University of Illinois Medical Center, Department of Physiology, 901 South Wolcott Street, Chicago, Illinois, 60680.
* This work was supported by U.S. Public Health Service Grants NS-12759 and AA-2696 and a Campus Research Board Grant from the University of Illinois. B.T. is a Schweppe Foundation Fellow.
The neurochemical components of the addiction syndrome referred to as alcoholism, most probably result from the interaction of this drug with neurons which under normal circumstances are tightly controlled by feedback regulatory systems. The presence of ethanol in the cellular milieu for extended periods and the resultant alterations in neuronal function may produce a resetting of the regulatory systems to compensate for the presence of ethanol. Such a conceptualization of the effects of ethanol on control systems is similar to the conceptual framework within which the study of other addictive agents has been carried out (1,2). The prior theories relating to the development of physical dependence have envisioned a continuum between the development of tolerance and physical dependence. However, as will be discussed in the following paragraphs and as previously noted by Kissin (3), the development of tolerance and physical dependence to ethanol may not be due to a common singular mechanism. Tolerance and physical dependence may, however, be causally related phenomena; i.e., the development of tolerance in a susceptible individual may simply allow for imbibing of sufficiently large doses of ethanol such that physical dependence can become manifest.
Dependence and tolerance have been studied on various levels of physiological complexity. At the neuronal level, chronic consumption of ethanol has been thought to alter the cybernetic capacity of the cells. If one considers that the final information-containing event in neuronal transmission can be quantitatively expressed as the amount of transmitter released at terminals (4), one can easily envision several points of interaction where ethanol may alter such output. The interactions between ethanol and the neuron may be direct or indirect. Direct interactions are exemplified by the interaction of ethanol with cellular membranes (5), which can lead to a block in neuronal conduction (6). The ability of ethanol to block conduction seems to be inversely related to the size of the neuron (7) and one should consider that this selective action of ethanol also imparts a concentration-dependent specificity to the actions of ethanol. Other direct effects of ethanol may be exemplified by changes in receptor sensitivity induced by ethanol (8). The receptors affected may be the post-synaptic excitatory or inhibitory receptors, or the recently described presynaptic receptors which control the overflow release of transmitter (9). On the other hand, the effects of ethanol may be indirect such as those producing changes in input to a particular neuron (10). Inputs travelling multisynaptic paths, as those determining evokes cortical potentials, seem to be more susceptible to the actions of ethanol (7) than inputs traveling direct paths. All the above examples lead one to expect that the interaction between ethanol and neurons would change the output of neurons, and such changes in output would activate feedback controls which would try to compensate for the effects of ethanol. One can then expect that the examination of changes in the turnover of the various neurotransmitter substances in the CNS during chronic ethanol treatment and during withdrawal, may lend insight into the functional changes responsible for the development of tolerance and physical dependence.
THE "ANIMAL" MODEL FOR ANALYZING THE EFFECTS OF ETHANOL ON BRAIN FUNCTION
The necessity for examining, chemically, brain material during the development of physical dependence to ethanol has led to the recent proliferation of animal models of the disease (11,12). Certain criticisms have been leveled at the applicability of such models to the study of alcoholism, since some authors have experienced difficulty in establishing drug-seeking behavior in animals when ethanol is used as a positive reinforcement (13). However, this difficulty was not evident in all studies and ethanol has been demonstrated by other investigators to be a strong reinforcer of behavior (14) leading to intake of increasing amounts of ethanol (15). Although controversy remains regarding the induction of alcohol-seeking behavior in animals, it has been clearly demonstrated that animals do become physically dependent on ethanol if dependence is defined in terms of the occurrence of withdrawal symptoms after removal of ethanol from the animals' diets (11). Many of the overt symptoms evidenced during the early period of withdrawal in animals bear a resemblance to the symptoms seen in humans (Table 1).
