Dendrodendritic inhibition in the olfactory bulb is driven by NMDA receptors.

At many central excitatory synapses, AMPA receptors relay the electrical signal, whereas activation of NMDA receptors is conditional and serves a modulatory function. We show here quite a different role for NMDA receptors at dendrodendritic synapses between mitral and granule cells in the rat olfactory bulb. In whole-cell patch-clamp recordings in bulb slices, stimulation of mitral cells elicited slowly decaying, GABAA receptor-mediated reciprocal IPSCs that reflected prolonged GABA release from granule cells. Although granule cells had a normal complement of AMPA and NMDA receptors, the IPSC was completely blocked by the NMDA receptor antagonist D,L-AP-5, suggesting that NMDA receptor activation is an absolute requirement for dendrodendritic inhibition. The AMPA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX) had no effect on IPSCs in the absence of extracellular magnesium but modestly reduced IPSCs in 1 mM magnesium, indicating that the primary effect of the AMPA receptor-mediated depolarization was to facilitate the unblocking of NMDA receptors. Granule cell voltage recordings indicated that effective spike stimulation in granule cells depended on the slow NMDA receptor kinetics. Granule cells also showed a pronounced delay between synaptic stimulation and action potential generation, suggesting that their intrinsic membrane properties underlie the ineffectiveness of brief AMPA receptor-mediated EPSPs. NMDA receptors also seem to have a central role in dendrodendritic inhibition in vivo, because intraperitoneal dizocilpine maleate (MK-801) injection in young adult rats resulted in disinhibition of mitral cells as measured by the generation of c-fos mRNA. The unique dependence of dendrodendritic inhibition on slow EPSPs generated by NMDA receptors suggests that olfactory information processing depends on long-lasting reciprocal and lateral inhibition.

Immunolocalization of metabotropic glutamate receptor 7 in the rat olfactory bulb.

Metabotropic glutamate receptors (mGluRs) constitute a large family of G-protein-coupled receptors that are subdivided into three groups based on sequence similarity, pharmacological profiles, and coupling to second messengers. Although mRNAs for seven of the eight mGluRs are expressed in the olfactory system, the localization and function of specific subtypes have not been fully characterized. Mitral cells of the olfactory bulb express mRNA for several mGluRs, including mGluR7, which has been suggested as a presynaptic glutamate autoreceptor. To investigate the immunolocalization of mGluR7 in the olfactory system, we used a polyclonal antiserum specific for the carboxy terminus of the receptor. Mitral cell somata and proximal dendrites were strongly labeled by the mGluR7 antibody. Electron microscopic analysis revealed that most of the mitral cell somatic staining was cytoplasmic. In olfactory bulb glomeruli, immunoreactivity was present in axons and dendrites. In the piriform cortex, diffuse staining was present in layer Ia that was markedly reduced following bulbectomy, consistent with expression of mGluR7 in mitral cell axon terminals. Electron microscopic analysis of this region confirmed the presence of mGluR7 in multiple axon terminals. Distinct labeled fibers in all levels of layer I appeared to originate from labeled piriform cortex pyramidal cells in layers II and III. Our results indicate that mGluR7 is primarily presynaptic at olfactory bulb synapses. However, the postsynaptic localization of mGluR7 at selected synapses indicates that mGluR7 is not targeted exclusively to axonal compartments.

Cloning and expression of rat metabotropic glutamate receptor 8 reveals a distinct pharmacological profile.

