Basis of the gabamimetic profile of ethanol.

This article summarizes the proceedings of a symposium held at the 2005 Research Society on Alcoholism meeting. The initial presentation by Dr. Wallner provided evidence that selected GABA(A) receptors containing the delta subunit display sensitivity to low intoxicating ethanol concentrations and this sensitivity is further increased by a mutation in the cerebellar alpha6 subunit, found in alcohol-hypersensitive rats. Dr. Mameli reported that ethanol affects gamma-aminobutyric acid (GABA) function by affecting neural circuits that influence GABA release. Dr. Parsons presented data from electrophysiological and microdialysis investigations that ethanol is capable of releasing GABA from presynaptic terminals. Dr. Morrow demonstrated that systemic ethanol increases neuroactive steroids in brain, the absence of which alters various functional responses to ethanol. Dr. Criswell presented evidence that the ability of ethanol to increase GABA was apparent in some, but not all, brain regions indicative of regional specificity. Further, Dr. Criswell demonstrated that neurosteroids alone and when synthesized locally by ethanol act postsynaptically to enhance the effect of GABA released by ethanol in a region specific manner. Collectively, this series of reports support the GABAmimetic profile of acutely administered ethanol being dependent on several specific mechanisms distinct from a direct effect on the major synaptic isoforms of GABA(A) receptors.

Nociceptin reduces epileptiform events in CA3 hippocampus via presynaptic and postsynaptic mechanisms.

The opiate-like peptide nociceptin/orphanin FQ (Noc) and its receptor [opiate receptor-like receptor (ORL-1)] are highly expressed in the hippocampus. Noc has inhibitory postsynaptic actions in CA1, CA3, and the dentate and seems to lack the disinhibitory, excitatory actions demonstrated for some opiate peptides in the hippocampus. The CA3 hippocampal region is important in the generation of hippocampal seizures. Therefore, we tested the action of Noc on spontaneous epileptiform activity recorded extracellularly or intracellularly in CA3 and generated by removal of Mg(2+) from the bathing solution or by raising extracellular K(+) from 3.5 to 7.5 mm. Superfusion of Noc robustly depressed spontaneous bursting without desensitization. The ORL-1 antagonist [Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) (1-2 microm) greatly attenuated the reduction of spontaneous bursting by Noc. To characterize the cellular mechanism of action of Noc, we recorded intracellularly from CA3 pyramidal neurons. Noc reduced EPSCs evoked by stimulating either mossy or associational/commissural fibers. Analysis of miniature EPSCs using whole-cell voltage-clamp recording suggests that Noc acts presynaptically to inhibit glutamate release. This is the first demonstration of a presynaptic effect for Noc in the hippocampus. Noc also increased K(+) currents in CA3 pyramidal neurons, including the voltage-sensitive M-current. Blocking the M-current with linopirdine increased the duration of individual CA3 bursts but did not attenuate Noc-mediated inhibition of bursting. Thus, Noc acts via multiple mechanisms to reduce excitation in CA3. However, Noc inhibition of epileptiform events is not dependent on augmentation of the M-current.

Ethanol inhibition of N-methyl-D-aspartate responses involves presynaptic gamma-aminobutyric acid(B) receptors.

Ethanol alters N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid subtype A (GABA(A)) receptor-mediated neurotransmission. We have previously demonstrated that GABA(B) receptor blockade uncovers ethanol enhancement of GABA(A) responses in the hippocampus. Therefore, we evaluated in vivo and in vitro the role of GABA(B) receptors in ethanol-induced inhibition of neuronal activity as well as NMDA responses in the hippocampus, ventral tegmental area (VTA), and nucleus accumbens (NAcc), three brain areas with known sensitivity to low doses of ethanol. In vivo, in situ microelectrophoretic application of ethanol enhanced inhibition of VTA GABA neuron firing rate by the GABA(B) agonist baclofen and reduced inhibition of VTA GABA firing rate by the GABA(A) agonist muscimol. The GABA(B) antagonist CGP35348 blocked baclofen- and ethanol-induced, but not muscimol-induced, reduction of NMDA-activated firing of hippocampal hilar mossy cells, hilar interneurons, and VTA GABA neurons, as well as ethanol inhibition of NMDA receptor-sensitive, amygdala-driven NAcc neurons. We performed in vitro studies in NAcc slices to evaluate the mechanism of GABA(B) receptor-mediated ethanol inhibition of NMDA neurotransmission. In the presence of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and the GABA(A) receptor antagonist bicuculline, superfusion of the GABA(B) antagonist CGP55845 blocked ethanol (66 mM) inhibition of evoked NMDA receptor-mediated excitatory postsynaptic potentials. However, CGP55845 did not significantly affect ethanol inhibition of NMDA currents produced by pressure application of NMDA or non-NMDA glutamatergic excitatory postsynaptic potentials evoked in the presence of the bicuculline and the NMDA antagonist DL-2-amino-5-phosphonovalerate. Taken together, these findings suggest that the sensitivity of NMDA receptor-mediated neurotransmission to ethanol is regulated by GABA(B) receptors, possibly at presynaptic sites.

