In vivo chronic intermittent ethanol exposure reverses the polarity of synaptic plasticity in the nucleus accumbens shell

Glutamatergic synaptic plasticity in the nucleus accumbens (NAc) is implicated in response to sensitization to psychomotor-stimulating agents, yet ethanol effects here are undefined. We studied the acute in vitro and in vivo effects of ethanol in medium spiny neurons from the shell NAc subregion of slices of C57BL/6 mice by using whole-cell voltage-clamp recordings of α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) excitatory postsynaptic current (EPSCs). Synaptic conditioning (low-frequency stimulation with concurrent postsynaptic depolarization) reliably depressed AMPA EPSCs by nearly 30%; this accumbal long-term depression (LTD) was blocked by a nonselective N-methyl-D-aspartate (NMDA) receptor antagonist (DL-2-amino-5-phosphonovaleric acid) and a selective NMDA receptor 2B antagonist [R-(R*,S*)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propanol]. Acute ethanol exposure inhibited the depression of AMPA EPSCs differentially with increasing concentrations, but this inhibitory action of ethanol was occluded by a D1-selective dopamine receptor agonist. Ethanol dependence was elicited in C57BL/6 mice by two separate 4-day bouts of chronic intermittent ethanol (CIE) vapor exposure. When assessed 24 h after a single bout of in vivo CIE vapor exposure, NAc LTD was absent, and instead NMDA receptor-dependent synaptic potentiation [long-term potentiation (LTP)] was reliably observed. It is noteworthy that both LTP and LTD were completely absent after an extended withdrawal (72 h) after a single 3-day CIE vapor bout. These observations demonstrate that 1) accumbal synaptic depression is mediated by NR2B receptors, 2) accumbal synaptic depression is highly sensitive to both acute and chronic ethanol exposure, and 3) alterations in this synaptic process may constitute a neural adaptation that contributes to the induction and/or expression of ethanol dependence.     

Cocaine induces apoptosis in fetal myocardial cells through a mitochondria-dependent pathway

The ability of subanesthetic doses of N-methyl-D-aspartate (NMDA) antagonists to induce positive, negative, and cognitive schizophrenia-like symptoms suggests that reduced NMDA receptor function may contribute to the pathophysiology of schizophrenia. An increasing body of evidence indicates that antipsychotic drugs, especially those with "atypical" properties, can antagonize the effects of NMDA antagonists in a variety of experimental paradigms. We demonstrated previously that clozapine, the prototype of atypical antipsychotics, but not haloperidol, the typical antipsychotic, blocked ketamine-induced alterations in brain metabolism. In this study, effects of clozapine were compared with two of the newer atypical antipsychotic drugs, risperidone and olanzapine, on ketamine-induced alterations in regional [(14)C]2-deoxyglucose (2-DG) uptake. A subanesthetic dose of ketamine (25 mg/kg) induced robust increases in 2-DG uptake in limbic cortical regions, hippocampal formation, nucleus accumbens, and basolateral amygdala. Pretreatment of rats with risperidone (0.3 mg/kg) before ketamine administration did not alter the effects of ketamine. These data suggest that novel pharmacological properties may contribute to the effects of clozapine in this model, in addition to the well characterized actions at D(2) and 5HT(2A) receptors. In contrast to the results with risperidone, olanzapine blocked ketamine-induced increases in 2-DG uptake. However, a higher dose of olanzapine (10 mg/kg) was required to completely block the effects of ketamine than would be expected if D(2) and 5HT(2) receptor blocking properties of the drug were solely responsible for its action. The results suggest that the ketamine challenge 2-DG paradigm may be a useful model to identify antipsychotic drugs with atypical characteristics and to explore mechanisms of atypical antipsychotic action.     

Group III metabotropic glutamate receptor agonists selectively suppress excitatory synaptic currents in the rat prefrontal cortex induced by 5-hydroxytryptamine2A receptor activation

