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The hypothalamus exhibits electrophysiologic evidence for morphine tolerance and dependence
EXPERIMENTAL
NEUROLOGY
77.66-17
(1982)
The Hypothalamus Exhibits Electrophysiologic Evidence Morphine Tolerance and Dependence NACHUM Department
of Neurobiology
Received
DAFNY’
and Anatomy, The University of Texas Houston, Houston, Texas 77030
September
for
10, 1981; revision
received
February
Medical
Il.
School
at
1982
The electrophysiologic effects of morphine (10 mg/kg) were studied in naive rats and after chronic morphine treatment for 5 days. Spontaneous multiunit activity was recorded from the ventromedial hypothalamus of unanesthetized, freely behaving rats implanted previously with permanent electrodes. In the naive condition, firing rates were altered by morphine in 60 of 73 multiunits. After development of tolerance, 28 of multiunits responded to the morphine challenge dose, in 22 of which the direction of change was opposite that observed in the naive condition. The other six were not affected by morphine on day 1 of the experiment and became sensitive to the morphine challenge dose only after the animal had become physically dependent on morphine. Predrug baseline activity in morphine physically dependent rats was altered in 23 recordings compared to the predrug activity obtained in morphine naive animals. Based on effects of morphine and naloxone, responses could be grouped into six electrophysiologic patterns which provide for separation of tolerance from the dependence phenomena.
INTRODUCTION Chronic administration of morphine or other opiate drugs leads to profound behavioral changes which are most prominently characterized by the development of tolerance and physical dependence. Systematic neurophysiologic investigations of the properties and parameters of these interesting pharmacologic properties of the narcotic analgesic agents, however, have been few. Opiate administration has been reported to elicit Abbreviations: MBH-medial basal hypothalamus, VMH-ventromedial hypothalamus. ’ This work was supported by U.S. Public Health Service grant DA 00803. The author is grateful to Dr. T. F. Burks for his advice and suggestions during the experiments, M. Brown for technical assistance, D. Parker for secretarial assistance, and Endo Laboratories for the naloxone. 66 0014-4886/82/010066-12$02.00/O Copyright 63 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
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alterations in neuroendocrine activities, possibly caused by effects in the ventromedial hypothalamus (VMH) (l-4, 15-18, 21,23, 28, 30). Kerr and Pozuelo (25) suggested, in fact, that morphine dependence in part results from functional reorganization of hypothalamic neurons. Lesions in the ventromedial hypothalamus attenuated withdrawal symptoms, abolished or depressed morphine dependence, and enhanced the sensitivity to morphine, which suggests that the VMH is either a main locus or a relay site for physical-depedence mechanisms and plays a primary role in the morphine-addiction phenomenon (25). In morphine-dependent rats, morphologic changes were identified in VMH synaptic vesicles, representing an exaggeration and alteration of VMH structural characteristics (15). Because it seems susceptible to morphine chronic effects, the VMH was chosen as the target site to study the electrophysiologic consequences of morphine challenge in the same animals when naive to morphine and after development of physical dependence. The objective of the present study was to determine whether neuronal activity recorded from the VMH exhibits neurophysiologic characteristics related to tolerance and/or dependence phenomena induced by chronic morphine treatment. Experiments in both naive and morphine-dependent rats were carried out in freely behaving animals previously implanted with permanent electrodes. Multiunit recording techniques were used because this procedure allows for sampling from the same neuronal population during several days (12, 13,32,35), which is necessary in studying the electrophysiologic properties that underlie the development of tolerance and/or dependence to morphine. MATERIALS
AND
METHODS
Experiments were carried out on 85 male rats (Sprague-Dawley) weighing 250 to 350 g. The methods of animal preparation and of inserting electrodes into specific brain sites were similar to those described elsewhere (12, 13). With the animals anesthetized with pentobarbital (50 mg/kg, i.p.), bilateral electrodes composed of 62-pm Diamel-insulated (except at the tip) Nichrome wire were placed stereotaxically in the ventromedial hypothalamus (VMH) [A 4.380 pm, L 0.5 mm, H -3.6 mm) (27)]. A reference electrode was placed in the frontal sinus. Multiunit activity was monitored during placement of the electrodes. When the signal-to-noise ratio was at least 3:1, the electrodes were fixed to the skull with dental cement and attached to terminals on an Amphenol plug. Several days later, the rats were placed in plastic cages within a sound-attenuated electrophysiologic test chamber. The electrodes were connected to Grass P511 preamplifiers by means of low-noise leads attached to terminals on the Amphenol plug. A commutator and counterbalanced arm allowed the an-
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TABLE
1
Injection Schedule for the Development of Physical Morphine Dependence and Tolerance”
Day
Morning dose
Challenge doseb or midday dose
1 2 3 4 5
20 30 40 50
lob 20 30 40 lob
Evening dose 20 30 40 50
a All doses are mgfkg, i.p. b Recordings were for 30 min before and during the challenge dose and after naloxone, I .O w/kg.
