Mapping the agonist-binding site of GABAB type 1 subunit sheds light on the activation process of GABAB receptors.
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The gamma-amino-n-butyric acid type B (GABA(B)) receptor is composed of two subunits, GABA(B)1 and GABA(B)2, belonging to the family 3 heptahelix receptors. These proteins possess two domains, a seven transmembrane core and an extracellular domain containing the agonist binding site. This binding domain is likely to fold like bacterial periplasmic binding proteins that are constituted of two lobes that close upon ligand binding. Here, using molecular modeling and site-directed mutagenesis, we have identified residues in the GABA(B)1 subunit that are critical for agonist binding and activation of the heteromeric receptor. Our data suggest that two residues (Ser(246) and Asp(471)) located within lobe I form H bonds and a salt bridge with carboxylic and amino groups of GABA, respectively, demonstrating the pivotal role of lobe I in agonist binding. Interestingly, our data also suggest that a residue within lobe II (Tyr(366)) interacts with the agonists in a closed form model of the binding domain, and its mutation into Ala converts the agonist baclofen into an antagonist. These data demonstrate the pivotal role played by the GABA(B)1 subunit in the activation of the heteromeric GABA(B) receptor and are consistent with the idea that a closed state of the binding domain of family 3 receptors is required for their activation. (+info)
Inhibition suppresses transmission of tonic vibrissa-evoked activity in the rat ventrobasal thalamus.
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Previous studies have demonstrated that tonic responses of trigeminal ganglion neurons to maintained whisker deflections are transformed to mainly phasic responses in thalamocortical neurons. The high tonic responsiveness of thalamic reticular neurons suggests that thalamic inhibition may contribute to this suppression of tonic activity. To test this hypothesis we recorded responses of thalamocortical neurons in the ventroposterior medial (VPm) nucleus to 200 and 400 msec sustained whisker deflections during simultaneous microiontophoresis of the GABA receptor antagonists bicuculline and phaclofen. Under control conditions, VPm units responded to deflection plateaus with mean activities of only 18 spikes/sec, compared with 16 spikes/sec spontaneous firing. A minority of cells (5/19) had significantly greater plateau than spontaneous activity, and these cells were classified as tonic; the other 14/19 were considered phasic. Under GABA receptor antagonism, however, mean plateau activity increased to 53 spikes/sec compared with 30 spikes/sec spontaneous activity, and 7 of the 14 phasic units became tonically responsive. Increases in plateau activity were significantly greater, by both absolute and relative measures, than increases in spontaneous activity. Transient responses to stimulus onsets and offsets also increased in magnitude 4.0- and 2. 9-fold, attributable mainly to their increased duration. These data indicate that VPm neurons receive tonic excitatory inputs that under normal conditions are masked by inhibition. Suppression of tonic activity in VPm by inhibitory thalamic reticular neurons may reduce tonic inhibition in cortical layer IV circuits, preserving their responsiveness to transient signals. (+info)
Involvement of the nucleus accumbens and medial prefrontal cortex in the expression of conditioned hyperactivity to a cocaine-associated environment in rats.
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This study examined the roles of the nucleus accumbens (NAc), medial prefrontal cortex, basolateral amygdala, and ventral subiculum of the hippocampus in the expression of Pavlovian conditioned hyperactivity responses to cocaine-related stimuli. This was accomplished by pharmacologically inhibiting these regions prior to drug-free tests for conditioned hyperactivity in an environment previously associated with cocaine. The results indicate that conditioned hyperactivity could be disrupted by infusions of the GABA-B agonist, baclofen (0.2 nmol/0.5 microl/side) into the NAc, or completely blocked by infusions of the GABA-A agonist, muscimol (0.1 and 0.2 nmol/0.5 microl/side) into the medial prefrontal cortex. In contrast, conditioned hyperactivity was unaffected by pharmacological inhibition of the basolateral amygdala, the ventral subiculum, or sites dorsal to the NAc or prefrontal cortex. These findings suggest that the NAc and the prefrontal cortex are crucial elements of the neural circuitry underlying the expression of Pavlovian conditioned responses to cocaine-related stimuli. (+info)
Slow synaptic inhibition mediated by metabotropic glutamate receptor activation of GIRK channels.
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Glutamate is the predominant excitatory neurotransmitter in the vertebrate CNS. Ionotropic glutamate receptors mediate fast excitatory actions whereas metabotropic glutamate receptors (mGluRs) mediate a variety of slower effects. For example, mGluRs can mediate presynaptic inhibition, postsynaptic excitation, or, more rarely, postsynaptic inhibition. We previously described an unusually slow form of postsynaptic inhibition in one class of projection neuron in the song-control nucleus HVc of the songbird forebrain. These neurons, which participate in a circuit that is essential for vocal learning, exhibit an inhibitory postsynaptic potential (IPSP) that lasts several seconds. Only a portion of this slow IPSP is mediated by GABA(B) receptors. Since these cells are strongly hyperpolarized by agonists of mGluRs, we used intracellular recording from brain slices to investigate the mechanism of this hyperpolarization and to determine whether mGluRs contribute to the slow synaptic inhibition. We report that mGluRs hyperpolarize these HVc neurons by activating G protein-coupled, inwardly-rectifying potassium (GIRK) channels. MGluR antagonists blocked this response and the slow synaptic inhibition. Thus, glutamate can combine with GABA to mediate slow synaptic inhibition by activating GIRK channels in the CNS. (+info)
The human GABA(B1b) and GABA(B2) heterodimeric recombinant receptor shows low sensitivity to phaclofen and saclofen.
