Drug and Alcohol Dependence, 4 (1979) 307 - 319 @ Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

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CYCLIC NUCLEOTIDES IN THE DEVELOPMENT OF ALCOHOL TOLERANCE AND DEPENDENCE: A COMMENTARY

GEORGE ROBERT SIGGINS

Arthur V. Davis Center for Behavioral Neurobiology, The Salk Institute, San Diego, California 92112 (U.S.A.)

Summary

This paper aims to outline some of the caveats inherent in studies on first and second messengers in the central nervous system as related to tolerance and dependence. Analysis of systems based on measures of first and second messengers may be an over-simplification, since the actual mole- cular consequences of neurotransmission might result in a multilevel cascade of many sequential messages including protein kinase activation and protein phosphorylation and/or dephosphorylation.

Introduction

Implicit in current concepts of cyclic nucleotides in normal brain func- tion is their role as mediators or “second messengers” of first messages com- municated by agents such as neurotransmitters, hormones or “local modula- tors” [ 1 - 41. Although alcohol might readily exert direct effects on cyclic nucleotides independent of any action on these “first messengers”, initially it would seem helpful to develop criteria for testing those first messages most likely to utilize cyclic nucleotides as their mediators under normal condi- tions. Armed with these criteria we might then proceed to analyze those neurons or brain structures for which there is strongest evidence of cyclic nucleotide involvement in expression of the first message and then to seek possible alterations in these systems by alcohol. Among such future analyses, which will rely heavily on biochemical (for details see reviews [ 1, 21) and electrophysiological [ 3, 41 data, are the personal and circumscribed views I present here.

First messenger criteria

Because the mammalian nervous system is a frustratingly complex system of heterogeneous, interconnected neurons and associated neuroglia,

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the analysis of how nucleotides might participate in synaptic transmission should be undertaken along tactical lines in which the criteria for the identi- fication of a synaptic transmitter intersect the criteria for the mediation of that transmitter by a cyclic nucleotide. The most important criteria for identification of a neurotransmitter as a first messenger may be paraphrased and condensed from those previously suggested [ 51 for establishing a neuro- transmitter:

(1) Neuronal localization of the substance and its enzymes of synthesis and degradation.

(2) Release of the substance upon selective activation of a specific neuronal pathway.

(3) Identical physiological response to exogenously applied transmitter and to activation of the pathway.

(4) Identical action of pharmacological agents (antagonists, etc.) on res- ponses to pathway activation and to exogenous transmitter.

With regard to second messenger mediation by cyclic nucleotides, the central catecholamine-containing pathways merit the greatest consideration because they satisfy three practical requirements: (1) catecholamines meet most or all of the criteria above for a first messenger as well as for a synaptic transmitter [6] ; (2) catecholamines are known to influence adenylate cyclase or cyclic AMP levels in various discrete regions of the nervous system by definable receptors [ 1, 21; and (3) the source neurons and target neurons of the central catecholamine pathways have been sufficiently characterized so that their effects can be determined and related to the effects of cyclic nucleotides and related substances [ 61.

Second messenger criteria

The second messenger concept as currently applied to brain has evolved from the mediator role of cyclic AMP in peripheral hormonal responses, as first suggested by the Sutherland group [ 71. Modified for neuronal trans- mission, this concept may be summarized (Fig. 1) as follows: Synaptically released neurotransmitter could act at certain pre- or postsynaptic receptors to activate the synthesis of cyclic AMP within the target neurons. Intra- cellular cyclic AMP would then initiate subsequent enzymatic or molecular events, which, among other actions, would result in changes in membrane potential and cell discharge rate. Four major criteria may be paraphrased from Sutherland’s [ 71 criteria for hormones to establish that the action of a transmitter is mediated by cyclic AMP [ 4, 81 :

(1) Exogenous neurotransmitter substance and activation of the synaptic pathway both regulate intracellular levels of cyclic nucleotide in the post- synaptic cell.

(2) The change in intracellular cyclic nucleotide content should precede “the biological event” triggered by the transmitter or nerve pathway.

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Fig. 1. Scheme of criteria for second messenger mediation, using cyclic AMP as an example of a second messenger mediating norepinephrine first messages postsynaptically. Reproduced from Siggins [ 41.

(3) Responses to the transmitter or nerve pathway should be logically altered by drugs that specifically interact with the nucleotide cyclase or that inhibit the appropriate phosphodiesterase.