Activation and blockade of prefrontal cortical 5-hydroxytryptamine2A (5-HT2A) receptors have been linked to the action of hallucinogenic and antidepressant/antipsychotic drugs; these effects may involve modulation of glutamate release from thalamocortical afferents. Although activation of metabotropic glutamate 2 (mGlu2) receptors may suppress 5-HT-induced excitatory postsynaptic currents (EPSCs), group III mGlu receptors (mGlu4/7/8) also are expressed in the thalamus and may suppress 5-HT-induced EPSCs. We have found by intracellular recordings from layer V pyramidal cells of the medial prefrontal cortex (mPFC) that group III mGlu receptor agonists (R,S)-4-phosphonophenylglycine (PPG), L-4-phosphono-2-aminobutyric acid (L-AP4), L-serine-O-phosphate (L-SOP), and (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) preferentially suppress 5-HT-induced EPSCs compared with excitatory postsynaptic potentials evoked by electrical stimulation of the white matter. A number of pharmacological features [e.g., the rank order of agonist potency; MAP4 partial agonist action; differential potency for the group III mGlu receptor antagonist (R,S)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) in blocking the suppressant action of PPG or MAP4; and a relatively low potency of 2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid (LY341495) in blocking the suppressant action of PPG or L-SOP] suggest that activation of both mGlu4 and mGlu8 receptors may play a role in suppressing 5-HT-induced EPSCs. Furthermore, L-SOP did not alter the synaptic currents or steady-state inward current induced by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid. Thus, although both group III and group II mGlu receptor agonists suppress the frequency of 5-HT-induced EPSCs in the mPFC, they differ in that the group III mGlu receptor agonists appear to have relatively minimal effects on glutamate released by sources other than thalamocortical afferents.     

Somatostatin-induced activation and up-regulation of N-methyl-D-aspartate receptor function: mediation through calmodulin-dependent protein kinase II, phospholipase C, protein kinase C, and tyrosine kinase in hippocampal noradrenergic nerve endings

Somatostatin receptors and glutamate N-methyl-D-aspartate (NMDA) receptors coexist on hippocampal noradrenergic axon terminals. Activation of somatostatin receptors was previously found to positively influence the function of NMDA receptors regulating norepinephrine release. The somatostatin receptors involved were pharmacologically characterized as sst5 type in experiments in Mg2+-free solutions. Here, we first confirm the pharmacology of these receptors using selective sst5 ligands in Mg2+-containing solutions. Moreover, we show by Western blot that the sst5 protein exists on purified hippocampal synaptosomal membranes. We then investigated the pathways connecting the two receptors using as a functional response the release of norepinephrine from rat hippocampal synaptosomes in superfusion. The release of norepinephrine evoked by somatostatin-14 plus NMDA/glycine was partly prevented by the protein kinase C inhibitor GF109203X [dihydrochloride3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione] and by the nonreceptor tyrosine kinase (Src) inhibitors PP2 [3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine] and lavendustin A; it was largely and almost totally abolished by the phospholipase C inhibitor U73122 [1-(6-[([17beta]-3-methoxyextra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione] and by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 [N-(2-[N-[4-chlorocinnamyl]-N-methyl-amino-methyl]phenyl)-N-(2-hydroxyethyl)-4-methoxy-benzene-sulfonamide-phosphate salt], respectively; and it was unaffected by the protein kinase A inhibitor H89 [N-(2-[p-bromocinnamylamino]ethyl)5-isoquinolinesulfonamide hydrochloride]. The norepinephrine release evoked by somatostatin-14/NMDA/glycine was inhibited when anti-phosphotyrosine antibodies had been entrapped into synaptosomes. Entrapping the recombinant activated tyrosine kinase pp60(c-Src) strongly potentiated the release of norepinephrine elicited by NMDA/glycine in Mg2+-free medium but failed to permit NMDA receptor activation in presence of external Mg2+ ions. The results suggest the involvement of CaMKII in the sst5 receptor-mediated activation of NMDA receptors in presence of Mg2+ and of the PLC/PKC/Src pathway in the up-regulation of the ongoing NMDA receptor activity.     

Physiological antagonism between 5-hydroxytryptamine(2A) and group II metabotropic glutamate receptors in prefrontal cortex

In prefrontal cortex, 5-hydroxytryptamine(2A) (5-HT(2A)) receptors have been linked to the action of hallucinogens and atypical antidepressant/antipsychotic drugs. Previously, we have shown in cortical layer V pyramidal cells that a nonselective metabotropic glutamate (mGlu) receptor agonist suppresses the induction of excitatory postsynaptic potentials/currents (EPSPs/EPSCs) via activation of 5-HT(2A) receptors. In this study, we tested the ability of the selective mGlu2/3 agonist (1S,2S,5R, 6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and the selective mGlu2/3 antagonist 2S-2-amino-2-(1S, 2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid (LY341495) to modulate serotonin(5-HT)-induced EPSPs and electrically evoked EPSPs by using intracellular recording from layer V pyramidal cells in medial prefrontal cortex. The mGlu2/3 antagonist LY341495 increased the frequency and amplitude of 5-HT-induced EPSCs, suggesting a role for mGlu2/3 receptors in mediating the action of endogenous glutamate on autoreceptors. Conversely, the mGlu2/3 agonist LY354740 was highly effective and potent (EC(50) = 89 nM) in suppressing glutamate release induced by 5-HT(2A) receptor activation in the medial prefrontal cortex, probably via a presynaptic mechanism. The mGlu2/3 antagonist LY341495 potently blocked the suppressant effect of LY354740 on 5-HT-induced EPSCs as well as electrically evoked early EPSPs. Autoradiography with the radioligands [(3)H]LY354740 and [(125)I](+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane showsa striking overlap of the laminar distribution of mGlu2/3 and 5-HT(2A) receptors in the medial prefrontal cortex that is not apparent in other cortical regions. These findings suggest a close coupling between mGlu2/3 and 5-HT(2A) receptors in the prefrontal cortex that may be relevant for novel therapeutic approaches in the treatment of neuropsychiatric syndromes such as depression and schizophrenia.     

