Nerve cells (i.e., neurons) communicate via a combination of electrical and chemical signals. Within the neuron, electrical signals driven by charged particles allow rapid conduction from one end of the cell to the other. Communication between neurons occurs at tiny gaps called synapses, where specialized parts of the two cells (i.e., the presynaptic and postsynaptic neurons) come within nanometers of one another to allow for chemical transmission. The presynaptic neuron releases a chemical (i.e., a neurotransmitter) that is received by the postsynaptic neuron’s specialized proteins called neurotransmitter receptors. The neurotransmitter molecules bind to the receptor proteins and alter postsynaptic neuronal function. Two types of neurotransmitter receptors exist—ligand-gated ion channels, which permit rapid ion flow directly across the outer cell membrane, and G-protein–coupled receptors, which set into motion chemical signaling events within the cell. Hundreds of molecules are known to act as neurotransmitters in the brain. Neuronal development and function also are affected by peptides known as neurotrophins and by steroid hormones. This article reviews the chemical nature, neuronal actions, receptor subtypes, and therapeutic roles of several transmitters, neurotrophins, and hormones. It focuses on neurotransmitters with important roles in acute and chronic alcohol effects on the brain, such as those that contribute to intoxication, tolerance, dependence, and neurotoxicity, as well as maintained alcohol drinking and addiction.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was John M. Littleton. The presentations were (1) Examination of ethanol spermine and acamprosate actions on native and recombinant NMDA receptors, by David Lovinger; (2) Ethanol inhibition of NMDA neurotoxicity on the polyamine site in cerebellar granule cells, by Sture Liljequist; (3) Alterations in expression of NMDA receptor subunits during ethanol exposure and withdrawal, by Raj Ticku; (4) Alterations in polyamine synthesis and release as a potential mechanism for ethanol dependence and withdrawal, by Izuru Matsumoto; (5) The role of polyamines in neurotoxicity induced by alcohol withdrawal in vitro, by John Littleton; and (6) Agmatine reduces some of the effects of "third trimester" alcohol exposure using a rodent model, by Susan Barron.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Toshio Narahashi and Kinya Kuriyama. The presentations were (1) Modulation of neuroreceptors and ion channels by alcohol, by T. Narahashi; (2) Inhibition by ethanol of NMDA and AMPA receptor-channels, by P. Illes, K. Wirkner, W. Fischer, K. Mühlberg, P. Scheibler, and C. Allgaier; (3) Effects of ethanol on metabotropic glutamate receptors, by K. Minami; (4) Acute alcohol actions on the 5-HT3 ligand-gated ion channel, by D. Lovinger; (5) Inhibition of NMDA receptors by MK801 attenuates ethanol-induced taurine release from the hippocampus, by F. Lallemand, R.J. Ward, and P. DeWitte; and (6) Effect of ethanol on voltage-operated Ca2+ channels in hepatic stellate cells, by T. Itatsu, Y. Takei, H. Oide, M. Hirose, X. E. Wang, S. Watanabe, M. Tateyama, R. Ochi, and N. Sato.

The neurotransmitter-gated ion channels form a superfamily of neurotransmitter receptors specialized for recognizing transmitters and rapidly gating ion channels that are contained within the same holoprotein complex. A large body of research indicates that alcohols alter the function of this class of receptor at concentrations relevant to the intoxicating and anesthetic effects of the alcohols. In addition, studies have implicated several types of neurotransmitter-gated channels in intoxicating and anesthetic alcohol actions. All of the neurotransmitter-gated ion channels contain conserved features such as N-terminal ligand binding domains, hydrophobic membrane spanning domains and charged pore-lining domains. However, at least two, and possibly three families of receptors have been identified within the superfamily, including the nicotinic ACh-like receptors, the ionotropic glutamate receptors and the ATP-gated ion channels. This review will begin with a brief overview of the structural features of the different receptors, with an emphasis on comparing and contrasting features of the different families of neurotransmitter-gated channels. The emphasis will be mostly on the nicotinic-like receptors and the iGluRs, since more is known about these receptors than about other types of ligand-activated cation channels. The remainder of the review focuses on the latest studies aimed at determining the mechanism of alcohol actions on this superfamily of receptor-channels as well as the relationship between the molecular structure of these channels and the effects of alcohols on channel function. In addition, emerging directions for future study of these effects of alcohols and possible regions of the protein that may be altered during alcohol exposure are discussed.