imals to move freely. Spontaneous multiunit activity recorded from one or both electrodes in each animal was monitored with a multibeam oscilloscope and simultaneously was fed to window discriminators. The amplitude and fall time of the spikes were used to define discriminator settings for spike counting by the computer (Interdata 70). Simultaneously, the outputs from the discriminators were connected with an integrator and fed to a Beckman Model 411 polygraph to plot the frequency activity (spikes per second) on-line (40). Ten rats were used as a noninjected time control group, another 10 as a saline (injection) control group, and 69 animals as the morphine-treated experimental group. Each rat participated in two recording sessions 4 days apart; each session consisted of three periods of 30 min each. After 1 h of equilibration within the test chamber, recordings were taken for 30 min before and 30 min after an injection of morphine (10 mg/kg, i.p.) or saline (1 ml/kg) and for an additional 30 min after injection of naloxone (1 mg/ kg, i.p.) or saline. Drugs used were morphine sulfate (Merck) and naloxone hydrochloride (Endo Laboratories). The drugs were dissolved in 0.9% sodium chloride. The drug doses indicated refer to the salt form. All injections were administered in a volume of 1 ml/kg. The time control group also participated in three consecutive 30-min recording sessions, but was not injected with morphine or saline. After the recordings were obtained on day 1, animals in the morphine group were injected with incremental multiple doses of additional morphine (Table 1) (morning, noon, and evening) for 5 days to induce tolerance and physical dependence ( 13,29). Recording procedures identical to those on day 1 were resumed at noon on day 5, 4 h after the last morphine treatment, which is approximately 1 h before rats will exhibit initial behavioral withdrawal symptoms (unpublished observations). The mean and standard error were calculated for the frequency
HYPOTHALAMUS
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TABLE
2
Summary of Changes in Averaged Spontaneous Firing Rate Recorded from the Medial Basal Hypothalamus” Day 1
Day 5
O-30 Min Multi-unit no.
d
2SE
Time elfectb 1 2 3 4 5 6 7 8 9 10
2.8 3.4 5.7 8.2 10.5 15.4 17.2 17.5 24.3 21.6
0.8 1.3 0.6 3.1 1.2 2.3 2.1 4.6 3.8 4.4
O-30 Min 30-60 Min
-
T
-
1.2 0.4 0.2 1.6 2.1 1.7 2.5 2.2 3.7 5.7
-
-
First Saline effect’ 1 1.2 2 3.1 3 3.4 4 3.7 5 5.2, 6 8.3 7 13.5 8 14.9 9 19.6 10 35.4
60-90 Min 2SE
2.3 3.8 5.9 5.4 9.5 18.7 15.8 14.4 26.3 25.1
1.2 1.1 1.5 2.6 3.2 4.1 2.6 3.2 2.8 2.9
Injection Second
-
30-60 Min x
-
-
T
-
-
First 2.0 2.8 4.1 3.7 4.8 7.3 12.8 14.4 21.3 30.0
0.8 1.3 2.6 0.5 1.1 2.4 1.8 3.6 2.5 4.2
60-90 Min
Injection Second
T -
’ Each line represents the direction of responses of a particular recording compared with its own control recording. T, significant increase in firing; -, no change; SE, standard error from the mean (2 SE = P 5 0.05). * Time effect represents noninjected time control group. ’ Saline effect represents saline (injection) control group.