Antagonist functional selectivity: 5-HT2A serotonin receptor antagonists differentially regulate 5-HT2A receptor protein level in vivo

Dysregulation of the 5-HT(2A) receptor is implicated in both the etiology and treatment of schizophrenia. Although the essential role of 5-HT(2A) receptors in atypical antipsychotic drug actions is widely accepted, the contribution of 5-HT(2A) down-regulation to their efficacy is not known. We hypothesized that down-regulation of cortical 5-HT(2A) receptors contributes to the therapeutic action of atypical antipsychotic drugs. To test this hypothesis, we assessed the effect of chronically administered antipsychotics (clozapine, olanzapine, and haloperidol) and several 5-HT(2A) antagonists [ketanserin, altanserin, α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (M100907), α-phenyl-1-(2-phenylethyl)-4-piperidinemethano (M11939), 4-[(2Z)-3-{[2-(dimethylamino)ethoxy]amino}-3-(2-fluorophenyl)prop-2-en-1-ylidene]cyclohexa-2,5-dien-1-one (SR46349B), and pimavanserin], on the phencyclidine (PCP)-induced hyperlocomotor response and cortical 5-HT(2A) receptor levels in C57BL/6J mice. Clozapine and olanzapine, but not haloperidol, induced receptor down-regulation and attenuated PCP-induced locomotor responses. Of the selective 5-HT(2A) antagonists tested, only ketanserin caused significant receptor protein down-regulation, whereas SR46349B up-regulated 5-HT(2A) receptors and potentiated PCP-hyperlocomotion; the other 5-HT(2A) receptor antagonists were without effect. The significance of these findings with respect to atypical antipsychotic drug action is discussed.     

Effects of acute and subchronic administration of typical and atypical antipsychotic drugs on the neurotensin system of the rat brain

The acute and subchronic effects of a variety of doses of a prototype typical (haloperidol) or one of several atypical antipsychotic drugs (clozapine, olanzapine, risperidone, quetiapine, or sertindole) on regional brain neurotensin (NT) tissue concentrations, and NT receptor binding were examined. Acute administration of haloperidol, clozapine, olanzapine, and risperidone dose-dependently increased NT tissue concentrations in the nucleus accumbens. Haloperidol, olanzapine, risperidone, and sertindole also increased NT tissue concentrations in the caudate nucleus. NT tissue concentrations in the nucleus accumbens and caudate remained elevated after 14-day administration of haloperidol, olanzapine, sertindole, and risperidone. In contrast, at the doses studied, quetiapine decreased NT tissue concentrations in the nucleus accumbens; clozapine had no effect. Haloperidol significantly increased NT receptor binding in the substantia nigra after 14-day administration. All of the atypical antipsychotic drugs decreased NT receptor binding in the nucleus accumbens and in the substantia nigra. Although these studies do not conclusively support the hypothesis that increased NT neurotransmission is involved in the clinically relevant effects of all antipsychotic drugs, the extant evidence clearly suggests that further study is warranted. Inconsistencies in the data suggest that differential effects of antipsychotic drug administration on subpopulations of NT neurons must be scrutinized further.     

