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Effects of 6-hydroxydopamine or 5,7-dihydroxy-tryptamine on the development of physical dependence on ethanol
Drug and Alcohol Dependence, 2 (1977) 349 - 359 @ Elsevier Sequoia S.A ., Lausanne - Printed in the Netherlands
349
EFFECTS OF 6-HYDROXYDOPAMINE OR 5,7-DIHYDROXYTRYPTAMINE ON THE DEVELOPMENT OF PHYSICAL DEPENDENCE ON ETHANOL GERALD D. FRYE*
and FRED W. ELLIS
Department of Pharmacology, 27514 (U.S.A.)
University of North Carolina, Chapel Hill, North Carolina
Summary Infant rats, treated intracistemally with 6-hydroxydopamine or 5,7dihydroxytryptamine, alone or in combination with desmethylimipramine or pargyline, at 5 to 7 days of age, had significant specific depletions of brain norepinephrine, dopamine, both of these amines, or serotonin at 2.5 months of age. Despite apparent long-term depletions of brain biogenic amines, susceptibility to audiogenically-induced seizures following chronic ethanol withdrawal in these animals was similar to that of controls. Amine-depleted rats also displayed spontaneous withdrawal-induced tremors, spastic motor activity and irritability. The interpretation of these preliminary findings with regard to the proposed role of the biogenic amines in the development of physical dependence on ethanol is discussed.
Introduction It has been proposed that various adaptive processes in central biochemical and physiological mechanisms are involved in the development of functional tolerance to and physical dependence on central nervous system (CNS) depressant drugs like ethanol [ 1,2]. A variety of mechanisms have been postulated by which drugs might interact with feedback systems regulating the activity of enzymes [ 3,4] or the sensitivity of receptors [ 5,6] for putative neurotransmitter substances in the brain to produce tolerance and physical dependence. This has led to the examination of the putative neurotransmitters norepinephrine (NE), dopamine (DA) and serotonin (5-HT), which have been shown to be important in CNS function [ 71, for possible involvement in the development of physical dependence on ethanol. Early studies designed to look for changes in the levels of brain biogenic amines during both acute and chronic ethanol treatment reported conflicting *Now at the Biological Sciences Research Chapel Hill, North Carolina 27514 (U.S.A.)
Center,
University of North Carolina,
results and failed to produce a consistent picture of interaction [ 8. 91. However, more recent studies using techniques that examine biogenic amine turnover [ 10 - 121 now suggest that acute ethanol treatment first accelerates and then depresses turnover of brain NE. Later, during continued ethanol treatment NE turnover is once again accelerated and then increased further following ethanol withdrawal. Studies examining DA and 5HT turnover have continued to produce conflicting results [ 91. The pharmacological manipulation of central catecholamine and serotonin systems results in changes during the ethanol abstinence syndrome suggesting their involvement in withdrawal reactions. Drugs which interfere with the function of catecholamine neurons have been shown to aggravate the severity of convulsions on handling mice which were made physically dependent after exposure to ethanol vapors [ 13, 141. Others have found that drugs facilitating the activity of NE neurons reduced head twitches in mice during ethanol withdrawal [ 151. However, results from studies in which brain serotonin was manipulated imply that this neurochemical may be involved in withdrawal-induced head twitches [ 151, but not in convulsions on handling ethanol-dependent mice [ 13, 161. Thus, recent biochemical and pharmacological studies of chronic ethanol intoxication and withdrawal strongly suggest an ethanol effect on NE neurons in the brain, while effects on DA and 5-HT containing neurons are less clear. However, these results may not necessarily indicate that NE is involved in the adaptive processes which are responsible for the development of tolerance to and physical dependence on ethanol. For example, changes in the turnover of NE in the brain during chronic ethanol exposure may simply reflect intoxication and withdrawal-induced stress or may be due secondarily to more direct actions of the drug to alter the function of other neurons. Also acute pharmacological manipulation of biogenic amines resulting in modification of withdrawal reactions does not necessarily indicate that these substances are involved in the mechanisms underlying physical dependence development. They may be involved primarily in systems mediating the display or modulation of withdrawal reactions. Recently we have attempted to examine more closely the hypothesis that the biogenic amines may play a major role in the underlying adaptive mechanisms leading to physical dependence on ethanol. It was assumed, perhaps rather simplistically, that if NE, DA, or 5-HT were involved in the principal mechanism of ethanol action to produce physical dependence, then destruction of these respective transmitter systems in the brain prior to chronic ethanol treatment should markedly and dramatically reduce the severity of the resulting withdrawal syndrome. Conversely, if these systems were not involved directly or simply acted as modulators of ongoing withdrawal reactions, much less dramatic changes in the severity of the withdrawal syndrome might be expected. The present report indicates results of preliminary experiments designed to test this hypothesis. Effects of chronic ethanol consumption on rats pre-treated with neurocytotoxic drugs to produce long-term depletions of brain biogenic amines were examined.
351
TABLE
1
Treatments
to deplete
norepinephrine,
dopamine,
and serotonin
in infant
rats
Treatmenta .~
Day 5
Day 7
Control NE down DA down CA down 5-HT down
Saline 13 pg 6-OHDA 100 pg 6-OHDA 100 E.cg6-OHDA 100 pg 5,7-DHT
-
aVolume
of the intracisternal
injections
+ 20 mg/kg
DMI
+ 30 mg/kg
pargyline
15 pg 6-OHDA -
was 10 ~1.
