Reduced sedation and increased ethanol consumption in knock-in mice expressing an ethanol insensitive alpha 2 subunit of the glycine receptor.

Previous studies have shown the presence of several subunits of the inhibitory glycine receptor (GlyR) in the reward system, specifically in medium spiny neurons (MSNs) of the nucleus Accumbens (nAc). It was suggested that GlyR α1 subunits regulate nAc excitability and ethanol consumption. However, little is known about the role of the α2 subunit in the adult brain since it is a subunit highly expressed during early brain development. In this study, we used genetically modified mice with a mutation (KR389-390AA) in the intracellular loop of the GlyR α2 subunit which results in a heteromeric α2ß receptor that is insensitive to ethanol. Using this mouse model denoted knock-in α2 (KI α2), our electrophysiological studies showed that neurons in the adult nAc expressed functional KI GlyRs that were rather insensitive to ethanol when compared with WT GlyRs. In behavioral tests, the KI α2 mice did not show any difference in basal motor coordination, locomotor activity, or conditioned place preference compared with WT littermate controls. In terms of ethanol response, KI α2 male mice recovered faster from the administration of ataxic and sedative doses of ethanol. Furthermore, KI α2 mice consumed higher amounts of ethanol in the first days of the drinking in the dark protocol, as compared with WT mice. These results show that the α2 subunit is important for the potentiation of GlyRs in the adult brain and this might result in reduced sedation and increased ethanol consumption.

Presence of ethanol-sensitive glycine receptors in medium spiny neurons in the mouse nucleus accumbens.

KEY POINTS: The nucleus accumbens (nAc) is involved in addiction-related behaviour caused by several drugs of abuse, including alcohol. Glycine receptors (GlyRs) are potentiated by ethanol and they have been implicated in the regulation of accumbal dopamine levels. We investigated the presence of GlyR subunits in nAc and their modulation by ethanol in medium spiny neurons (MSNs) of the mouse nAc. We found that the GlyR α1 subunit is preferentially expressed in nAc and is potentiated by ethanol. Our study shows that GlyR α1 in nAc is a new target for development of novel pharmacological tools for behavioural intervention in drug abuse. ABSTRACT: Alcohol abuse causes major social, economic and health-related problems worldwide. Alcohol, like other drugs of abuse, increases levels of dopamine in the nucleus accumbens (nAc), facilitating behavioural reinforcement and substance abuse. Previous studies suggested that glycine receptors (GlyRs) are involved in the regulation of accumbal dopamine levels. Here, we investigated the presence of GlyRs in accumbal dopamine receptor medium spiny neurons (MSNs) of C57BL/6J mice, analysing mRNA expression levels and immunoreactivity of GlyR subunits, as well as ethanol sensitivity. We found that GlyR α1 subunits are expressed at higher levels than α2, α3 and ß in the mouse nAc and were located preferentially in dopamine receptor 1 (DRD1)-positive MSNs. Interestingly, the glycine-evoked currents in dissociated DRD1-positive MSNs were potentiated by ethanol. Also, the potentiation of the GlyR-mediated tonic current by ethanol suggests that they modulate the excitability of DRD1-positive MSNs in nAc. This study should contribute to understanding the role of GlyR α1 in the reward system and might help to develop novel pharmacological therapies to treat alcoholism and other addiction-related and compulsive behaviours.

Associative and sensorimotor cortico-basal ganglia circuit roles in effects of abused drugs.

The mammalian forebrain is characterized by the presence of several parallel cortico-basal ganglia circuits that shape the learning and control of actions. Among these are the associative, limbic and sensorimotor circuits. The function of all of these circuits has now been implicated in responses to drugs of abuse, as well as drug seeking and drug taking. While the limbic circuit has been most widely examined, key roles for the other two circuits in control of goal-directed and habitual instrumental actions related to drugs of abuse have been shown. In this review we describe the three circuits and effects of acute and chronic drug exposure on circuit physiology. Our main emphasis is on drug actions in dorsal striatal components of the associative and sensorimotor circuits. We then review key findings that have implicated these circuits in drug seeking and taking behaviors, as well as drug use disorders. Finally, we consider different models describing how the three cortico-basal ganglia circuits become involved in drug-related behaviors. This topic has implications for drug use disorders and addiction, as treatments that target the balance between the different circuits may be useful for reducing excessive substance use.