Preclinical pharmacology of FMPD [6-fluoro-10-[3-(2-methoxyethyl)-4-methyl-piperazin-1-yl]-2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulene]: a potential novel antipsychotic with lower histamine H1 receptor affinity than olanzapine

FMPD [6-fluoro-10-[3-(2-methoxyethyl)-4-methyl-piperazin-1-yl]-2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulene] is a potential novel antipsychotic with high affinity for dopamine D2 (Ki= 6.3 nM), 5-HT(2A) (Ki= 7.3 nM), and 5-HT6 (Ki= 8.0 nM) human recombinant receptors and lower affinity for histamine H1 (Ki= 30 nM) and 5-HT2C (Ki= 102 nM) human recombinant receptors than olanzapine. Oral administration of FMPD increased rat nucleus accumbens 3,4-dihyroxyphenylacetic acid concentrations (ED200 = 6 mg/kg), blocked 5-HT2A agonist-induced increases in rat serum corticosterone levels (ED50= 1.8 mg/kg), and inhibited the ex vivo binding of [125I]SB-258585 [4-iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulfonamide] to striatal 5-HT6 receptors (ED50= 10 mg/kg) but failed to inhibit ex vivo binding of [3H]pyrilamine to hypothalamic histamine H1 receptors at doses of up to 30 mg/kg. In electrophysiology studies, acute administration of FMPD selectively elevated the number of spontaneously active A10 (versus A9) dopamine neurons and chronic administration selectively decreased the number of spontaneously active A10 (versus A9) dopamine neurons. FMPD did not produce catalepsy at doses lower than 25 mg/kg p.o. In Fos-induction studies, FMPD had an atypical antipsychotic profile in the striatum and nucleus accumbens and increased Fos expression in orexin-containing neurons of the hypothalamus. FMPD produced only a transient elevation of prolactin levels. These data indicate that FMPD is an orally available potent antagonist of dopamine D2, 5-HT2A, and 5-HT6 receptors and a weak antagonist of H1 and 5-HT2C receptors. FMPD has the potential to have efficacy in treating schizophrenia and bipolar mania with a low risk of treatment-emergent extrapyramidal symptoms, prolactin elevation, and weight gain. Clinical trials are needed to test these hypotheses.     

Clozapine, but not haloperidol, prevents the functional hyperactivity of N-methyl-D-aspartate receptors in rat cortical neurons induced by subchronic administration of phencyclidine

Repeated exposure of rats to the psychotomimetic drug phencyclidine (PCP) markedly increased the response of prefrontal cortical neurons to the glutamate agonist N-methyl-D-aspartate (NMDA) relative to agonist alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. Moreover, acute challenge by PCP produced a significantly reduced block of NMDA-induced current. In addition, the subchronic administration of PCP reduced significantly the paired-pulse facilitation, accompanied by a significant increase of excitatory postsynaptic current variance. These results suggest that repeated exposure to PCP increased evoked release of excitatory amino acids. The enhanced release of excitatory amino acids evoked by NMDA could explain, at least partly, a hypersensitive response to NMDA and a reduced blockade of the NMDA responses by a PCP challenge in rats exposed repeatedly to PCP. Pretreatment with the atypical antipsychotic drug clozapine, but not the typical antipsychotic drug haloperidol, attenuates the repeated PCP-induced effect. Our results support the hypothesis that clozapine may facilitate NMDA receptor-mediated neurotransmission to improve schizophrenic-negative symptoms and cognitive dysfunction. This novel approach is useful for evaluating the cellular mechanisms of action of atypical antipsychotic drugs.     

Aberrant synaptic activation of N-methyl-D-aspartate receptors underlies ethanol withdrawal hyperexcitability

Chronic ethanol exposure may induce neuroadaptive responses in N-methyl-d-aspartate (NMDA) receptors, which are thought to underlie a variety of alcohol-related brain disorders. Here, we demonstrate that hyperexcitability triggered by withdrawal from chronic ethanol exposure is associated with increases in both synaptic NMDA receptor expression and activation. Withdrawal from chronic ethanol exposure (75 mM ethanol, 5-9 days) elicited robust and prolonged epileptiform activity in CA1 pyramidal neurons from hippocampal explants, which was absolutely dependent upon NMDA receptor activation but independent of chronic inhibition of protein kinase A (PKA). Analysis of Sr(2+)-supported asynchronous NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) was employed to assess changes in NMDA neurotransmission. After chronic exposure, ethanol withdrawal was associated with an increase in mEPSC amplitude 3.38-fold over that after withdrawal from acute ethanol exposure. Analysis of paired evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid EPSCs and spontaneous mEPSCs indicated that withdrawal after chronic exposure was also associated with a selective increase in action potential evoked but not spontaneous transmitter release probability. Immunoblot analysis revealed significant increases in total NR1, NR2A, and NR2B subunit expression after chronic exposure and unaffected by PKA-inhibition manner. Confocal imaging studies indicate that increased NR1 subunit expression was associated with increased density of NR1 expression on dendrites in parallel with a selective increase in the size of NR1 puncta on dendritic spines. Therefore, neuroadaptation to chronic ethanol exposure in NMDA synaptic transmission is responsible for aberrant network excitability after withdrawal and results from changes in both postsynaptic function as well as presynaptic release.     