Methods Male infant Long-Evans rats, 5 to 7 days old, were selectively depleted of brain NE (NE down), DA (DA down), both these catecholamines (CA down), or 5-HT (5-HT down). Selective depletions of the amines had been attempted as reported earlier [ 17, 181 using intracisternal administration of the neurocytotoxic drugs 6-hydroxydopamine (6-OHDA) or 5,7-dihydroxytryptamine (5,7-DHT) alone, in combination with desmethylimipramine (DMI) or with pargyline (Table 1). Litters of ten animals with a foster mother were housed under normal colony conditions (7 AM - 7 PM, light-dark cycle, 22 - 24 “C, ad Iibitum Purina Chow and water) until weaned at 30 days of age, then housed separately. Animals in each of the five different treatments were then divided into two groups at 45 days of age. One group received 6 - 8 g/100 ml ethanol incorporated into a nutritionally complete liquid diet prepared fresh in the laboratory from purified materials [ 191. The other served as control and was pair-fed the same diet except that ethanol was replaced isocalorically with dextrose. Both groups were acclimated to the control diet for 3 days before ethanol treatment began. Of the total calories in the control diet, 67.8% were derived from dextrose, 24.9% from soluble protein (lactalbumin), 4.9% from lipid (corn oil) in addition to essential vitamins and soluble mineral salts. Animals received the diets fresh daily between 12 - 1 PM in clean graduated cylinders fitted with drinking spouts designed to reduce spillage, for a period of 11 days. Previously, we had shown that rats treated over a period of up to 15 days with this ethanol diet exhibited excessive ethanol consumption (mean range of 10 - 17 g/kg per 24 h) and maintained elevated blood ethanol concentrations (BEC; mean range of 100 - 300 mg/lOO ml) when sampled at 12 noon [ 19,201. In addition, these animals remained in good physical condition and continued to gain weight, showing no ill effects from the treatment until ethanol was withdrawn. Following removal of ethanol from the diet, frequent spontaneous tremors, spasticity, irritability and occasional convulsions over a period of 1 - 4 days were observed which were similar to
352
those reported by others [21, 231. Tremors occurred in as many as 90 100% of the animals during withdrawal, while spontaneous convulsions were infrequent (20% or less). We also found that rats undergoing ethanol withdrawal were susceptible to audiogenic seizures induced by ringing an electric bell (98 db) for one minute [ 19,201, even though they were not susceptible when initially screened before the start of treatment. Audiogenic seizures were observed with much greater frequency than spontaneous ones (essentially 100% for animals which maintained the higher mean BEC). None of the controls to both audiogenic and exhibited audiogenic seizures. Susceptibility spontaneous convulsions generally lasted only 24 hours. We attempted to quantitate the severity and latency of audiogenic seizures by determining an audiogenic response score (Table 2). The scoring TABLE
2
Designated audiogenic response scores according to severity of the response Audiogenic 0 3-l 6-4 9-7 12 - 10 15 - 13 18 - 16
response
score
for observed
Audiogenic
audiogenic
withdrawal
withdrawal
responses
responses
No response or normal behavioral activity First wild running phase Additional wild running phase(s) Wild running phase(s) + clonic convulsive activity Wild running phase(s) + clonic--tonic convulsive activity Wild running phase(s) + tonic convulsive activity Wild running phase(s + convulsive activity + death
Components of the audiogenic withdrawal responses listed above were defined as follows: (1) wild running - at least 3 cycles of rapid running activity where the phases were separated by at least 10 s of quiescent stationary behavior; (2) clonic conuukion - 75% or more of the seizure activity observed as that of hind limb clonus; (3) clonic tonic conuulsion - less than 75% of seizure activity as either hind limb clonus or tonus; (4) tonic convulsion - 75% or more of seizure activity as hind limb tonus; (5) death due to audiogenie seizure - death occurred within 30 minutes after sound testing. Animals which underwent spontaneous convulsions while being handled or transported to the sound test cage received a score of 12 points, while those which died as a result of their spontaneous convulsion received a maximum score of 18 points.
system was comprised of an increasing set of arbitrary values reflecting increasing susceptibility to audiogenic seizures. Scores ranged from a minimum of 0 points for a normal response to a maximum of 18 points for audiogenic seizures followed by death. The range of values at a given level of severity also reflected the latency of that response during the 90-second test interval. For example, a clonic audiogenic seizure which occurred during the first 30 seconds received the highest value, 9 points. One point was deducted from the score for each 30 seconds that the seizure reponse was delayed.
353 TABLE 3 Effects of specific 6-hydroxydopamine whole brain biogenic amines Treatments
Control CA down NE down DA down 5-HT down “p < 0.001,
or 5,7-dihydroxytryptamine
treatments to deplete
Whole brain biogenic amines (ng/g) Norepinephrine
Dopamine
Serotonin
N
532.5 129.8 343.9 552.4 652.5
797.1 148.5 688.1 174.8 888.9
462.3 514.1 472.4 496.6 95.3
5 5 5 5 5
?- 64.4 + 25.1a _+30.0b + 32.6 f 47.4
when compared with controls.
+ ? i + +
53.6 13.6a 99.9 17.3a 32.2
+ 12.3 +_16.2 t 25.4 i 17.5 i 21.5a
bp < 0.025, when compared with controls.
Prior to ethanol treatment all rats in the present study were screened for susceptibility to audiogenic seizures and reactive animals were eliminated from the study. During the llday treatment period ethanol consumption and BEC of aminedenleted rats were monitored at 12 - 1 PM daily. The concentration of ethanol in duplicate samples of venous blood, taken from the tip of the tail, was measured according to the automated enzymatic method of Ellis and Hill [ 241 as modifed by Payne and Ellis [ 251. Following withdrawal of ethanol the animals were observed at frequent intervals for the appearance of spontaneous withdrawal reactions including tremor, spastic motor movements and irritability. Susceptibility to audiogenie seizures was also measured at 8, 24, and 53 hours after ethanol withdrawal. When the animals were 2.5 months of age, which corresponded to 2 weeks after ethanol withdrawal, the concentrations of whole brain biogenic amines were determined as previously reported [26 - 281. A more detailed account of the experimental design will be reported elsewhere [ 291. Values of samples for particular treatment groups were expressed as the sample mean f the standard error of the mean (s.e.m.) for that group. Statistical comparison of the differences between two group means was carried out by calculation of an independent t statistic, with a one-tailed test at a 0.05 level of significance and Nr + Nz - 2 degrees of freedom.
Results and Discussion Determination of whole brain biogenic amine content at 2.5 months of age (2 weeks after chronic ethanol treatment) indicated rather specific longterm depletions of the amines had been produced with 6-OHDA or 5,7-DHT (Table 3). For example, the brains of animals which were pre-treated with 6-OHDA to deplete specifically both NE and DA (see CA down group, Table 1) contained significantly less of these biogenic amines than did controls (Table 3). However, levels of serotonin were not affected. Similarly,
354
animals pre-treated with 5,7-DHT in combination with pargyline (5-HT down group, Table 1) had significantly lower levels of 5-HT than had controls, while levels of NE and DA were not altered. These long-term depletions of specific biogenic amines in developing animals were in agreement with previous observations of Breese and Traylor [ 171 and Smith et al. [ 181. Such depletions of NE and/or DA or 5-HT after 60HDA or 5,7-DHT, respectively, have been correlated with destruction of biogenic amine neurons in the brain [30] . Deficits in the behavior of animals with selective depletions of brain serotonin or catecholamines have been interpreted as evidence of a role for these compounds in specific CNS functions such as the control of temperature, ingestion and operant behaviours, as well as the behavioral effects of amphetamine [30] . Rats which had received intracisternal injections of 6-OHDA or 5,7DHT as infants weighed significantly less than saline controls at 45 days of age (Table 4). Animals in the CA down groups gained the least weight and weighed significantly less than animals in all other groups. This finding was consistent with the observations of others [ 17, 31, 321 who have described a prominent reduction in the growth of animals which received similar neurocytotoxic treatments to deplete NE, DA, or 5-HT. None of the animals in any of the treatment groups displayed susceptibility to audiogenic seizures when exposed to sound stimulation at this time. During the period of liquid diet treatment ethanol consumption was similar for controls, NE down, DA down, and 5-HT down animals (Table 4). However, CA down rats consumed, on a weight basis, significantly more ethanol each day (13.8 + 0.1 g/kg per 24 h compared with 12.8 f 0.2 g/kg per 24 h for controls). Combined mean BEC values measured across days 3,6,9, and 11 of the treatment period (Table 4) were also similar for all treatment groups, with the exception of the CA down animals. Controls maintained a BEC of 202.7 + 19.2 mg/dl when sampled on these 4 days at 12 - 1 PM, while CA down rats had significantly lower levels (116.0 + 22.4 mg/dl) despite greater ethanol consumption. The reason for this discrepancy with CA down rats is not apparent. It is possible that for some unknown TABLE Effects
4 of brain biogenic
Depletion treatment
Control NE down DA down CA down 5-HT down ‘p < 0.001,
amine depletion
in rats under ethanol
treatment
Body weight at 45 days of life
Ethanol consumption
Mean BEC across days 3,6, 9, and 11
(g)
(g/kg per 24 h)
(mg/dl)
12.8 12.8 12.5 13.8 12.9
202.7 191.5 173.4 116.0 198.9
168.4 144.8 128.0 97.0 146.8
? 2.4 i 4.5a _+6.1a * 8.2a r 4.3a
when compared
with control.