A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus.

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg(2+)-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg(2+)-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

Dynamic actin filaments are required for stable long-term potentiation (LTP) in area CA1 of the hippocampus.

The hypothesis that dynamic actin filaments participate in specific aspects of synaptic plasticity was investigated at the Schaffer-collateral-CA1 pyramidal cell synapse of mouse hippocampus. Low concentrations (0.01-1 microM) of compounds that inhibit actin filament assembly were bath applied to hippocampal slices during extracellular recording of field excitatory postsynaptic potentials. Cytochalasin D, cytochalasin B, and latrunculin A all impaired the maintenance of LTP induced by brief high-frequency stimulation. This effect on LTP maintenance was specific, because none of the compounds affected basal synaptic transmission, paired-pulse facilitation, LTP induction, or post-tetanic potentiation. The effect of cytochalasin B was reversible. The results are consistent with a model in which dynamic actin filaments play an essential role in the molecular mechanisms underlying the early maintenance phase of LTP, such as growth of new synaptic connections or conversion of silent synapses.

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.

Opioid enhancement of calcium oscillations and burst events involving NMDA receptors and L-type calcium channels in cultured hippocampal neurons.

Opioid receptor agonists are known to alter the activity of membrane ionic conductances and receptor-activated channels in CNS neurons and, via these mechanisms, to modulate neuronal excitability and synaptic transmission. In neuronal-like cell lines opioids also have been reported to induce intracellular Ca(2+) signals and to alter Ca(2+) signals evoked by membrane depolarization; these effects on intracellular Ca(2+) may provide an additional mechanism through which opioids modulate neuronal activity. However, opioid effects on resting or stimulated intracellular Ca(2+) levels have not been demonstrated in native CNS neurons. Thus, we investigated opioid effects on intracellular Ca(2+) in cultured rat hippocampal neurons by using fura-2-based microscopic Ca(2+) imaging. The opioid receptor agonist D-Ala(2)-N-Me-Phe(4),Gly-ol(5)-enkephalin (DAMGO; 1 microM) dramatically increased the amplitude of spontaneous intracellular Ca(2+) oscillations in the hippocampal neurons, with synchronization of the Ca(2+) oscillations across neurons in a given field. The effects of DAMGO were blocked by the opioid receptor antagonist naloxone (1 microM) and were dependent on functional NMDA receptors and L-type Ca(2+) channels. In parallel whole-cell recordings, DAMGO enhanced spontaneous, synaptically driven NMDA receptor-mediated burst events, depolarizing responses to exogenous NMDA and current-evoked Ca(2+) spikes. These results show that the activation of opioid receptors can augment several components of neuronal Ca(2+) signaling pathways significantly and, as a consequence, enhance intracellular Ca(2+) signals. These results provide evidence of a novel neuronal mechanism of opioid action on CNS neuronal networks that may contribute to both short- and long-term effects of opioids.

Dynorphin selectively augments the M-current in hippocampal CA1 neurons by an opiate receptor mechanism.