Human withdrawal symptomatology has been separated by some authors (16) into an early phase and a late phase known as delerium tremens, and concordant with such a progression, French and his coworkers (17) have also reported a secondary phase of withdrawal in animals. This phase included hyperkinesia which developed approximately three days after ethanol withdrawal, and was equated by the authors to the delerium tremens state of human withdrawal. In the past, one of the drawbacks in human and animal experiments has been the lack of continuous quantitative measures of the severity and time course of ethanol withdrawal. Recently a good behavioral and physiologic method of analysis of the human syndrome has been proposed by Gross et al. (18), and we have found that body temperature may serve as a good quantitative means of assessing withdrawal severity in some animals (19). However, one must be careful not to depend on only one symptom to monitor withdrawal, since different biochemical events may be responsible for the various components of withdrawal as exemplified by studies of opiate withdrawal (20). For example Cox et al. (20) found that hypothermia and "wet" shakes occuring during morphine withdrawal were controlled by dopaminergic neurons, while other symptoms such as diarrhea, head shakes and sneezing were not at all affected by manipulation of the dopaminergic systems in brain.
ALTERATIONS IN NEUROTRANSMITTER LEVELS AND TURNOVER PRODUCED BY ACUTE AND CHRONIC ETHANOL INTAKE
Several authors (21,22,23) have attempted to analyze, by use of pharmacologic agents, which neuronal systems are of primary importance in determining the severity of ethanol withdrawal symptoms. These studies have produced somewhat contradictory results. The contradictions within these studies are probably due to the nonequivalence of the symptoms examined by the various authors (21,23). Thus, while the studies of Goldstein (21) and Blum (22) indicated that catecholaminergic neurons may play a determinant role in producing convulsions due to handling (11) in mice undergoing withdrawal, the studies of Collier (23) indicate a primary role for serotonergic systems in determining withdrawal symptomatology. However, Collier (23) monitored head twitching as a primary measure of withdrawal. Alterations in GABA metabolism by pharmacologic agents have also been reported to produce changes in the severity of withdrawal convulsions (21) and this transmitter was suggested as a candidate for determining withdrawal symptomatology. The use of pharmacologic agents (eg. reserpine, picrotoxin) which in themselves predispose animals to seizures, may lead to ambiguous results. The systems affected by the drugs may simply prove to be synergistic with other undefined systems which are actually responsible for the symptoms of ethanol withdrawal. Pharmacologic manipulations should be correlated with actual changes in neurotransmitter utilization occuring in the CNS prior to and during withdrawal before any definitive conclusions may be reached as to which systems are determining the manifestations of physical dependence.
Many studies on both the levels and the "turnover" of various postulated transmitter substances have appeared in the literature and a representative sampling of the conclusions is presented in Table 2. These results indicate a lack of agreement on what changes occur in the various measures of serotonergic, dopaminergic and GABA-dependent function in the CNS of animals treated chronically with ethanol. Some of these differences are probably due to such variables as time of testing and the degree of tolerance and physical dependence developed in the animals. In addition, the necessity of assessing whether animals are still intoxicated or are in partial (24) or total withdrawal is paramount. Another major problem is that different methods of testing neurotransmitter "turnover" in the CNS may in themselves lead to different results. We found this to be the case with measures of serotonin turnover (25). If serotonin turnover was measured by the method of Neff and Tozer (26) using the monoamine oxidase inhibitor pargyline, no differences in serotonin turnover were evident between ethanol withdrawn or control animals. On the other hand, measuring turnover by monitoring the conversion of administered 14C-tryptophan to serotonin and 5-hydroxyindoleacetic acid demonstrated a significant decrease in the utilization of serotonin in brains of mice undergoing withdrawal (25). Such disparate results may be explained by the known inhibitory effect of monoamine oxidase inhibitors on discharge rate of serotonergic neurons (27).
In examining the published results on the effects of ethanol on trasmitter turnover, one area of agreement does, however, become evident. An acute dose of ethanol decreases the turnover of norepinephrine (NE) in brain while chronic ethanol administration produces increases in NE turnover which are evident throughout the period of withdrawal (see Tables 3 and 4).