The metabotropic glutamate receptor (mGluR) cDNAs were originally cloned from rat, except for the mouse cDNA clone encoding mGluR8. Mouse mGluR8 couples weakly to the inhibition of adenylate cyclase, thus hindering the characterization of its pharmacological properties. We isolated a rat mGluR8 cDNA that encodes a protein of 908 amino acids. In situ hybridization revealed prominent mGluR8 mRNA expression in olfactory bulb, pontine gray, lateral reticular nucleus of the thalamus, and piriform cortex. Less abundant expression was detected in cerebral cortex, hippocampus, cerebellum, and mammillary body. Glutamate evoked pertussis toxin-sensitive potassium currents in Xenopus laevis oocytes coexpressing mGluR8 and G protein-coupled inwardly rectifying potassium channels. mGluR8 was also activated by the group III-specific agonist L-2-amino-4-phosphonobutyric acid; (2(S), 1'(S), 2'(S)]- 2-(carboxycyclopropyl)glycine, which has been frequently used as a selective group II agonist; and the nonselective agonist (1(S), 3(R)]-1-aminocyclopentane-1,3-dicarboxylic acid but not by the group I-specific agonist 3,5-dihydroxyphenylglycine or the group II-specific agonist [2(S), 1'(R), 2(R), 3'(R)]-2-(2, 3-dicarboxycyclopropyl)glycine. The agonist profile in order of potency was [2(S), 1'(S), 2'(S)]-2-(carboxycyclopropyl)glycine approximately L-2-amino-4-phosphonobutyric acid > glutamate > > [1(S), 3(R)]-1-aminocyclopentane-1, 3-dicarboxylic acid, with EC50 values of 0.63, 0.67, 2.5, and 47 microM, respectively. Both the group I/II-specific antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine and the group III-specific antagonist alpha-methyl-amino-phosphonobutyrate inhibited mGluR8. The pharmacological profile of mGluR8 is distinct among mGluRs but closely matches that of presynaptic inhibition in some central nervous system pathways. Thus, cellular responses mediated by both group II and III agonists may in some cases reflect activation of mGluR8 rather than multiple mGluR subtypes.

Small-conductance, calcium-activated potassium channels from mammalian brain.

Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium-activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance, calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

Distribution of metabotropic glutamate receptor 7 messenger RNA in the developing and adult rat brain.

The large number of metabotropic glutamate receptor subtypes suggests diverse roles in brain function, although specific distribution patterns can give clues to subtype-specific functions [Hayashi Y. et al. (1993) Nature 366, 687-690; Nakajima Y. et al. (1993) J. biol. Chem. 268, 11868-11873; Nomura A. et al. (1994) Cell 77, 361-369; Ohishi H. et al. (1993), 1009-1018]. The metabotropic glutamate receptor mGluR7 is sensitive to the agonist L-2-amino-4-phosphonobutyric acid, a presynaptic inhibitor of neurotransmitter release. We examined the anatomic distribution of mGluR7 messenger RNA expression by in situ hybridization in the developing and adult rat central nervous systems. Our results demonstrate that mGluR7 messenger RNA is among the most widely distributed of metabotropic glutamate receptors in both the developing and adult rat nervous system and that mGluR7 messenger RNA is expressed in most neuronal groups known to respond to L-2-amino-4-phosphonobutyric acid, including mitral cells of the olfactory bulb, granule cells of the dentate gyrus and neurons of the entorhinal cortex and dorsal root ganglion. mGluR7 exhibits preferential expression in sensory afferent pathways and is highly represented in the periventricular zone of the hypothalamus, the latter implying a modulatory role for mGluR7 in neuroendocrine pathways. Most strikingly, the majority of neurons at all levels of olfactory circuitry are among the areas of highest mGluR7 messenger RNA content. The anatomic distribution of mGluyR7 messenger RNA suggests that mGluR7 activation may participate in the processing of hippocampal, sensory and olfactory information.

Cloning and expression of a new member of the L-2-amino-4-phosphonobutyric acid-sensitive class of metabotropic glutamate receptors.

Despite the cloning of several metabotropic glutamate receptors (mGluR1-6), the activity and localization of the cloned mGluRs do not account for the action of L-2-amino-4-phosphonobutyric acid (L-AP4) on mitral/tufted cells in the rat olfactory bulb. Thus, we screened a rat olfactory bulb library for novel cDNA clones, using probes derived from mGluR1 and mGluR4. A full length cDNA clone encoding a metabotropic receptor (mGluR7) whose sequence was 69% identical to that of mGluR4 was isolated. Stimulation of mGluR7 with L-AP4 and glutamate (each at 1 mM) in stably transfected baby hamster kidney cells inhibited forskolin-stimulated cAMP formation, whereas ACPD (1 mM) and quisqualate (0.5 mM) were less effective. Inhibition of cAMP required high concentrations of agonist in the transfected cells, suggesting that inhibition of adenylate cyclase may not be the predominant transduction mechanism for this receptor in neurons. RNA blot analysis and in situ hybridization revealed that mGluR7 has an expression pattern in the central nervous system distinct from that of other L-AP4-sensitive mGluRs. Double-labeling with probes for mGluR1 and mGluR7 revealed that individual mitral/tufted neurons in the olfactory bulb expressed both mRNAs. The expression pattern and L-AP4 sensitivity of mGluR7 suggest that it mediates inhibition of transmitter release at selected glutamatergic synapses. The coexpression of multiple mGluR mRNAs in single neurons indicates that the cellular effects of mGluR activation are likely to result from the integrated action of several receptor subtypes.