of electrical discharges (spikes per second) for each of the six 30-min recording periods in the two experimental days (days 1 and 5). Several comparisons were made. On day 1 of the experiment, the discharges after morphine, naloxone, or saline were compared with those of the previous period; differences equal to or greater than two standard errors were con-
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sidered significant changes induced by the treatment and were classified as an increase or decrease in firing rate. Identical calculations and comparisons were made for the second recording session on day 5 of the experiment. Comparisons were made between the two recording sessions (day 1 compared with day 5) using the mean + 2 SE as a criterion to classify differences between the recording sessions induced by the treatments (Table 3). The first 30 min of the recording for each multiunit was used as a control for the statistical comparisons. Similar procedures (recording and calculation) were carried out for the two control groups (Table 2). In the time control group, three periods of 30-min recording were obtained on days 1 and 5, but no injections were administered. In the saline control group, additional saline injections (1 ml/kg each) were given morning, noon, and evening, as in the morphine group (Table 1). On day 5, the recordings were taken before and after two saline injections as with the morphine treatment group (Table 2). At the end of each experiment, the rats were killed for histological verification of electrode sites (9-12). Twenty-five percent of the electrode placements were scattered around the VMH, e.g., in the arcuate nucleus, and no significant differences in the recording could be identified from those placed in the target site (VMH). We grouped all our data and classified our experimental neuronal population recordings as obtained from the medial basal hypothalamus (MBH). RESULTS The activity from 93 multiunits was recorded twice: once on day 1 of the experiment and again on day 5. These recordings included 10 multiunits from the untreated time control group, 10 from the saline-injected control , TABLE
3
Summary of the Significant Changes Elicited by the Drugs in Averaged Spontaneous Firing Rate Recorded from the Medial Basal Hypothalamus” Day 1 Group
N
M
I
8 10 20 22
t t 1 1
II
IIIa IIIb IV V
6 I
-
Day 5 Nal
M
Nal
T T
1 T T 1 T
t t T T
-
-
Spon day 5 Sport day 1 T t T T
-
a M, morphine, 10 mg/kg, i.p.: Nal, naloxone, I .O mg/kg, i.p.; T, increase in firing; 1, decrease in firing; -, no.change; N, number of recording multiunits. Further explanation in text.
HYPOTHALAMUS
N=23,1
N=1613
0
128 rnY
2%
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0
128 ran
25%
FIG. I. Time interval histograms of spontaneous (control) multiunit activity before and after drug treatments. N, total number of spikes/5 min. The 5-min data were taken 15 to 20 min after each treatment. Left--control, middle-morphine (10 mg/kg), right-naloxone (1 .O w/b).