(4) Exogenous cyclic nucleotides should elicit the biological event caused by the transmitter or nerve pathway.

These criteria are schematized in Fig. 1.

Central norepinephrine as a first messenger for cyclic AMP second messages

Considerable data exist for norepinephrine (NE) as a neurotransmitter in several brain regions, including, but not limited to, cerebellum, hippo- campus and cerebral cortex [6, 81. However, attempts to satisfy the second messenger criteria for central neurons meet with considerable technical obstacles, such as the indirect actions of systemic drugs, blood-brain barriers to systemic agents, slow nucleotide sampling and measurement times com- pared to fast synaptic events, and relative impermeability of cyclic nucleo- tides into target cells. Several of these obstacles can be partially overcome in the central nervous system by the techniques of microiontophoresis and electrophysiology, as has been applied to several brain areas.

From the electrophysiologist’s point of view, the cerebellar Purkinje cell (P-cell) is the best candidate for a target neuron that receives a nora- drenergic input (from the nucleus locus coeruleus, LC) which is capable of generating cyclic AMP postsynaptically. The data reinforcing this notion have been reviewed in detail elsewhere [2 - 4,6, 81, but may be summarized as follows: (1) catecholamines elevate cyclic AMP levels and increase adenyl- ate cyclase activity in cerebellum in vitro, and exogenous NE and stimulation of the LC increase cyclic AMP histochemical immunoreactivity in P-cells in uivo; (2) the increase in cyclic AMP immunoreactivity is detectable at a time when the electrophysiological effects of LC stimulation are apparent; (3) the

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inhibitory effects on P-cells of LC stimulation or of NE iontophoresis are potentiated by several phosphodiesterase (PDE) inhibitors and antagonized by agents such as PGEl and Ez , MJ-1999, fluphenazine, known to block NE-elevated cyclic AMP levels in uitro; (4) responses to iontophoresis of cyclic AMP and several more potent synthetic analogues generally mimic the inhibitory or hyperpolarizing action of iontophoretic NE and LC stimulation, as well as evoking the novel increases in membrane resistance usually seen with such noradrenergic stimuli.

The criteria for second messenger mediation appear to be largely sa- tisfied for the inhibitory NE input to P-cells, except for the technical inabi- lity to detect postsynaptic increases in cyclic AMP at a time prior to the “biological event” triggered by NE. Although some disagreement exists as to the exact percentage of P-cells inhibited by iontophoretic cyclic AMP [ 9, lo] , this discrepancy can be explained by poor cell penetrability and other tech- nical considerations [ll, 121. Moreover, iontophoresis of derivatives of cyclic AMP (for example 8-p-chlorophenyl cyclic AMP) known to have a greater action on the protein kinase enzyme (the intracellular “receptor” for cyclic AMP) can depress the activity of up to 90% of P-cells [13]. In addi- tion, the strong correlation between the percentage of P-cells depressed and the potency of several derivatives in activating protein kinase argues for an involvement of cyclic AMP-dependent protein kinase in the depressant responses.

Similar evidence exists for cyclic AMP mediation of the inhibitory nore- pinephrine input to hippocampal and cerebral cortical pyramidal cells [ 14 - 161. Here again, exogenous catecholamine elevates cyclic AMP in uitro, the inhibitory effects of LC activation or NE iontophoresis are affected in a predictable way by drugs which interact with the cyclic AMP system, and iontophoresis of cyclic AMP generally mimics the inhibitory action of LC stimulation and iontophoretic NE. However, immunohistochemical methods for discrete in uiuo localization of cyclic AMP to the pyramidal cells have not yet been applied to the hippocampus or cortex, nor has it been possible to detect cyclic AMP in these structures prior to the NE-induced biological event.

Central dopamine as a first messenger for cyclic AMP

Although there are many similarities between the central NE- and dopamine-regulated cyclic AMP systems in vitro, there are some important differences, including the involvement of different receptors (beta-adrenergic us. dopamine receptors), and the greater viability of the DA systems with homogenization [ 11. To date the best central model system for DA-activated cyclic AMP is the mammalian caudate nucleus. However, strict proof of cyclic nucleotide mediation of the profuse DA input to this structure suffers from the present inability to activate selectively the nigrostriatal dopamine pathway [ 17,181. Although exogenous DA and apomorphine elevate cyclic

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AMP levels in striatal homogenates [ 1, 191, the effects of synaptically re- leased DA are controversial [ 18, 201. Moreover, immunohistochemical methods have not yet been applied to this structure. Electrophysiological proof of cyclic AMP mediation of DA effects here rests largely on ionto- phoresis and pharmacological experiments in rat showing: (1) blockade of the inhibitory effects of dopamine by phenothiazines, which block DA- induced cyclic AMP increases; (2) potentiation [21] of DA inhibitory res- ponses by several PDE inhibitors and PGE (which elevates striatal cyclic AMP in uitro); and (3) the close mimicry between inhibitory responses to iontophoretic DA, apomorphine, cyclic AMP and monobutyryl cyclic AMP [ 17,211.