? f ? f t
0.2 0.3 0.3 O.lb 0.4
bp < 0.05,
when compared
* i f i +
19.2 30.9 27.0 22.4= 34.3
with control.
355
reason the capacity of CA down animals for ethanol clearance or metabolism may be greater than that of controls. Significant differences in body weight between CA down animals and controls may also partially explain the observed reverse correlation between ethanol consumption and BEC in control and CA down groups, since ethanol consumption measurements based on body weight may have been distorted. Studies are currently underway to examine this possibility. At the end of the 11-day treatment period, when ethanol was withdrawn at 9 AM, BEC were found to be similar for all groups (Table 5). The TABLE 5 Effects of brain biogenic amine depletion in rats on the subsequent development of ethanol-withdrawal hyperexcitability Treatment
BEC at withdrawal (mg/dl)
ARS8h post-withdrawal
ARS 24 h post-withdrawal
Post-convulsive lethality (%)
Control NE down DA down CA down 5-HT down
239.7 236.2 203.8 160.9 213.1
8.33 10.50 9.62 6.55 8.62
00.00 2.16 f 1.79 1.16 f 1.16 00.00 00.00
44.3 33.3 33.3 o.oa 44.4
‘p < 0.001,
+ 41.4 f 25.1 + 46.5 + 26.6 + 62.0
+ r i r +
2.51 2.52 2.32 1.71 3.26
N 9 9 9 9 9
when compared with controls.
TABLE 6 Effect of biogenic amine depletion on the severity of audiogenically induced ethanol withdrawal responses Treatment
Wild running
Convulsions
Deaths
Control NE down DA down CA down 5-HT down
619 719
519 619
419 319
519
719
Olga
“p < 0.001,
719 519
619 519
319 419
when compared with control.
CA down animals once again had the lowest levels. Within 2 - 3 hours after withdrawal, tremors, spastic motor movements and irritability became apparent in all ethanol-treated rats, but not in pair-fed controls. These reactions appeared to attain maximum intensity within 5 - 8 hours and then they gradually diminished over the next 2 - 3 days, as previously reported for ethanol-withdrawn rats naive to 6-OHDA or 5,7-DHT treatment [20]. However, no attempt was made to quantitate and compare these spontaneous reactions between groups.
356
Exposure to standardized sound stimuli at 8 hours after withdrawal revealed susceptibility to audiogenic seizures in a majority of rats in all groups that received ethanol treatment (Table 6), but not in groups pairfed control diets. Of the rats receiving ethanol, there were no significant differences among the numbers of animals displaying wild running or convulsions in the presence of sound for any of the amine-depleting treatments as compared with controls. The numbers of rats that died following seizures were also similar among groups, except for the CA down group in which no rats had lethal reactions. Audiogenic response scores (ARS) calculated from these data (Table 5) indicated no significant differences between groups at any of the three time points studied. However, at the 8hour test period, CA down animals did possess the lowest test scores. The fact that CA down rats did not die as a result of withdrawal-related audiogenic seizures might suggest an involvement of one or both catecholamines in the mechanisms leading to seizure-induced lethality. However, it is more likely that this group did not develop a level of physical dependence sufficiently severe to result in post-convulsive lethality. The lower BEC observed during liquid diet treatment (Table 4) would support this view. Also depletion of either NE or DA alone had no apparent effect on the incidence of postconvulsive lethality in other groups (Tables 5 and 6). Because young rats pre-treated with the neurocytotoxic agents to deplete specifically NE and/or DA or 5-HT displayed ethanol withdrawal syndromes and were susceptible to audiogenic seizures in a manner similar to controls, no support for the proposed role of these biogenic amines in the mechanisms underlying the development of physical dependence on ethanol is provided by this study. However, these results are not necessarily in disagreement with the results of others [8 - 121 indicating changes in the biochemistry of brain amines after chronic ethanol treatment. Failure of 6-OHDA or 5,7-DHT treatments to block the development of physical dependence might suggest that observed changes in the turnover of NE in the brain [lo - 121 may be due to other effects of ethanol, such as stress or tolerance development. Recently, Ritzmann and Tabakoff [33] have shown that intraventricular injections of 6-OHDA, which altered levels of both NE and DA in the brain of mice, blocked the development of tolerance to narcotic doses of ethanol, but were without effect on physical dependence development. Others have reported that administration of drugs interfering with the homeostasis of NE, DA, or 5-HT during withdrawal aggravates the withdrawal syndrome [ 13 - 151. Such changes in withdrawal suggest a role of these amines as modulators of specific ongoing withdrawal reactions, probably independent of the general underlying mechanism responsible for physical dependence development. For example, drugs interfering with 5-HT systems have been used to modify withdrawal-induced head twitches in mice [ 161 , while having no effect on convulsions on handling the animals [ 13,161 . It is possible that the magnitude of NE, DA or 5-HT depletions in the developing rats was not sufficient to produce significant disruption of
357
function in biogenic amine pathways. Also, treatment with 6-OHDA has been shown to produce differential degrees of depletion in various brain areas [26,34,35] . However, similar depletions of NE and DA have been shown to block tolerance development to ethanol in mice [ 331.
In conclusion Evidence of an ethanol withdrawal syndrome in rats which had received prior treatment with neurocytotoxic drugs to produce long-term depletions of NE, DA, both of these amines, or 5-HT did not provide support for the proposed role of these biogenic amines in the mechanisms underlying physical dependence development. The results suggest that previously observed changes in biogenic amine biochemistry during ethanol treatment or modifications of withdrawal behaviors after pharmacological manipulation of biogenic amines may indicate an involvement of the amines in other effects of ethanol, such as stress and tolerance development, or as modulators of withdrawal reactions.
Acknowledgments The authors wish to thank Dr. George R. Breese for generously supplying 6-OHDA and 5,7-DHT treated rats and for the determination of brain biogenic amines. The research was supported in part by a grant from the North Carolina Alcoholism Research Authority.