Most electrophysiological studies of opioids on hippocampal principal neurons have found indirect actions, usually through interneurons. However, our laboratory recently found reciprocal alteration of the voltage-dependent K(+) current, known as the M-current (I(M)), by kappa and delta opioid agonists in CA3 pyramidal neurons. Recent ultrastructural studies have revealed postsynaptic delta opiate receptors on dendrites and cell bodies of CA1 and CA3 hippocampal pyramidal neurons (HPNs). Reasoning that previous electrophysiological studies may have overlooked voltage-dependent postsynaptic effects of the opioids in CA1, we reevaluated their role in CA1 HPNs using the rat hippocampal slice preparation for intracellular current- and voltage-clamp recording. None of the delta and mu; receptor-selective opioids tested, including [D-Pen(2,5)]-enkephalin (DPDPE), [D-Ala(2)]-deltorphin II (deltorphin), [D-Ala(2), NMe-Phe(4), Gly-ol]-enkephalin (DAMGO), and [D-Ala(2), D-Leu(5)] enkephalin (DADLE), altered membrane properties such as I(M) or Ca(2+)-dependent spikes in CA1 HPNs. The nonopioid, Des-Tyr-dynorphin (D-T-dyn), also had no effect. By contrast, dynorphin A (1-17) markedly increased I(M) at low concentrations and caused an outward current at depolarized membrane potentials. The opioid antagonist naloxone and the kappa receptor antagonist nor-binaltorphimine (nBNI) blocked the I(M) effect. However, the kappa-selective agonists U69,593 and U50,488h did not significantly alter I(M) amplitudes when averaged over all cells tested, although occasional cells showed an I(M) increase with U50,488h. Our results suggest that dynorphin A postsynaptically modulates the excitability of CA1 HPNs through opiate receptors linked to voltage-dependent K(+) channels. These findings also provide pharmacological evidence for a functional kappa opiate receptor subtype in rat CA1 HPNs but leave unanswered questions on the role of delta receptors in CA1 HPNs.

Nociceptin augments K(+) currents in hippocampal CA1 neurons by both ORL-1 and opiate receptor mechanisms.

We previously reported (see also the accompanying paper) that dynorphin A significantly enhanced the voltage-dependent K(+) M-current (I(M)) in CA3 and CA1 hippocampal pyramidal neurons (HPNs). Because the opioid-receptor-like-1 (ORL-1) receptor shares a high sequence homology with opioid receptors and is expressed in rat hippocampus, we examined the effects of orphanin FQ or nociceptin, the endogenous ligand for the ORL-1 receptor, using the rat hippocampal slice preparation and intracellular voltage-clamp recording. Current-voltage (I-V) relationships from CA1 HPNs revealed that nociceptin superfusion induced an outward current reversing near the equilibrium potential for K(+) ions. Ba(2+) (2 mM) blocked this effect. The nociceptin-induced current was largest at depolarized membrane potentials, where I(M) is largely activated. Nociceptin concentrations of 0.5-1 microM (but not 0.1 microM) significantly increased I(M) relaxation amplitudes with recovery on washout. Interestingly, both the general opiate antagonist naloxone and the kappa receptor antagonist nor-binaltorphimine (nBNI) inhibited the nociceptin-induced I(M) increases and outward currents in the depolarized range but not the inward current induced at hyperpolarized potentials. The putative ORL-1 receptor antagonist, [Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) (hereafter ORLAn), blocked most of the nociceptin current near rest but not the I(M) increase. However, ORLAn alone had direct effects similar to those of nociceptin, indicating that ORLAn might be a partial agonist. Our results suggest that nociceptin postsynaptically modulates the excitability of HPNs through ORL-1 and kappa-like opiate receptors linked to different K(+) channels.

Chronic morphine treatment alters NMDA receptor-mediated synaptic transmission in the nucleus accumbens.

In a study of a possible substrate underlying morphine addiction, we examined NMDA receptor-mediated synaptic transmission of core nucleus accumbens neurons after chronic morphine treatment, using intracellular recording in a slice preparation of rat. We evoked pharmacologically isolated NMDA EPSCs by local stimulation and elicited inward currents by NMDA superfusion. In control slices, Mg(2+) and phorbol 12,13-diacetate (PDAc), a protein kinase C activator, strongly inhibited and increased, respectively, NMDA EPSC amplitudes. The PDAc effects were likely postsynaptic because PDAc enhanced the currents evoked by superfused NMDA to the same extent that it did the NMDA EPSCs. Chronic morphine treatment significantly decreased NMDA EPSC amplitudes and the sensitivity of NMDA EPSCs to Mg(2+) and PDAc, as well as the kinetics of the decay (inactivation rate) of the EPSCs (from 97 +/- 2.5 msec in untreated rats to 78.7 +/- 1.8 msec in slices from treated rats). One week after withdrawal, the Mg(2+) and PDAc effects were still significantly less than those in control slices. Interestingly, 1 week of withdrawal led to an increased NMDA EPSC inactivation rate compared with controls. These data demonstrate that chronic morphine treatment significantly alters NMDA receptor-mediated synaptic transmission in the accumbens, and these effects persist 1 week after withdrawal. These long-term effects may represent an important neuroadaptation in opiate dependence.