Extrastriatal dopamine D2 receptors: distribution, pharmacological characterization and region-specific regulation by clozapine.

The distribution of dopamine D2 receptors in the rat brain was determined by quantitative autoradiography of the binding of [125I]epidepride and the effects of chronic drug administration on regulation of receptors in striatal and extrastriatal brain regions were characterized. [125I]Epidepride (2200 Ci/mmol) bound with high affinity to coronal tissue sections from the rat brain (Kd = 78 pM), and specific binding was detected in a number of discrete layers, nuclei or regions of the hippocampus, thalamus, cerebellum and other extrastriatal sites. Pharmacological analysis of radioligand binding to hippocampal and cerebellar membranes indicated binding to dopamine D2 receptors, and approximately 10% of the binding appeared to represent low affinity idazoxan-displaceable binding to alpha-2 adrenoceptors. The binding to extrastriatal regions resembled previously reported radioligand binding to dopamine D2 receptors in striatal and cortical membranes. Chronic (14 day) administration of two dopamine D2 receptor antagonists, either the typical neuroleptic haloperidol (1.5 mg/kg i.p.) or the atypical neuroleptic clozapine (30 mg/kg i.p.), caused a significant increase in the density of [125I]epidepride binding sites in the medial prefrontal cortex and parietal cortex. Only haloperidol caused a significant increase in the density of [3H]spiperone and [125I]epidepride binding sites in the striatum and a slight increase in [125I]epidepride binding sites in the hippocampus. Similar administration of amphetamine (5 mg/kg i.p.) had no significant effect on the density of dopamine D2 receptors in any brain region examined. In addition, no drug-induced changes in the characteristics of dopamine D2 receptors in discrete areas of the cerebellum were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-dependent isomerization of dopamine D-2 receptors characterized using [125I]epidepride, a high-affinity substituted benzamide ligand.

We have characterized the in vitro binding of a new ligand, [125I]epidepride, and used this substituted benzamide to assess the sensitivity of dopamine D-2 receptors to sodium. Both direct and indirect binding studies with [125I]epidepride and unlabeled epidepride, respectively, demonstrated that the affinity of D-2 receptors for the ligand was decreased from 20 to 30 pM in the presence of sodium to 350 to 500 pM in the absence of sodium. The density of binding sites for [125I]epidepride was identical in the presence and absence of NaCl. The time courses for association of [125I]epidepride to and dissociation from D-2 receptors in the presence of sodium were not consistent with simple bimolecular reactions, suggesting the possibility of a sodium-dependent ligand-induced receptor isomerization. Thus, dissociation of [125I]epidepride was biphasic in the presence of sodium, but monophasic in the absence of sodium. The rank order of potency for inhibition of [125I]epidepride binding by drugs was identical in rat striatum and cells expressing a D-2 receptor cDNA, and similar to the previously described pharmacological profile of D-2 receptors labeled by [3H]spiperone. [125I]Epidepride bound to two classes of binding sites in rat medial prefrontal cortex. One class, present at a density of 10 fmol/mg of protein and with a Kd value of approximately 40 pM, was pharmacologically indistinguishable from D-2 receptors in striatum and transfected cells. The pharmacological profile of the second class of sites was similar to that of alpha-2 adrenergic receptors. [125I]Epidepride had 50- to 100-fold lower affinity (approximately 2 nM) for alpha-2 receptors than for D-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Excretion and biotransformation of carboxymethyl-cysteine in rat, dog, monkey and man.

1. Following an oral dose of S-carboxymethyl [35S]cysteine monkey (rhesus and African green), rat, dog, and man excreted 77,88,95, and 100% respectively of the 35S radioactivity in urine and 7.0, 2.5, 0.7, and 0.3% in faeces during a 3 to 4 day period. 2. The principal drug-related components excreted were unchanged carboxymethylcysteine, dicarboxymethyl sulphide and inorganic sulphate. 3. Rat, dog, and man excreted primarily dicarboxymethyl sulphide and unchanged carboxymethylcysteine and no inorganic sulphate (rat, 7%). 4. Monkey excreted largely inorganic sulphate, moderate amounts of dicarboxymethyl sulphide and a trace of unchanged drug.