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1. The aim of this study was to characterize the pharmacological profile of the GABA(B1)/GABA(B2) heterodimeric receptor expressed in Chinese hamster ovary (CHO) cells. We have compared receptor binding affinity and functional activity for a series of agonists and antagonists. 2. The chimeric G-protein, G(qi5), was used to couple receptor activation to increases in intracellular calcium for functional studies on the Fluorimetric Imaging Plate Reader (FLIPR), using a stable GABA(B1)/GABA(B2)/G(qi5) CHO cell line. [(3)H]-CGP-54626 was used in radioligand binding studies in membranes prepared from the same cell line. 3. The pharmacological profile of the recombinant GABA(B1/B2) receptor was consistent with that of native GABA(B) receptors in that it was activated by GABA and baclofen and inhibited by CGP-54626A and SCH 50911. 4. Unlike native receptors, the GABA(B1)/GABA(B2)/G(qi5) response was not inhibited by high microMolar concentration of phaclofen, saclofen or CGP 35348. 5. This raises the possibility that the GABA(B1)/GABA(B2)/G(qi5) recombinant receptor may represent the previously described GABA(B) receptor subtype which is relatively resistant to inhibition by phaclofen. (+info)
Differential regulation of dopamine D2 and D3 receptors by chronic drug treatments.
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Regulation of the expression of dopamine D2 and D3 receptors in the rat brain was examined using quantitative autoradiography after chronic (14 day) drug treatments designed to increase or decrease dopamine receptor stimulation. Reserpine treatment depleted endogenous dopamine by more than 90% and significantly increased the binding of [(125)I]NCQ 298 to D2 receptors in the nucleus accumbens, ventral pallidum, and substantia nigra. In contrast, this treatment significantly decreased the binding of [(125)I]7-OH-PIPAT to D3 receptors in each of these regions. Chronic stimulation of D2-like receptors with quinpirole (1 mg/kg/day) or 7-OH-DPAT (1 mg/kg/day) produced decreases in [(125)I]NCQ 298 binding in the nucleus accumbens, ventral pallidum, and substantia nigra as expected. As with depletion, chronic stimulation elicited an opposite response from D3 receptors with significant increases observed in the ventral pallidum and substantia nigra. D3 receptor expression in the nucleus accumbens was unchanged. Baclofen (30 mg/kg/day) or continuous administration of the psychomotor stimulant cocaine (20 mg/kg/day) produced no significant changes in D2 or D3 receptor binding in any region examined. Acute administration of the irreversible antagonist EEDQ (10 mg/kg) nearly eliminated D2 receptor binding in all regions, but inactivated D3 receptors only in the VP and SN, suggesting subtype-specific and region-specific differences in receptor occupancy. The existence of regional and subtype-specific heterogeneities in the regulation of these receptors supports the contention that despite their similar pharmacological profiles, D2 and D3 receptors may mediate different functional responses. (+info)
The GABAB receptor interacts directly with the related transcription factors CREB2 and ATFx.
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gamma-Aminobutyric acid type B (GABA(B)) receptors mediate the metabotropic actions of the inhibitory neurotransmitter GABA. These seven-transmembrane receptors are known to signal primarily through activation of G proteins to modulate the action of ion channels or second messengers. The functional GABA(B) receptor is made up of a heterodimer consisting of two subunits, GABA(B)-R1 and GABA(B)-R2, which interact via coiled-coil domains in their C-terminal tails. By using a yeast two-hybrid approach, we have identified direct interactions between the C-terminal tails of GABA(B)-R1 and GABA(B)-R2 with two related transcription factors, CREB2 (ATF4) and ATFx. In primary neuronal cultures as well in recombinant Chinese hamster ovary cells expressing GABA(B) receptors, CREB2 is localized within the cytoplasm as well as the nucleus. Activation of the GABA(B) receptor by the specific agonist baclofen leads to a marked translocation and accumulation of CREB2 from the cytoplasm into the nucleus. We demonstrate that receptor stimulation results in activation of transcription from a CREB2 responsive reporter gene. Such a signaling mechanism is unique among Family C G protein-coupled receptors and, in the case of the GABA(B) receptor and CREB2, may play a role in long-term changes in the nervous system. (+info)
Light induces chromatin modification in cells of the mammalian circadian clock.
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The mammalian circadian clock resides in neurons of the hypothalamic suprachiasmatic nucleus (SCN). Light entrains phase resetting of the clock using the retino-hypothalamic tract, via release of glutamate. Nighttime light exposure causes rapid, transient induction of clock and immediate-early genes implicated in phase-shifting the pacemaker. Here we show that a nighttime light pulse caused phosphorylation of Ser10 in histone H3's tail, in SCN clock cells. The effect of light was specific, and the kinetics of H3 phosphorylation were characteristic of the early response, paralleling c-fos and Per1 induction. Using fos-lacZ transgenic mice, we found that H3 phosphorylation and Fos induction occurRed in the same SCN neurons. Systemic treatment with the GABAB receptor agonist baclofen prevented light-induced c-fos and Per1 expression and H3 phosphorylation, indicating that one signaling pathway governs both events. Our results suggest that dynamic chromatin remodeling in the SCN occurs in response to a physiological stimulus in vivo. (+info)