group, and 73 from the morphine-treated group. In the two control groups only a few random changes (4/80) in firing rates were observed. These changes occurred on day 1 or 5 of the recording sessions (Table 2). Morphine and Naloxone Efects in Naive Rats (Day I). On day 1 of the experiment, 73 multiunits were recorded from 69 animals before and after morphine (10 mg/kg) and naloxone (1.0 mg/kg) treatment. The spontaneous activity of 60/73 multiunits was modified significantly by the morphine challenge dose (Table 3, day 1). In the majority of the responsive multiunits (42/60), morphine induced a decrease in electrical discharges (Fig. 1); in the remaining multiunits (18/60), morphine caused a significant increase in firing rate (Table 3). Naloxone antagonized morphine effects only in those multiunits which exhibited a decrease in firing rate after
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morphine administration (Table 3, IIIa,b and Fig. l), and induced further increases (agonist effect) in about half the multiunits in which the morphine challenge dose caused an increase in activity (Table 3, II); in the other half of this neuronal population, naloxone failed to modify the morphine effects (Table 3, I). Six multiunits failed to modify their electrical discharges after morphine injection, but changed their activity after naloxone injection (Table 3, IV). Morphine and Naloxone Efects in Morphine Tolerant-Dependent Rats (Day 5). Thirty-eight of the 60 previously responsive multiunits failed to
respond to the morphine challenge dose on day 5 of the experiments (Table 3, I, II, and IIIa, day 5). In approximately half the multiunits from group III, however, morphine caused reversal effects (Fig. 1 and Table 3, IIIb, day 5), i.e., on day 1 morphine caused a decrease in activity, whereas in tolerant-dependent rats (day 5) the same dose of morphine induced an increase in electrical discharges. Moreover, those multiunits which altered their firing rates only after naloxone in naive rats (Table 3, IV, day 1) became sensitive to morphine in tolerant-dependent animals and altered their activities after the morphine challenge dose on day 5. Naloxone affected more neurons on day 5 (66/73) than on day 1 in naive animals (58/ 73). Naloxone on day 5 modified the activity of the recordings which were responsive to morphine on day 1, but which failed to alter their firing rates after the morphine challenge dose on day 5 (Table 3, I, II, and IIIa). Naloxone also antagonized morphine effects in the multiunits which were unaffected by morphine on day 5 (Table 3, IIIb) and exhibited similar effects in the six multiunits from group IV (Table 3, day 5). Seven multiunits did not change their electrical discharges after treatment with either drug (Table 3, group V). Comparison Between Activity Recorded in Premorphine Challenge Dose (Control) on Days 1 and 5 of the Experiment. Forty-four multiunits dis-
played significant modified predrug spike discharge rates on day 5 of the experiment compared with those obtained on day 1 (Table 3, Spon day 1 compared to Spon day 5). Behavioral Observations. In all animals, neither morphine (10 mg/kg) nor naloxone (1 mg/kg) treatments on day 1 of the experiment induced any gross behavioral changes. On days 3 to 5 the animals were waiting anxiously in their cages at the injection time (see Table 1) to get their morphine treatment, i.e., drug-seeking behavior. After they received the morphine injection, they returned to their “normal” behavior. On day 5, naloxone injections induced the expected behavioral withdrawal phenomena. All animals, whether they had responded electrophysioiogically or not to naloxone injections in day 5 of the experiment, behaviorally responded within 1 to 2 min by exhibiting withdrawal symptoms: wet-dog shakes,
HYPOTHALAMUS
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frequent stretching of their bodies, diarrhea, excitability, running, jumping, and hyperactivity to environmental stimuli outside their cages. DISCUSSION The phenomenon of tolerance has been under investigation for almost 100 years. However, systematic neurophysiological investigations of the properties and parameters of this interesting physiologic and pharmacologic property of the opiates have been few (31). We used multiunit recording techniques in freely behaving animals to examine the electrical discharges from medial basal hypothalamic neurons from the same neuronal pool after a challenge dose of morphine when the animal was naive to morphine and later when the same animal became physically dependent on morphine. The objective of the present study was to determine