Postsynaptic endocannabinoid release is critical to long-term depression in the striatum.

The striatum functions critically in movement control and habit formation. The development and function of cortical input to the striatum are thought to be regulated by activity-dependent plasticity of corticostriatal glutamatergic synapses. Here we show that the induction of a form of striatal synaptic plasticity, long-term depression (LTD), is dependent on activation of the CB1 cannabinoid receptor. LTD was facilitated by blocking cellular endocannabinoid uptake, and postsynaptic loading of anandamide (AEA) produced presynaptic depression. The endocannabinoid necessary for striatal LTD is thus likely to be released postsynaptically as a retrograde messenger. These findings demonstrate a new role for endocannabinoids in the induction of long-term synaptic plasticity in a circuit necessary for habit formation and motor control.

Activation of group I mGluRs is necessary for induction of long-term depression at striatal synapses.

Activation of metabotropic glutamate receptors (mGluRs), which are coupled to G proteins, has important roles in certain forms of synaptic plasticity including corticostriatal long-term depression (LTD). In the present study, extracellular field potential and whole cell voltage-clamp recording techniques were used to investigate the effect of mGluR antagonists with different subtype specificity on high-frequency stimulation (HFS)-induced LTD of synaptic transmission in the striatum of brain slices obtained from 15-to 25-day-old rats. Induction of LTD was prevented during exposure to the nonselective mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (500 microM). The group I mGluR-selective antagonists (S)-4-carboxy-phenylglycine (50 microM) and (RS)-1-aminoindan-1,5-dicarboxylic acid (100 microM) prevented induction of LTD when applied before and during HFS. The mGluR1-selective antagonist 7-(Hydroxyimino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (80 microM) also blocked LTD induction. Unexpectedly, the mGluR5-selective antagonist 2-methyl-6-(phenylethyl)-pyridine (10 microM) also prevented LTD induction. The group II mGluR antagonist LY307452 (10 microM) did not block LTD induction at corticostriatal synapses, but LY307452 was able to block transient synaptic depression induced by the group II agonist LY314593. None of the antagonists had any effect on basal synaptic transmission at the concentrations used, and mGluR antagonists did not reverse LTD when applied beginning 20 min after HFS. These results suggest that both group I mGluR subtypes contribute to the induction of LTD at corticostriatal synapses.

Magnesium potentiation of the function of native and recombinant GABA(A) receptors.

Mg2+ decreased basal and GABA-inhibited t-butylbicyclophosphoro[35S]thionate binding to GABAA receptor ion channels in rat brain sections up to 1 mM, but increased the binding at 10 mM. The Mg2+-effect was detectable in the presence of a specific GABA site competitive antagonist. Two-electrode voltage clamp recordings of recombinant alpha1beta2gamma2S, alpha1beta2, alpha2beta2gamma2S and alpha2beta2 GABAA receptors revealed a potentiation by 0.1-1 mM Mg2+ of EC20 GABA-evoked ion currents. At 10 mM, Mg2+ decreased the currents. In the absence of GABA, Mg2+ did not evoke any currents. The results show that physiologically relevant Mg2+ concentrations affect the GABA responses on GABAA receptors in native and the main recombinant receptor subtypes, suggesting putative Mg2+ binding sites on the receptor complex.

Dopamine-dependent synaptic plasticity in striatum during in vivo development.

The neurotransmitters dopamine (DA) and glutamate in the striatum play key roles in movement and cognition, and they are implicated in disorders of the basal ganglia such as Parkinson's disease. Excitatory synapses in striatum undergo a form of developmental plasticity characterized by a decrease in glutamate release probability. Here we demonstrate that this form of synaptic plasticity is DA and DA D2 receptor dependent. Analysis of spontaneous synaptic responses indicates that a presynaptic mechanism involving inhibition of neurotransmitter release underlies the developmental plasticity. We suggest that a major role of DA in the striatum is to initiate mechanisms that regulate the efficacy of excitatory striatal synapses, producing a decrease in glutamate release.

CB1 cannabinoid receptor inhibits synaptic release of glutamate in rat dorsolateral striatum.