Structural and functional neuropathology in transgenic mice with CNS expression of IFN-alpha.

Cytokines belonging to the type I interferon (e.g. interferon-alpha) family are important in the host response to infection and may have complex and broad ranging actions in the central nervous system (CNS) that may be beneficial or harmful. To better understand the impact of the CNS expression of the type I interferons (IFN), transgenic mice were developed that produce IFN-alpha(1) chronically from astrocytes. In two independent transgenic lines with moderate and low levels of astrocyte IFN-alpha mRNA expression respectively, a spectrum of transgene dose- and age-dependent structural and functional neurological alterations are induced. Structural changes include neurodegeneration with loss of cholinergic neurons, gliosis, angiopathy with mononuclear cell cuffing, progressive calcification affecting basal ganglia and cerebellum and the up-regulation of a number of IFN-alpha-regulated genes. At a functional level, in vivo and in vitro electrophysiological studies revealed impaired neuronal function and disturbed synaptic plasticity with pronounced hippocampal hyperexcitability. Severe behavioral alterations were also evident in higher expressor GFAP-IFNalpha mice which developed fatal seizures around 13 weeks of age precluding their further behavioral assessment. Modest impairments in discrimination learning were measured in lower expressor GFAP-IFNalpha mice at various ages (7-42 weeks). The behavioral and electrophysiological findings suggest regional changes in hippocampal excitability which may be linked to abnormal calcium metabolism and loss of cholinergic neurons in the GIFN mice. Thus, these transgenic mice provide a novel animal model in which to further evaluate the mechanisms that underlie the diverse actions of type I interferons in the intact CNS and to link specific structural changes with functional impairments.

Somatostatin acts in CA1 and CA3 to reduce hippocampal epileptiform activity.

Although the peptide somatostatin (SST) has been speculated to function in temporal lobe epilepsy, its exact role is unclear, as in vivo studies have suggested both pro- and anticonvulsant properties. We have shown previously that SST has multiple inhibitory cellular actions in the CA1 region of the hippocampus, suggesting that in this region SST should have antiepileptic actions. To directly assess the effect of SST on epileptiform activity, we studied two in vitro models of epilepsy in the rat hippocampal slice preparation using extracellular and intracellular recording techniques. In one, GABA-mediated neurotransmission was inhibited by superfusion of the GABAA receptor antagonist bicuculline. In the second, we superfused Mg2+-free artificial cerebrospinal fluid to remove the Mg2+ block of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We show here that SST markedly reduces the intensity of evoked epileptiform afterdischarges and the frequency of spontaneous bursts in both CA1 and CA3. SST appears to act additively in the two regions to suppress the transmission of epileptiform events through the hippocampus. We further examined SST's actions in CA3 and found that SST dramatically reduced the frequency of paroxysmal depolarizing shifts (PDSs) recorded intracellularly in current clamp, as well as increasing the threshold for evoking "giant" excitatory postsynaptic currents (EPSCs), large polysynaptically mediated EPSCs that are the voltage-clamp correlate of PDSs. We also examined the actions of SST on pharmacologically isolated EPSCs generated at both mossy fiber (MF) and associational/commissural (A/C) synapses. SST appears to act specifically to reduce recurrent excitation between CA3 neurons because it depresses A/C- but not MF-evoked EPSCs. SST also increased paired-pulse facilitation of A/C EPSCs, suggesting a presynaptic site of action. Reciprocal activation of CA3 neurons through A/C fibers is critical for generation of epileptiform activity in hippocampus. Thus SST reduces feedforward excitation in rat hippocampus, acting to "brake" hyperexcitation. This is a function unique from that described for other hippocampal neuropeptides, which affect more standard neurotransmission. Our results suggest that SST receptors could be a unique, selective clinical target for treatment of limbic seizures.

