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.

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.

Ethanol inhibits NMDA-activated ion current in hippocampal neurons.

The ion current induced by the glutamate receptor agonist N-methyl-D-aspartate (NMDA) in voltage-clamped hippocampal neurons was inhibited by ethanol (EtOH). Inhibition increased in a concentration-dependent manner over the range 5 to 50 mM, a range that also produces intoxication. The amplitude of the NMDA-activated current was reduced 61 percent by 50 mM EtOH; in contrast, this concentration of EtOH reduced the amplitude of current activated by the glutamate receptor agonists kainate and quisqualate by only 18 and 15 percent, respectively. The potency for inhibition of the NMDA-activated current by several alcohols is linearly related to their intoxicating potency, suggesting that alcohol-induced inhibition of responses to NMDA receptor activation may contribute to the neural and cognitive impairments associated with intoxication.

Translocation of protein kinase C activity may mediate hippocampal long-term potentiation.

Protein kinase C activity in rat hippocampal membranes and cytosol was determined 1 minute and 1 hour after induction of the synaptic plasticity of long-term potentiation. At 1 hour after long-term potentiation, but not at 1 minute, protein kinase C activity was increased twofold in membranes and decreased proportionately in cytosol, suggesting translocation of the activity. This time-dependent redistribution of enzyme activity was directly related to the persistence of synaptic plasticity, suggesting a novel mechanism regulating the strength of synaptic transmission.

Heterologous expression of metabotropic glutamate receptors in adult rat sympathetic neurons: subtype-specific coupling to ion channels.

A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGluR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-type Ca2+ channels through a pertussis toxin-sensitive pathway. Injection of mGluR1 alpha cRNA resulted in the expression of receptors that inhibited M-type K+ channels via a pertussis toxin-insensitive pathway. Coupling was restricted to specific transduction elements and effectors, since mGluR2 did not inhibit M channels and mGluR1 alpha had minimal effects on Ca2+ channels. These findings demonstrate that heterologously expressed, and thus unambiguously identified, mGluR subtypes modulate specific neuronal ion channels through discrete signal transduction pathways.

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.

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.

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.

Selective decline in protein F1 phosphorylation in hippocampus of senescent rats.

Certain forms of neuronal plasticity have been found to be expressed through alterations in brain protein phosphorylation, and its regulation by protein kinase activity. Of interest in this regard is the possibility that the decline in neuronal plasticity and cognitive function that occurs in advanced age may result in part from altered phosphorylation of specific proteins. As a first attempt to identify age-related changes in phosphoproteins, we assayed in vitro phosphorylation of proteins in hippocampus, cerebellum, entorhinal cortex, and frontal cortex from Fischer-344 rats of 5 months, 11 months, and 25 months of age. Compared to the middle-aged animals, the aged rats showed a selective 46% decline in phosphorylation of the 47 kDa protein (F1) in hippocampus, with no change in the phosphorylation of other proteins measured in this structure. Aged animals also showed decreased phosphorylation relative to young animals. No age-related change was observed in any protein band for the other brain areas examined. Since protein F1 is phosphorylated by protein kinase C (PKC), the cytosolic and membrane distribution of this enzyme was compared across age groups. The activity of PKC in hippocampus did not change across age. The explanation of this age-related decline in protein F1 phosphorylation is likely to be a decline in the substrate protein itself. The results are discussed in terms of protein F1's possible role in age-related decline of hippocampal synaptic plasticity.

Metabotropic glutamate receptor modulation of synaptic transmission in corticostriatal co-cultures: role of calcium influx.