Intrinsic efficacy of antipsychotics at human D2, D3, and D4 dopamine receptors: identification of the clozapine metabolite N-desmethylclozapine as a D2/D3 partial agonist

Drugs that antagonize D2-like receptors are effective antipsychotics, but the debilitating movement disorder side effects associated with these drugs cannot be dissociated from dopamine receptor blockade. The "atypical" antipsychotics have a lower propensity to cause extrapyramidal symptoms (EPS), but the molecular basis for this is not fully understood nor is the impact of inverse agonism upon their clinical properties. Using a cell-based functional assay, we demonstrate that overexpression of Galphao induces constitutive activity in the human D2-like receptors (D2, D3, and D4). A large collection of typical and atypical antipsychotics was profiled for activity at these receptors. Virtually all were D2 and D3 inverse agonists, whereas none was D4 inverse agonist, although many were potent D4 antagonists. The inverse agonist activity of haloperidol at D2 and D3 receptors could be reversed by mesoridazine demonstrating that there were significant differences in the degrees of inverse agonism among the compounds tested. Aripiprazole and the principle active metabolite of clozapine NDMC [8-chloro-11-(1-piperazinyl)-5H-dibenzo [b,e] [1,4] diazepine] were identified as partial agonists at D2 and D3 receptors, although clozapine itself was an inverse agonist at these receptors. NDMC-induced functional responses could be reversed by clozapine. It is proposed that the low incidence of EPS associated with clozapine and aripiprazole used may be due, in part, to these partial agonist properties of NDMC and aripiprazole and that bypassing clozapine blockade through direct administration of NDMC to patients may provide superior antipsychotic efficacy.     

Differential effects of antipsychotic drugs on serotonin-1A receptor-mediated disruption of prepulse inhibition

Serotonin-1A (5-HT(1A)) receptors have been implicated in the symptoms of schizophrenia. However, there is limited in vivo evidence for an interaction of antipsychotic drugs with 5-HT(1A) receptor-mediated behavioral effects. We therefore investigated in rats the action of several antipsychotic drugs on prepulse inhibition (PPI), a measure of sensorimotor gating that is deficient in schizophrenia. Disruption of PPI at the 100-ms interstimulus interval (ISI), but not the 30-ms ISI, was induced by treatment with 0.5 mg/kg 8-hydroxy-di-propylaminotetralin (8-OH-DPAT), the 5-HT(1A) receptor agonist. In rats pretreated with 0.25 mg/kg haloperidol (4-[-4-(p-chlorophenyl)-4-hydroxypiperidino]-4'-fluoro butyrophenone) or raclopride [3,5-dichloro-N-(1-ethylpyrrolidin-2-ylmethyl)-2-hydroxy-6-methoxybenzamide tartrate], the disruption of PPI was no longer significant. Of the atypical antipsychotic drugs clozapine (8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]-diazepine), olanzapine (2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine), risperidone [3-[2-[-4-(6-fluoro-1,2-benzisoxazol-3-yl) piperidino] ethyl-6,7,8,9-tetrahydro-2-methyl-4H-pyrido[1,2-a]pyrimidin-4-one)], amisulpride (4-amino-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(ethylsulfonyl)-o-anisamide), and aripiprazole (7-[4-[-4[-(2,3-dichlorophenyl)-1-piperazinyl]butoxy]-3,4-dihydrocarbostyrilor 7-[4-[4-(2,3-dichlorophenyl) piperazin-1-yl]butoxy]-1,2,3,4,-tetrahydroquinolin-2-one), only aripiprazole significantly reduced the effect of 8-OH-DPAT on PPI. This effect was mimicked by pretreatment with the 5-HT(1A) receptor partial agonist, buspirone [N-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione hydrochloride]. On the other hand, some of the antipsychotic drugs and other pretreatments showed complex, prepulse-dependent effects on their own. These data show little in vivo interaction of several atypical antipsychotic drugs with the disruption of PPI mediated by 5-HT(1A) receptor stimulation. The action of haloperidol and raclopride suggests a major involvement of dopamine D(2) receptors in this effect, possibly downstream from the initial serotonergic stimulation. The action of aripiprazole could be mediated by its partial agonist properties at 5-HT(1A) receptors or its dopamine D(2)-blocking properties.     