Chronic morphine treatment selectively augments metabotropic glutamate receptor-induced inhibition of N-methyl-D-aspartate receptor-mediated neurotransmission in nucleus accumbens.

We compared the effects of different metabotropic glutamate receptor (mGluR) agonists on pharmacologically isolated N-methyl-D-aspartate-excitatory postsynaptic currents (NMDA-EPSCs) in core nucleus accumbens neurons using conventional intracellular recording in untreated and morphine-treated rats. The rats were treated by s.c. implantation of two morphine pellets and studied over a 3- to 6-day period. This model is known to exhibit opiate tolerance and dependence. We elicited NMDA-EPSCs by stimulating locally in the presence of the alpha-amino-3-hydroxy-5-methly-4-isoxazolepropionic acid/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and the gamma-aminobutyric acid receptor antagonist bicuculline (15 microM). We found that trans-1-aminocyclopentane-1,3-decarboxylic acid, an agonist of group 1 and 2 mGluRs, decreased NMDA-EPSC areas (time-integrals) in a dose-dependent manner (1-10 microM) in slices taken from untreated rats. This inhibitory effect was significantly enhanced after chronic morphine treatment. In contrast, although the group 3 mGluR agonist L(+)-2-amino-4-phosphonobutyric acid also markedly reduced NMDA-EPSC areas, there was no apparent change in this effect after chronic morphine. We found that quisqualate, the group 1 mGluR agonist, failed to elicit any effect on NMDA-EPSCs in either untreated or chronically treated rats. Paired-pulse stimulation of core nucleus accumbens NMDA-EPSCs in slices from these groups showed that chronic morphine enhanced paired-pulse facilitation, consistent with a presynaptic reduction in glutamate release. Because of the relevance to opiate tolerance and dependence of the chronic model used, the brain region (accumbens), and the receptors studied, our data provide a cellular substrate that could account for some aspects of these phenomena.

Somatostatin increases a voltage-insensitive K+ conductance in rat CA1 hippocampal neurons.

Somatostatin (SST) is a neuropeptide involved in several central processes. In hippocampus, SST hyperpolarizes CA1 pyramidal neurons and augments the K+ M current (IM). However, the limited involvement of IM at resting potential in these cells suggests that the peptide also may modulate another channel to hyperpolarize hippocampal pyramidal neurons (HPNs). We studied the effect of SST on noninactivating conductances of rat CA1 HPNs in a slice preparation. Using MK886, a specific inhibitor of the enzymatic pathway that leads to the augmentation of IM by SST, we have uncovered and characterized a second conductance activated by the peptide. SST did not affect IM when applied with MK886 or the amplitudes of the slow Ca2+-dependent K+ afterhyperpolarization-current and the cationic Q current but still caused an outward current, indicating that SST acts upon another conductance. In the presence of MK886, SST elicited an outward current that reversed around -100 mV and that displayed a linear current-voltage relationship. Reversal potentials obtained in different external K+ concentrations are consistent with a conductance carried solely by K+ ions. The slope of the current-voltage relationship increased proportionately with the extracellular K+ concentration and remained linear. This suggests that SST opens a voltage-insensitive leak current (IK(L)) in HPNs not an inwardly rectifying K+ current as reported in other neuron types. A low concentration of extracellular Ba2+ (150 M) only slightly decreased the SST-induced effect in a voltage-independent manner, whereas a high concentration of Ba2+ (2 mM) completely blocked it. Extracellular Cs+ (2 mM) did not affect the outward SST current but inhibited the inward component. We conclude that SST inhibits HPNs by activating two different K+ conductances: the voltage-insensitive IK(L) and the voltage-dependent IM. The hyperpolarizing effect of SST at resting membrane potential appears to be mainly carried by IK(L), whereas IM dominates at slightly depolarized potentials.

Transgenic mice with cerebral expression of human immunodeficiency virus type-1 coat protein gp120 show divergent changes in short- and long-term potentiation in CA1 hippocampus.