Modulation of excitatory glutamatergic transmission at corticostriatal synapses by a metabotropic glutamate receptor (mGluR) was examined using a newly developed cell culture preparation in which small explants of cortical tissue are grown in co-culture with isolated striatal neurons. Electrical stimulation of cortical tissue evoked excitatory postsynaptic currents (eEPSCs) observed during tight-seal, whole-cell recordings from striatal neurons. Transmission was mediated by activation of AMPA/kainate-type glutamate receptors. The mGluR agonists, 1SR,3RS-ACPD and DCG-IV, reduced eEPSC amplitude. The effect of 1SR,3RS-ACPD increased in a concentration-dependent manner. Application of phorbol diacetate (PDAc) potentiated eEPSC amplitude and reduced the inhibitory effect of mGluR activation. Pretreatment with pertussis toxin (PTX) also reduced inhibition by 1SR,3RS-ACPD. Under conditions in which transmission was independent of the function of voltage-gated calcium channels, mGluR activation reduced the frequency of occurrence of miniature EPSCs (mEPSCs), but did not alter mEPSC amplitude. This effect of mGluR activation was reduced by PDAc treatment. mGluR activation modulates glutamatergic transmission via a presynaptic autoreceptor at corticostriatal synapses in this newly-developed corticostriatal co-culture preparation as in striatal slices. Modulation of transmission occurs whether or not transmission involves activation of voltage-gated calcium channels. Furthermore, many of the characteristics of mGluR modulation of eEPSCs are shared by mGluR modulation of mEPSCs. These findings indicate that mechanisms downstream from calcium entry may contribute to modulation of synaptic transmission by mGluR autoreceptors.

Alcohols potentiate ion current mediated by recombinant 5-HT3RA receptors expressed in a mammalian cell line.

The effect of acute exposure to alcohols on ion current mediated by recombinant 5-HT3RA receptors transiently expressed in human embryonic kidney 293 cells was investigated. Cells transfected with 5-HT3RA cDNA expressed receptors with pharmacological and functional properties similar to those of native 5-HT3 receptors. Potentiation of receptor-mediated cation current was observed in the presence of ethanol (10-100 mM), butanol (0.1-20 mM), isopentanol (0.01-25 mM) and trichloroethanol (0.5-25 mM). Potentiation increased in a concentration-dependent manner until saturation was achieved for all alcohols tested. The maximal efficacies of potentiation differed among the alcohols with isopentanol > butanol = trichloroethanol > ethanol. Potentiation by butanol and isopentanol appeared to show acute tolerance such that the percent increase in current amplitude was largest upon the first of a series of alcohol applications and decreased during subsequent applications. The effect of ethanol was variable with potentiation occurring in 74% of cells examined, but not in the remaining cells. These observations indicate that the potentiating action of alcohols is similar in recombinant receptors to that previously observed in neuroblastoma cells and neurons expressing native receptors. These findings indicate that this recombinant system is suitable for studying the molecular basis of alcohol actions on the 5-HT3 receptor.

Ifenprodil inhibition of the 5-hydroxytryptamine3 receptor.

The anti-hypertensive drug ifenprodil is known to interact potently with the alpha 1-adrenergic receptor as well as a number of other second messenger-linked receptors. In addition to these properties, ifenprodil has been shown to prevent glutamate-mediated excitotoxicity via non-competitive antagonism of NMDA receptors [Legendre and Westbrook (1991) Molec. Pharmac. 40: 289-298; Shalaby et al. (1992) J. Pharmac. Exp. Ther. 260: 925-932]. With these things in mind, we have begun to examine the specificity of ifenprodil for various ligand-gated ion channels using electrophysiological methods. While ifenprodil effectively inhibits NMDA-mediated currents in cortical neurons in culture, it does not interact with either kainate or GABA receptors. Surprisingly, ifenprodil also acts as a relatively potent antagonist of the 5-hydroxytryptamine3 (5-HT3) receptor in the NG108-15 neuroblastoma x glioma cell line. Furthermore, several aspects of ifenprodil action on the 5-HT3 receptor resemble its interaction with the NMDA receptor. Namely, inhibition of 5-HT3-mediated cation currents is readily reversible, has relatively slow onset, is non-competitive, and is not voltage dependent. Since most of the known 5-HT3 antagonists are competitive, it is possible that ifenprodil may define a unique modulatory site(s) on this neurotransmitter receptor.