DA-containing fibers also project from ventral tegmental areas to certain cortical regions [ 22, 231, where exogenous DA also inhibits neuronal activity [ 241 and elevates cyclic AMP levels [ 251. However, since the actions of iontophoretic cyclic AMP and of DA pathway stimulation in these areas have not been analyzed, criteria for cyclic AMP mediation here remain largely unsatisfied.

Central purines as first messengers for cyclic AMP second messages

Adenine nucleotides and nucleosides have received attention as possible neurotransmitters in peripheral nervous systems 126, 271. The exact action of the purines is somewhat unclear, although they appear to be inhibitory in many peripheral sites [ 26 - 281. In the central nervous system (CNS), the concept of purinergic neurotransmission gained support with the finding that exogenous adenosine and other adenine derivatives could evoke large in- creases in cyclic AMP levels in slices from several brain areas [29]. Since this effect of the adenine derivatives is blocked (rather than potentiated) by methyl xanthines [29], it is thought that an “adenosine receptor” exists which is functionally linked to adenylate cyclase. This concept has gained additional support from iontophoresis studies showing marked inhibition of neuronal activity by adenosine (similar to that produced by cyclic AMP) in cerebellum 130,311, cerebral cortex [32], and caudate nucleus [17].

Although the criteria for second messenger mediation of adenosine effects are mostly satisfied (except for detection of increased cyclic AMP prior to the biological effect), the case for adenosine and other purines as neurotransmitters merits other unique considerations. It is generally accepted [ 26, 271 that in peripheral systems: (1) ATP and its associated enzymes are localized in nerves, as in all cells; (2) ATP can be released from tissue by activation of neuronal pathways; (3) exogenous ATP and activation of the “purinergic” pathway have identical actions. However, proof of the hypo- thesis that purines function as neurotransmitters requires the development of truly specific ATP antagonists. Furthermore, the ubiquitous distribution of ATP as an energy form in all cell types, and the possible presence of ATP (as a binding agent) in adrenergic storage granules, raises questions as to whether

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or not ATP is released specifically from a discrete set of nerves containing only ATP as a neurotransmitter, as a “co-transmitter” [ 331 with some other substance such as NE or acetylcholine (ACh), or as an epiphenomenon of the release of the “real” transmitter. Moreover, in the CNS no electrophysiolo- gical effect has yet been detected with nerve-released purines for comparison with the effect of exogenous purines, and the ubiquitous distribution of ATP in brain complicates attempts to develop specific histochemical methods for localization of “purinergic” nerves. Although methyl xanthines might be used as specific antagonists of adenosine or possibly ATP receptors, the lack of histochemically defined “purinergic” nerve tracts for activation renders the xanthine effect useless in this regard. Thus, with the available evidence, adenosine and/or ATP might subserve a role as local neuromodulator (for example, released on aerobia, injury, or under certain ionic conditions), or as “co-transmitter”, as easily as a discrete neurotransmitter. Unfortunately, little work has been done to determine the effects of alcohol on such purines and their influence on the cyclic nucleotides.

Cyclic GMP as a second messenger in the CNS

The putative role for cyclic GMP in central synaptic transmission is more covert than for cyclic AMP, in part because of the insensitivity of guanylate cyclase in homogenates to neurotransmitters and because of the large number of neurotransmitters which increase cyclic GMP levels in brain- slice preparations [ 1, 21. Attempts have been made to link cyclic GMP with ACh in brain. However, early iontophoresis studies with cyclic GMP in rat cerebellum (which has high levels of endogenous cyclic GMP) showed no differences between P-cell inhibitory responses to cyclic AMP and cyclic GMP and no clear-cut correlation with neurotransmitter effects [ 341.