The human immunodeficiency virus type-1 envelope glycoprotein gp120 is shed from the virus and from infected cells and thus can diffuse and interact with a variety of central nervous system cells. Transgenic mice constitutively expressing glial fibrillary acidic protein-driven gp120 from brain astrocytes display neuronal and glial changes resembling abnormalities in human immunodeficiency virus type-1-infected human brains. To assess the neurophysiology of these transgenic mice and determine whether gp120 expression impairs synaptic plasticity, we examined CA1 population excitatory postsynaptic potentials in hippocampal slices from transgenic mice and from non-transgenic controls, using a double-blind protocol. Compared with slices from non-transgenic littermate controls, slices from gp120 transgenic mice showed four significant alterations: (i) increased mean slopes of normalized population excitatory postsynaptic potentials; (ii) larger paired-pulse facilitation after induction of long-term potentiation at 50 ms interpulse intervals; (iii) markedly elevated short-term potentiation after 10 and 20 shocks at 100 Hz; and (iv) a significant reduction in the magnitude of CA1 long-term potentiation. In slices from transgenic mice expressing Escherichia coli beta-galactosidase from the same promoter, paired-pulse facilitation and long-term potentiation were normal. These results indicate that brain slice preparations from gp120 transgenic mice can be used to assess pathophysiological effects of gp120 on neuronal networks. Because short-term potentiation involves presynaptic mechanisms, our results suggest that gp120 expression in these mice enhances either presynaptic glutamate release or postsynaptic glutamate receptor function, or both. These changes could lead to increased Ca2+ influx, thereby contributing to neuronal dysfunction and injury. As long-term potentiation is a cellular model of learning and memory, our results may be relevant to memory (cognitive) impairments seen in patients with AIDS.

Acamprosate enhances N-methyl-D-apartate receptor-mediated neurotransmission but inhibits presynaptic GABA(B) receptors in nucleus accumbens neurons.

Acamprosate (calcium acetylhomotaurine) is used therapeutically in Europe to reduce relapse in weaned alcoholics. However, the mechanisms of acamprosate action in the central nervous system are still obscure, although early studies suggested an action on GABA receptors. The nucleus accumbens (NAcc) is a brain region thought to underlie ethanol reinforcement. Recent studies from our laboratory have demonstrated that ethanol inhibits both N-methyl-D-aspartate (NMDA) and non-NMDA types of glutamatergic synaptic transmission in the NAcc. In the present study, we used voltage- and current-clamp intracellular recording of NAcc core neurons in a slice preparation to examine acamprosate actions on resting membrane properties and pharmacologically isolated synaptic responses. We isolated NMDA and non-NMDA receptor-mediated excitatory postsynaptic potentials or currents (EPSP/Cs) with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovalerate (d-APV), respectively. Bicuculline was also included to block GABA(A) receptors. Superfusion of acamprosate (5, 50, and 300 microM) did not alter the resting membrane properties of NAcc neurons. However, 300 microM acamprosate significantly increased the NMDA receptor-mediated components of EPSP/Cs (NMDA-EPSP/Cs) with recovery on washout. In contrast, 300 microM acamprosate had no significant effect on the non-NMDA receptor component of the EPSP/Cs (non-NMDA-EPSP/Cs). To test acamprosate actions on the GABA system, we superfused 60 microM d-APV and 20 microM CNQX to block glutamatergic transmission and evoked monosynaptic GABA(A) receptor-mediated synaptic responses within the NAcc. Acamprosate (300 microM) did not change these monosynaptic GABA(A)-IPSCs. We also used a paired-pulse paradigm to test whether acamprosate could act on presynaptic GABA(B) autoreceptors, in the presence of d-APV and CNQX to block glutamatergic transmission. Like 0.5 microM CGP 34358 (a GABA[B] receptor blocker), acamprosate significantly decreased the paired-pulse inhibition (PPI) of GABA(A)-IPSCs at short interstimulus intervals (ISIs). Thus, acamprosate may concomitantly enhance NMDA-EPSP/Cs while blocking presynaptic GABA(B) receptor-mediated inhibition of GABA release. These results suggest that acamprosate's clinical efficacy in preventing relapse in weaned alcoholics could derive from its interactions with both the glutamatergic and GABAergic systems in the NAcc.

