Prefrontal synaptic markers of cocaine addiction-like behavior in rats.

Defining the drug-induced neuroadaptations specifically associated with the behavioral manifestation of addiction is a daunting task. To address this issue, we used a behavioral model that differentiates rats controlling their drug use (Non-Addict-like) from rats undergoing transition to addiction (Addict-like). Dysfunctions in prefrontal cortex (PFC) synaptic circuits are thought to be responsible for the loss of control over drug taking that characterizes addicted individuals. Here, we studied the synaptic alterations in prelimbic PFC (pPFC) circuits associated with transition to addiction. We discovered that some of the changes induced by cocaine self-administration (SA), such as the impairment of the endocannabinoid-mediated long-term synaptic depression (eCB-LTD) was similarly abolished in Non-Addict- and Addict-like rats and thus unrelated to transition to addiction. In contrast, metabotropic glutamate receptor 2/3-mediated LTD (mGluR2/3-LTD) was specifically suppressed in Addict-like rats, which also show a concomitant postsynaptic plasticity expressed as a change in the relative contribution of AMPAR and NMDAR to basal glutamate-mediated synaptic transmission. Addiction-associated synaptic alterations in the pPFC were not fully developed at early stages of cocaine SA, when addiction-like behaviors are still absent, suggesting that pathological behaviors appear once the pPFC is compromised. These data identify specific synaptic impairments in the pPFC associated with addiction and support the idea that alterations of synaptic plasticity are core markers of drug dependence.

Group 2 metabotropic glutamate receptors induced long term depression in mouse striatal slices.

We studied the roles of mGlu2/3 receptors (mGlu2/3) in glutamatergic transmission at corticostriatal synapses in mice brain slices. Perfusion of the selective mGlu2/3 agonists LY354740 and L-CCG1 caused the long term depression (LTD) of evoked synaptic responses. Photonic and electronic microscopy showed mGlu2/3 on axonal fibers and glial processes but not on striatal dendrites. mGlu2/3-LTD was independent of synaptic activity and insensitive to specific antagonists of dopamine D1, D2, GABA(B), N-methyl-D-aspartate or adenosine A1 receptors. Manipulation of the cAMP/protein kinase A cascade had no effect on the mGlu2/3-LTD. In contrast, MEK1-2 inhibitors reduced both mGlu2/3 initial depression and LTD suggesting the involvement of the mitogen activated kinase pathway in mGlu2/3-LTD.

Cannabinoids inhibit GABAergic synaptic transmission in mice nucleus accumbens.

In mice nucleus accumbens slices, whole-cell patch clamp recording of medium-spiny neurons revealed that cannabimimetics ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone) (WIN-2) and ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) inhibit stimulus-evoked gamma-aminobutyric acid mediated inhibitory post-synaptic currents (IPSC). The actions of WIN-2 were reversed by the selective cannabinoid CB(1) receptor antagonist [N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride] (SR141716A). WIN-2 modified paired-pulse ratio of evoked IPSCs and decreased miniature IPSC frequency indicating a presynaptic localization of cannabinoid CB(1) receptors.

Localization and mechanisms of action of cannabinoid receptors at the glutamatergic synapses of the mouse nucleus accumbens.

Despite the role of excitatory transmission to the nucleus accumbens (NAc) in the actions of most drugs of abuse, the presence and functions of cannabinoid receptors (CB1) on the glutamatergic cortical afferents to the NAc have never been explored. Here, immunohistochemistry has been used to show the localization of CB1 receptors on axonal terminals making contacts with the NAc GABAergic neurons. Electrophysiological techniques in the NAc slice preparation revealed that cannabimimetics [WIN 55,212,2 (WIN-2) and CP55940] strongly inhibit stimulus-evoked glutamate-mediated transmission. The inhibitory actions of WIN-2 were dose-dependent (EC(50) of 293 +/- 13 nm) and reversed by the selective CB1 antagonist SR 141716A. In agreement with a presynaptic localization of CB1 receptors, WIN-2 increased paired-pulse facilitation, decreased miniature EPSC (mEPSC) frequency, and had no effect on the mEPSCs amplitude. Perfusion with the adenylate cyclase activator forskolin enhanced glutamatergic transmission but did not alter presynaptic CB1 actions, suggesting that cannabinoids inhibit glutamate release independently from the cAMP-PKA cascade. CB1 did not reduce evoked transmitter release by inhibiting presynaptic voltage-dependent Ca(2+) currents through N-, L-, or P/Q-type Ca(2+) channels, because CB1 inhibition persisted in the presence of omega-Conotoxin-GVIA, nimodipine, or omega-Agatoxin-IVA. The K(+) channel blockers 4-aminopyridine (100 micrometer) and BaCl(2) (300 micrometer) each reduced by 40-50% the inhibitory actions of WIN-2, and their effects were additive. These data suggest that CB1 receptors are located on the cortical afferents to the nucleus and can reduce glutamate synaptic transmission within the NAc by modulating K(+) channels activity.