if the neuronal population in this central site exhibited neurophysiologic evidence of tolerance or dependence to morphine. From the present observations we conclude that the observed changes in neuronal activity were induced mainly by the treatments with morphine. Neither time nor saline treatment in the two control groups elicited detectable systematic effects on MBH neurons, whereas morphine injections affected the vast majority of the neuronal population. Moreover, in the majority of the recordings, the morphine antagonist, naloxone, reversed the morphine-induced effects. In previous experiments (13, 29) and the present one, naloxone injection on day 5 induced behavioral signs of withdrawal (24) within 1 to 2 min. Based on the standard criterion, the animals in our study on day 5 of the experiment were morphine dependent. Moreover, the present study demonstrated that spontaneous discharges from MBH neurons exhibit six types (Table 3) of responses to morphine and naloxone treatments. These data suggest that the effects observed are direct and specific because nonspecific responses are less variable (8). Furthermore, these findings are consistent with the current theories suggesting that there are multiple sites of opiate receptors (22, 33, 39). Three types of physiologic-pharmacologic phenomena were observed in the present study. Changes in prechallenge spontaneous activity on day 5 compared with the activity obtained in the premorphine period on day 1 recorded from the same animal and the same locus, can be related to the physiologic alterations underlying dependence. The effects elicited by the morphine challenge dose in naive animals compared with the responses to morphine challenge in the same animals on day 5 can be related to tolerance, which is commonly defined as loss of responsiveness to a particular dose. Responses to naloxone in animals after chronic morphine treatment can be related to withdrawal. In the present study, a large fraction of the
14
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neuronal population exhibited changes in premorphine challenge activity recorded on day 5 of the experiment compared with the recordings obtained in the premorphine period on day 1 of the experiment. It is possible to assume that the “new” baseline activity is related to the electrophysiologic properties underlying the development of dependence. The neurons may require additional morphine to maintain this “new” baseline activity, and this modified activity in the physically dependent animals may be the electrophysiologic correlate of the dependence phenomenon. There has been great interest in what happens to opiate receptors as a result of the development of a dependency on opiates. Theories have abounded for years (5-7, 37, 38) that changes in receptor numbers or receptor properties are involved in these processes. Efforts to demonstrate such changes have been made (26, 34), but neither the number nor the binding affinity of receptors appeared to be altered. It was postulated by Collier (6, 7), however, that chronic opiate treatment can alter the concentration of neurotransmitter, providing two explanations or possible ways to interpret our data: chronic opiate treatment leads to (i) increases in activity of excitatory neurotransmitters that elicit the increase in firing discharges observed, or (ii) decreases in the inhibitory neurotransmitters (disinhibition) induce this excitation. It is interesting in this context that 38 recordings (Table 3; I, II, and IIIa) in which predrug firing rates were higher on day 5 than on day 1 of the experiment, failed to respond to morphine on day 5 of the experiment, whereas on day 1 morphine induced in these units significant alterations in their electrical discharges. This observation is in agreement with Collier’s statement (7) that physical dependence is always accompanied by tolerance. Thirty-eight of 60 multiunits failed to respond to the morphine challenge dose on day 5; whereas, on day 1 significant alterations were elicited by the same dose of morphine (Table 3; I, II, and IIIa). Because the tolerance phenomenon is regarded as a reduction in the pharmacologic response after repeated use of a drug (24), these neurons recorded from the MBH exhibited the classical sign of tolerance. The multiunits represented in Table 3 as group IIIb represent what we believe is another phenomenon, i.e., the direction of their firing rates induced by morphine on day 5 was opposite that observed on day 1 of the experiment. It has been reported ( 19, 3 1, 36) that in the absence of the original morphine response, tolerance may be expressed as a reversal in response direction due to the selective loss of a previous influence. Other experiments suggest that reversal of direction of morphine responses after development of tolerance may not be due solely to loss of one component of morphine action, but may be caused by supersensitivity to an opposing action. Frequently, high doses of morphine produce responses which are opposite those induced by low doses (9- 11,