CB1 cannabinoid receptors in the neostriatum mediate profound motor deficits induced when cannabinoid drugs are administered to rodents. Because the CB1 receptor has been shown to inhibit neurotransmitter release in various brain areas, we investigated the effects of CB1 activation on glutamatergic synaptic transmission in the dorsolateral striatum of the rat where the CB1 receptor is highly expressed. We performed whole cell voltage-clamp experiments in striatal brain slices and applied the CB1 agonists HU-210 or WIN 55,212-2 during measurement of synaptic transmission. Excitatory postsynaptic currents (EPSCs), evoked by electrical stimulation of afferent fibers, were significantly reduced in a dose-dependent manner by CB1 agonist application. EPSC inhibition was accompanied by an increase in two separate indices of presynaptic release, the paired-pulse response ratio and the coefficient of variation, suggesting a decrease in neurotransmitter release. These effects were prevented by application of the CB1 antagonist SR141716A. When Sr(2+) was substituted for Ca(2+) in the extracellular solution, application of HU-210 (1 microM) significantly reduced the frequency, but not amplitude, of evoked, asynchronous quantal release events. Spontaneous release events were similarly decreased in frequency with no change in amplitude. These findings further support the interpretation that CB1 activation leads to a decrease of glutamate release from afferent terminals in the striatum. These results reveal a novel potential role for cannabinoids in regulating striatal function and thus basal ganglia output and may suggest CB1-targeted drugs as potential therapeutic agents in the treatment of Parkinson's disease and other basal ganglia disorders.

Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice.

We have recently disrupted Slc12a2, the gene encoding the secretory Na-K-2Cl cotransporter in mice (NKCC1) (Delpire et al., 1999). Gramicidin perforated-patch and whole-cell recordings were performed to study GABA-induced currents in dorsal root ganglion (DRG) neurons isolated from wild-type and homozygote NKCC1 knock-out mice. In wild-type DRG neurons, strong GABA-evoked inward current was observed at the resting membrane potential, suggesting active accumulation of Cl(-) in these cells. This GABA-induced current was blocked by picrotoxin, a GABA(A) receptor blocker. The strong Cl(-) accumulation that gives rise to depolarizing GABA responses is caused by Na-K-2Cl cotransport because reduction of external Cl(-) or application of bumetanide induced a decrease in [Cl(-)](i), whereas an increase in external K(+) caused an apparent [Cl(-)](i) accumulation. In contrast to control neurons, little or no net current was observed at the resting membrane potential in homozygote NKCC1 mutant DRG neurons. E(GABA) was significantly more negative, demonstrating the absence of Cl(-) accumulation in these cells. Application of bumetanide induced a positive shift of E(GABA), suggesting the presence of an outward Cl(-) transport mechanism. In agreement with an absence of GABA depolarization in DRG neurons, behavioral analysis revealed significant alterations in locomotion and pain perception in the knock-out mouse. Our results clearly demonstrate that the Na-K-2Cl cotransporter is responsible for [Cl(-)](i) accumulation in DRG neurons and that via regulation of intracellular Cl(-), the Na-K-2Cl cotransporter participates in the modulation of GABA neurotransmission and sensory perception.

Regional and postnatal heterogeneity of activity-dependent long-term changes in synaptic efficacy in the dorsal striatum.

High-frequency activation of excitatory striatal synapses produces lasting changes in synaptic efficacy that may contribute to motor and cognitive functions. While some of the mechanisms responsible for the induction of long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic responses at striatal synapses have been characterized, much less is known about the factors that govern the direction of synaptic plasticity in this brain region. Here we report heterogeneous activity-dependent changes in the direction of synaptic strength in subregions of the developing rat striatum. Neurons in the dorsolateral region of the anterior striatum tended to express LTD after high-frequency afferent stimulation (HFS) in slices from animals aged P15-P34. However, HFS in dorsolateral striatum from P12-P14 elicited an N-methyl-D-aspartate (NMDA) receptor-dependent form of LTP. Synapses in the dorsomedial anterior striatum exhibited a propensity to express an NMDA-receptor dependent form of LTP across the entire developmental time period examined. The NMDA receptor antagonist (+/-)-2-amino-5-phosphopentanoic acid (APV) inhibited evoked excitatory postsynaptic potentials recorded in striatum obtained from P12-P15 rats but had little effect in striatum from older animals. The expression of multiple forms of synaptic plasticity in the striatum suggests mechanisms by which this brain region plays pivotal roles in the acquisition or encoding of some forms of motor sequencing and stereotypical behaviors.