Ethanol enhances gamma-aminobutyric acid responses in a subpopulation of nucleus accumbens neurons: role of metabotropic glutamate receptors

The nucleus accumbens (NAcc) may be a key area in the rewarding effects of abused drugs. We previously showed that low ethanol concentrations decreased both N-methyl-D-aspartate (NMDA)-induced and kainate-induced currents in NAcc core neurons. To explore the effects of ethanol on gamma-aminobutyric acid (GABA) responses in NAcc, we used intracellular voltage-clamp recordings and locally applied GABA in a slice preparation containing the NAcc. Ethanol (11-200 mM) had no effect on resting membrane properties, but 11, 22, 44, 100, and 200 mM ethanol increased GABA currents in 17, 33, 45, 50, and 22% of cells, respectively. Superfusion of low glutamate concentrations that had no direct effect on membrane properties enhanced ethanol potentiation of GABA currents in more than half the NAcc cells. Neither alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptor nor NMDA receptor antagonists affected the percentage of cells showing ethanol enhancement of GABA responses or the degree of ethanol enhancement of GABA currents in NAcc neurons. However, in ethanol-sensitive cells, the metabotropic receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG) blocked the ethanol enhancement of GABA currents. In addition, the metabotropic receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid enhanced GABA responses in 50% of cells tested, an effect blocked by MCPG. These data suggest that NAcc core neurons possess both ethanol-sensitive and -insensitive GABA receptors and that glutamate can mimic and enhance the ethanol potentiation of GABA currents in many of these neurons. Furthermore, the ethanol potentiation of GABA currents may involve metabotropic glutamate receptors, perhaps via a phosphorylation mechanism that regulates ethanol sensitivity of GABA receptors in some NAcc neurons.     

Tachykinin gene expression in rat limbic nuclei: modulation by dopamine antagonists

The content and nature of the preprotachykinin (PPT; i.e., substance P/neurokinin A-encoding) messenger RNAs (mRNAs) present in rat brain striatum and limbic tissues were determined by RNA protection experiments. The rank order of PPT mRNA concentration was striatum greater than nucleus accumbens much greater than bed nucleus of the stria terminalis greater than hypothalamus, amygdala and septum. The proportion of beta-(full length) to gamma-(minus exon 4) PPT mRNA was invariant (40/60) among the tissues tested. Because these brain regions receive prominent dopaminergic innervations, the effects of repeated treatment with dopamine antagonists (antipsychotic drugs) on PPT gene expression were assessed. The prototypical dopamine antagonists haloperidol and chlorpromazine decreased striatal PPT mRNA, had no effect on PPT mRNA in the nucleus accumbens or bed nucleus of the stria terminalis, and increased septal PPT mRNA levels. In contrast, the atypical antipsychotic drugs clozapine and l-sulpiride did not alter striatal or septal PPT mRNA, but increased PPT mRNA content in the nucleus accumbens and bed nucleus. The correlation between the effects of typical and atypical antipsychotic drugs on rat striatal and limbic PPT gene expression and their clinical side effects and therapeutic efficacy is discussed.     

A novel selective allosteric modulator potentiates the activity of native metabotropic glutamate receptor subtype 5 in rat forebrain

We found that N-[4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl]-2-hydroxybenzamide (CPPHA), is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). CPPHA alone had no agonist activity and acted as a selective positive allosteric modulator of human and rat mGluR5. CPPHA potentiated threshold responses to glutamate in fluorometric Ca(2+) assays 7- to 8-fold with EC(50) values in the 400 to 800 nM range, and at 10 microM shifted mGluR5 agonist concentration-response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine (DHPG) 4- to 7-fold to the left. The only effect of CPPHA on other mGluRs was weak inhibition of mGluR4 and 8. Neither CPPHA nor the previously described 3,3'-difluorobenzaldazine (DFB) affected [(3)H]quisqualate binding to mGluR5, but although DFB partially competed for [(3)H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding, CPPHA had no effect on the binding of this 2-methyl-6-(phenylethynyl)-pyridine analog to mGluR5. Although the binding sites for the two classes of allosteric modulators seem to be different, these different allosteric sites can modulate functionally and mechanistically similar allosteric effects. In electrophysiological studies of brain slice preparations, it had been previously shown that activation of mGluR5 receptors by agonists increased N-methyl-D-aspartate (NMDA) receptor currents in the CA1 region of hippocampal slices. We found that CPPHA (10 microM) potentiated NMDA receptor currents in hippocampal slices induced by threshold levels of DHPG, whereas having no effect on these currents by itself. Similarly, 10 microM CPPHA also potentiated mGluR5-mediated DHPG-induced depolarization of rat subthalamic nucleus neurons. These results demonstrate that allosteric potentiation of mGluR5 increases the effect of threshold agonist concentrations in native systems.     