References
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
J. Cochin, Fed. Proc., 29 (1970) 19 - 27. W. R. Martin, Fed. Proc., 29 (1970) 13 - 18. L. Schuster, Nature, 189 (1961) 314 - 315. D. B. Goldstein and A. Goldstein, Biochem. Pharmacol., 8 (1961) 48. H. 0. J. Collier, Nature, 205 (1965) 181 - 182. J. H. Jaffe and S. K. Sharpless, J. Pharmacol. Exp. Ther., 150 (1965) 140 - 145. B. B. Brodie, S. Spector and S. L. Silver, Pharmacol. Rev., 11 (1959) 548 - 564. A. Feldstein, in B. Kissin and H. Begleiter (eds.), The Biology of Alcoholism, Vol. 1, Plenum Press, New York, 1971, pp. 127 - 160. R. A. Deitrich, Psychoneuropharmacology, 1 (1976) 325 - 346. L. A. Pohorecky, J. Pharmacol. Exp. Ther., 189 (1974) 380 - 391. W. A. Hunt and E. Majchrowicz, J. Neurochem., 23 (1974) 549 - 552. L. Ahtee and M. Svartstrhm-Fraser, Acta Pharmacol. Toxicol., 36 (1975) 289 - 298. D. B. Goldstein, J. Pharmacol. Exp. Ther., 186 (1973) 1 - 9. P. J. Griffiths, J. M. Littleton and A. Ortiz, Brit. J. Pharmacol., 50 (1974) 489 - 498. H. 0. J. Collier, Brit. J. Pharmacol., 51 (1974) 310 - 311. P. J. Griffith, J. M. Littleton and A. Ortiz, Brit. J. Pharmacol., 51 (1974) 307 - 309. G. R. Breese and T. D. Traylor, Brit. J. Pharmacol., 44 (1972) 210 - 222.
358 18 R. D. Smith, B. R. Cooper and G. R. Breese, J. Pharmacol. Exp. Ther., 185 (1973) 609 - 619. 19 G. D. Frye, R. G. Reitz, F. W. Ellis and J. Thompson, in preparation. 20 G. D. Frye and F. W. Ellis, Pharmacologist, 17 (1975) 197. 21 E. Majchrowicz, Psychopharmacologia (Berlin), 43 (1975) 245 - 254. 22 B. E. Hunter, C. A. Boast, D. W. Walker and S. F. Zornetzer, Pharmacol. Biochem. Behavior, 1 (1973) 719 - 725. 23 G. Freund, in E. Majchrowicz (ed.), Biochemical Pharmacology of Ethanol, Plenum Press, New York, 1975, pp. 311 . 325. 24 F. W. Ellis and J. B. Hill, Clin. Chem., 15 (1969) 91 - 101. 25 D. W. Payne and F. W. Ellis, in Advances in Automated Analysis, Meidad Inc., White Plains, 1973. 26 G. R. Breese and T. D. Traylor, J. Pharmacol. Exp. Ther., 174 (1970) 413 - 420. 27 G. R. Breese and T. D. Traylor, Brit. J. Pharmacol., 42 (1971) 88 - 99. 28 G. R. Breese, B. R. Cooper, L. D. Grant and R. D. Smith, Neuropharmacology, 13 (1974) 177 - 187. 29 G. D. Frye, G. R. Breese and F. W. Ellis (in preparation). 30 G. R. Breese, in L. L. Iversen, S. D. Iversen and S. H. Snyder (eds.), Handbook of Psychopharmacology, Vol. 1, Plenum Press, New York, 1975, pp. 127 - 189. 31 L. Lytle, W. Shoemaker, K. Cottman and R. Wurtman, J. Pharmacol. Exp. Ther., 183 (1972) 56 - 64. 32 G. R. Breese and B. R. Cooper, Brain Res., 98 (1975) 517 - 527. 33 R. F. Ritzmann and B. Tabakoff, Nature, 263 (1976) 418 - 419. 34 L. L. Iversen and N. J. Uretsky, Brain Res., 24 (1970) 364 - 367. 35 L. L. Iversen and N. J Uretsky, in T. Malmfors and H. Thoenen (eds.), B-Hydroxydopamine and Catecholamine Neurons, North Holland, Amsterdam, 1971, pp. 171 186.
Discussion SMITH - The fact that you can demonstrate that dependence, measured by withdrawal symptomatology, is unaffected by depletion of amines, tends to diminish the likelihood that catecholamine alkaloids, for example, are involved in the process of dependence. FRYE - Our results do demonstrate that lowering the amines, and thus making them less available for these types of reactions, does not affect dependence. COHEN - I’d like to point out that in your experiments you’re knocking out various tracts. However, in any specific neuron that was left, amine levels are normal and therefore the condensation reactions could still take place in particular neurons. I’m trying to find a way out of the idea that catecholamines and 5-HT are not involved in dependence. We should remember that it takes a great deal of destruction in order to get a functional change. I have two questions. First, do you think that supersensitivity following degeneration may offset any effect you might see? Secondly, since when you lesion with 6-GHDA or 5,7-DHT you can get reinnervation, do you think that animals which have 30 - 40% of normal levels of catecholamines may have these catecholamines localized in different areas from those of normal animals? FRYE - It’s likely that there is increased receptor sensitivity which could affect our results. Regarding regeneration, we have observed this phenomenon; however, whether the axons that sprout from functional connections is not known. Two and one half months after treatment amine levels are still lowered, although eventually norepinephrine levels do begin to come back towards normal values.
359
DEITRICH - I think that it’s important that you have standardized your technique for inducing audiogenic seizures and also that you screened your animals for susceptibility to these seizures prior to ethanol treatment. FRYE - It is important to screen animals for their susceptibility to audiogenic seizures. We have found that about 15% of our Sprague-Dawley animals are normally susceptible to audiogenic seizures while Long- Evans animals are not. POHORECKY - Your results indicating that amine depletion has little effect on withdrawal symptoms may not be so surprising since it is known that functions which are influenced by monoamines, although they are initially quite impaired in animals treated with neurotoxins, eventually return to normal. M. COLLINS - In some cases the drugs that you use such as o-methyl-p-tyrosine may affect ethanol metabolism or ethanol may affect the uptake of the drug into brain. So you may have a different situation using a drug plus ethanol rather than the drug itself. FRYE
- Yes, these are real problems.
TRUITT - There are many factors that have to be considered in these types of studies. For example, the time course of the study, the strain of animal used, and whether turnover or levels of amines are measured. The role of acetaldehyde is also important and may also depend on the animal used or the time that the study is done. One should also consider the relationship of various biogenic amines to each other rather than each amine in isolation. THURMAN - In the course of the Symposium we’ve heard a couple of reports on increases in cyclic nucleotide levels during withdrawal. You see no change in neurotransmitter levels. What model would you propose to account for changes in nucleotide levels? FRYE - Steady-state levels don’t tell the whole story. There is evidence for changes in turnover of transmitters which may be correlated with the changes in cyclic nucleotide levels. KALANT - We have found no change in 5-HT turnover with either acute or chronic ethanol treatment or during withdrawal. We did find that when we used PCPA to severely deplete serotonin levels we could prevent the development of ethanol tolerance. We were using a lower dose of ethanol than you and we did not see signs of withdrawal. We found an important interaction between PCPA and the type of tolerance that is measured: using a behavioral test for tolerance we could block tolerance with PCPA. However, PCPA had no effect when sleep time was used to measure tolerance. The apparent reason is that no nervous system tolerance for sleep time developed with the type of chronic treatment that we used. If anything, PCPA prolonged sleep time after ethanol or barbiturate treatment and any tolerance that did occur was metabolic. It’s important, in determining whether tolerance and dependence are separable and whether 5-HT is involved in these phenomena, what dose of ethanol is used to determine tolerance and how much of tolerance is metabolic and how much is functional. TABAKOFF - In our studies in which 6-OHDA was used to separate the development of tolerance from the development of dependence we used three doses of ethanol, all in the sedative range, to measure functional tolerance. We found little metabolic tolerance. It’s interesting that 5-HT may affect one type of tolerance and possibly noradrenergic systems another type, since tolerance to the hypnotic effects of ethanol as measured by sleep time is affected by 6-OHDA but not by PCPA pretreatment.