A more encouraging relationship between ACh and cyclic GMP has been reported for pyramidal cells of rat cerebral cortex, where there is good evidence for a cholinergic projection. In brief, Stone and co-workers [15, 161 report that those neurons responding to iontophoretic ACh with excita- tion generally respond equivalently to cyclic GMP, and phosphodiesterase inhibitors potentiate excitations to both agents. However, rigorous proof of cyclic GMP as a second messenger for cholinergic neurotransmission awaits fulfillment of the other second messenger criteria, such as localization and stimulation of the ACh pathway and detection of increases in cyclic GMP in pyramidal cells prior to the biological event.

A similar situation exists for hippocampal pyramidal cells where a choli- nergic input projects from the septal nucleus. In the hippocampus in situ [ 351 and in intraocular transplants [36] both ACh and cyclic GMP are strongly excitatory, and may even cause paroxysmal discharges or frank seizures [ 361. Again, no biochemical or immunocytochemical studies have been done to detect or localize cyclic GMP changes in hippocampus with cholinergic stimulation, although pharmacological data with PDE inhibitors

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point to a link between ACh and cyclic GMP excitations in hippocampus in oculo [36].

The hippocampal transplant model represents one of the more relevant subjects for analysis of the role of cyclic GMP (and perhaps cyclic AMP as well) in alcohol tolerance and development, and especially with reference to the motor seizures seen upon withdrawal from chronic alcohol treatment. As reported elsewhere in this volume [37] hippocampal transplants in oculo also develop epileptiform activity after termination of chronic treat- ment of the host animal with alcohol, Although the relationship of the cyclic GMP-induced seizures (see above) and alcohol-induced seizures in this struc- ture are at present only correlative, the biochemical data showing increased brain cyclic GMP upon withdrawal from chronic alcohol ([38, 391, but see ref. 40) reinforces the notion that cyclic GMP might have something to do with alcohol dependence.

However, the fact that no specific agonists or antagonists of guanylate cyclase have been found may be significant in itself. It is possible that cyclic GMP may increase as a result of, or be a mediator of, non-specific membrane depolarization. This hypothesis is supported by studies showing dramatic increases in cyclic GMP in cortical areas exhibiting penicillin-induced epilepti- form seizures [41]. Although the increases in cerebellar cyclic GMP with harmaline treatment [ 421 are interpreted as implicating cyclic GMP mediation of possible amino acid- (for example glutamate) transmitted climbing fiber responses, it also seems reasonable that cyclic GMP might increase as a result of (rather than mediate) non-specific depolarization of Purkinje cells by any means. The same mechanism could account for the reported reciprocal relationship between cerebellar cyclic GMP and GABA [43] , a potent hyperpolarizing neurotransmitter. The answer to this dilemma hinges on whether the second criterion for cyclic nucleotide mediation, namely detec- tion of cyclic GMP before depolarization, can be satisfied.

The data of Murad et al. [ 441, showing a relationship between brain redox potentials and cyclic GMP levels, also suggest a role for cyclic GMP in processes other than simple neurotransmission per se. The possible involve- ment of redox changes in the effects of alcohol on brain cyclic GMP levels is strongly implied by the data of Breese [45]. His finding that cyclic GMP changes inversely with tissue oxygen tension could also implicate non-specific membrane depolarization (due to oxygen lack) as causative in these elevations in cyclic GMP. Again the answer to this speculation may derive from fulfill- ment of the second criterion for cyclic nucleotide mediation, namely, detection of cyclic GMP changes before membrane depolarization produced by these various means. Electrophysiological recording of neuronal activity during redox manipulations and acute and chronic alcohol administration might provide correlative data in this regard.

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Modulation of adenylate cyclase by altering catecholamines

Biochemical studies in the last decade have revealed the interesting concept that the catecholamine-cyclic AMP system is not a static entity, but can be modulated by changes in the functionality of the endogenous catecholamine systems. In general, chronic reductions in catecholamine innervation or release (for example, by disruption with B-hydroxydo- pamine; 6-OHDA) bring about an increase in responsivity of brain adenylate cyclase to fl-adrenergic or dopaminergic stimulation, while pharmacological manipulations designed to elevate synaptic catecholamines (for example with re-uptake blockers such as desipramine, DMI) reduce sensitivity to these agonists [46] . These findings of a dynamic inverse relationship between catecholamine levels and the sensitivity of the associated adenylate cyclases present important implications for the possible involvement of cyclic AMP in chronic alcohol consumption. Thus, reports of enhanced catecholamine turnover (and therefore release) with chronic alcohol treatment [47] would seem to predict that catecholamine-stimulated adenylate cyclases would be “down-regulated”. Reports to that effect have recently appeared in the literature [ 48,491. Whether such modulations could constitute the molecular substrate for alcohol tolerance or dependence is an exciting but speculative possibility.