Evidence for the role of metabotropic glutamate (mGlu)2 not mGlu3 receptors in the preclinical antipsychotic pharmacology of the mGlu2/3 receptor agonist (-)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039)

(-)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039) is a potent and selective group II metabotropic glutamate [(mGlu)2 and mGlu3] receptor agonist for which its prodrug LY2140023 [(1R,4S,5S,6S)-2-thiabicyclo[3.1.0]-hexane-4,6-dicarboxylic acid,4-[(2S)-2-amino-4-(methylthio)-1-oxobutyl]amino-, 2,2-dioxide monohydrate] has recently been shown to have efficacy in the treatment of the positive and negative symptoms of schizophrenia. In this article, we use mGlu receptor-deficient mice to investigate the relative contribution of mGlu2 and mGlu3 receptors in mediating the antipsychotic profile of LY404039 in the phencyclidine (PCP) and d-amphetamine (AMP) models of psychosis. To further explore the mechanism of action of LY404039, we compared the drugs' ability to block PCP-induced hyperlocomotion to that of atypical antipsychotics in wild-type and mice lacking mGlu2/3 receptors. In wild-type animals, LY404039 (3-30 mg/kg i.p.) significantly reversed AMP (5 mg/kg, i.p.)-induced increases in ambulations, distance traveled, and reduced time spent at rest. LY404039 reversed PCP (7.5 mg/kg i.p.)-evoked behaviors at 10 mg/kg. The antipsychotic-like effects of LY404039 (10 mg/kg i.p.) on PCP and AMP-evoked behavioral activation were absent in mGlu2 and mGlu2/3 but not in mGlu3 receptor-deficient mice, indicating that the activation of mGlu2 and not mGlu3 receptors is responsible for the antipsychotic-like effects of the mGlu2/3 receptor agonist LY404039. In contrast, the atypical antipsychotic drugs clozapine and risperidone inhibited PCP-evoked behaviors in both wild-type and mGlu2/3 receptor-deficient mice. These data demonstrate that the antipsychotic-like effects of the mGlu2/3 receptor agonist LY404039 in psychostimulant models of psychosis are mechanistically distinct from those of atypical antipsychotic drugs and are dependent on functional mGlu2 and not mGlu3 receptors.     

Effect of the {mu} opioid on excitatory and inhibitory synaptic inputs to periaqueductal gray-projecting neurons in the amygdala

Opioids are potent analgesics, but the sites of their action and cellular mechanisms are not fully understood. The central nucleus of the amygdala (CeA) is important for opioid analgesia through the projection to the periaquaductal gray (PAG). In this study, we examined the effects of mu opioid receptor stimulation on inhibitory and excitatory synaptic inputs to PAG-projecting CeA neurons retrogradely labeled with a fluorescent tracer injected into the ventrolateral PAG of rats. Whole-cell voltage-clamp recordings were performed on labeled CeA neurons in brain slices. The specific mu opioid receptor agonist, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM), significantly reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs) without altering the amplitude and decay constant of mIPSCs in 47.6% (10 of 21) of cells tested. DAMGO also significantly decreased the peak amplitude of evoked IPSCs in 69% (9 of 13) of cells examined. However, DAMGO did not significantly alter the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of evoked EPSCs in 69% (9 of 13) and 83% (10 of 12) of labeled cells, respectively. The IPSCs were blocked by the GABA(A) receptor antagonist bicuculline, whereas the EPSCs were largely abolished by the non-N-methyl-d-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The immunoreactivity of mu opioid receptors was colocalized with synaptophysin, a presynaptic marker, in close appositions to labeled CeA neurons. These results suggest that activation of mu opioid receptors on presynaptic terminals primarily attenuates GABAergic synaptic inputs to PAG-projecting neurons in the CeA.     