349
EFFECTS OF 6-HYDROXYDOPAMINE OR 5,7-DIHYDROXYTRYPTAMINE ON THE DEVELOPMENT OF PHYSICAL DEPENDENCE ON ETHANOL GERALD D. FRYE*
and FRED W. ELLIS
Department of Pharmacology, 27514 (U.S.A.)
University of North Carolina, Chapel Hill, North Carolina
Summary Infant rats, treated intracistemally with 6-hydroxydopamine or 5,7dihydroxytryptamine, alone or in combination with desmethylimipramine or pargyline, at 5 to 7 days of age, had significant specific depletions of brain norepinephrine, dopamine, both of these amines, or serotonin at 2.5 months of age. Despite apparent long-term depletions of brain biogenic amines, susceptibility to audiogenically-induced seizures following chronic ethanol withdrawal in these animals was similar to that of controls. Amine-depleted rats also displayed spontaneous withdrawal-induced tremors, spastic motor activity and irritability. The interpretation of these preliminary findings with regard to the proposed role of the biogenic amines in the development of physical dependence on ethanol is discussed.
Introduction It has been proposed that various adaptive processes in central biochemical and physiological mechanisms are involved in the development of functional tolerance to and physical dependence on central nervous system (CNS) depressant drugs like ethanol [ 1,2]. A variety of mechanisms have been postulated by which drugs might interact with feedback systems regulating the activity of enzymes [ 3,4] or the sensitivity of receptors [ 5,6] for putative neurotransmitter substances in the brain to produce tolerance and physical dependence. This has led to the examination of the putative neurotransmitters norepinephrine (NE), dopamine (DA) and serotonin (5-HT), which have been shown to be important in CNS function [ 71, for possible involvement in the development of physical dependence on ethanol. Early studies designed to look for changes in the levels of brain biogenic amines during both acute and chronic ethanol treatment reported conflicting *Now at the Biological Sciences Research Chapel Hill, North Carolina 27514 (U.S.A.)
Center,
University of North Carolina,
results and failed to produce a consistent picture of interaction [ 8. 91. However, more recent studies using techniques that examine biogenic amine turnover [ 10 - 121 now suggest that acute ethanol treatment first accelerates and then depresses turnover of brain NE. Later, during continued ethanol treatment NE turnover is once again accelerated and then increased further following ethanol withdrawal. Studies examining DA and 5HT turnover have continued to produce conflicting results [ 91. The pharmacological manipulation of central catecholamine and serotonin systems results in changes during the ethanol abstinence syndrome suggesting their involvement in withdrawal reactions. Drugs which interfere with the function of catecholamine neurons have been shown to aggravate the severity of convulsions on handling mice which were made physically dependent after exposure to ethanol vapors [ 13, 141. Others have found that drugs facilitating the activity of NE neurons reduced head twitches in mice during ethanol withdrawal [ 151. However, results from studies in which brain serotonin was manipulated imply that this neurochemical may be involved in withdrawal-induced head twitches [ 151, but not in convulsions on handling ethanol-dependent mice [ 13, 161. Thus, recent biochemical and pharmacological studies of chronic ethanol intoxication and withdrawal strongly suggest an ethanol effect on NE neurons in the brain, while effects on DA and 5-HT containing neurons are less clear. However, these results may not necessarily indicate that NE is involved in the adaptive processes which are responsible for the development of tolerance to and physical dependence on ethanol. For example, changes in the turnover of NE in the brain during chronic ethanol exposure may simply reflect intoxication and withdrawal-induced stress or may be due secondarily to more direct actions of the drug to alter the function of other neurons. Also acute pharmacological manipulation of biogenic amines resulting in modification of withdrawal reactions does not necessarily indicate that these substances are involved in the mechanisms underlying physical dependence development. They may be involved primarily in systems mediating the display or modulation of withdrawal reactions. Recently we have attempted to examine more closely the hypothesis that the biogenic amines may play a major role in the underlying adaptive mechanisms leading to physical dependence on ethanol. It was assumed, perhaps rather simplistically, that if NE, DA, or 5-HT were involved in the principal mechanism of ethanol action to produce physical dependence, then destruction of these respective transmitter systems in the brain prior to chronic ethanol treatment should markedly and dramatically reduce the severity of the resulting withdrawal syndrome. Conversely, if these systems were not involved directly or simply acted as modulators of ongoing withdrawal reactions, much less dramatic changes in the severity of the withdrawal syndrome might be expected. The present report indicates results of preliminary experiments designed to test this hypothesis. Effects of chronic ethanol consumption on rats pre-treated with neurocytotoxic drugs to produce long-term depletions of brain biogenic amines were examined.
351
TABLE
1
Treatments
to deplete
norepinephrine,
dopamine,
and serotonin
in infant
rats
Treatmenta .~
Day 5
Day 7
Control NE down DA down CA down 5-HT down
Saline 13 pg 6-OHDA 100 pg 6-OHDA 100 E.cg6-OHDA 100 pg 5,7-DHT
-
aVolume
of the intracisternal
injections
+ 20 mg/kg
DMI
+ 30 mg/kg
pargyline
15 pg 6-OHDA -
was 10 ~1.