An initial increase in turnover of NE seen after an acute dose of ethanol by Hunt and Majchrowicz (28) (Table 3) may be a result of the early excitant, disinhibiting effects of ethanol in the CNS 129) or possibly due to the release of catecholamines in the CNS (30) by an early infiltration of brain tissue with acetaldehyde. Since the availability of NA& is the rate-limiting step in ethanol metabolism (31), the administration of large doses of ethanol could result in an initial rapid generation of acetaldehyde while NAD+ is available for ethanol oxidation. The initial increase in NE turnover after acute ethanol administration is, however, soon supplanted by a period of decreased noradrenergic activity which is evident for the remainder of the period of intoxication (28,32,33).
In contrast to the effects of an acute dose of ethanol, chronic ethanol administration results in a prolonged and sustained period of increased NE turnover (Table 4). Whether this increased turnover is responsible for certain of the withdrawal symptoms or is a response elicited by the withdrawal symptomatology remains an enigma. Another possibility is that alterations in NE metabolism may be related to the, development of tolerance rather than physical dependence.
MECHANISMS BY WHICH ETHANOL MAY ALTER NE TURNOVER--ROLE OF CALCIUM
Before speculating on these subjects one should consider the mechanisms by which chronic ethanol intake may alter the functional aspects of NE metabolism. The rate-limiting enzyme in the synthesis of NE is tyrosine hydroxylase (34). The activity of this enzyme is coupled to impulse flow in noradrenergic neurons (35) and to concentrations of certain components (eg. Cat+, catechols) which comprise the intracellular milieu in which the enzyme resides (36). The enzyme is quite sensitive to feedback inhibition by NE (37), and control of enzyme activity has been postulated to be maintained by changes in a small extravesicular pool of NE during impulse flow (37). Thus, depletion of such a pool of NE would decrease the feedback inhibition and increase the activity of tryosine hydroxylase. The block in conduction in neurons after administration of ethanol could result in an accumulation of presynaptic NE and produce a decrease in enzyme activity which would be reflected in the decrease in NE turnover measured after acute ethanol administration. A more current proposal on the control of tyrosine hydroxylase involves the effects of Ca++ and cyclic adenosine mono-phosphate (c-AMP) on enzyme activity (36,38). The addition of Ca++ and c-AMP to assay mixtures for tyrosine hydroxylase has been shown to increase the affinity of the enzyme for its cosubstrates (reduces pteridine and tyrosine) and to significantly decrease the affinity of the enzyme for norepinephrine. The decreased affinity for the natural feedback inhibitor of the enzyme results in an effective increase in enzyme activity even in the presence of NE. Since increases in intracellular Ca++ and c-AMP can be coupled to impulse flow (39), the levels of these compounds may synchronize enzyme activity with the need for transmitter during neuronal activity. Seeman et al. (40) lave shown that a significant amount of Cat+ is segregated from the free form onto cellular membranes in the presence of ethanol, and this phenomenon could remove the Ca++ necessary for optimum activity of tyrosine hydroxylase. In addition, Ross et al. (41) have demonstrated that total brain Ca++ levels are actually decreased after a single dose of ethanol. The decreases in free Ca++ and total brain Ca++ coupled with decreased impulse flow could sum to produce the significant decrease in turnover of NE witnessed after a single intoxicating dose of ethanol.
Brain Ca++ levels have been postulated to be controlled by active transport systems residing in the choroid plexus (42) and our studies (43,44) have shown that ethanol both in vivo and in vitro inhibits certain active transport systems of the choroid plexus. Although the transport systems shown to be inhibited by ethanol were those removing organic acids from brain, the transport of organic acids has been shown to be coupled to inorganic ion [including Ca++ (45)] transport within the choroid plexus (46). Thus, the effects of acute ethanol administration on brain Ca++ levels (41) may be directly related to the inhibition of choroid plexus transport systems by ethanol (43).
To account for the increased NE turnover after chronic ethanol intake, one would have to predict that a tolerance develops to the membrane and Ca++-lowering effects of ethanol. Interestingly, Ross and coworkers have recently reported (47) that chronic exposure of animals to ethanol results in increased Ca++ content of synaptosomal membranes as opposed to the lowered content seen after a single dose of ethanol.