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13, 29). Often, low doses of morphine reduce and high doses increase activity. In the present experiments, reversal of responses after development of tolerance could be explained either by loss of responsiveness to inhibitory effects of morphine or by an increase in responsiveness to excitatory effects of morphine. One striking difference in responsiveness to morphine suggests the presence of two populations of functionally distinct neurons in the MBH. The two populations are presented as groups IIIa,b in Table 3. Both populations responded on day 1 with decreases in firing rates, but differed on day 5 in the following respects: group IIIa displayed increased predrug activity, increase in firing rates after morphine, and decreased activity after naloxone. The differences cannot be explained on the basis of a failure of group IIIb multiunits to develop tolerance, as the direction of response to morphine was reversed on day 5. The altered responses to morphine challenge injections in physically morphine-dependent animals are probably related to tolerance. Tolerance is generally regarded as a reduction in the pharmacologic response after repeated use of a drug. After the original morphine response is lost, tolerance may be expressed either as no response to a challenge dose of morphine on day 5 of the experiment, or as a reversal in direction of the response due to selective loss of previously dominant influences (24, 3 1). All animals exhibited withdrawal symptoms 1 to 2 min after naloxone injection. Responses to naloxone on day 5 can be used as an indication of withdrawal and the degree of dependence (20). In physically morphinedependent animals, naloxone affected more recording sites than was observed on day 1 of the experiment (Table 3). The improved efficacy of naloxone was suggested to be a sensitive indicator of the initiation and development of narcotic tolerance (20). Moreover, it was suggested that morphine tolerance-dependence is usually associated with an increase in the antagonist potency of naloxone (14,20). Using morphine and naloxone in morphine naive and physically dependent animals makes it possible to identify as many as six response patterns that can be correlated with different functional properties of neurons and it provides a model for the separation of tolerance from dependence in further investigations. REFERENCES 1. BORELL, J., I. LLORENS, AND S. BORRELL. 1974. Study of the effects of morphine on adrenal corticosteroids, ascorbic acid and catecholamines in unanesthetized and anesthetized cats. Horm. Res. 5: 351-358. 2. BRUNI, J. F., D. VAN VUGT, S. MARSHALL, AND J. MEITES. 1977. Effects of naloxone, morphine and methionine enkephalin on serum prolactin, luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone and growth hormone. Life Sci. 21: 461-466.
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3. CICERO, T. J., E. R. MEYER, R. D. BELL, AND G. A. KOCH. 1976. Effects of morphine and methadone on serum testosterone and luteinizing hormone levels and on the secondary sex organs of the male rat. Endocrinology 98: 367-372. 4. CICERO, T. J., C. E. WILCOX, R. D. BELL, AND E. R. MAYER. 1976. Acute reductions in serum testosterone levels by narcotics in the male rat: stereospecificity, blockade by naloxone and tolerance. J. Pharmacol. Exp. Ther. 198: 340-346. 5. COCHIN, J., AND B. E. MUSHLIN. 1976. Effects of agonist-antagonist interaction on the development of tolerance and dependence. Ann. N. Y. Acad. Sci. 281: 244-25 1. 6. COLLIER, H. 0. J. 1965. A general theory on the genesis of drug dependence by induction of receptors. Nature (London) 205: 181-182. 7. COLLIER, H. 0. J. 1966. Tolerance, physical dependence and receptors. Adv. Drug Res. 3: 171-188. 8. DAFNY, N. 1978. Neurophysiological approach as a tool to study the effect of drugs on the central nervous system. I. Dose effect of pentobarbital. Exp. Neurof. 59: 263-274. 9. DAFNY, N., AND T. F. BURKS. 1976. Opiate and endocrine interaction: morphine effects on hypothalamus and pineal body. Neuroendocrinology 22: 72-88. 10. DAFNY, N., AND T. F. BURKS. 1976. Neurophysiological changes in caudate nucleus and substantia nigra following morphine treatment. Neuropharmacology 15: 547-554. 