Methods Male infant Long-Evans rats, 5 to 7 days old, were selectively depleted of brain NE (NE down), DA (DA down), both these catecholamines (CA down), or 5-HT (5-HT down). Selective depletions of the amines had been attempted as reported earlier [ 17, 181 using intracisternal administration of the neurocytotoxic drugs 6-hydroxydopamine (6-OHDA) or 5,7-dihydroxytryptamine (5,7-DHT) alone, in combination with desmethylimipramine (DMI) or with pargyline (Table 1). Litters of ten animals with a foster mother were housed under normal colony conditions (7 AM - 7 PM, light-dark cycle, 22 - 24 “C, ad Iibitum Purina Chow and water) until weaned at 30 days of age, then housed separately. Animals in each of the five different treatments were then divided into two groups at 45 days of age. One group received 6 - 8 g/100 ml ethanol incorporated into a nutritionally complete liquid diet prepared fresh in the laboratory from purified materials [ 191. The other served as control and was pair-fed the same diet except that ethanol was replaced isocalorically with dextrose. Both groups were acclimated to the control diet for 3 days before ethanol treatment began. Of the total calories in the control diet, 67.8% were derived from dextrose, 24.9% from soluble protein (lactalbumin), 4.9% from lipid (corn oil) in addition to essential vitamins and soluble mineral salts. Animals received the diets fresh daily between 12 - 1 PM in clean graduated cylinders fitted with drinking spouts designed to reduce spillage, for a period of 11 days. Previously, we had shown that rats treated over a period of up to 15 days with this ethanol diet exhibited excessive ethanol consumption (mean range of 10 - 17 g/kg per 24 h) and maintained elevated blood ethanol concentrations (BEC; mean range of 100 - 300 mg/lOO ml) when sampled at 12 noon [ 19,201. In addition, these animals remained in good physical condition and continued to gain weight, showing no ill effects from the treatment until ethanol was withdrawn. Following removal of ethanol from the diet, frequent spontaneous tremors, spasticity, irritability and occasional convulsions over a period of 1 - 4 days were observed which were similar to
352
those reported by others [21, 231. Tremors occurred in as many as 90 100% of the animals during withdrawal, while spontaneous convulsions were infrequent (20% or less). We also found that rats undergoing ethanol withdrawal were susceptible to audiogenic seizures induced by ringing an electric bell (98 db) for one minute [ 19,201, even though they were not susceptible when initially screened before the start of treatment. Audiogenic seizures were observed with much greater frequency than spontaneous ones (essentially 100% for animals which maintained the higher mean BEC). None of the controls to both audiogenic and exhibited audiogenic seizures. Susceptibility spontaneous convulsions generally lasted only 24 hours. We attempted to quantitate the severity and latency of audiogenic seizures by determining an audiogenic response score (Table 2). The scoring TABLE
2
Designated audiogenic response scores according to severity of the response Audiogenic 0 3-l 6-4 9-7 12 - 10 15 - 13 18 - 16
response
score
for observed
Audiogenic
audiogenic
withdrawal
withdrawal
responses
responses
No response or normal behavioral activity First wild running phase Additional wild running phase(s) Wild running phase(s) + clonic convulsive activity Wild running phase(s) + clonic--tonic convulsive activity Wild running phase(s) + tonic convulsive activity Wild running phase(s + convulsive activity + death
Components of the audiogenic withdrawal responses listed above were defined as follows: (1) wild running - at least 3 cycles of rapid running activity where the phases were separated by at least 10 s of quiescent stationary behavior; (2) clonic conuukion - 75% or more of the seizure activity observed as that of hind limb clonus; (3) clonic tonic conuulsion - less than 75% of seizure activity as either hind limb clonus or tonus; (4) tonic convulsion - 75% or more of seizure activity as hind limb tonus; (5) death due to audiogenie seizure - death occurred within 30 minutes after sound testing. Animals which underwent spontaneous convulsions while being handled or transported to the sound test cage received a score of 12 points, while those which died as a result of their spontaneous convulsion received a maximum score of 18 points.
system was comprised of an increasing set of arbitrary values reflecting increasing susceptibility to audiogenic seizures. Scores ranged from a minimum of 0 points for a normal response to a maximum of 18 points for audiogenic seizures followed by death. The range of values at a given level of severity also reflected the latency of that response during the 90-second test interval. For example, a clonic audiogenic seizure which occurred during the first 30 seconds received the highest value, 9 points. One point was deducted from the score for each 30 seconds that the seizure reponse was delayed.
353 TABLE 3 Effects of specific 6-hydroxydopamine whole brain biogenic amines Treatments
Control CA down NE down DA down 5-HT down “p < 0.001,
or 5,7-dihydroxytryptamine
treatments to deplete
Whole brain biogenic amines (ng/g) Norepinephrine
Dopamine
Serotonin
N
532.5 129.8 343.9 552.4 652.5
797.1 148.5 688.1 174.8 888.9
462.3 514.1 472.4 496.6 95.3
5 5 5 5 5
?- 64.4 + 25.1a _+30.0b + 32.6 f 47.4
when compared with controls.
+ ? i + +
53.6 13.6a 99.9 17.3a 32.2
+ 12.3 +_16.2 t 25.4 i 17.5 i 21.5a
bp < 0.025, when compared with controls.
Prior to ethanol treatment all rats in the present study were screened for susceptibility to audiogenic seizures and reactive animals were eliminated from the study. During the llday treatment period ethanol consumption and BEC of aminedenleted rats were monitored at 12 - 1 PM daily. The concentration of ethanol in duplicate samples of venous blood, taken from the tip of the tail, was measured according to the automated enzymatic method of Ellis and Hill [ 241 as modifed by Payne and Ellis [ 251. Following withdrawal of ethanol the animals were observed at frequent intervals for the appearance of spontaneous withdrawal reactions including tremor, spastic motor movements and irritability. Susceptibility to audiogenie seizures was also measured at 8, 24, and 53 hours after ethanol withdrawal. When the animals were 2.5 months of age, which corresponded to 2 weeks after ethanol withdrawal, the concentrations of whole brain biogenic amines were determined as previously reported [26 - 281. A more detailed account of the experimental design will be reported elsewhere [ 291. Values of samples for particular treatment groups were expressed as the sample mean f the standard error of the mean (s.e.m.) for that group. Statistical comparison of the differences between two group means was carried out by calculation of an independent t statistic, with a one-tailed test at a 0.05 level of significance and Nr + Nz - 2 degrees of freedom.
Results and Discussion Determination of whole brain biogenic amine content at 2.5 months of age (2 weeks after chronic ethanol treatment) indicated rather specific longterm depletions of the amines had been produced with 6-OHDA or 5,7-DHT (Table 3). For example, the brains of animals which were pre-treated with 6-OHDA to deplete specifically both NE and DA (see CA down group, Table 1) contained significantly less of these biogenic amines than did controls (Table 3). However, levels of serotonin were not affected. Similarly,
354
animals pre-treated with 5,7-DHT in combination with pargyline (5-HT down group, Table 1) had significantly lower levels of 5-HT than had controls, while levels of NE and DA were not altered. These long-term depletions of specific biogenic amines in developing animals were in agreement with previous observations of Breese and Traylor [ 171 and Smith et al. [ 181. Such depletions of NE and/or DA or 5-HT after 60HDA or 5,7-DHT, respectively, have been correlated with destruction of biogenic amine neurons in the brain [30] . Deficits in the behavior of animals with selective depletions of brain serotonin or catecholamines have been interpreted as evidence of a role for these compounds in specific CNS functions such as the control of temperature, ingestion and operant behaviours, as well as the behavioral effects of amphetamine [30] . Rats which had received intracisternal injections of 6-OHDA or 5,7DHT as infants weighed significantly less than saline controls at 45 days of age (Table 4). Animals in the CA down groups gained the least weight and weighed significantly less than animals in all other groups. This finding was consistent with the observations of others [ 17, 31, 321 who have described a prominent reduction in the growth of animals which received similar neurocytotoxic treatments to deplete NE, DA, or 5-HT. None of the animals in any of the treatment groups displayed susceptibility to audiogenic seizures when exposed to sound stimulation at this time. During the period of liquid diet treatment ethanol consumption was similar for controls, NE down, DA down, and 5-HT down animals (Table 4). However, CA down rats consumed, on a weight basis, significantly more ethanol each day (13.8 + 0.1 g/kg per 24 h compared with 12.8 f 0.2 g/kg per 24 h for controls). Combined mean BEC values measured across days 3,6,9, and 11 of the treatment period (Table 4) were also similar for all treatment groups, with the exception of the CA down animals. Controls maintained a BEC of 202.7 + 19.2 mg/dl when sampled on these 4 days at 12 - 1 PM, while CA down rats had significantly lower levels (116.0 + 22.4 mg/dl) despite greater ethanol consumption. The reason for this discrepancy with CA down rats is not apparent. It is possible that for some unknown TABLE Effects
4 of brain biogenic
Depletion treatment
Control NE down DA down CA down 5-HT down ‘p < 0.001,
amine depletion
in rats under ethanol
treatment
Body weight at 45 days of life
Ethanol consumption
Mean BEC across days 3,6, 9, and 11
(g)
(g/kg per 24 h)
(mg/dl)
12.8 12.8 12.5 13.8 12.9
202.7 191.5 173.4 116.0 198.9
168.4 144.8 128.0 97.0 146.8
? 2.4 i 4.5a _+6.1a * 8.2a r 4.3a
when compared
with control.
