Olfactory bulb interneurons are continuously generated throughout development and in adulthood. These neurons are born in the subventricular zone (SVZ) and migrate along the rostral migratory stream ...into the olfactory bulb where they differentiate into local interneurons. To investigate the differentiation of GABAergic interneurons of the olfactory bulb we used a transgenic mouse which expresses green fluorescent protein (GFP) under the control of the glutamic acid decarboxylase 65 kDa (GAD65) promoter. During development and in adulthood GFP was expressed by cells in the SVZ and along the entire length of its rostral extension including the distal portion within the olfactory bulb. The occurrence of GAD65 mRNA in these zones was confirmed by PCR analysis on microdissected regions along the pathway. Polysialic acid neural cell adhesion molecule, a marker of migrating neuroblasts in adults, was coexpressed by the majority of the GFP‐positive SVZ‐derived progenitor cells. Cell tracer injections into the SVZ indicated that ≈ 26% of migrating progenitor cells expressed GFP. These data show the early differentiation of migrating SVZ‐derived progenitors into a GAD65–GFP‐positive phenotype. These cells could represent a restricted lineage giving rise to GAD65‐positive GABAergic olfactory bulb interneurons.
Abstract Methamphetamine produces locomotor activation and typical stereotyped motor patterns, which are commonly related with increased catecholamine activity within the basal ganglia, including the ...dorsal and ventral striatum. Since the cerebellum is critical for movement control, and for learning of motor patterns, we hypothesized that cerebellar catecholamines might be a target of methamphetamine. To test this experimental hypothesis we injected methamphetamine into C57 Black mice at the doses of 5 mg/kg two or three times, 2 h apart. This dosing regimen is known to be toxic for striatal dopamine terminals. However, we found that in the cerebellum, methamphetamine increased the expression of the primary transcript of the tyrosine hydroxylase (TH) gene, followed by an increased expression of the TH protein. Increased TH was localized within Purkinje cells, where methamphetamine increased the number of TH-immunogold particles, and produced a change in the distribution of the enzyme by increasing the cytoplasmic percentage. Increased TH expression was accompanied by a slight increase in noradrenaline content. This effect was highly site-specific for the cortex of posterior vermal lobules, while only slight effects were detectable in the hemispheres. The present data indicate that the cerebellum does represent a target of methamphetamine, which produces specific and fine alterations of the catecholamine system involving synthesis, amount, and compartmentalization of TH as well as increased noradrenaline levels. This may be relevant for motor alterations induced by methamphetamine. In line with this, inherited cerebellar movement disorders in various animal species including humans are associated with increased TH immunoreactivity within intrinsic neurons of the same lobules of the cerebellar cortex.
Using a competitive polymerase chain reaction assay, we have quantitated the absolute amounts of mRNA encoding 14 distinct subunits of the γ-aminobutyric acid type A (GABAA) receptor in primary ...cultures of rat cerebellar granule neurons and cerebellar astrocytes. We found that the total amount of GABAAreceptor subunit mRNA in astrocytes was 2 orders of magnitude lower than in neuronal cells. Furthermore, granule cell cultures expressed all 14 different GABAAsubunit mRNAs, while the astroglial cultures contained detectable amounts of all the subunits expressed by granule cells except the α6 and the γ2L subunits. Of the α subunit family members, the α1, α5, and α6 mRNAs were prominent in granule cells, while the α1 and α2 mRNAs were abundant in astrocytes. Of the β receptor subunit mRNAs, the β1 and β3 mRNAs were abundantly expressed in both cultures. The γ 2S and γ mRNAs constituted the great majority of γ subunit mRNAs in neurons, while the γ1 subunit mRNA was the most abundant γ subunit mRNA in astrocytes. When various allosteric modulators of GABAAreceptors were tested electrophysiologically, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) was the only one to modulate chloride currents elicited by GABA in a significantly different manner in granule cells (negative modulation) compared with astrocytes (positive modulation). The latter effect was previously observed in transiently expressed recombinant GABAAreceptors containing a γ1 instead of a γ2 subunit. Our quantitative mRNA results suggest that an important molecular determinant responsible for the DMCM-positive modulatory effect on astroglial native GABAAreceptors is the presence of the γ1 subunit in the receptor assembly.
We studied the expression and distribution of the polypeptide diazepam binding inhibitor (DBI) in rat peripheral organs by immunocytochemistry, radioimmunoassay, Northern blot analysis and binding ...assay. Variable amounts of the DBI peptide and DBI mRNA were found in all the tissues examined (liver, duodenum, testis, kidney, adrenal gland, heart, ovary, lung, skeletal muscle and spleen), with the highest level of expression in liver (220 pmol of DBI/mg protein) and the lowest in spleen (11 pmol of DBI/mg protein). A good correlation between DBI-like immunoreactivity (DBI-LI) and mRNA content was found in all tissues except the heart. The immunohistochemical analysis revealed discrete localization of DBI-LI in cell types with specialized functions: for example, the highest DBI-LI content was found in steroid-producing cells (glomerulosa and fasciculata cells of adrenal cortex, Leydig cells of testis); lower DBI-LI immunostaining was found in epithelial cells specialized for water and electrolyte transport (intestinal mucosa, distal convoluted tubules of kidney). Hepatic cells contained moderate immunoreactivity however the total content of DBI in liver is relatively high and is due to the diffuse presence of DBI in every hepatocyte. Cells with high expression of DBI have been shown to contain a high density of mitochondrial benzodiazepine (BZ) binding sites. This observation led us to perform a competitive binding assay between DBI and 3HPK11195 (a ligand for the mitochondrial BZ binding sites) on mitochondrial membranes of adrenal cortical cells. In this experiment, DBI yielded an apparent competitive inhibition of the binding of PK11195 to the BZ binding sites. Our data support a possible role for DBI as endogenous regulator of intracellular metabolic functions, such as steroidogenesis, via the mitochondrial BZ receptors.
In this study, we analysed the molecular heterogeneity and synaptic localization of the N-methyl-D-aspartate receptor subunit 1 and the alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) ...receptor subunit 1 in the olfactory bulb glomerular synaptic circuitry. Semiquantitative reverse transcriptase polymerase chain reaction showed that approximately 40% of the N-methyl-D-aspartate receptor subunit 1 messenger RNA splice variants contain the N1 exon, which conveys specific functional properties on the channel. In other forebrain and hindbrain regions that we examined, the ratio of the N1-containing (receptor subunit 1 sub(1XX)) to N1-lacking (receptor subunit 1 sub(0XX)) N-methyl-D-aspartate receptor subunit 1 messenger RNAs varied considerably. The cellular and subcellular distribution of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 was investigated with antibodies generated against the C-terminal domain of the individual subunits. Both N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 were localized to the postsynaptic density of asymmetric synapses established by olfactory receptor neuron terminals with the dendrites of mitral and tufted cells. Not all of these synapses, however, were labelled. These results are consistent with the notion that glutamate is the neurotransmitter at the olfactory nerve to mitral and tufted cell synapses, and suggest a high heterogeneity in the expression of the postsynaptic glutamate receptors.
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