5-HT(3) receptor function and potentiation by alcohols in frontal cortex neurons from transgenic mice overexpressing the receptor.

The function of 5-hydroxytryptamine (5-HT)(3) receptors was examined by whole-cell patch-clamp recording in dissociated frontal cortex neurons from 5-HT(3) receptor overexpressing transgenic, and wild-type mice. The effect of acute exposure to alcohols on the 5-HT(3) receptor-mediated ion current was also investigated. The 5-HT(3) receptors expressed on frontal cortex neurons in transgenic mice were activated by 5-HT and a selective 5-HT(3) receptor agonist, 2-methyl-5-HT. This current was blocked by zacopride, a specific 5-HT(3) receptor antagonist. Dissociated frontal cortex neurons from wild-type mice exhibited little or no 5-HT(3) receptor-mediated current. Ethanol (EtOH) and trichloroethanol (TCEt) potentiated the function of 5-HT(3) receptors overexpressed in transgenic mice. This is the first evidence that 5-HT(3) receptors exhibit sensitivity to alcohols when expressed by a central neuron.

Interaction of acamprosate with ethanol and spermine on NMDA receptors in primary cultured neurons.

The N-methyl-D-aspartate (NMDA) receptor has been implicated as a putative sight of action for acamprosate, a novel drug that reduces craving for alcohol. The purpose of this study was to assess the effect of acamprosate on the function of native NMDA receptors expressed in primary cultured striatal and cerebellar granule cells, as well as ethanol inhibition and spermine modulation of these receptors, using whole-cell patch-clamp electrophysiological techniques. Under all circumstances, acamprosate (0.1-300 microM) did not alter NMDA- or glutamate-induced currents. Acamprosate did not alter the inhibitory effects of ethanol (10-100 mM) on receptor function. In a subpopulation of striatal neurons, acamprosate did reverse the potentiating effects of spermine. These findings indicate that although acamprosate may modify polyamine modulation of the NMDA receptor, acamprosate alone does not alter receptor function nor does it modify ethanol inhibition of this receptor expressed in primary cultured striatal and cerebellar granule neurons.

Ethanol and trichloroethanol alter gating of 5-HT3 receptor-channels in NCB-20 neuroblastoma cells.

Alcohol potentiation of 5-HT3 receptors was examined in NCB-20 neuroblastoma cells using whole-cell patch-clamp electrophysiological techniques. Activation of the receptor with the weak partial agonist dopamine (DA) was used to examine alcohol effects under conditions of full agonist occupancy, but low probability of channel opening. Dopamine activation of the receptor increased in a concentration-dependent manner (EC50=0.28 mM), and on average maximal responses to DA were 8.0+/-0.8% of the maximal response to 5-HT. Ethanol (EtOH) and trichloroethanol (TCEt) potentiated DA-activated ion current mediated by 5-HT3 receptors. Potentiation of responses to a maximally effective dopamine concentration averaged 52.0+/-8.0% for EtOH and 567+/-43% for TCEt, which was comparable to the potentiation observed when receptors were activated by a low concentration of 5-HT. The alcohols increased both the potency and efficacy with which dopamine activated the receptor. The observation that alcohols increase the maximal efficacy of dopamine activation of the receptor indicates that one action of alcohols on the 5-HT3 receptor is to increase the probability of channel opening independent of any effect on agonist affinity.

Role of pertussis toxin-sensitive G-proteins in synaptic transmission and plasticity at corticostriatal synapses.