? f ? f t
0.2 0.3 0.3 O.lb 0.4
bp < 0.05,
when compared
* i f i +
19.2 30.9 27.0 22.4= 34.3
with control.
355
reason the capacity of CA down animals for ethanol clearance or metabolism may be greater than that of controls. Significant differences in body weight between CA down animals and controls may also partially explain the observed reverse correlation between ethanol consumption and BEC in control and CA down groups, since ethanol consumption measurements based on body weight may have been distorted. Studies are currently underway to examine this possibility. At the end of the 11-day treatment period, when ethanol was withdrawn at 9 AM, BEC were found to be similar for all groups (Table 5). The TABLE 5 Effects of brain biogenic amine depletion in rats on the subsequent development of ethanol-withdrawal hyperexcitability Treatment
BEC at withdrawal (mg/dl)
ARS8h post-withdrawal
ARS 24 h post-withdrawal
Post-convulsive lethality (%)
Control NE down DA down CA down 5-HT down
239.7 236.2 203.8 160.9 213.1
8.33 10.50 9.62 6.55 8.62
00.00 2.16 f 1.79 1.16 f 1.16 00.00 00.00
44.3 33.3 33.3 o.oa 44.4
‘p < 0.001,
+ 41.4 f 25.1 + 46.5 + 26.6 + 62.0
+ r i r +
2.51 2.52 2.32 1.71 3.26
N 9 9 9 9 9
when compared with controls.
TABLE 6 Effect of biogenic amine depletion on the severity of audiogenically induced ethanol withdrawal responses Treatment
Wild running
Convulsions
Deaths
Control NE down DA down CA down 5-HT down
619 719
519 619
419 319
519
719
Olga
“p < 0.001,
719 519
619 519
319 419
when compared with control.
CA down animals once again had the lowest levels. Within 2 - 3 hours after withdrawal, tremors, spastic motor movements and irritability became apparent in all ethanol-treated rats, but not in pair-fed controls. These reactions appeared to attain maximum intensity within 5 - 8 hours and then they gradually diminished over the next 2 - 3 days, as previously reported for ethanol-withdrawn rats naive to 6-OHDA or 5,7-DHT treatment [20]. However, no attempt was made to quantitate and compare these spontaneous reactions between groups.
356
Exposure to standardized sound stimuli at 8 hours after withdrawal revealed susceptibility to audiogenic seizures in a majority of rats in all groups that received ethanol treatment (Table 6), but not in groups pairfed control diets. Of the rats receiving ethanol, there were no significant differences among the numbers of animals displaying wild running or convulsions in the presence of sound for any of the amine-depleting treatments as compared with controls. The numbers of rats that died following seizures were also similar among groups, except for the CA down group in which no rats had lethal reactions. Audiogenic response scores (ARS) calculated from these data (Table 5) indicated no significant differences between groups at any of the three time points studied. However, at the 8hour test period, CA down animals did possess the lowest test scores. The fact that CA down rats did not die as a result of withdrawal-related audiogenic seizures might suggest an involvement of one or both catecholamines in the mechanisms leading to seizure-induced lethality. However, it is more likely that this group did not develop a level of physical dependence sufficiently severe to result in post-convulsive lethality. The lower BEC observed during liquid diet treatment (Table 4) would support this view. Also depletion of either NE or DA alone had no apparent effect on the incidence of postconvulsive lethality in other groups (Tables 5 and 6). Because young rats pre-treated with the neurocytotoxic agents to deplete specifically NE and/or DA or 5-HT displayed ethanol withdrawal syndromes and were susceptible to audiogenic seizures in a manner similar to controls, no support for the proposed role of these biogenic amines in the mechanisms underlying the development of physical dependence on ethanol is provided by this study. However, these results are not necessarily in disagreement with the results of others [8 - 121 indicating changes in the biochemistry of brain amines after chronic ethanol treatment. Failure of 6-OHDA or 5,7-DHT treatments to block the development of physical dependence might suggest that observed changes in the turnover of NE in the brain [lo - 121 may be due to other effects of ethanol, such as stress or tolerance development. Recently, Ritzmann and Tabakoff [33] have shown that intraventricular injections of 6-OHDA, which altered levels of both NE and DA in the brain of mice, blocked the development of tolerance to narcotic doses of ethanol, but were without effect on physical dependence development. Others have reported that administration of drugs interfering with the homeostasis of NE, DA, or 5-HT during withdrawal aggravates the withdrawal syndrome [ 13 - 151. Such changes in withdrawal suggest a role of these amines as modulators of specific ongoing withdrawal reactions, probably independent of the general underlying mechanism responsible for physical dependence development. For example, drugs interfering with 5-HT systems have been used to modify withdrawal-induced head twitches in mice [ 161 , while having no effect on convulsions on handling the animals [ 13,161 . It is possible that the magnitude of NE, DA or 5-HT depletions in the developing rats was not sufficient to produce significant disruption of
357
function in biogenic amine pathways. Also, treatment with 6-OHDA has been shown to produce differential degrees of depletion in various brain areas [26,34,35] . However, similar depletions of NE and DA have been shown to block tolerance development to ethanol in mice [ 331.
In conclusion Evidence of an ethanol withdrawal syndrome in rats which had received prior treatment with neurocytotoxic drugs to produce long-term depletions of NE, DA, both of these amines, or 5-HT did not provide support for the proposed role of these biogenic amines in the mechanisms underlying physical dependence development. The results suggest that previously observed changes in biogenic amine biochemistry during ethanol treatment or modifications of withdrawal behaviors after pharmacological manipulation of biogenic amines may indicate an involvement of the amines in other effects of ethanol, such as stress and tolerance development, or as modulators of withdrawal reactions.
Acknowledgments The authors wish to thank Dr. George R. Breese for generously supplying 6-OHDA and 5,7-DHT treated rats and for the determination of brain biogenic amines. The research was supported in part by a grant from the North Carolina Alcoholism Research Authority.
References
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
J. Cochin, Fed. Proc., 29 (1970) 19 - 27. W. R. Martin, Fed. Proc., 29 (1970) 13 - 18. L. Schuster, Nature, 189 (1961) 314 - 315. D. B. Goldstein and A. Goldstein, Biochem. Pharmacol., 8 (1961) 48. H. 0. J. Collier, Nature, 205 (1965) 181 - 182. J. H. Jaffe and S. K. Sharpless, J. Pharmacol. Exp. Ther., 150 (1965) 140 - 145. B. B. Brodie, S. Spector and S. L. Silver, Pharmacol. Rev., 11 (1959) 548 - 564. A. Feldstein, in B. Kissin and H. Begleiter (eds.), The Biology of Alcoholism, Vol. 1, Plenum Press, New York, 1971, pp. 127 - 160. R. A. Deitrich, Psychoneuropharmacology, 1 (1976) 325 - 346. L. A. Pohorecky, J. Pharmacol. Exp. Ther., 189 (1974) 380 - 391. W. A. Hunt and E. Majchrowicz, J. Neurochem., 23 (1974) 549 - 552. L. Ahtee and M. Svartstrhm-Fraser, Acta Pharmacol. Toxicol., 36 (1975) 289 - 298. D. B. Goldstein, J. Pharmacol. Exp. Ther., 186 (1973) 1 - 9. P. J. Griffiths, J. M. Littleton and A. Ortiz, Brit. J. Pharmacol., 50 (1974) 489 - 498. H. 0. J. Collier, Brit. J. Pharmacol., 51 (1974) 310 - 311. P. J. Griffith, J. M. Littleton and A. Ortiz, Brit. J. Pharmacol., 51 (1974) 307 - 309. G. R. Breese and T. D. Traylor, Brit. J. Pharmacol., 44 (1972) 210 - 222.
