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Central Neuronal Communication, Drugs of Abuse, and Neuropathology

G.R. Siggins, P. Schweitzer, S. Madamba, Z. Nie, G. Martin, M. Tallent, T. Krucker, I. Petrou, W. Zieglgänsberger,* F. Berton,** W. Francesconi,** R. Przewlocki,*** D. Piomelli****

* Max Planck Institut für Psychiatrie, Munich, Germany
** University of Pisa, Pisa, Italy
*** Polish National Academy of Sciences, Krakow, Poland
**** The Neurosciences Institute, San Diego, CA

Our in vitro physiology group studies mechanisms of neuronal communication in the CNS and the effects of drugs of abuse and other neuropathologic events on neuronal function. We use electrophysiologic recordings of neurons from normal or transgenic animals and apply drugs, transmitters, cytokines, and viral products. We also stimulate inhibitory and excitatory synaptic transmission via electrodes.

We previously reported that the neuropeptide somatostatin inhibits neuronal firing by hyperpolarizing hippocampal pyramidal neurons (HPNs) by opening 2 potassium channels, the M-channel and the "leak" channel, and by depressing excitatory postsynaptic potentials (EPSPs) mediated by N-methyl-d-aspartate (NMDA) and non-NMDA glutamate receptors. Pharmacologic studies indicated that this depressant synaptic effect of somatostatin occurred presynaptically via reduction of glutamate release, through activation of sst₂ receptors linked to a G_i/G_o GTP-binding protein. Somatostatin particularly depressed polysynaptic (hyperexcitable) events. More recent studies showed that somatostatin selectively depresses feedforward excitatory connections between HPNs; such connections are critical for hippocampal seizure activity. Therefore, we used 2 in vitro models of epilepsy and showed that somatostatin significantly depressed epileptiform activity in the hippocampus, suggesting a function for endogenous somatostatin and a therapy for limbic seizures.

We have continued studies of drugs of abuse in the hippocampus and nucleus accumbens, regions thought to play major roles in drug abuse. We tested anandamide, a putative endogenous ligand for brain cannabinoid receptors, and other cannabinoids on HPNs. The results are presented in the report by P. Schweitzer, Department of Neuropharmacology.

We also continue to examine the role of endogenous opioids in the hippocampus, where dynorphin, enkephalins, and orphanin FQ (nociceptin) exist in interneurons and in afferent pathways. As reported previously, or µ opiate receptor agonists had no effect on membrane currents in HPNs, but the agonists dynorphin and U50,488h increased M-channel activity. Nociceptin mimicked this effect but also opened leak potassium channels. The opiate receptor antagonist naloxone and a receptor antagonist reversed the M-channel effect of all 3 ligands but not the leak channel effect of nociceptin, which was blocked by an antagonist for orphanin receptors. These findings suggest that peptides selective for and orphanin receptors have an inhibitory role and that nociceptin can postsynaptically modulate the excitability of HPNs through both opiate and nonopiate receptors linked to different potassium channels.

Previously, we showed that naloxone blocked a form of long-term depression, a cellular model of synaptic plasticity, in the hippocampus. These data suggested that the stimulation used for this long-term depression released opioid peptides that act on orphanin or receptors to play a role in long-term plasticity. Furthermore, in light of the role of calcium in plasticity, findings from our collaboration with D. Gruol, Department of Neuropharmacology, that opioids alter intracellular mobilization of calcium in cultured hippocampal neurons, have implications for the intracellular mediators of this plasticity.

We previously reported studies of the effects of alcohol in the hippocampus in which we used pharmacologically isolated inhibitory postsynaptic potentials (IPSPs) to evaluate alcohol enhancement of the effects of -aminobutyric acid (GABA). We found that ethanol augmented GABA_A receptor--mediated IPSPs only if GABA_B receptors were blocked. To determine the generality of this complex interaction between ethanol and GABA receptors, we repeated these studies in the nucleus accumbens and had equivalent results.

However, in the nucleus accumbens, unlike in the hippocampus, ethanol often enhanced the currents evoked by exogenous GABA, without blocking of GABA_B receptors. Superfusion of glutamate or of a metabotropic glutamate receptor agonist also augmented GABA responses, and a metabotropic receptor antagonist inhibited these effects and ethanol enhancement of GABA currents. An activator of protein kinase C also augmented ethanol-GABA interactions. These data suggest that ethanol enhancement of GABAergic neurotransmission in the nucleus accumbens is regulated by multiple metabotropic influences, including postsynaptic enhancement of the interaction between ethanol and GABA receptors by metabotropic glutamate receptors acting through protein kinase C.

The nucleus accumbens also contains opioid peptides. Both opiates and ethanol presynaptically decrease glutamatergic EPSPs, and naloxone reverses some of the ethanol effect, suggesting that the addictive properties of alcohol could involve opiate receptors. Ethanol also postsynaptically reduced responses to exogenous NMDA. Furthermore, GABA_B (metabotropic) receptor antagonists blocked the ethanol reduction of NMDA receptor--mediated EPSPs (NMDA-EPSPs), another example of metabotropic regulation of the effects of ethanol. These multiple ethanol actions and metabotropic regulations could represent neuroadaptations that underlie alcohol craving.

