Behavioral sensitization to cocaine is associated with increased glutamate receptor trafficking to the postsynaptic density after extended withdrawal period.

Glutamatergic signaling plays an important role in the behavioral and molecular plasticity observed in behavioral sensitization to cocaine. Redistribution of the glutamate receptors in the synaptosomal membrane fraction was investigated in the nucleus accumbens, dorsolateral striatum, and ventral tegmental area at 1 or 21 days of withdrawal in behaviorally sensitized rats. At 1 day of withdrawal, there were no changes in either tissue level or redistribution of glutamate receptors in nucleus accumbens core and shell and ventral tegmental area. At 21 days of withdrawal, there was a decrease in the expression of mGluR2/3 protein in core and shell, an increase in GluR1 and a decrease in Homer1b/c proteins in the nucleus accumbens core tissue. In dorsolateral striatum, the tissue level of NMDAR2B protein was increased. Moreover, there was an augmented presence of AMPA (GluR1, GluR2), NMDA (NMDAR1, 2A, 2B), and group 1 metabotropic glutamate receptor (mGluR5) proteins in the synaptosomal fraction in core and shell of the nucleus accumbens. There was also an increase in synaptosomal mGluR2/3 protein in nucleus accumbens core. The redistribution of glutamate receptors was selective for nucleus accumbens since no changes were observed in the dorsolateral striatum and ventral tegmental area. While the tissue level of the Homer1b/c protein was selectively reduced in nucleus accumbens core, that of PSD95, PICK1, and actin was not changed in any of the brain regions examined. However, the synaptosomal membrane fraction level of Homer1b/c and PSD95 was increased in nucleus accumbens core and shell, with no changes in PICK1, and a decrease in actin protein. These observations suggest that significant redistribution of glutamate receptors and postsynaptic scaffolding proteins into synaptosomal membrane fraction is associated with withdrawal from behavioral sensitization to cocaine.

The monoamine neurons of the rat brain preferentially express a splice variant of alpha1B subunit of the N-type calcium channel.

The N-type voltage-dependent calcium channels play a significant role in neurotransmitter release. The alpha1B subunit of the N-type calcium channel functions as the primary subunit that forms the pore and contains the structural motifs that mediate the pharmacological and gating properties of the channel. We report on an isoform of the alpha1B subunit that is preferentially expressed by the monoaminergic neurons of the rat brain. This isoform contains a 21-amino acid cassette in the synprint site present in the cytoplasmic loop between domains IIS6 and IIIS1. RT-PCR of micropunched tissue was used to show preferential expression of this isoform in regions of the brain containing monoaminergic neurons and to a lesser extent in the cerebellum. Double-label in situ hybridization was used to show expression of this isoform mRNA in dopaminergic neurons of the ventral mesencephalon. The expression of two distinct N-type calcium channels containing these alpha1B subunit isoforms by the monoaminergic neurons may provide for synapse-specific regulation of neurotransmitter release.

Regional distribution and cellular localization of gamma-aminobutyric acid subtype 1 receptor mRNA in the rat brain.

The distribution of gamma-aminobutyric acid (GABA) receptor subtype B1 (GABA(B1)) mRNA-containing cells in the brain of adult rats was determined with in situ hybridization histochemistry. The vast majority of neurons expressed GABA(B1) receptor mRNA. However, there were nuclei of relative high density, and, in some nuclei, the majority of neurons did not express detectable levels of GABA(B1) receptor transcripts. Areas where the majority of neurons expressed a high density of mRNA included the medial habenula; the septohippocampal, periventricular, suprachiasmatic, and supraoptic nuclei; Purkinje cells in the cerebellum; and pyramidal and granule cells of the hippocampus and dentate gyrus, respectively. Also, brainstem nuclei containing monoaminergic neurons and neurons in the thalamic motor nuclei contained relatively high levels of expression. mRNA was low or absent in neuronal populations in regions with well-developed cytoarchitecture, such as the stratum radiatum and stratum oriens of the hippocampus, the molecular and granular layers of the cerebellum, and the molecular layer of the cortex. Low expression was observed also in many extrapyramidal nuclei, such as the globus and ventral pallidum and the substantia nigra, pars reticulata. Expression also was low in the reticular thalamic nucleus and zona incerta. Neurons lacking detectable GABA(B1) receptor mRNA were generally in nuclei that contained largely GABAergic neurons.

The regulation of dopamine transmission by metabotropic glutamate receptors.