Gabapentin affects glutamatergic excitatory neurotransmission in the rat dorsal horn

We investigated the effects of gabapentin (GBP) on glutamatergic synaptic transmission in the dorsal horn of the rat spinal cord. Patch clamp whole cell recordings were made from superficial and deep dorsal horn neurons of rat spinal cord slices. In the majority of neurons in the superficial lamina, GBP decreased the amplitudes of evoked excitatory postsynaptic currents (evoked EPSCs) mediated by either non-NMDA or NMDA receptors. In contrast, neurons in the deep lamina showed variable effects, with a lower incidence of decrease in amplitude of evoked EPSCs and a subset of neurons showing an increase in amplitude of evoked NMDA receptor-mediated EPSCs. No enhancement of evoked non-NMDA receptor-mediated EPSCs was observed in either lamina. To determine whether the observed effects of GBP are presynaptic and/or postsynaptic, spontaneous miniature excitatory postsynaptic currents (mEPSCs) were studied. In neurons that showed a decrease in its frequency of mEPSCs by GBP, no change in the amplitude or shape accompanied the effect. On the other hand, in neurons that showed an increase in the frequency of NMDA receptor-mediated mEPSCs, the effect accompanied an increase in amplitude. These results suggest that GBP presynaptically inhibits glutamatergic synaptic transmission predominantly in the superficial lamina, while postsynaptically enhancing NMDA receptor-mediated transmission in some neurons of the deep lamina. The antinociceptive effects of GBP may involve the inhibition of the release of excitatory amino acids from presynaptic terminals.     

WAY-163909 [(7bR,10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole]: A novel 5-hydroxytryptamine 2C receptor-selective agonist with preclinical antipsychotic-like activity.

Serotonin-2C (5-HT2C) receptor antagonists and agonists have been shown to affect dopamine (DA) neurotransmission, with agonists selectively decreasing mesolimbic DA. As antipsychotic efficacy is proposed to be associated with decreased mesolimbic DA neurotransmission by virtue of DA D2 receptor antagonism, the 5-HT2C-selective receptor agonist, WAY-163909 [(7bR,10aR)-1,2, 3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7, 1hi]indole], was evaluated in animal models of schizophrenia and in vivo microdialysis and electrophysiology to determine the effects on mesolimbic and nigrostriatal DA neurotransmission. Similar to clozapine, WAY-163909 (1.7-30 mg/kg i.p.) decreased apomorphine-induced climbing with little effect on stereotypy and no significant induction of catalepsy. WAY-163909 (0.3-3 mg/kg s.c.) more potently reduced phencyclidine-induced locomotor activity compared with d-amphetamine with no effect on spontaneous activity. WAY-163909 (1.7-17 mg/kg i.p.) reversed MK-801 (5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate)- and DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]-disrupted prepulse inhibition of startle (PPI) and improved PPI in DBA/2N mice. In conditioned avoidance responding, WAY-163909 (0.3-3 mg/kg i.p.; 1-17 mg/kg p.o.) reduced avoidance responding, an effect blocked by the 5-HT(2B/2C) receptor antagonist SB 206553 [5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole]. WAY-163909 (10 mg/kg s.c.) selectively decreased extracellular levels of DA in the nucleus accumbens without affecting the striatum. Likewise, in vivo electrophysiological recordings showed a decrease in the number of spontaneously firing DA neurons in the ventral tegmental area but not in the substantia nigra with both acute and chronic (21-day) administration of WAY-163909 (1-10 mg/kg i.p.). Thus, the profile of the 5-HT2C selective receptor agonist WAY-163909 is similar to that of an atypical antipsychotic and additionally may have rapid onset properties.     

Electrophysiological evidence for expression of glycine receptors in freshly isolated neurons from nucleus accumbens

In the course of studying N-methyl-D-aspartate (NMDA) receptors of the nucleus accumbens (NAcc), we found that 20% of freshly isolated medium spiny neurons, as well as all interneurons, responded in an unexpected way to long (5-s) coapplication of NMDA and glycine, the coagonist of NMDA receptors. Whereas the reversal potential of the peak NMDA current of this subset of neurons was still around 0 mV, the desensitizing current became outward at hyperpolarized potentials around -30 mV. A Cl(-)-free solution shifted the equilibrium potentials of the desensitized currents to around 0 mV. This outward current was not blocked by a Ca(2+)-free, Ba(2+)-containing solution, suggesting that the anionic conductance was not activated by Ca(2+) influx through NMDA receptor channels. Interestingly, glycine alone also evoked a current with a similar hyperpolarized reversal potential in this subset of neurons. The glycine current reversed around -50 mV, rectified outwardly, and inactivated strongly. Its desensitization was best fitted with a double exponential. Only the slow desensitization showed clear voltage dependence. The glycine current was not blocked by 200 microM picrotoxin and 10 microM zinc, was weakly antagonized by 1 microM strychnine, and was not enhanced by 1 microM zinc. In addition, 1 mM taurine, but not GABA, inactivated glycine currents, and 1 mM glycine occluded 10 mM taurine-mediated currents. These data indicate that a subset of nucleus accumbens neurons expresses glycine receptors and that either glycine or taurine could be an endogenous agonist for these receptors.