mGluR1 in cerebellar Purkinje cells essential for long-term depression, synapse elimination, and motor coordination.

Targeted deletion of metabotropic glutamate receptor-subtype 1 (mGluR1) gene can cause defects in development and function in the cerebellum. We introduced the mGluR1alpha transgene into mGluR1-null mutant [mGluR1 (-/-)] mice with a Purkinje cell (PC)-specific promoter. mGluR1-rescue mice showed normal cerebellar long-term depression and regression of multiple climbing fiber innervation, events significantly impaired in mGluR1 (-/-) mice. The impaired motor coordination was rescued by this transgene, in a dose-dependent manner. We propose that mGluR1 in PCs is a key molecule for normal synapse formation, synaptic plasticity, and motor control in the cerebellum.

Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2.

Subtype 2 of the metabotropic glutamate receptor (mGluR2) is expressed in the presynaptic elements of hippocampal mossy fiber-CA3 synapses. Knockout mice deficient in mGluR2 showed no histological changes and no alterations in basal synaptic transmission, paired-pulse facilitation, or tetanus-induced long-term potentiation (LTP) at the mossy fiber-CA3 synapses. Long-term depression (LTD) induced by low-frequency stimulation, however, was almost fully abolished. The mutant mice performed normally in water maze learning tasks. Thus, the presynaptic mGluR2 is essential for inducing LTD at the mossy fiber-CA3 synapses, but this hippocampal LTD does not seem to be required for spatial learning.

Molecular cloning and tissue distribution of a receptor for pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PA-CAP) is a polypeptide hormone related to vasoactive intestinal polypeptide (VIP). Rat PACAP receptor cDNA was isolated from a brain cDNA library by cross-hybridization with rat VIP receptor cDNA. The recombinant PACAP receptor expressed in COS cells bound PACAP with about 1000 times higher affinity than VIP, and PACAP stimulated adenylate cyclase through the cloned PACAP receptor. The rat PACAP receptor consists of 495 amino acids, contains seven transmembrane segments, and has a significant similarity with other Gs-coupled receptors, such as VIP, glucagon, and secretin receptors. PACAP receptor mRNA was abundantly expressed in the brain, but not in the peripheral tissues except for the adrenal gland. In situ hybridization revealed a high level of expression of PACAP receptor mRNA in the hippocampal dentate gyrus, olfactory bulb, and cerebellar cortex.

A family of metabotropic glutamate receptors.

Three cDNA clones, mGluR2, mGluR3, and mGluR4, were isolated from a rat brain cDNA library by cross-hybridization with the cDNA for a metabotropic glutamate receptor (mGluR1). The cloned receptors show considerable sequence similarity with mGluR1 and possess a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR2 is expressed in some particular neuronal cells different from those expressing mGluR1 and mediates an efficient inhibition of forskolin-stimulated cAMP formation in cDNA-transfected cells. The mGluRs thus form a novel family of G protein-coupled receptors that differ in their signal transduction and expression patterns.

Antibodies inactivating mGluR1 metabotropic glutamate receptor block long-term depression in cultured Purkinje cells.

Antibodies were raised against two distinct extracellular sequences of the rat mGluR1 metabotropic glutamate receptor expressed as bacterial fusion proteins. Both antibodies specifically reacted with mGluR1 in the rat cerebellum and inhibited the mGluR1 activity as assessed by the analysis of glutamate-stimulated inositol phosphate formation in CHO cells expressing mGluR1. Using these antibodies, we examined the role of mGluR1 in the induction of long-term depression in cultured Purkinje cells. In voltage-clamped Purkinje cells, current induced by iontophoretically applied glutamate was persistently depressed by depolarization of the Purkinje cells in conjunction with the glutamate application. The mGluR1 antibodies completely blocked the depression of glutamate-induced current. The results indicate that activation of mGluR1 is necessary for the induction of cerebellar long-term depression and that these mGluR1 antibodies can be used as selective antagonists.

Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide.