Electrophysiological methods have also been brought to bear on this “homeostatic” phenomenon. For example, previous studies showed that disruption of the dopamine input to the caudate nucleus by 6-OHDA re- sulted in a supersensitivity of caudate neurons to iontophoretically applied dopamine and therefore apomorphine [ 17, 211, thus presenting a cellular correlation of the behavioral (rotatory) supersensitivity to systemically injected apomorphine [ 501. Recent studies in our laboratory suggest that the converse electrophysiological experiment also supports the principle of cyclic AMP homeostasis. Acute application of DMI by iontophoresis potentiates the effects of NE on P-cells [ 511 presumably by blocking NE uptake into noradrenergic terminals. In contrast, chronic treatment of rats with DMI (30 - 50 mg/kg per day for 5 - 10 days) significantly reduces the responsivity of cerebellar P-cells to iontophoretically applied NE (Table 1)

TABLE 1

The effect of lithium and DMI on Purkinje-cell thresholds to norepinephrine

Iontophoretic current (nA; mean + s.e.m.)

Controls Lithium DMI 5-Day withdrawal

Acute Chronic

NE Thresholds 24 + 3.7 19 +z 3.4 1.6 ?; 2.9 43 i 3.6** 31 ? 5.9*

No. P-cells studied 26 21 18 28 22

l-Way analysis of variance,p = 0.05*; Newman-Keuls analysis,p < O.Ol**

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as determined by the elevation of the average ejection current required to evoke threshold depressions of spontaneous activity [ 521.

It is interesting that lithium, another anti-depressant agent effective in bipolar manic-depressive syndrome, actually antagonizes noradrenergic responses when given acutely (Siggins and Henriksen, in preparation) while having no effect on NE responses with chronic treatment (Table 1). One must assume that the adrenergic receptor or the associated cyclase com- pensates to overcome the blocking action of lithium. Thus, these electro- physiological data reinforce the biochemical findings which point to the dynamic nature of the catecholamine-cyclic AMP system in brain.

Conclusions, caveats and speculations

Results of electrophysiological tests, taken with biochemical and cytochemical data, satisfy most first messenger or neurotransmitter criteria for NE and dopamine in brain. Studies summarized in this commentary also suggest that these two catecholamines may also serve as first messengers for postsynaptic generation of a cyclic nucleotide second messenger, especially in the cerebellum, hippocampus, caudate and cerebral cortex. Future studies attempting to forge a link between cyclic AMP and alcohol tolerance and dependence would do well to concentrate initially on these structures.

However, strict proof of a second messenger mediation in all neuro- transmitter systems still awaits satisfaction of the criterion that increases in cyclic AMP or cyclic GMP occur prior to the physiological response. Future biochemical and immunohistochemical studies should be directed toward detecting cyclic nucleotides within the msec or tens-of-msec domain likely to represent the synaptic delay in central neurotransmission.

The concept of adenosine, and perhaps adenine nucleotides as well, as first messengers for cyclic AMP second messages also seems likely, although the question of whether the purines function as distinct neurotransmitters or as local modulators remains to be explored. Since “adenosinergic” path- ways have not been selectively localized and/or stimulated in brain, fulfill- ment of second messenger criteria for adenosine is incomplete. Still, the lack of detailed biochemical data concerning the effects of alcohol on central adenosine, other purines and adenosine-stimulated adenylate cyclase suggests a new avenue of exploration relevant to alcohol tolerance and dependence.

To date, fulfillment of second messenger criteria for cyclic GMP has been difficult, principally owing to the paucity of pharmacological agents which interact with guanylate cyclase in homogenates and the inability to localize and activate the central pathway(s) for endogenous release of the relevant first messenger(s). The hypothesis is extended that neuronal de- polarization per se could non-specifically elevate intracellular cyclic GMP. The best identified neurotransmitter candidates for cyclic GMP mediation in the CNS are the cholinergic excitations in hippocampal pyramidal cells and cerebral cortical pyramidal cells. Future studies on the interaction of

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alcohol and the ACh-cyclic GMP system should initially concentrate on these neuronal systems.