1I. DAFNY, N., AND T. F. BURKS. 1976. Opiate-dependent effects of naloxone on the central nervous system-neurophysiological approach. Exp. Neural. 53: 633-645. 12. DAFNY, N., M. I. PHILLIPS, A. N. TAYLOR, AND S. GILMAN. 1973. Dose effects of cortisol on single unit activity in hypothalamus, reticular formation, and hippocampus of freely behaving rats correlated with plasma steroid levels. Brain Res. 59: 257-272. 13. DAFNY, N., M. BROWN, T. F. BURKS, AND B. M. RIGOR. 1979. Morphine tolerance and dependence: sensitivity of caudate nucleus neurons. Brain Rex 54: 216-224. 14. EIDELBERG, E., AND M. L. BOND. 1972. Effects of morphine and antagonists on hypothalamic cell activity. Arch. Int. Pharmacol. 196: 16-24. 15. FORD, D. H., K. VOELLER, B. CALLEGARI, AND E. GRESIK. 1974. Changes in neurons of the median eminence-arcuate region of rats. Neurobiology 4: 1- 11. 16. GEORGE, R. 1971. Hypothalamus: anterior pituitary gland. Pages 283-299, in D. H. CLOUET, Ed., Narcotic Drugs: Biochemical Pharmacology. Plenum, New York. 17. GEORGE, R. 1973. Effects of narcotic analgesics on hypothalamus-pituitary-thyroid function. Prog. Brain Res. 39: 339-345. 18. GOLD, E. M., AND W. F. GANONG. 1967. Effects of drugs on neuroendocrine processes, Pages 377-437 in L. MARTINI AND W. F. GANONG, Eds., Neuroendocrinology, Vol. 2. Academic Press, New York. 19. GUNNE, L. M. 1960. The temperature response in rats during acute and chronic morphine administration: a study of morphine tolerance. Arch. In?. Pharmacodyn. Ther. 79: 416428. 20. HARRIS,
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R. A., H. H. LOH, AND E. L. WAY. 1976. Alterations in the efficacy of naloxone induced by stress, cyclic adenosine monophosphate and morphine tolerance. Eur. J. Pharmacol. 39: l-10. HAYWARD, J. N. 1977. Functional and morphological aspects of hypothalamic neurons. Physiol. Rev. 57: 574-658. HILLER, J. M., L. M. ANSEL, AND E. J. SIMON. 1981. Multiple opiate receptors: alcohol selectively inhibits binding to delta receptors. Science 214: 468-469. Ho, W. K. K., S. LAM, K. C. LEUNG, K. K. Au, H. K. WONG, Y. F. TSANG, AND H. L. WEN. 1978. Effect of naloxone on morphine-induced changes in ACTH, corticosterone and cyclic nucleotides. Neuropharmacology 17: 397-400. JAFFE, J. H., AND W. R. MARTIN. 1980. Opioid analgesics and antagonists. Pages 494-
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534 in L. S. GOODMAN AND A. GILMAN, Eds., The Pharmacological Basis of Therapeutics, 6th ed. MacMillan, New York. 25. KERR, F. W. L., AND J. POZUELO. 1971. Suppression of physical dependence and induction of hypersensitivity to morphine by stereotaxic hypothalamic lesions in addicted rats. Mayo
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26. KLEE, W. A., AND R. A. STREATY. 1974. Narcotic receptor sites in morphine-dependent rats. Nature (London) 248: 61-63. 27. KOENIG,J. F. R., AND R. A. KLIPPEL. 1970. The Rat Brain: A Stereotoxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Krieger, New York. 28. KOKKA, N., J. F. GARCIA, AND H. W. ELLIOTT. 1973. Effect of acute and chronic administration of narcotic analgesics on growth hormone and corticotrophin (ACTH) secretion of rats. Prog. Brain Res. 39: 347-358. 29. MCCLUNG, R. E., T. F. BURKS, AND N. DAFNY. 1977. Neurophysiological assessment of site specific effects of chronic morphine administration in freely behaving rats. Arch. Int. Pharmacol. Ther. 229: 144-156. 30. MUNSON, P. L. 1973. Effects of morphine and related drugs on the corticotrophin (ACTH) stress reaction. Proc. Brain Res. 39: 361-372. 31. MUSHLIN, B. E., R. GRELL, AND J. COCHIN. 1976. Studies on tolerance. I. The role of the interval between doses on the development of tolerance to morphine. J. Pharmacol. Exp.
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32. OLDS, M. E., AND R. NIENHNIS. 1979. Depressant effects of topical morphine on selfstimulation-related units in hypothalamus. Neuropharmacology 18: 801-812. 33. OLSON, G. A., R. D. OLSON, A. J. KASTIN, AND H. COY. 1980. Endogenous opiates. Peptides
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34. PERT, C. B., G. PASTERNAK, AND S. H. SNYDER. 1973. Opiate agonists and antagonists discriminated by receptor binding in brain. Science 182: 1359-l 361. 35. PETERS, R. I., AND W. R. KLEMM. 1977. Electrophysiological signs of differential tolerance development to morphine in selected areas of the rabbit brain. LiJe Sci. 20: 85-94. 36. ROSENFIELD, G. C., AND T. F. BURKS. 1977. Single-dose tolerance to morphine hypothermia in the rat: differentiation of acute from long-term tolerance. J. Pharmacol. Exp. Ther.