Receptor subtype nonselective metabotropic glutamate receptor (mGluR) agonists have been shown to regulate the release of dopamine. The eight mGluR subtypes have been pharmacologically categorized into three groups, and the present study used in vivo microdialysis to examine the capacity of mGluR subgroup-selective drugs to modulate the extracellular levels of dopamine in the nucleus accumbens. By administering the drugs in the dialysis buffer, it was found that the group 3 mGluR agonist L-amino-4-phosphonobutyrate produced a dose-dependent reduction in extracellular dopamine, whereas the group 1 agonist 3,5-dihydroxyphenylglycine was ineffective. The group 2 agonist (2S,1'R,2'R,3'R)-2-(2, 3-dicarboxycyclopropyl)glycine produced a reduction that was biphasic with respect to dose. The group 2/3 antagonist alpha-methyl-4-phosphnophenylglycine elicited a dose-dependent increase in extracellular dopamine that was antagonized by coperfusion with either the L-type calcium channel blocker diltiazem or the group 3 agonist L-amino-4-phosphonobutyrate. These data demonstrate that group 3 and to a lesser extent group 2 mGluR may presynaptically regulate dopamine release or reuptake. Moreover, there exists significant in vivo glutamatergic tone on group 2/3 mGluRs to suppress extracellular dopamine levels.

Expression of glutamate receptor subunit/subtype messenger RNAS for NMDAR1, GLuR1, GLuR2 and mGLuR5 by accumbal projection neurons.

Nucleus accumbens neurons are the targets of glutamatergic inputs. By coupling in situ hybridization for glutamate receptor mRNAs with retrograde transport of Fluoro-Gold, the present study examined the relationship between the distribution patterns of glutamate receptor subtypes/subunits and the output pathways of the nucleus accumbens to the ventral pallidum and ventral tegmental area. Following iontophoretic deposits of Fluoro-Gold into the ventral pallidum, neurons in both the nucleus accumbens shell and core were retrogradely labeled. A high percentage of accumbens neurons retrogradely labeled from the ventral pallidum were double-labeled for mRNAs encoding for mGluR5 (82+/-4.1%), NMDAR1 (71+/-3.5%), GluR1 (70+/-6.1%) and GluR2 (76+/-3.6%). No significant difference in the proportion of double-labeled neurons between the core and shell was observed. Following the deposit of Fluoro-Gold into the ventral tegmental area, only the accumbens shell neurons were retrogradely labeled. The proportion of neurons expressing NMDAR1, GluR1 and GluR2 were somewhat less in the projection to the ventral tegmental area compared to the ventral pallidum since approximately 60% of the neurons retrogradely-labeled from the ventral tegmental area expressed these transcripts. In contrast to the high proportion of mGluR5-containing neurons in the nucleus accumbens innervating the ventral pallidum, only half of the neurons projecting to the ventral tegmental area expressed mGluR5. These data show that accumbens neurons innervating the ventral pallidum and ventral tegmental area differ in the relative proportion of expressed mRNA encoding mGluR5, implying differential postsynaptic impact by glutamate transmission on neurons contributing to the two major efferent pathways of the nucleus accumbens.

Neuroadaptations in ionotropic and metabotropic glutamate receptor mRNA produced by cocaine treatment.

The expression of glutamate receptor/subunit mRNAs was examined 3 weeks after discontinuing 1 week of daily injections of saline or cocaine. The level of mRNA for GluR1-4, NMDAR1, and mGluR5 receptors was measured with in situ hybridization and RT-PCR. In nucleus accumbens, acute cocaine treatment significantly reduced the mRNA level for GluR3, GluR4, and NMDAR1 subunits, whereas repeated cocaine reduced the level for GluR3 mRNA. Acute cocaine treatment also reduced the NMDAR1 mRNA level in dorsolateral striatum and ventral tegmental area. In prefrontal cortex, repeated cocaine treatment significantly increased the level of GluR2 mRNA. The GluR2 mRNA level was not changed by acute or repeated cocaine in any other brain regions examined. Repeated cocaine treatment also significantly increased mGluR5 mRNA levels in nucleus accumbens shell and dorsolateral striatum. Functional properties of the ionotropic glutamate receptors are determined by subunit composition. In addition, metabotropic glutamate receptors can modulate synaptic transmission and the response to stimulation of ionotropic receptors. Thus, the observed changes in levels of AMPA and NMDA receptor subunits and the mGluR5 metabotropic receptor may alter excitatory neurotransmission in the mesocorticolimbic dopamine system, which could play a significant role in the enduring biochemical and behavioral effects of cocaine.

Role of transforming growth factor (TGF)-beta Type I and TGF-beta type II receptors in the TGF-beta1-regulated gene expression in pituitary prolactin-secreting lactotropes.