This hypothesis is consistent with our findings in studies of neurons taken from the nucleus accumbens of rats withdrawn from several weeks of ethanol treatment. Compared with findings in control neurons, the dose-response curve for exogenous NMDA in these neurons was shifted to the left, indicating that long-term exposure to ethanol sensitizes NMDA receptors. This change could underlie the craving or relapse of abstaining alcoholics.

Studies with acamprosate, a new drug that reduces relapse in abstaining alcoholics, support this possibility. Acamprosate increased NMDA-EPSPs in both hippocampal and accumbens neurons in vitro, an indication that its clinical efficacy may be due to modulation of NMDA receptors. We also found that acamprosate inhibits presynaptic GABA_B receptors, an action that also could play a role in its clinical effects.

Because opiates reduce glutamatergic EPSPs, the latter may be major sites in the rewarding effects of heroin. Data from other laboratories suggest that NMDA receptors play some role in opiate tolerance and dependence. We found that activation of µ opiate receptors in the nucleus accumbens decreased amplitudes of glutamatergic EPSPs yet enhanced currents evoked by exogenous NMDA. Furthermore, metabotropic glutamate receptor agonists reduced NMDA-EPSPs via both presynaptic and postsynaptic metabotropic receptors.

Further, we found that the presynaptic inhibitory effect of a group 2 metabotropic agonist, but not the postsynaptic group 3 agonists, on the size of NMDA-EPSPs was increased in neurons from the nucleus accumbens of morphine-treated rats. Changes in paired-pulse facilitation of the NMDA-EPSPs further suggested a presynaptic effect of morphine treatment. Also, augmentation of the NMDA-EPSPs by a protein kinase C activator was significantly reduced in neurons from morphine-treated rats. These data suggest that both NMDA and metabotropic glutamate receptors may be involved in morphine tolerance and dependence.

Studies on viral infection, cytokines, and brain function continue. One hypothesis suggests that AIDS-related dementia is due to neurotoxic effects of the HIV coat protein gp120. Therefore, we studied synaptic transmission and plasticity in hippocampal slices from transgenic mice that express gp120. These slices had greater paired-pulse facilitation and short-term potentiation than did slices from littermate controls, but the magnitude of long-term potentiation (LTP) was reduced. Superfused gp120 also decreased LTP, suggesting that the effects of the transgene were not developmentally compensatory. Moreover, NMDA-EPSPs were increased in gp120 transgenic mice. These alterations by gp120 model memory impairment in AIDS patients and support the HIV coat-protein hypotheses of AIDS-related dementia.

In collaboration with I. Campbell, Department of Neuropharmacology, we used transgenic mice that overexpress IL-6 or IFN- to investigate the role of cytokines in neuropathologic events. Compared with hippocampal slices from littermate controls, slices from IL-6 transgenic mice showed decreased of LTP in both the dentate and the CA1 and decreased excitability in the CA1. In contrast, CA1 hippocampal slices from transgenic mice that overexpress IFN- showed increased LTP and synaptic excitability, in accord with the overt epileptic seizures seen in these animals. These studies suggest that cytokines can disrupt neuronal function in the hippocampus.

PUBLICATIONS

Berton, F., Francesconi, W., Madamba, S., Siggins, G.R. Acamprosate enhances the N-methyl-d-aspartate receptor-mediated neurotransmission but inhibits presynaptic GABA_B receptors in nucleus accumbens neurons. Alcohol. Clin. Exp. Res. 22:183, 1998.

Krucker, T., Toggas, S.M., Mucke, L., Siggins, G.R. Dissociation of short-term and long-term potentiation in CA1 hippocampus of transgenic mice with cerebral expression of the HIV-1 coat protein. Neuroscience 83:691, 1998.

Martin, G., Siggins, G.R. Metabotropic glutamate receptors regulate N-methyl-d-aspartate-receptor-mediated responses in nucleus accumbens neurons in vitro. J. Neurophysiol. 78:3028, 1997.

Schweitzer, P., Madamba, S., Siggins, G.R. Somatostatin increases a voltage-insensitive potassium conductance in rat CA1 hippocampal neurons. J. Neurophysiol. 79:1230, 1998.

Siggins, G., Nie, Z., Madamba, S. A metabotropic hypothesis for ethanol sensitivity of GABAergic and glutamatergic central synapses. In: The 'Drunken' Synapse: Studies of Alcohol Related Disorders. Liu, Y. (Ed.). Plenum, New York, in press.

Tallent, M., Siggins, G.R. Somatostatin depresses excitatory but not inhibitory neurotransmission in rat CA1 hippocampus. J. Neurophysiol. 78:3008, 1997.