MECHANISMS BY WHICH ETHANOL MAY ALTER NE TURNOVER--ROLE OF TIQ ALKALOIDS ETHANOL DEPENDENCE
There are, however, several other phenomena which may occur during the chronic intake of ethanol and contribute to the observed increase in NE turnover. One of the widely debated (48,49) possibilities is the formation of tetrahydroisoquinoline alkaloids (TIQ's) in the CNS during ethanol intoxication. These alkaloids have been demonstrated to arise from the spontaneous condensation of either acetaldehyde (eq. salsolinol) or the aldehyde derivatives of the monoamines (eg. tetrahydropapaveroline) with the neuroamine transmitters. Although these condensation products are isolated in quantity from in vitro incubation systems (50), only two reports (51,52) have appeared on the isolation of products resembling the TIQ's from brain of animals receiving ethanol. In one report (51), the formation of salsolinol was found to be dependent on the concomitant administration of pyrogallol. Pyrogallol was shown to act not only as a catechol-o-methyl transferase inhibitor but also as an inhibitor of aldehyde dehydrogenase (53). Inhibition of aldehyde dehydrogenase led to an increase in circulating acetaldehyde when rats received both ethanol and pyrogallol and such increased acetaldehyde levels may have contributed to the formation of salsolinol in these animals (54). Turner et al. (52) have shown that tetrahydropapaveroline can also be isolated from brain, but ethanol administration did not increase the brain levels of this alkaloid. Since the presence--of acetaldehyde in brain is a prerequisite for the formation of the TIQ alkaloids, it is of interest that animals injected with ethanol were found to have little or no acetaldehyde present in brain unless blood acetaldehyde levels were particularly high (55,56). It has been postulated that aldehyde dehydrogenase in brain quickly metabolizes most incoming acetaldehyde (55). One has to, however, consider that although brain may have a high capacity for metabolizing acetaldehyde (56), it is precisely such metabolism which would compete (57) for the normal paths of disposition of biogenic aldehydes and lead to formation of TIQ's such as tetrahydropapaveroline and other aberrant reactions (58).
Chronic ethanol administration may, in addition, alter the normal capacity of tissues to metabolize acetaldehyde. Korsten et al. (59) have noted that alcoholics have significantly higher blood acetaldehyde levels than non-alcoholics after receiving equivalent amounts of ethanol. We have also noted (Anderson, Ritzmann and Tabakoff, in preparation) that mice chronically imbibing ethanol are found to have higher circulating levels of acetaldehyde during the later phases of the period of ethanol consumption. Increased blood acetaldehyde levels, if reflected in brain, could lead to increased formation of the TIQ alkaloids.
Thus, although ethanol-dependent formation of TIQ's has yet to be unequivocally demonstrated, one should consider that these compounds may form more readily during chronic ethanol ingestion. These alkaloids have diverse pharmacologic properties, acting as MAO inhibitors (60), catecholamine uptake blockers (61), and releasers of stored catecholamines (62). The TIQ's, particularly the tetrahydropapaverolines, have also been shown to have agonist properties on beta-adrenergic receptors (63). The interaction of the TIQ's with postsynaptic receptors (assuming that they are only weak agonists) could induce activity in a feedback loop which would in turn activate synthesis. On the other hand, the release of TIQ derivatives into the synaptic cleft could result in an interaction with presynaptic alpha receptors, which have been shown by several groups to control the release of NE into the cleft (9,64). Blockade of these alpha receptors would result in overflow release of NE from presynaptic terminals. The depletion of presynaptic stores would also activate amine synthesis.
Alterations in receptor responsiveness to catecholamines in the presence of TIQ alkaloids, and alterations in tyrosine hydroxylase activity which are dependent on changes in the intracellular distribution of Ca++ in the presence of ethanol, could, I feel, provide the explanation for the increased turnover of NE noted after the chronic ethanol consumption'.