Regulation of central synaptic transmission by 5-HT(1B) auto- and heteroreceptors.

Although 5-HT(1B) receptors are believed to be expressed on nerve terminals, their precise mode of action is not fully understood because of the lack of selective antagonists. The 5-HT(1B) receptor knockout mouse was used in the present investigation to assess the function of 5-HT(1B) receptors in the modulation of synaptic transmission in three areas of the central nervous system: the dorsal raphe, the ventral midbrain, and the nucleus accumbens. N-(3-Trifluoromethylphenyl)piperazine, a 5-HT(1B) receptor agonist, potently inhibited 5-HT(1A) receptor-mediated slow inhibitory postsynaptic potentials (IPSPs) in the dorsal raphe of wild-type but not knockout mice. Both synaptically released 5-HT and exogenous 5-HT caused a presynaptic inhibition that outlasted the postsynaptic hyperpolarization only in wild-type mice. In the ventral midbrain, 5-HT(1B) receptor-dependent inhibition of gamma-aminobutyric acid(B) IPSPs in dopamine neurons was present in wild-type animals and absent in knockout animals. Similar results were obtained in the nucleus accumbens measuring glutamate-mediated excitatory postsynaptic currents in medium spiny neurons. Finally, cocaine, which blocks 5-HT uptake, inhibited IPSPs in the dorsal raphe and the ventral midbrain of wild-type but not knockout mice, whereas cocaine produced comparable inhibition of excitatory postsynaptic currents in the nucleus accumbens of both types of animals. These results indicate that 5-HT(1B) receptors function as autoreceptors and heteroreceptors to exert presynaptic inhibition of transmitter release in the central nervous system. Furthermore, this study underscores the role played by presynaptic 5-HT(1B) receptors in mediating the effects of cocaine on synaptic transmission.

Presynaptic regulation of glutamate release in the ventral tegmental area during morphine withdrawal.

The regulation of glutamate (Glu) release from the excitatory input to dopamine cells in the ventral tegmental area (VTA) during acute withdrawal from morphine was studied in slices from animals treated for 6-7 d with morphine. EPSCs were inhibited by opioid agonists acting at micro-subtype receptors but not by selective delta- or kappa-subtype agonists. The opioid inhibition was reduced by 65% with the potassium channel blocker 4-aminopyridine (4-AP; 100 microM) and a 12-lipoxygenase inhibitor, baicalein (5 microM), suggesting that opioids acted via a transduction pathway involving activation of a voltage-dependent potassium conductance by lipoxygenase metabolites as has been shown in the periaqueductal gray (). During withdrawal, neither the potency nor the efficacy of D-Ala-Met-enkephalin-Gly-ol (DAMGO) were changed; however, the blockade of micro-opioid inhibition by both 4-AP and baicalein was reduced. In addition, the potency of baclofen to depress EPSCs by GABA-B receptors and the effects of the GABA-uptake inhibitor NO-711 (10 microM) were increased in withdrawn rats. Finally, group 2 (but not group 4 or 1) metabotropic glutamate receptor-mediated presynaptic inhibition was also enhanced in morphine-withdrawn rats. These results suggest that one of the consequences of withdrawal from chronic morphine is an enhanced presynaptic inhibition of the excitatory inputs to the dopamine cells of the VTA. Inhibition of glutamate release during acute withdrawal would add to the inhibition of dopamine cells that is mediated by an augmented release of GABA ().

Pharmacology of metabotropic glutamate receptors at the mossy fiber synapses of the guinea pig hippocampus.