The role of pertussis toxin (PTX)-sensitive G-proteins in corticostriatal synaptic transmission and long-term synaptic depression (LTD) was examined using extracellular field potential and whole cell voltage-clamp recordings in striatal slices. High-frequency stimulation (HFS) produced LTD, defined as long-lasting decreases both in synaptically driven population spikes (PSs) measured with field potential recording and in excitatory postsynaptic currents (EPSCs) measured with whole cell recording. Striatal LTD could not be induced in slices obtained from rats that had received a unilateral intrastriatal injection of PTX. However, LTD could be induced in slices obtained from paired control slices. Furthermore, striatal LTD was prevented by pretreatment with N-ethylmaleimide (NEM), another compound that disrupts the function of PTX-sensitive G-proteins. NEM, itself, also potentiated PS and EPSC amplitudes. In addition, NEM increased the frequency and amplitude of both spontaneous and miniature EPSCs and decreased the paired-pulse facilitation ratio, suggesting that it may act on both pre- and postsynaptic sites. The findings suggest that PTX-sensitive G-proteins have multiple roles at corticostriatal synapses, including regulation of synaptic transmission at both pre- and postsynaptic sites, and a key role in striatal LTD.

5-HT3 receptors and the neural actions of alcohols: an increasingly exciting topic.

The 5-HT3 receptor is a ligand-gated ion channel activated by the neurotransmitter serotonin. Receptors of this subtype have been localized to several regions of the brain, and appear to be involved in many neuronal functions including responses to alcohol and other drugs of abuse. There is an extensive and growing literature indicating that 5-HT3 receptors are involved in several facets of alcohol seeking behavior, alcohol intoxication and addiction. In addition, there is strong evidence that alcohols, including ethanol, alter the function of the 5-HT3 receptor, possibly through actions on the receptor protein itself. In this article, our current understanding of the role of the 5-HT3 receptor in alcohol abuse and alcoholism will be reviewed. In addition, an overview of current understanding of the mechanism of alcohol actions of the receptor is provided.

Factors that enhance ethanol inhibition of N-methyl-D-aspartate receptors in cerebellar granule cells.

The objective of this study was to identify factors that influence ethanol (EtOH) inhibition of the N-methyl-D-aspartate receptor (NMDAR) in primary cultured cerebellar granule cells. Several factors contributing to the inhibitory effects of EtOH on NMDAR function were assessed using both whole-cell and perforated patch-clamp recordings. The NMDAR subunit composition was examined by Western blot analysis using NR2 subunit-specific antibodies and pharmacological manipulation with the NR2B-specific antagonist infenprodil. Western blot analysis indicated that NMDAR subunit composition changed from a combination of NR2A and NR2B containing NMDARs to primarily NR2A with increasing days in vitro (DIV). Although the NR2B subunit was detectable until 21 DIV, there was a significant decrease in ifenprodil sensitivity after 7 DIV. EtOH sensitivity did not change with an increasing DIV. A high concentration of glycine reversed EtOH inhibition of steady-state, but not peak, NMDA-induced current during whole-cell recordings. Significant glycine reversal of effects of a low concentration of EtOH on peak current was observed under perforated patch-clamp conditions. A 30-s EtOH pretreatment significantly enhanced EtOH inhibition of NMDA-induced peak current. Collectively, these results indicate that EtOH sensitivity of the NMDAR in primary cultured cerebellar granule cells is not related to subunit composition nor ifenprodil sensitivity, involves a kinetic interaction with glycine, and can be enhanced by a slowly developing transduction mechanism that occurs within tens of seconds.

Characterization of (S)-des-4-amino-3-[125I]iodozacopride ([125I]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine3 receptors.

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.

Ethanol sensitivity and subunit composition of NMDA receptors in cultured striatal neurons.

Assessment of ethanol (EtOH) sensitivity was combined with analysis of N-methyl-D-aspartate (NMDA) NR1-NR2 subunit composition in primary cultured striatal neurons. Subunit composition was determined by western blot analysis; assessment of ifenprodil and spermine sensitivity during whole-cell patch-clamp recordings. From 3-21 days in culture, NR2B was the only NR2 subunit detected using NR2 subunit specific antibodies; NMDA-induced currents were strongly inhibited by the NR2B-selective antagonist ifenprodil. Two populations of neurons were identified at all ages in culture: those in which NMDA-induced current was or was not potentiated by 100 microM spermine. This suggested that the striatal neurons expressed functional NMDARs which lacked or contained the NR1 N-terminal cassette. The EtOH sensitivity did not differ between these two populations of neurons nor did it change with age in culture at all concentrations of EtOH studied. Human embryonic kidney (HEK) 293 cells containing NR1-1a or NR1-1b with either the NR2A or NR2B subunits did not differ in their EtOH sensitivity. Thus, it would appear that the presence or absence of the N-terminal cassette does not affect the EtOH sensitivity of recombinant NMDARs and native NMDARs expressed in cultured striatal neurons.

