouse is thus identical to that of the normal mouse in spite of the formers cytoarchitectonic abnormalities.

Immunohistochemical analysis on the role of adenosine A1 receptors in epilepsy

Adenosine has an anticonvulsant effect in various models of epilepsy. This effect appears to be mediated through the activation of adenosine A1 receptors (A1Rs). We immunohistochemically investigated the changes of A1Rs expression in kainate-treated and hippocampus-kindled rats as chronic models of epilepsy. In the normal hippocampus, a predominant expression of A1Rs was detected in the CA2/CA3a field. The A1Rs immunoreactivity in this field began to decline drastically approximately 4 weeks after kainate treatment and remained minimal 8 weeks after treatment. In the hippocampus-kindled animals, A1Rs expression was minimal in the stimulated side but remained high in the nonstimulated side. The reduced expression of A1Rs in the CA2/CA3a field may be related to chronic epileptogenesis.

Characterization and autophosphorylation of Ca2+/calmodulin-dependent protein kinase in the postsynaptic density of the rat forebrain

The enzymatic and regulatory properties of Ca2+/calmodulin-dependent protein kinase in the postsynaptic density (mPSDp CaM kinase) of the rat forebrain was compared with those of soluble Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). mPSDp CaM kinase was different from soluble CaM kinase II in terms of substrate specificity, regulatory consequences and sites of autophosphorylation. Both soluble and PSD kinases generated Ca(2+)-independent activity by autophosphorylation and Ca(2+)-independent activity almost reached the maximum during the first minute of autophosphorylation. Ca(2+)-independent activity of mPSDp CaM kinase was more stable than that of the soluble kinase under autophosphorylating conditions. Autophosphorylation of the kinases decreased the mobility of the kinases on SDS-polyacrylamide gels. The mobility shift and determination of 32P phosphate incorporation into the kinases demonstrated that there were three species in mPSDp CaM kinase alpha isoform: two active forms with and without the mobility shift (about 22 and 19%, respectively), and an inactive form (about 59%). However, there was only one species in the soluble kinase alpha isoform, which was active. The maximum incorporation of 32P phosphate into mPSDp CaM kinase alpha isoform was less than that of the soluble kinase. Tryptic peptide analysis indicated that the phosphorylation sites of mPSDp CaM kinase alpha isoform differed from those of the soluble kinase.

Immunocytochemical localization of calcium/calmodulin-dependent protein kinase II isoforms in the ganglion cells of the rat retina: immunofluorescence histochemistry combined with a fluorescent retrograde tracer

To determine whether or not calcium/calmodulin-dependent protein kinase II (CaM kinase II) is localized in the ganglion cells in the rat retina, we labeled ganglion cells by injection of Fast blue (FB) into the lateral geniculate nucleus and then stained the retina immunohistochemically with monoclonal antibodies which react specifically with the alpha and beta isoforms of CaM kinase II. Eighty and 90% of the FB-labeled ganglion cells in the ganglion cell layer were immunoreactive with the alpha and beta antibodies, respectively, suggesting that both alpha and beta isoforms of CaM kinase II are expressed in most ganglion cells which project to the lateral geniculate nucleus.

Musculotopic organization in the motor trigeminal nucleus of the reeler mutant mouse

We examined the musculotopic organization in the motor trigeminal nucleus and the somatotopical arrangement in the trigeminal ganglion of the normal and reeler mice. To determine whether or not masticatory motoneurons are malpositioned in the reeler mouse, we injected horseradish peroxidase (HRP) into the masticatory muscles of normal and reeler mice. Injections of HRP into the jaw-closing muscles, i.e., the masseter and temporalis muscles, labeled large multipolar neurons in the dorsolateral division of the motor trigeminal nucleus of both normal and reeler mice. Similar injections into the jaw-opening muscles, i.e., the anterior belly of the digastric muscle and mylohyoid muscle, labeled large multipolar neurons in the ventromedial division of the motor trigeminal nucleus of both mouse strains. Thus, the normal myotopical arrangement of the masticatory muscles on the motor trigeminal nucleus is preserved in the reeler mouse. However, detailed analysis revealed that jaw-opening motoneurons were more widely scattered in the reeler mouse than in the normal control. To examine the somatotopical arrangement of the first-order sensory neurons in the trigeminal ganglion of the normal and reeler mice, we subcutaneously injected Fast blue (FB) into the mental region and Diamidino yellow (DY) into the vibrissal region of the same animals. No differences in distribution patterns of FB-labeled and DY-labeled neurons in the whole-mounted trigeminal ganglion could been seen between these two strains, suggesting that migration of trigeminal ganglion cells, which are derived from the neural crest and placode, is not deranged by the reeler genetic locus.

Alpha calcium/calmodulin-dependent protein kinase II immunoreactivity in corticospinal neurons: combination of axonal transport method and immunofluorescence

A combination of either retrograde or anterograde fluorescent tracer and immunofluorescence histochemistry using the monoclonal antibody specific for the alpha isoform of calcium/calmodulin-dependent protein kinase II (CaM kinase IIα) was employed to test whether CaM kinase IIα is expressed in somata of corticospinal neurons and their axons over their whole course. After the injection of carbocyanine dye DiI into the hindlimb area of the primary motor cortex of the rat, corticospinal axons and their terminal arbors were anterogradely labeled: DiI-labeled corticospinal fibers proceeded caudally in the ipsilateral internal capsule, cerebral peduncle and medullary pyramid, crossed at the pyramidal decussation and descended in the ventralmost area of the contralateral dorsal funiculus of the spinal cord. These DiI-labeled corticospinal axons expressed strong CaM kinase IIα immunoreactivity along their course. However, their terminal arbors within the gray matter of the lumbar cord were very weakly immunostained. With the injection of Fast Blue into the lumbar enlargement of the rat, somata of corticospinal neurons in layer V of the motor cortex were retrogradely labeled. The subsequent immunofluorescent histochemistry revealed that more than 80% of Fast Blue-labeled corticospinal neurons were immunostained with CaM kinase IIα antibody. The present immunohistochemical study demonstrated that CaM kinase IIα is strongly expressed in both somata and axons of a majority of corticospinal neurons, although we could not detect this enzyme in the corticospinal terminals in the spinal target areas.

Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase II in rat retina

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) consisting of alpha and beta isoforms is highly expressed in the central nervous system and is implicated in the regulation of various Ca(2+)-dependent physiological processes. We investigated the immunohistochemical distribution of the alpha and beta isoforms of this enzyme in the rat retina, using highly specific monoclonal antibodies which recognize each isoform. Immunoblotting revealed that not only the alpha but also the beta isoform of CaM kinase II were expressed in the retina. The immunohistochemical study showed that highly alpha-immunoreactive products were localized in amacrine cells in the inner nuclear layer and displaced amacrine cells and ganglion cells in the ganglion cell layer. In addition, two well-defined bands within the inner plexiform layer were densely stained with the anti-alpha antibody. By contrast, immunoreactivity against the anti-beta antibody was very weak in the same neuronal components of the retina. beta-Immunoreactive products were homogeneously distributed throughout the inner plexiform layer and no well-defined bands were detected in this layer. Glial cells such as Müller cells were immunoreactive neither to alpha nor beta antibody. A possible co-existence of choline acetyl transferase (ChAT) within CaM kinase II alpha-immunopositive neurons was examined by evaluating adjacent sections stained with anti-CaM kinase II alpha antibody and anti-ChAT antibody, respectively. The distribution of CaM kinase II alpha immunoreactivity in the rat retina was remarkably similar to that of ChAT immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)