358 18 R. D. Smith, B. R. Cooper and G. R. Breese, J. Pharmacol. Exp. Ther., 185 (1973) 609 - 619. 19 G. D. Frye, R. G. Reitz, F. W. Ellis and J. Thompson, in preparation. 20 G. D. Frye and F. W. Ellis, Pharmacologist, 17 (1975) 197. 21 E. Majchrowicz, Psychopharmacologia (Berlin), 43 (1975) 245 - 254. 22 B. E. Hunter, C. A. Boast, D. W. Walker and S. F. Zornetzer, Pharmacol. Biochem. Behavior, 1 (1973) 719 - 725. 23 G. Freund, in E. Majchrowicz (ed.), Biochemical Pharmacology of Ethanol, Plenum Press, New York, 1975, pp. 311 . 325. 24 F. W. Ellis and J. B. Hill, Clin. Chem., 15 (1969) 91 - 101. 25 D. W. Payne and F. W. Ellis, in Advances in Automated Analysis, Meidad Inc., White Plains, 1973. 26 G. R. Breese and T. D. Traylor, J. Pharmacol. Exp. Ther., 174 (1970) 413 - 420. 27 G. R. Breese and T. D. Traylor, Brit. J. Pharmacol., 42 (1971) 88 - 99. 28 G. R. Breese, B. R. Cooper, L. D. Grant and R. D. Smith, Neuropharmacology, 13 (1974) 177 - 187. 29 G. D. Frye, G. R. Breese and F. W. Ellis (in preparation). 30 G. R. Breese, in L. L. Iversen, S. D. Iversen and S. H. Snyder (eds.), Handbook of Psychopharmacology, Vol. 1, Plenum Press, New York, 1975, pp. 127 - 189. 31 L. Lytle, W. Shoemaker, K. Cottman and R. Wurtman, J. Pharmacol. Exp. Ther., 183 (1972) 56 - 64. 32 G. R. Breese and B. R. Cooper, Brain Res., 98 (1975) 517 - 527. 33 R. F. Ritzmann and B. Tabakoff, Nature, 263 (1976) 418 - 419. 34 L. L. Iversen and N. J. Uretsky, Brain Res., 24 (1970) 364 - 367. 35 L. L. Iversen and N. J Uretsky, in T. Malmfors and H. Thoenen (eds.), B-Hydroxydopamine and Catecholamine Neurons, North Holland, Amsterdam, 1971, pp. 171 186.
Discussion SMITH - The fact that you can demonstrate that dependence, measured by withdrawal symptomatology, is unaffected by depletion of amines, tends to diminish the likelihood that catecholamine alkaloids, for example, are involved in the process of dependence. FRYE - Our results do demonstrate that lowering the amines, and thus making them less available for these types of reactions, does not affect dependence. COHEN - I’d like to point out that in your experiments you’re knocking out various tracts. However, in any specific neuron that was left, amine levels are normal and therefore the condensation reactions could still take place in particular neurons. I’m trying to find a way out of the idea that catecholamines and 5-HT are not involved in dependence. We should remember that it takes a great deal of destruction in order to get a functional change. I have two questions. First, do you think that supersensitivity following degeneration may offset any effect you might see? Secondly, since when you lesion with 6-GHDA or 5,7-DHT you can get reinnervation, do you think that animals which have 30 - 40% of normal levels of catecholamines may have these catecholamines localized in different areas from those of normal animals? FRYE - It’s likely that there is increased receptor sensitivity which could affect our results. Regarding regeneration, we have observed this phenomenon; however, whether the axons that sprout from functional connections is not known. Two and one half months after treatment amine levels are still lowered, although eventually norepinephrine levels do begin to come back towards normal values.
359
DEITRICH - I think that it’s important that you have standardized your technique for inducing audiogenic seizures and also that you screened your animals for susceptibility to these seizures prior to ethanol treatment. FRYE - It is important to screen animals for their susceptibility to audiogenic seizures. We have found that about 15% of our Sprague-Dawley animals are normally susceptible to audiogenic seizures while Long- Evans animals are not. POHORECKY - Your results indicating that amine depletion has little effect on withdrawal symptoms may not be so surprising since it is known that functions which are influenced by monoamines, although they are initially quite impaired in animals treated with neurotoxins, eventually return to normal. M. COLLINS - In some cases the drugs that you use such as o-methyl-p-tyrosine may affect ethanol metabolism or ethanol may affect the uptake of the drug into brain. So you may have a different situation using a drug plus ethanol rather than the drug itself. FRYE
- Yes, these are real problems.
TRUITT - There are many factors that have to be considered in these types of studies. For example, the time course of the study, the strain of animal used, and whether turnover or levels of amines are measured. The role of acetaldehyde is also important and may also depend on the animal used or the time that the study is done. One should also consider the relationship of various biogenic amines to each other rather than each amine in isolation. THURMAN - In the course of the Symposium we’ve heard a couple of reports on increases in cyclic nucleotide levels during withdrawal. You see no change in neurotransmitter levels. What model would you propose to account for changes in nucleotide levels? FRYE - Steady-state levels don’t tell the whole story. There is evidence for changes in turnover of transmitters which may be correlated with the changes in cyclic nucleotide levels. KALANT - We have found no change in 5-HT turnover with either acute or chronic ethanol treatment or during withdrawal. We did find that when we used PCPA to severely deplete serotonin levels we could prevent the development of ethanol tolerance. We were using a lower dose of ethanol than you and we did not see signs of withdrawal. We found an important interaction between PCPA and the type of tolerance that is measured: using a behavioral test for tolerance we could block tolerance with PCPA. However, PCPA had no effect when sleep time was used to measure tolerance. The apparent reason is that no nervous system tolerance for sleep time developed with the type of chronic treatment that we used. If anything, PCPA prolonged sleep time after ethanol or barbiturate treatment and any tolerance that did occur was metabolic. It’s important, in determining whether tolerance and dependence are separable and whether 5-HT is involved in these phenomena, what dose of ethanol is used to determine tolerance and how much of tolerance is metabolic and how much is functional. TABAKOFF - In our studies in which 6-OHDA was used to separate the development of tolerance from the development of dependence we used three doses of ethanol, all in the sedative range, to measure functional tolerance. We found little metabolic tolerance. It’s interesting that 5-HT may affect one type of tolerance and possibly noradrenergic systems another type, since tolerance to the hypnotic effects of ethanol as measured by sleep time is affected by 6-OHDA but not by PCPA pretreatment.