Alcohols potentiate the function of 5-HT3 receptor-channels on NCB-20 neuroblastoma cells by favouring and stabilizing the open channel state.

1. 5-HT3 receptor-mediated ion current was recorded from NCB-20 neuroblastoma cells using the whole-cell patch-clamp technique. Rapid drug superfusion was used to study the mechanism of alcohol potentiation of 5-HT3 receptor function and to analyse effects of alcohols on receptor-channel kinetics in detail. 2. Trichloroethanol (TCEt) increased in a dose-dependent way the initial slope, 20-80% rise time and measured desensitization rate of the current induced by low concentrations (1-2 microM) of 5-HT. Ethanol (EtOH) and butanol (ButOH) had similar effects on the 5-HT3 receptor-induced current. 3. TCEt and ButOH decreased the measured desensitization rate of current induced by 10 microM 5-HT, a maximally effective concentration of agonist. These alcohols also increased the relative amplitude of steady state to peak current induced by 2 or 10 microM 5-HT, indicating a possible decrease in the intrinsic rate of desensitization. 4. TCEt also decreased the deactivation rate of the current activated by 2 microM 5-HT after a short pulse of agonist application. 5. Current sweeps generated by 1 microM 5-HT in the presence or absence of 10 mM TCEt or 100 mM EtOH were well fitted using a modified standard kinetic model derived from the nicotinic acetylcholine receptor. This analysis indicated that potentiation by alcohols could be accounted for by increases in the association rate constant coupled with decreases in the dissociation and desensitization rate constants. 6. This study suggests that alcohols potentiate 5-HT3 receptor-mediated current by both increasing the rate of channel activation and stabilizing the open state by decreasing the rates of channel deactivation and desensitization.

Rat group I metabotropic glutamate receptors inhibit neuronal Ca2+ channels via multiple signal transduction pathways in HEK 293 cells.

We have shown previously that metabotropic glutamate receptors with group I-like pharmacology couple to N-type and P/Q-type calcium channels in acutely isolated cortical neurons using G proteins most likely belonging to the Gi/Go subclass. To better understand the potential mechanisms forming the basis for group I mGluR modulation of voltage-gated calcium channels in the CNS, we have examined the ability of specific mGluRs to couple to neuronal N-type (alpha1B-1/alpha2delta/beta1b) and P/Q-type (alpha1A-2/alpha2delta/beta1b) voltage-gated calcium channels in an HEK 293 heterologous expression system. Using the whole cell patch-clamp technique where intracellular calcium is buffered to low levels, we have shown that group I receptors inhibit both N-type and P/Q-type calcium channels in a voltage-dependent fashion. Similar to our observations in cortical neurons, this voltage-dependent inhibition is mediated almost entirely by N-ethylmaleimide (NEM)-sensitive heterotrimeric G proteins, strongly suggesting that these receptors can use Gi/Go-like G proteins to couple to N-type and P/Q-type calcium channels. However, inconsistent with the apparent NEM sensitivity of group I modulation of calcium channels, modulation of N-type channels in group I mGluR-expressing cells was only partially sensitive to pertussis toxin (PTX), indicating the potential involvement of both PTX-sensitive and -resistant G proteins. The PTX-resistant modulation was voltage dependent and entirely resistant to NEM and cholera toxin. A time course of treatment with PTX revealed that this toxin caused group I receptors to slowly shift from using a primarily NEM-sensitive G protein to using a NEM-resistant form. The PTX-induced switch from NEM-sensitive to -resistant modulation was also dependent on protein synthesis, indicating some reliance on active cellular processes. In addition to these voltage-dependent pathways, perforated patch recordings on group I mGluR-expressing cells indicate that another slowly developing, calcium-dependent form of modulation for N-type channels may be seen when intracellular calcium is not highly buffered. We conclude that group I mGluRs can modulate neuronal Ca2+ channels using a variety of signal transduction pathways and propose that the relative contributions of different pathways may exemplify the diversity of responses mediated by these receptors in the CNS.