We have tested the ability of several specific agonists of glutamate metabotropic receptors (mGluRs) to depress synaptic transmission at mossy fiber synapses in the CA3 region of the guinea pig hippocampus. 1S,3R-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD) reversibly inhibited monosynaptic mossy fiber field potentials, presumably by a presynaptic mechanism, with an EC50 of 2.0 +/- 0.4 microM (n = 3), suggesting the presence of mGluRs on mossy fiber synaptic terminals of the group 1 or 2 category. L-2-amino-4-phosphono butanoate (L-AP4) also inhibited responses with an EC50 of 1.1 +/- 0.2 microM suggesting that mGluRs of the group 3 (mGluR4, 6, 7 and 8) category of receptors are also present on mossy fiber terminals. Both (2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1) and (S)-4-carboxy-3-hydroxy phenylglycine (4C3HPG) were also efficacious at blocking mossy fiber transmission, with an EC50 of 1.1 +/- 0.1 microM (n = 4) and 4.8 +/- 0.6 microM (n = 3) respectively. The latter finding indicates the involvement of mGluRs belonging to the group 2 (mGluR2, 3) category of receptors. The effects of L-AP4 and L-CCG1 were both antagonized by (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG]. MAP4, an antagonist of group 3 mGluRs in other systems, blocked the effect of L-AP4, but not the effect of L-CCG1, while MCCG, an antagonist of group 2 mGluRs in other systems, blocked the effect of L-CCG1, but not the effect of L-AP4. These pharmacological findings provide strong evidence for the coexistence of group 2 and 3 mGluRs on the terminals of mossy fibers in the guinea pig.

Release of adenosine by activation of NMDA receptors in the hippocampus.

Adenosine is present in the mammalian brain in large amounts and has potent effects on neuronal activity, but its role in neural signaling is poorly understood. The glutamate receptor agonist N-methyl-D-aspartate (NMDA) caused a presynaptic depression of excitatory synaptic transmission in the CA1 region of guinea pig hippocampal slices. This depression was blocked by an adenosine A1 receptor antagonist, which suggests that activation of the NMDA subtype of glutamate receptor raises the concentration of extracellular adenosine, which acts on presynaptic inhibitory A1 receptors. Strong tetanic stimulation caused a heterosynaptic inhibition that was blocked by both NMDA and A1 receptor antagonists. Enkephalin, which selectively inhibits interneurons, antagonized the heterosynaptic inhibition. These findings suggest that synaptically released glutamate activates NMDA receptors, which in turn releases adenosine, at least in part from interneurons, that acts at a distance to inhibit presynaptically the release of glutamate from excitatory synapses. Thus, interneurons may mediate a widespread purinergic presynaptic inhibition.

MCPG antagonizes metabotropic glutamate receptors but not long-term potentiation in the hippocampus.

In the CA1 and CA3 regions of the guinea pig hippocampus, we have tested the ability of the new antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) to inhibit the well-known effects of (trans)-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), a specific agonist of glutamate metabotropic receptors. Whole-cell recordings showed that MCPG was able to antagonize the blocking action of ACPD on IAHP in the CA1 region. In addition, we report here that MCPG also antagonized the presynaptic inhibitory actions of ACPD on field excitatory postsynaptic potentials in both areas CA1 and CA3. Thus, MCPG proved to be an effective tool for determining physiological roles of the glutamate metabotropic receptors in synaptic transmission in the hippocampus. We next tested the possible effects of this antagonist on long-term potentiation (LTP). In completely blind experiments MCPG was without effect on LTP in both areas CA1 and CA3. In conclusion, our results suggest that, although MCPG is a valuable antagonist of the ACPD-sensitive receptors, it has no inhibitory effect on LTP.

beta-N-methylamino-L-alanine is a low-affinity agonist of metabotropic glutamate receptors.

beta-N-methylamino-L-alanine (L-BMAA) is an excitotoxin whose neurodegenerative effects are associated with its agonist properties at the N-methyl-D-aspartate (NMDA) receptor. We measured the effects of L-BMAA on inositol phosphate (InsP) formation in primary cultured striatal neurons. This culture is almost devoid of glial cells and the pharmacology of glutamate receptors is well-defined. This allowed us to show that L-BMAA induced InsP formation via a direct action at the glutamate metabotropic (Qp) receptors coupled to InsP formation. We demonstrated that L-BMAA is a full-agonist of the Qp receptor, but with a low potency. Therefore, the neurotoxic properties of L-BMAA might implicate the activation of the Qp receptor in association with the NMDA receptor.

Pharmacological characterization of the quisqualate receptor coupled to phospholipase C (Qp) in striatal neurons.