Transgenic models to assess the pathogenic actions of cytokines in the central nervous system.

In order to better understand the actions of proinflammatory cytokines in the mammalian CNS, a transgenic approach was employed in which the expression of IL-6, IL-3 or TNF-alpha was targeted to astrocytes in the intact CNS of mice. Transgenic mice exhibited distinct chronic-progressive neurological disorders with neurodegeneration and cognitive decline due to IL-6 expression, macrophage/microglial-mediated primary demyelination with motor impairment due to IL-3 expression and lymphocytic meningoencephalomyelitis with paralysis induced by TNF-alpha expression. Thus, expression of specific cytokines alone in the intact CNS results in unique neuropathological alterations and functional impairments, thereby directly implicating these mediators in the pathogenesis of CNS disease.

mu-Opioid receptors modulate NMDA receptor-mediated responses in nucleus accumbens neurons.

The nucleus accumbens (NAcc) may play a major role in opiate dependence, and central NMDA receptors are reported to influence opiate tolerance and dependence. Therefore, we investigated the effects of the selective mu-opioid receptor agonist [D-Ala2-N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) on membrane properties of rat NAcc neurons and on events mediated by NMDA and non-NMDA glutamate receptors, using intracellular recording in a brain slice preparation. Most NAcc neurons showed a marked inward rectification (correlated with Cs+- and Ba2+-sensitive inward relaxations) when hyperpolarized, as well as a slowly depolarizing ramp with positive current pulses. Superfusion of DAMGO did not alter membrane potential, input resistance, or the inward relaxations. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) used to block non-NMDA glutamate receptors and bicuculline to block GABAA receptors, EPSPs evoked by local stimulation displayed characteristics of an NMDA component: (1) long duration, (2) voltage sensitivity, and (3) blockade by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (D-APV). DAMGO (0.1-1 muM) significantly decreased both NMDA- and non-NMDA-EPSP amplitudes with reversal of this effect by naloxone and the mu-selective antagonist [Cys2-Tyr3-Orn5-Pen7]-somatostatinamide (CTOP). To assess a postsynaptic action of DAMGO, we superfused slices with tetrodotoxin and evoked inward currents by local application of glutamate agonists. Surprisingly, 0.1-1 microM DAMGO markedly enhanced the NMDA currents (with reversal by CTOP) but reduced the non-NMDA currents. At higher concentrations (5 microM), DAMGO reduced NMDA currents, but this effect was enhanced, not blocked, by CTOP. These results indicate a complex DAMGO modulation of the NMDA component of glutamatergic synaptic transmission in NAcc: mu receptor activation decreases NMDA-EPSP amplitudes presynaptically yet increases NMDA currents postsynaptically. These new data may provide a cellular mechanism for the previously reported role of NMDA receptors in opiate tolerance and dependence.

Naloxone blocks long-term depression of excitatory transmission in rat CA1 hippocampus in vitro.

In rat hippocampal slices, high intensity tetanic stimulation (two 1 s trains of 100 Hz separated by 20 s, 3-5X intensity of the test stimulus) of the Schaffer collateral-commissural (SCC) fibers induced a long-term depression (LTD) of the negative field excitatory postsynaptic potentials (fEPSP) in stratum radiatum of the CA1 region. The initial slope of the fEPSP, evoked by a single test shock applied to the SCC fibers, was depressed for a period longer than 40 min following such high intensity tetanic stimulation to this fiber system. However, the same tetanic stimulation delivered at low (test) intensity induced long-term potentiation (LTP) of the fEPSPs. Thus, similar patterns of stimulation can induce either LTP or LTD, depending on whether low- or high-intensity tetanic stimuli are delivered. The LTD induced by high strength tetanic stimulation was clearly blocked by the opioid antagonist naloxone (1 microM); however, the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoate (AP5; 50 microM) had no effect on the LTD. Our data suggest that the strong stimulation used for LTD induction may have activated other afferent fiber systems and/or local interneurons in addition to SCC fibers, such as the enkephalin-containing terminals of the perforant path (PP) projecting to the stratum lacunosum moleculare or opioid peptide-containing interneurons. Thus, the resulting release of endogenous opioid peptides could play a role in the cellular mechanisms involved in some forms of long-term synaptic depression.