Vasoactive intestinal polypeptide (VIP), a 28 amino acid peptide hormone, plays many physiological roles in the peripheral and central nerve systems. A functional cDNA clone of the VIP receptor was isolated from a rat lung cDNA library by cross-hybridization with the secretin receptor cDNA. VIP bound the cloned VIP receptor expressed in mouse COP cells and stimulated adenylate cyclase through the cloned receptor. The rat VIP receptor consists of 459 amino acids with a calculated Mr of 52,054 and contains seven transmembrane segments. It is structurally related to the secretin, calcitonin, and parathyroid hormone receptors, suggesting that they constitute a new subfamily of the Gs protein-coupled receptors. VIP receptor mRNA was detected in various rat tissues including liver, lung, intestines, and brain. In situ hybridization revealed that VIP receptor mRNA is widely distributed in neuronal cells of the adult rat brain, with a relatively high expression in the cerebral cortex and hippocampus.

Immunohistochemical localization of metabotropic glutamate receptors, mGluR2 and mGluR3, in rat cerebellar cortex.

The distribution of the metabotropic glutamate receptors mGluR2 and mGluR3 was immunohistochemically examined in the rat cerebellar cortex at both light and electron microscope levels. An antibody was raised against a fusion protein containing a C-terminal portion of mGluR2. On immunoblot, the antibody reacted with both mGluR2 and mGluR3 in rat brain. mGluR2/3 immunoreactivity was expressed in cell bodies, dendrites, and axon terminals of Golgi cells, as well as in presumed glial processes. Golgi axon terminals with mGluR2/3 immunoreactivity were often encountered in the vicinity of glutamatergic mossy fiber terminals. The results suggest that transmitter glutamate may exert control influences upon Golgi cells not only through dendritic mGluR2/3, but also through axonal mGluR2/3.

Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site.

Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission.

Morphology and synaptic input of substance P receptor-immunoreactive interneurons in control and epileptic human hippocampus.

Substance P (SP) is known to be a peptide that facilitates epileptic activity of principal cells in the hippocampus. Paradoxically, in other models, it was found to be protective against seizures by activating substance P receptor (SPR)-expressing interneurons. Thus, these cells appear to play an important role in the generation and regulation of epileptic seizures. The number, distribution, morphological features and input characteristics of SPR-immunoreactive cells were analyzed in surgically removed hippocampi of 28 temporal lobe epileptic patients and eight control hippocampi in order to examine their changes in epileptic tissues. SPR is expressed in a subset of inhibitory cells in the control human hippocampus, they are multipolar interneurons with smooth dendrites, present in all hippocampal subfields. This cell population is considerably different from SPR-positive cells of the rat hippocampus. The CA1 (cornu Ammonis subfield 1) region was chosen for the detailed morphological analysis of the SPR-immunoreactive cells because of its extreme vulnerability in epilepsy. The presence of various neurochemical markers identifies functionally distinct interneuron types, such as those responsible for perisomatic, dendritic or interneuron-selective inhibition. We found considerable colocalization of SPR with calbindin but not with parvalbumin, calretinin, cholecystokinin and somatostatin, therefore we suppose that SPR-positive cells participate mainly in dendritic inhibition. In the non-sclerotic CA1 region they are mainly preserved, whereas their number is decreased in the sclerotic cases. In the epileptic samples their morphology is considerably altered, they possessed more dendritic branches, which often became beaded. Analyses of synaptic coverage revealed that the ratio of symmetric synaptic input of SPR-immunoreactive cells has increased in epileptic samples. Our results suggest that SPR-positive cells are preserved while principal cells are present in the CA1 region, but show reactive changes in epilepsy including intense branching and growth of their dendritic arborization.

Distribution of the glucose transporters in human brain tumors.