Norepinephrine contributes to antinociceptive, sedative, and sympatholytic responses in vivo, and a2 adrenergic receptor (a2AR) agonists are used clinically to mimic these effects. Lack of subtype-specific agonists has prevented elucidation of the role that each a2AR subtype (a2A, a2B, and a2C) plays in these central effects. Here we demonstrate that a2AR agonist-elicited sedative, anesthetic-sparing, and analgesic responses are lost in a mouse line expressing a subtly mutated a2AAR, D79N a2AAR, created by two-step homologous recombination. These functional changes are accompanied by failure of the D79N a2AAR to inhibit voltagegated Ca21 currents and spontaneous neuronal firing, a measure of K1 current activation. These results provide definitive evidence that the a2AAR subtype is the primary mediator of clinically important central actions of a2AR agonists and suggest that the D79N a2AAR mouse may serve as a model for exploring other possible a2AAR functions in vivo. a2-adrenergic receptors (a2ARs) present in the central nervous system (CNS) respond to norepinephrine (NE) and epinephrine and mediate sympatholytic, sedative-hypnotic, analgesic, anesthetic-sparing, hypotensive, and anxiolytic responses (1). Many of these responses are therapeutically useful and are exploited clinically, for example, during anesthesia and to attenuate the symptoms of opioid withdrawal (2). Three a2AR subtypes have been revealed by pharmacological (a2AAR, a2BAR, and a2CAR) and molecular cloning (a2aAR, a2bAR, and a2cAR) strategies (3), and all couple, via pertussis toxin-sensitive GiyGo proteins, to attenuation of adenylyl cyclase, suppression of voltage-gated Ca21 channels, and activation of inwardly rectifying K1 channels (4). Multiple experimental limitations have precluded clarifying the involvement of each a2AR subtype in catecholaminemediated physiological responses in the CNS. Subtype-specific a2AR agonists and antagonists are not available (5); even when subtype selectivity has been noted in vitro, varying and unknown in vivo bioavailability precludes confident correlation of the administered dose with the amount of drug at the receptor site. Previous studies to explore a2AR involvement in various responses have used prazosin to block catecholamine responses mediated by a1 adrenergic receptors (a1AR); however, it is now known that the a2BAR and a2CAR subtypes also are blocked by prazosin (5), thus confounding the interpretations of these earlier studies. In addition, because a1AR can functionally antagonize a2AR-mediated responses in some settings, a2AR responses in the presence of prazosin (added to block a1AR, a2BAR, and a2CAR) may reflect the disturbance of the balance between the functionally antagonistic a2AR and a1AR systems rather than provide insights concerning the role of the a2AAR subtype. Consequently, we manipulated the mouse genome to provide definitive evidence regarding the role of the a2AAR subtype in CNS responses. We used the ‘‘hit and run’’ targeting variant of homologous recombination (6, 7) to substitute a subtle mutation of the a2aAR, D79N into the mouse genome as a tool to explore the role of the a2aAR in vivo (8). The aspartate residue at position 79 (D79) is highly conserved in a topologically identical position in the second transmembrane span in a large subset of G protein-coupled receptors (9). Mutation of this residue has been shown to eliminate allosteric regulation of receptor binding by monovalent cations (10–13) and to perturb receptor–G protein–effector coupling (14–17) in heterologous expression systems. Thus, the animals expressing the D79N a2aAR provide the opportunity to examine the functional importance of the a2AAR in a variety of complex physiological and behavioral responses. EXPERIMENTAL PROCEDURES Mouse Lines. The D79N mouse line was created using a hit and run gene targeting strategy (18), as described (8). Male chimeras were mated with C57BLy6 mice to generate heterozygous mice for intercrosses. B6,129 hybrid offspring of wild-type (WT) and D79N breeding pairs were used in the present studies. Male chimeras also were mated with 129ySv females to establish the D79N mutation on a pure 129ySv background. These 129ySv D79N mice showed binding properties indistinguishable from those in the mixed genetic background (other functions not evaluated). In addition, the B6,129 heterozygous offspring of a chimera have been backcrossed against C57BLy6 to establish the D79N mutation on a pure C57BLy6 background (10 generations), and the mice have been made available to Jackson Laboratories (designated Adra2atm1Lel; #2–777). The purebred C57BLy6 animals showed changes in receptor binding and in vivo sedative The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y949950-6$2.00y0 PNAS is available online at http:yywww.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: a2AR, a2-adrenergic receptor; cDNA or genes encoding the a2AR subtypes are designated as a2aAR, a2bAR, and a2cAR; the pharmacologically defined subtype proteins are designated as a2AAR, a2BAR, and a2CAR; CNS, central nervous system; WT, wild-type; dex, dexmedetomidine; LC, locus ceruleus; LORR, loss of righting reflex; MHPG, 3-methoxy-4-hydroxyphenylethylene glycol; NE, norepinephrine; SCG, superior cervical ganglion; DAMGO, [D-Ala2, N-McPhe4, Gly5-ol]enkephalin. *P.P.L. and L.B.M. contributed equally to this study. ¶To whom reprint requests should be addressed. e-mail: Lee.Limbird@mcmail.vanderbilt.edu.

1. Protein kinase C (PKC) stimulators, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or cis-unsaturated fatty acid (UFA), have been shown to prolong synaptic enhancement induced by long-term potentiation (LTP). This observation suggests a role for PKC in the biochemical mechanisms underlying maintained enhancement. 2. To determine if PKC stimulators prolong LTP by acting selectively at synapses given high-frequency stimulation or by actions that are not synapse-specific (e.g. increased postsynaptic excitability) we examined the effect of TPA or UFA on input-selective enhancement. Population EPSPs, evoked in the same granule cell population by either the medial (MPP) or lateral (LPP) perforant path, can be selectively enhanced leaving the other perforant path input which receives only low-frequency stimulation as an internal control for PKC stimulator effects not specific to enhanced synapses. 3. Synapse-specific effects were in fact observed, as UFA or TPA selectively prolonged MPP enhancement following two trains of high-frequency MPP stimulation, without affecting responses evoked by the LPP. A similar synapse selectivity of PKC stimulator action was seen following high-frequency LPP stimulation. 4. These findings suggest that PKC stimulators prolong enhancement by acting specifically at high-frequency-stimulated synapses. PKC stimulators do not appear to affect either postsynaptic neurone excitability or synapses given only low-frequency stimulation. This provides further evidence that PKC acts synergistically with the consequences of repetitive synaptic activation to maintain enhancement.

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.