NEUROTRANSMITTER INVOLVEMENT IN THE DEVELOPMENT OF TOLERANCE AND DEPENDENCE
At this point I would like to return and attempt to answer the questions posed above as to whether the increase NE turnover during withdrawal is 1) responsible for the symptoms of withdrawal, 2) a response to the stress of withdrawal, or 3) more related to the manifestations of tolerance to ethanol. One finds that two types of published experiments related to such questions lead to different answers. The time course of the increase in NE turnover as shown in Table 4, demonstrates the presence of the increased turnover at times when no withdrawal signs are present. Such data indicates that increased NE turnover is not simply a protective response to the stress of withdrawal. On the other hand, several authors (65,66) have shown that increased noradrenergic activity in the CNS protects aiimals from drug, electrically and sound-induced seizures and three sets of authors (21,22,67) have demonstrated that blocking noradrenergic systems and depletion NE potentiates the hyperreactivity and seizures seen during withdrawal. Thus the questions of the exact role of NE in determining withdrawal symptomatology remains unanswered.
Some current results from laboratory (68), however, indicate that an intimate relationship exists between the noradrenergic system and the development of tolerance to ethanol after chronic treatment. We placed control mice and mice pretreated with 6-hydroxydopamine (6-OHDA) in a situation in which they were provided with only one source of food and water, and this source was a liquid diet containing seven percent ethanol. Prior to being placed on this diet and at various intervals after being placed on the diet, the mice were injected with a 3 g/kg challenge dose of ethanol and their temperatures and behavioral responses were monitored. We found that control animals consuming the ethanol-containing diet became quite tolerant to the effects of ethanol, while the animals treated with 6-OHDA prior to chronic ethanol administration did not develop any tolerance to the temperature-lowering or hypnotic effects of ethanol (see Figure 1). On the other hand, no difference in the withdrawal symptomatology was evident between the 6-OHDAtreated animals and the control physically dependent animals (68), thus indicating that 6-OHDA did not affect the development of physical dependence but did affect the development of tolerance.
A fruitful model for the development of tolerance may be the following: an acute dose of ethanol, possibly by depressing transport systems in the choroid plexus, lowers brain Ca++ levels. This in turn decreases tyrosine hydroxylase activity which coupled with decreased impulie flow would result in a decreased amount of NE in the synaptic cleft. The hypothermia and behavioral depression concomitant with acute ethanol ingestion may be directly or secondarily coupled to this noradrenergic deficit.
On chronic ethanol administration, a tolerance develops to the Ca++-lowering effect of ethanol, and in fact Ca++ levels were found to be increased in synaptic areas of brain (47). This increase in Ca++ could result in increased tyrosine hydroxylase activity leading to increase in NE available for release with impulse flow and thus result in a tolerance to the depressant effects of ethanol.
Our studies described above also indicate that tolerance and dependence may not be part of a unitary biochemical phenomenon. I do not, however, feel that a single transmitter is responsible for all the concomitants of tolerance and another for physical dependence. Interaction between noradrenergic and serotonergic (69) and noradrenergic and cholinergic (70) systems has been demonstrated on both biochemical and behavioral levels. Thus, a more complete neurochemical profile of tolerant and addicted animals and humans will be invaluable in further explaining the total gamut of symptoms resulting from chronic ethanol ingestion.
Insert A--mean sleep times (minutes) in control (0-0), Group 3 (0-0) and Group 4 (X-X) animals after ethanol injection. Statistical comparisons at each dose were made using Student t-test: *-p < 0.05; **-p < 0.01; ***-p < 0.001; comparison between Group 3 and Group 4 animals. No significant differences were found between Group 4 and control animals.
Insert B--Rectal temperature was monitored at fifteen and thirty minute intervals for three hours after ethanol injection. The bars represent the mean ± S.D. of the greatest drop (°C) from preinjection temperatures recorded during this period. The nadir for the 3 and 4 g/kg dose occurred at thirty minutes and for the 5 g/kg dose at sixty minutes after injection. Comparison between groups at each dose was made using the t-test and starred (*) groups were significantly different from each of the other groups (p < 0.01) while the unstarred groups were not significantly (p > 0.2) different from one another.
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