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.

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.

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.

Involvement of N- and non-N-type calcium channels in synaptic transmission at corticostriatal synapses.

Calcium channels participate in the events linking axon terminal depolarization to neurotransmitter secretion. We wished to evaluate the role of N-type and non-N-type calcium channels in glutamatergic transmission at corticostriatal synapses, since this is a well defined excitatory synapse. In addition, these synapses are subject to a variety of forms of presynaptic modulation, some of which may involve alterations in calcium channel function. Application of the selective N-type channel blocker omega-conotoxin GVIA produced an irreversible depression of excitatory synaptic transmission in rat neostriatal slices shown by a decrease of approximately 50% in the amplitude of the synaptically driven population spike during field potential recording and a similar decrease in the amplitude of excitatory postsynaptic potentials during whole-cell recording. The component of transmission which was resistant to omega-conotoxin GVIA was blocked by omega-conotoxin MVIIC. omega-Agatoxin IVA had little effect on transmission. Activation of a presynaptic metabotropic glutamate receptor depressed transmission to a similar extent before and after omega-conotoxin GVIA treatment. Likewise, protein kinase C-activating phorbol esters potentiated transmission to the same extent before and after omega-conotoxin GVIA treatment. N-type calcium channels appear to be crucial for a component of excitation-secretion coupling at corticostriatal synapses. A component of transmission involves non-N-, non-L-type high-voltage-activated calcium channels. The effects of presynaptic metabotropic receptors and protein kinase C activation cannot be accounted for solely by alterations in the N-type channel function.

Ethanol inhibition of N-methyl-D-aspartate-activated current in mouse hippocampal neurones: whole-cell patch-clamp analysis.

1. The action of ethanol on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. 2. Ethanol inhibited NMDA-activated current in a voltage-independent manner, and did not alter the reversal potential of NMDA-activated current. 3. Concentration-response analysis of NMDA- and glycine-activated current revealed that ethanol decreased the maximal response to both agonists without affecting their EC50 values. 4. The polyamine spermine (1 microM) increased amplitude of NMDA-activated current but did not alter the percentage inhibition of ethanol. 5. Compared to an extracellular pH of 7.0, pH 6.0 decreased and pH 8.0 increased the amplitude of NMDA-activated current, but these changes in pH did not significantly alter the percentage inhibition by ethanol. 6. The sulphydryl reducing agent dithiothreitol (2 mM) increased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 7. Mg2+ (10, 100, 500 microM), (5, 20 microM) or ketamine (2, 10 microM) decreased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 8. The observations are consistent with ethanol inhibiting the function of NMDA receptors by a non-competitive mechanism that does not involve several modulatory sites on the NMDA receptor-ionophore complex.

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.

Alcohol and anesthetic actions on excitatory amino acid-activated ion channels.

The actions of alcohol and anesthetics have been studied on excitatory amino acid activated ion channels in mammalian neurons. Ethanol inhibits NMDA-activated current over a concentration range that produces intoxication, and the potency of several alcohols for inhibiting the NMDA-activated current is correlated with their intoxicating potency, suggesting that alcohol-induced inhibition of responses to NMDA receptor activation may contribute to the neural and cognitive impairments associated with intoxication. Studies on the mechanism of ethanol inhibition of NMDA-activated current indicate that ethanol does not appear to block the ion channel, alter the ion selectivity of the channel, or interact with previously described binding sites on the NMDA receptor/ionophore complex. The linear relation between the potency of several alcohols for inhibiting the NMDA-activated current and the hydrophobicity of the alcohols suggests that ethanol may inhibit the NMDA-activated ion current by a novel type of interaction with a hydrophobic site associated with the NMDA channel. In addition, different types of general anesthetic agents exhibit different inhibitory actions on NMDA-, kainate-, and quisqualate-activated currents, suggesting that differences in the profile of inhibition of excitatory amino acid neurotransmission in the CNS among different classes of general anesthetics may contribute to the differences in their behavioral and physiological effects.

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.