Transforming growth factor beta1 (TGF-beta1) inhibits pituitary lactotrope proliferation and secretion of PRL in an autocrine/paracrine manner. In this study, the role of TGF-beta1 type I (TbetaR-I) and TGF-beta type II (TbetaR-II) receptors in TGF-beta1-regulated gene expression in lactotropes was determined using anterior pituitary cells known to be responsive to TGF-beta1 growth inhibition and using a transformed PR1 cell line known to be nonresponsive to TGF-beta1 growth inhibition. Treatment with TGF-beta1 inhibited cell proliferation and decreased PRL mRNA levels in anterior pituitary cells, but in PR-1 cells, the treatment caused only decreased PRL mRNA levels. Affinity labeling of TGF-beta binding proteins indicated that anterior pituitary cells contain several TGF-beta-binding protein complexes, including the 65 kDa size TbetaR-I and 95 kDa size TbetaR-II. In the PR1 cells, the major complex found was similar to the 65 kDa size of TbetaR-I. Immunocytochemistry identified TbetaR-I and TbetaR-II receptor proteins in lactotropes but detected primarily TbetaR-I receptor protein in PR1 cells. RT-PCR detection of TbetaR-I and TbetaR-II mRNA identified both receptor mRNA transcripts in anterior pituitary cells and in PR1 cells but the levels of TbetaR-II and TbetaR-I mRNA transcripts in PR1 cells was much lower than that in anterior pituitary cells. Determination of the TGF-beta1 gene responses in PR1 cells following TbetaR-I and TbetaR-II gene transfection indicated that PR1 cells transactivate transcription of the TGF-beta-responsive p3TP-Lux reporter in the absence of cotransfected TbetaR-II receptor. The introduction of the TbetaR-II receptor alone or in combination with TbetaR-I confer ligand-independent reporter transactivation in these cells. When only TbetaR-I was introduced along with reporter, a ligand-dependent transactivation was observed. These data suggest for the first time that the TGF-beta1-mediated transcriptional activation response can be distinguished from the growth response in lactotropes. Furthermore, the TGF-beta1 gene-transcription response is less dependent on TbetaR-II receptor expression than is the TGF-beta1 growth-inhibitory response.

Expression of D1 receptor, D2 receptor, substance P and enkephalin messenger RNAs in the neurons projecting from the nucleus accumbens.

In situ hybridization was combined with FluoroGold retrograde labelling to determine the distribution of messenger RNAs for the D1 dopamine receptor, D2 dopamine receptor, beta-preprotachykinin or preproenkephalin in the neurons projecting from the nucleus accumbens to the ventral pallidum and the ventral tegmental area. Neurons were quantified in both the core and the shell of the nucleus accumbens to estimate the proportion of neurons projecting to the ventral pallidum or ventral tegmental area that contain transcripts for D1 receptors, D2 receptors, beta-preprotachykinin or preproenkephalin. Following the deposition of FluoroGold into the central ventral pallidum, both the core and the shell of the nucleus accumbens were retrogradely labelled, while deposits into the ventral tegmental area selectively labelled cells in the shell. A high percentage of nucleus accumbens neurons innervating the ventral tegmental area expressed messenger RNAs for D1 receptors (72%) and beta-preprotachykinin (62%), while less than 3% of the neurons contained messenger RNAs for preproenkephalin or D2 receptors. The neurons projecting to the ventral pallidum did not show the discrete distribution of transcripts as was observed in the accumbens-ventral tegmental area projection. Preproenkephalin messenger RNA was identified in 46% of the neurons innervating the ventral pallidum, and D2 receptor messenger RNA was found in approximately 40% of the cells. A large minority of neurons projecting from the nucleus accumbens to the ventral pallidum also expressed messenger RNAs for D1 receptors (37%) and beta-preprotachykinin (35%). While a higher percentage of D1 receptor, and beta-preprotachykinin messenger RNA expressing cells were located in the shell than in the core of the nucleus accumbens, the percentage tended to be higher in the core for cells expressing D2 receptors or preproenkephalin messenger RNA. These data indicate that messenger RNAs for D2 receptors and enkephalin are selectively expressed in the accumbens-pallidal projection while transcripts encoding D1 receptors and substance P are contained in the efferent projections to both the ventral pallidum and ventral tegmental area. The presence of D1 receptor and beta-preprotachykinin messenger RNAs in both mesencephalic and pallidal projections contrasts output from the striatum where the expression of D1 receptor and beta-preprotachykinin messenger RNAs is primarily restricted to the mesencephalic projection.