A detailed pharmacological characterization of the quisqualate (QA) receptor coupled to phospholipase C (Qp) was performed in striatal neurons. The experiments were carried out in the presence of the ionotropic antagonists MK-801 (1 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM), concentrations that block N-methyl-D-aspartate (NMDA) or alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in these cells. QA, ibotenate and trans-1-aminocyclopentyl-1,3-dicarboxylate (ACPD) evoked dose-dependent inositol phosphate formations with EC50 values of 0.3, 6.7 and 29 microM, respectively. QA and ibotenate had the same maximal effect (295.7 +/- 17.9% of basal, n = 6) whereas the efficacy of ACPD was somewhat lower (70.2 +/- 8.9% of the maximal quisqualate effect, n = 4). The QA-, ibotenate- and ACPD-induced maximal effects were not additive, and the inositol phosphate formations induced by high concentrations of L-aspartate (L-ASP), AMPA, kainate (KA) and domoate (DO) (100 microM or higher) were also not additive. The inositol phosphate responses induced by all these agonists were totally blocked by the phorbol ester phorbol 12,13-dibutyrate (PdBu), but not by atropine or prazosin suggesting that all these substances were able to stimulate the Qp excitatory amino acid receptor in striatal neurons. Of the excitatory amino acid receptor antagonists tested, only D,L-2-amino-3-phosphonopropionate (D,L-AP3) inhibited QA-induced InsP formation in a competitive manner (mean pKi = 4.45 +/- 0.43, n = 4). However, this drug was also a partial agonist of the Qp receptor since it stimulated the inositol phosphate formation. We found that D,L-AP3 also inhibited NMDA-induced calcium increase, in a competitive manner (mean pIC50 = 4.34 +/- 0.22, n = 8, and mean pKi = 3.7 +/- 0.11, n = 5). The Qp excitatory amino acid receptor in striatal neurons therefore closely resembles Qp receptors with high potency for agonists as described in striatal and retinal slices and synaptoneurosomes, and has several pharmacological differences compared to the Qp receptors which have low potency for agonists described in hippocampal and cortical slices, cerebellar granule cells, astrocytes and rat brain mRNA-injected oocytes.

Cholinergic inositol phosphate formation in striatal neurons is mediated by distinct mechanisms.

In murine striatal neurons devoid of functional synapses (6 days in vitro) the cholinergic agonists carbachol and arecoline evoked dose-dependent inositol phosphate (InsP) responses with mean log EC50s of -4.1 +/- 0.5 and -4.48 +/- 0.1, respectively. Carbachol (1 mM) and arecoline (1 mM) responses were insensitive to tetrodotoxin, a voltage-sensitive Na+ channel blocker, and were blocked by pirenzepine with relatively low affinity (logIC50 = -5.9 +/- 0.3 for the carbachol response and logIC50 = -5.8 +/- 0.3 for the arecoline response). After synaptogenesis (13 days in vitro) the maximal carbachol effect doubled whereas the arecoline response remained unchanged. This additional effect was sensitive to tetrodotoxin and the voltage-dependent Ca2+ channel blocker, omega-conotoxin. The tetrodotoxin-sensitive carbachol response was blocked by lower concentrations of pirenzepine than the tetrodotoxin-insensitive carbachol response. More than 75% of the InsP response evoked by low concentrations of muscarine (1 and 10 microM) was sensitive to tetrodotoxin whereas only 38% of the InsP response stimulated by 1 mM of muscarine could be blocked by tetrodotoxin. These results suggest that there are at least two different mechanisms (depending on the stage of development), activated most probably by two different muscarinic receptors responsible for the carbachol-induced InsP formation in striatal neurons.

The glutamate receptor of the Qp-type activates protein kinase C and is regulated by protein kinase C.

In striatal neurons in primary culture quisqualate potently stimulated the formation of inositol phosphates via a metabotropic receptor we recently termed Qp in order to distinguish it from the classical ionotropic quisqualate receptor termed Qi. Here we show that 10 microM of quisqualate activated in a rapid and transient manner protein kinase C as assessed by its translocation from the cytosolic to the membrane fraction. As 10 microM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), the Qi specific agonist, was without effect, this translocation was most probably mediated by the Qp receptor. Phorbol 12,13-dibutyrate blocked in a dose-dependent manner the Qp receptor-induced inositol phosphate formation (IC50 = 2 +/- 0.4 nM). The inactive ester 4 alpha-phorbol-12,13-didecanoate was without effect. Very low concentrations of staurosporine completely reversed the phorbol 12,13-dibutyrate-induced blockade (IC50 = 2.2 +/- 1.3 nM). It can therefore be concluded that the Qp receptor is able to activate protein kinase C and that the activity of this metabotropic receptor is regulated by protein kinase C.