At this point it may be well to insert a cautionary note on the signi- ficance of negative findings in biochemical measurements of regional cyclic nucleotide levels. It is well known that most brain regions are composed of several groups of different neuron types, each of which could contribute disproportionate amounts of cyclic nucleotide to the total assayed for that region. The result might be a dilution effect in which a large neuron popula- tion with constant high levels of nucleotide (for example, cyclic AMP in cerebellar granule cells) overpowers the expression of drug-induced changes in a smaller population of neurons (for example, the cerebellar P-cell). Alternatively, reciprocal changes in two or more populations of a region in response to a drug (alcohol) could also yield confusing data. Thus, it becomes important to attempt finer localizations of nucleotide changes at the cellular level, by any of several means such as microdissection, immunohistochemistry [53] and the use of animals with deficiencies in specific cell types (for example, neurological mutants [ 541 and animals X-irradiated at birth [ 551).

The “homeostatic” modulations of the NE-adenylate cyclase systems with alterations of functional NE levels, suggested by biochemical [46] and electrophysiological experiments, presents a hypothetical model for alcohol tolerance and dependence. The subsensitivity of NE-stimulated adenylate cyclase [56, 571 with chronic DMI treatment parallels the physiological tolerance which develops to chronic alcoho1 treatment, while the molecular events leading to alcohol dependence might be analogous to the low adrener- gic “tone” which would likely persist beyond the cessation of DMI treat- ment. One difficulty in applying this model directly to alcohol tolerance and dependence arises from our ignorance of whether the primary CNS effects of alcohol are exerted directly by alcohol (perhaps via a hypothetical “al- cohol receptor”) or indirectly through some other first or second messenger(s) such as normal neurotransmitters or tetrahydroisoquinolines [ 581.

Although it is understood that other neurotransmitter systems (or their receptor homeostases) are likely to be involved in alcohol effects, it may be instructive to follow a hypothetical alcohol model of NE and ACh interactions in the hippocampus. In this structure it is known that NE normally inhibits and ACh excites pyramidal neurons, to the extent that NE can prevent and ACh augment or even trigger epileptiform activity [ 14,361. These responses are thought to be mediated by cyclic AMP and cyclic GMP, respectively [ 14, 361. Alcohol presented to this well-balanced system could then cause an increased release of NE (as suggested by turnover studies [47] ) and diminished ACh release (cf. ref. 59). With continued alcohol adminis- tration, the NE-receptor-adenylate cyclase system would respond by down- regulation to adjust to the increased local NE, while converse changes might occur in the ACh-guanylate cyclase system. Abrupt withdrawal of alcohol treatment would then find the diminished NE-adenylate cyclase system in- capable of dampening the enhanced excitatory ACh system, with the result that seizures develop. Similar sequelae might apply to the cerebral cortex,

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with the development of motor convulsions. Although this mcdel is entirely empirical, it agrees with pharmacological studies indicating that decreases in brain NE exacerbate the convulsions following alcohol withdrawal [ 60,611. Furthermore, intraventricular administration of dibutyryl cyclic GMP increases the severity of alcohol withdrawal symptoms in mice, while dibutyryl cyclic AMP administration decreases the symptoms [62].

In the final analysis, the concept of first and second messengers may be an oversimplification in neuronal systems, since the actual molecular conse- quences of neurotransmission might be represented as a multilevel cascade of many sequential messages, including protein kinase activation and protein phosphorylation and/or dephosphorylation. Such a cascade in the nervous system would allow for an enormous amplification of the original first signal, such that a few neurotransmitter molecuies could ultimately affect the opening or closing of a multitude of ionic gates, or the activation of many ionic carriers or pumps. The advantages of such an amplification could in- clude reducing the need for axonal transport of many transmitter molecules (or their enzymes of synthesis) and the development of a high safety factor at the synaptic cleft. Perhaps more important are the multiple points at which external modulation may be superimposed. However, such a complex cascade obscures cause-and-effect relationships in alcohol actions, and underlines the need for multidimensional analyses of all components of the system. Neglected areas of investigation include analysis of alcohol actions on cyclic AMP dependent protein kinases (cfi ref. 63) and protein phos- phatases. Obviously a great deal of research remains to delineate the func- tional significance of such a complexity of linkages in alcohol tolerance and dependence.

Acknowledgements

I wish to thank Dr. Floyd Bloom for critical evaluation of this manus- cript and Nancy Callahan for secretarial skills. Parts of this study were supported by NIMH grant 29466 and NIAAA grants 03119 and 03504.

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