In the present study, we have investigated the expression of both the erythrocyte-type (GLUT1) and the brain-type (GLUT3) glucose transporter isoforms in primary human brain tumors. In situ hybridization made it possible to localize and semiquantify both GLUT1 and GLUT3 mRNAs of individual cells in all 18 samples examined. More signals for GLUT3 mRNA than for GLUT1 mRNA were found over astrocytoma cells, while the reverse was the case in all 6 meningiomas. In astrocytomas, for both mRNAs, the density of silver grains over tumor cells was well correlated with the malignancy of the cells. This correlation was, as was also confirmed by Northern blot analysis, more marked with GLUT3 mRNA than with GLUT1 mRNA. In 2 of 5 anaplastic astrocytomas and in all 3 glioblastomas, numerous tumor cells with large amounts of both mRNAs tended to surround the perivascular regions. "Tumor vessels" with endothelial proliferation, an almost pathognomonic feature of glioblastomas, expressed much GLUT3 mRNA but no significant GLUT1 mRNA, while a single- or a few-layered capillary endothelium expressed much GLUT1 mRNA. The distribution of both mRNAs was in good accordance with that of both proteins. Our results suggest that the expression of both glucose transporter isoforms may contribute to the maintenance of human brain tumors and that the expression of the GLUT3 isoform may be closely related to the malignant change of astrocytomas and particularly related to the aberrant neovascularization which accompanies glioblastomas.

Selective blockade of P/Q-type calcium channels by the metabotropic glutamate receptor type 7 involves a phospholipase C pathway in neurons.

Although presynaptic localization of mGluR7 is well established, the mechanism by which the receptor may control Ca(2+) channels in neurons is still unknown. We show here that cultured cerebellar granule cells express native metabotropic glutamate receptor type 7 (mGluR7) in neuritic processes, whereas transfected mGluR7 was also expressed in cell bodies. This allowed us to study the effect of the transfected receptor on somatic Ca(2+) channels. In transfected neurons, mGuR7 selectively inhibited P/Q-type Ca(2+) channels. The effect was mimicked by GTPgammaS and blocked by pertussis toxin (PTX) or a selective antibody raised against the G-protein alphao subunit, indicating the involvement of a G(o)-like protein. The mGuR7 effect did not display the characteristics of a direct interaction between G-protein betagamma subunits and the alpha1A Ca(2+) channel subunit, but was abolished by quenching betagamma subunits with specific intracellular peptides. Intracellular dialysis of G-protein betagamma subunits did not mimic the action of mGluR7, suggesting that both G-protein betagamma and alphao subunits were required to mediate the effect. Inhibition of phospholipase C (PLC) blocked the inhibitory action of mGluR7, suggesting that a coincident activation of PLC by the G-protein betagamma with alphao subunits was required. The Ca(2+) chelator BAPTA, as well as inhibition of either the inositol trisphosphate (IP(3)) receptor or protein kinase C (PKC) abolished the mGluR7 effect. Moreover, activation of native mGluR7 induced a PTX-dependent IP(3) formation. These results indicated that IP(3)-mediated intracellular Ca(2+) release was required for PKC-dependent inhibition of the Ca(2+) channels. Possible control of synaptic transmission by the present mechanisms is discussed.

Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7.

To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7(-/-)). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7(-/-), but not in mGluR7(+/-), mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist (R,S)-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7(-/-) mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs.

Glutamate and GABA receptor signalling in the developing brain.

Our understanding of the role played by neurotransmitter receptors in the developing brain has advanced in recent years. The major excitatory and inhibitory neurotransmitters in the brain, glutamate and GABA, activate both ionotropic (ligand-gated ion channels) and metabotropic (G protein-coupled) receptors, and are generally associated with neuronal communication in the mature brain. However, before the emergence of their role in neurotransmission in adulthood, they also act to influence earlier developmental events, some of which occur prior to synapse formation: such as proliferation, migration, differentiation or survival processes during neural development. To fulfill these actions in the constructing of the nervous system, different types of glutamate and GABA receptors need to be expressed both at the right time and at the right place. The identification by molecular cloning of 16 ionotropic glutamate receptor subunits, eight metabotropic glutamate receptor subtypes, 21 ionotropic and two metabotropic GABA receptor subunits, some of which exist in alternatively splice variants, has enriched our appreciation of how molecular diversity leads to functional diversity in the brain. It now appears that many different types of glutamate and GABA receptor subunits have prominent expression in the embryonic and/or postnatal brain, whereas others are mainly present in the adult brain. Although the significance of this differential expression of subunits is not fully understood, it appears that the change in subunit composition is essential for normal development in particular brain regions. This review focuses on emerging information relating to the expression and role of glutamatergic and GABAergic neurotransmitter receptors during prenatal and postnatal development.