Multiplicity of glutamate receptor subunits in single striatal neurons: an RNA amplification study.

The RNA amplification technique was used to examine the pattern of coexpression of mRNAs encoding 16 subtypes/subunits of the glutamate receptor (GluR) in acutely dissociated neurons from adult rat striata. THe signal intensity for each mRNA varied within single neurons, but the general pattern of low versus high expression signals was similar among neurons, except for the GluR4 subunit of the (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. The mRNAs for GluR1-3 subunits of the AMPA receptor were present in all cells, with the signal intensity of GluR1 mRNAs usually the lowest. The kainate receptor subunit mRNAs (GluR5-7) were present in most neurons, and the signal intensity for GluR6 mRNA was the highest. The signals for N-methyl-D-aspartate (NMDA)R1 and NMDAR2B mRNAs were high in most neurons; however, NMDAR2A and NMDAR2C mRNAs gave low or undetectable signals. For mRNAs encoding metabotropic GluRs (mGluRs), signals for mGluR1, mGluR2, and mGluR3 mRNAs were low or undetectable, whereas mGluR4 and mGluR5 mRNA signals were high in most neurons. In most cases (12 of 16 mRNAs), the results agreed with data from in situ hybridization experiments in which individual mRNAs were examined. All neurons expressed subtypes/subunits mRNAs for all four types of GluRs; however, there were differences in the relative intensity of the mRNA signals detected in individual cells, suggesting that these receptors could exist in various combinations within individual neurons and thus confer synapse-specific function for information processing in the striatum.

Real-time monitoring of electrically stimulated norepinephrine release in rat thalamus: I. Resolution of transmitter and metabolite signal components.

Electrical stimulation of an ascending path of the locus ceruleus-norepinephrine system was used to elicit release of norepinephrine at noradrenergic terminal fields of the rat thalamus. Overflow into the extracellular fluid space was measured by fast in vivo chronoamperometry. At pretreated carbon fibers, the electrochemical signal consists of a sharp peak of approximately 20-30 s duration followed by a slower, plateau-like decay to baseline. The peak, characterized by a variety of pharmacological manipulations and dialysis perfusion, is primarily due to norepinephrine. The plateau was shown to correspond to metabolite efflux of 3,4-dihydroxy-phenylacetic acid. By varying the degree of electrochemical pretreatment, the response time and sensitivity of the fibers can be tuned to follow the entire signal or to select the separate components for detailed evaluation. This approach can be used to provide new information on the spatial and temporal characteristics of stimulated neurotransmitter release.

Real-time monitoring of electrically stimulated norepinephrine release in rat thalamus: II. Modeling of release and reuptake characteristics of stimulated norepinephrine overflow.

As in the preceding study, electrical stimulation was used to effect release overflow of norepinephrine in the rat thalamus. Using a weak electrochemical pretreatment of a carbon fiber electrode, it was possible to "tune in" the electrochemical response signal for norepinephrine without metabolite interference. This reasonably selective signal was then used to study the degradation of norepinephrine release ability caused by prolonged stimulation. Further, the signals were modeled by the method used successfully for stimulated dopamine overflow, providing hitherto unavailable information on the temporal and spatial characteristics of norepinephrine release overflow. Pertinent comparisons between the release characteristics of the dopamine and norepinephrine systems show that the half-life for norepinephrine in the extracellular fluid space is approximately 1 s in thalamus compared with 33 ms for dopamine in caudate.

Intracerebral infusion of DOPAC decreases striatal dopamine.

The purpose of this study was to determine whether elevated levels of 3,4-dihydroxyphenylacetic acid (DOPAC), the major metabolite of dopamine (DA) in the brain, could decrease the DA content in the striatum. Levels of DA were determined by high pressure liquid chromatography with electrochemical detection (HPLC-EC) in the striatum of male rats 24 h following a single intracerebral administration of DOPAC into the right striatum. DOPAC at 16.8 micrograms reduced the DA content of the infused side by 17%, p = 0.01. In contrast, infusion of 1.68 micrograms of DOPAC or the vehicle had no effect on striatal DA levels. Coapplication of the antioxidant, ascorbic acid, at 0.2 mg/ml with 16.8 micrograms of DOPAC prevented the decrease in DA content. Furthermore, infusion of 18.2 micrograms of homovanillic acid (HVA), the product of DOPAC methylation, had no effect on striatal DA. These results indicate that DOPAC may undergo autoxidation in vivo to produce neurotoxic species which may result in reduction of striatal DA. Formation of such an autoxidation product(s) of endogenous DOPAC was verified in the extracellular fluid of striatal slices in vitro.