The regional distribution and cellular localization of mRNA encoding rat prostacyclin synthase.

The cloned cDNA for rat prostacyclin synthase was found to contain a 1503-bp open reading frame which encoded a 501-amino acid protein sharing 84.0% identity with the human enzyme. RNA blot analysis revealed that the rat prostacyclin synthase mRNA, as a single species of 2.1 kb, is expressed abundantly in the aorta and uterus. High levels of expression were also observed in the stomach, lung, heart, testis, liver, and skeletal muscle. Low but significant expression was also seen in the brain and kidney. Furthermore, the regional distribution and cellular localization of prostacyclin synthase mRNA were examined by in situ hybridization analysis of rat tissue sections. The definitive signals for the mRNA were localized in smooth muscle cells of the arteries, bronchi and uterus, and in the cells of the fibrous tunic surrounding the seminiferous tubules, which are characterized as smooth muscle cells. Besides smooth muscle cells, signals were also detected in the fibroblasts of the heart myocardium, lung parenchyma cells and kidney inner medulla tubules and interstitial cells.

Distinct tissue distribution and cellular localization of two messenger ribonucleic acids encoding different subtypes of rat endothelin receptors.

Endothelins (ETs) are very potent vasoconstrictive peptides and have diverse functions in both vascular and nonvascular tissues. This investigation concerns the tissue distribution and cellular localization of rat mRNAs encoding two different subtypes of ET receptors (ETA and ETB). We isolated 46 cDNA clones from a rat lung cDNA library by hybridization with the bovine ETA cDNA. The characterization of these cDNA clones indicated that they represent either the ETA or ETB cDNA. In situ and blot hybridization analyses revealed that the rat ETA mRNA is predominantly expressed in vascular smooth muscle cells of a variety of tissues, bronchial smooth muscle cells, myocardium, and the pituitary gland. There is no significant expression of ETB mRNA in vascular smooth muscle cells, and ETA, thus, plays a primary role in ET-induced vascular contraction. ETB mRNA is more widely distributed in various cell types of many tissues. Its prominent expression is seen in glial cells throughout the brain regions, epithelial cells of the choroid plexus, ependymal cells lining the ventricle, myocardium, endothelial cells of glomeruli, and epithelial cells of the thin segments of Henle's loops. Our investigation demonstrates that the mRNAs for the two subtypes of rat ET receptors show specialized expression patterns of cell types in both brain and peripheral tissues.

Cellular and subcellular distribution of substance P receptor immunoreactivity in the dorsal vagal complex of the rat and cat: a light and electron microscope study.

Immunoreactivity for the substance P receptor (NK1 receptor) has been investigated by light and electron microscopy in the dorsal vagal complexes of adult rats and cats. The general pattern of NK1 immunoreactivity was similar for both rat and cat. Numerous NK1-immunoreactive neurons were present in the area postrema, the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus nerve. The density of labelled neurons differed between the subnuclei of the nucleus of the solitary tract. Overall, the efferent neurons of the dorsal motor nucleus of the vagus nerve highly expressed NK1 when compared to neurons in the nucleus of the solitary tract. The results are discussed with reference to the viscerotopic organisation of the dorsal vagal complex. Ultrastructural analysis demonstrated that NK1 immunoreactivity was present only at the membrane surface of somatic and dendritic profiles of neurons. No labelling was found in axon terminals, axons, or glial processes. NK1 immunoreactivity, as revealed by a preembedding immunogold technique in serial ultrathin sections, was preferentially located at nonsynaptic sites. A semiquantitative study suggested that the density of NK1 receptors is statistically higher at membrane sites free of any contact (synaptic or not) with axon terminals. The subcellular localisation of NK1 immunoreactivity was similar for neurons of both rat and cat. These results suggest that in the dorsal vagal complex, substance P might act on NK1 receptors through a process of volume transmission.

Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: an in situ hybridization study in adult and developing rat.

Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1), which is linked to phosphoinositide (PI) hydrolysis, was investigated in adult and developing rat central nervous system (CNS) by in situ hybridization. Transcripts of mGluR1 were specifically localized to neurons and widely distributed throughout the adult rat brain. Most intensely labeled neurons were Purkinje cells of the cerebellum, mitral and tufted cells of the olfactory bulb, and neurons in the hippocampus, lateral septum, thalamus, globus pallidus, entopeduncular nucleus, ventral pallidum, magnocellular preoptic nucleus, substantia nigra, and dorsal cochlear nucleus. Moderately labeled neurons were seen in high density in the dentate gyrus, striatum, islands of Calleja, superficial layers of the retrosplenial, cingulate and entorhinal cortices, mammillary nuclei, red nucleus, and superior colliculus. In the developing rat brain, the level of mGluR1 expression gradually increased during early postnatal days in accordance with the maturation of neuronal elements. These results show prominent expression of mGluR1 in the major targets of putative glutamatergic pathways and unique distribution pattern of mGluR1 distinct from those reported for ionotropic subtypes of glutamate receptors, suggesting specific roles of mGluR1 in the glutamatergic system.

Distribution of the mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ hybridization study.

Distribution of the mRNA for a metabotropic glutamate receptor, mGluR3, which is coupled to the inhibitory cAMP cascade, was examined in the central nervous system of the adult albino rat by in situ hybridization. The hybridization signals of mGluR3 were detected not only on neuronal cells but also on many glial cells throughout the brain and spinal cord. In the neuronal cells, prominent expression of mGluR3 mRNA was seen in the thalamic reticular nucleus. Moderately labeled neurons were seen in the anterior olfactory nucleus, cerebral neo- and mesocortical regions, lateral amygdaloid nucleus, ventral part of the basolateral amygdaloid nucleus, dorsal endopiriform nucleus, supraoptic nucleus, superficial layers of the superior colliculus, inferior colliculus, interpeduncular nucleus, superior olivary nuclei, and Golgi cells in the cerebellar cortex. Weakly labeled neurons were observed in the striatum, nucleus accumbens, ventral pallidum, globus pallidus, entopeduncular nucleus, lateral hypothalamic area, hypothalamic paraventricular nucleus, medial habenular nucleus, anterior pretectal nucleus, Barrington's nucleus, Nucleus O, paragenual nucleus, trigeminal sensory complex, cochlear nuclei, dorsal motor nucleus of the trigeminal nerve, dorsal cap of the inferior olive, spinal dorsal horn, and lamina X of the spinal cord. The stellate cells in the cerebellar cortex, and neurons in the deep cerebellar nuclei were also labeled weakly. The granule cell layer of the dentate gyrus, as a whole, appeared to be labeled intensely, but each of the granule cells was labeled only weakly. No significant labeling was detected in the mitral and tufted cells in the olfactory bulb, hippocampal pyramidal cells, Purkinje and granule cells in the cerebellar cortex, or somatic motoneurons. The distribution of mGluR3 mRNA in particular neurons and glial cells indicates specific roles of mGluR3 in the glutamatergic system of the central nervous system.

Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats.

Five N-methyl-D-aspartate (NMDA) receptor subunits have been identified thus far: NR1, NR2A, NR2B, NR2C, and NR2D. Here, we have analyzed the expression patterns of mRNAs for the NMDA receptor subunits in the developing and adult rats by in situ hybridization. The developmental changes of the expression patterns were most salient in the cerebellum. In the external granular layer, hybridization signals of mRNAs for NR1, NR2A, NR2B, and NR2C appeared by postnatal day 3, but no NR2D mRNA was expressed at any developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stages examined. The signals for the NR2A mRNA appeared in Purkinje cells and granule cells during the second postnatal week. The signals for the NR2B mRNA in granule cells were seen transiently during the first 2 weeks after birth. The signals for NR2C mRNA appeared in granule cells and glial cells during the second postnatal week. The signals for NR2D mRNA appeared transiently in Purkinje cells during the first 8 postnatal days; in adult rats, these were seen in stellate and Golgi cells. In the cerebellar nuclei, mRNAs for NR1, NR2A, NR2B, and NR2D were more or less expressed on postnatal day 0, while expression signals for the NR2C mRNA were first detected in postnatal day 14. Thus, the most conspicuous changes of expression patterns were observed in the cerebellar cortex during the first 2 weeks after birth, when development and maturation of the cerebellum proceed most rapidly.