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for qualitative and quantitative unit
NEUROLOGY
77.66-17
(1982)
The Hypothalamus Exhibits Electrophysiologic Evidence Morphine Tolerance and Dependence NACHUM Department
of Neurobiology
Received
DAFNY’
and Anatomy, The University of Texas Houston, Houston, Texas 77030
September
for
10, 1981; revision
received
February
Medical
Il.
School
at
1982
The electrophysiologic effects of morphine (10 mg/kg) were studied in naive rats and after chronic morphine treatment for 5 days. Spontaneous multiunit activity was recorded from the ventromedial hypothalamus of unanesthetized, freely behaving rats implanted previously with permanent electrodes. In the naive condition, firing rates were altered by morphine in 60 of 73 multiunits. After development of tolerance, 28 of multiunits responded to the morphine challenge dose, in 22 of which the direction of change was opposite that observed in the naive condition. The other six were not affected by morphine on day 1 of the experiment and became sensitive to the morphine challenge dose only after the animal had become physically dependent on morphine. Predrug baseline activity in morphine physically dependent rats was altered in 23 recordings compared to the predrug activity obtained in morphine naive animals. Based on effects of morphine and naloxone, responses could be grouped into six electrophysiologic patterns which provide for separation of tolerance from the dependence phenomena.
INTRODUCTION Chronic administration of morphine or other opiate drugs leads to profound behavioral changes which are most prominently characterized by the development of tolerance and physical dependence. Systematic neurophysiologic investigations of the properties and parameters of these interesting pharmacologic properties of the narcotic analgesic agents, however, have been few. Opiate administration has been reported to elicit Abbreviations: MBH-medial basal hypothalamus, VMH-ventromedial hypothalamus. ’ This work was supported by U.S. Public Health Service grant DA 00803. The author is grateful to Dr. T. F. Burks for his advice and suggestions during the experiments, M. Brown for technical assistance, D. Parker for secretarial assistance, and Endo Laboratories for the naloxone. 66 0014-4886/82/010066-12$02.00/O Copyright 63 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
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67
alterations in neuroendocrine activities, possibly caused by effects in the ventromedial hypothalamus (VMH) (l-4, 15-18, 21,23, 28, 30). Kerr and Pozuelo (25) suggested, in fact, that morphine dependence in part results from functional reorganization of hypothalamic neurons. Lesions in the ventromedial hypothalamus attenuated withdrawal symptoms, abolished or depressed morphine dependence, and enhanced the sensitivity to morphine, which suggests that the VMH is either a main locus or a relay site for physical-depedence mechanisms and plays a primary role in the morphine-addiction phenomenon (25). In morphine-dependent rats, morphologic changes were identified in VMH synaptic vesicles, representing an exaggeration and alteration of VMH structural characteristics (15). Because it seems susceptible to morphine chronic effects, the VMH was chosen as the target site to study the electrophysiologic consequences of morphine challenge in the same animals when naive to morphine and after development of physical dependence. The objective of the present study was to determine whether neuronal activity recorded from the VMH exhibits neurophysiologic characteristics related to tolerance and/or dependence phenomena induced by chronic morphine treatment. Experiments in both naive and morphine-dependent rats were carried out in freely behaving animals previously implanted with permanent electrodes. Multiunit recording techniques were used because this procedure allows for sampling from the same neuronal population during several days (12, 13,32,35), which is necessary in studying the electrophysiologic properties that underlie the development of tolerance and/or dependence to morphine. MATERIALS
AND
METHODS
Experiments were carried out on 85 male rats (Sprague-Dawley) weighing 250 to 350 g. The methods of animal preparation and of inserting electrodes into specific brain sites were similar to those described elsewhere (12, 13). With the animals anesthetized with pentobarbital (50 mg/kg, i.p.), bilateral electrodes composed of 62-pm Diamel-insulated (except at the tip) Nichrome wire were placed stereotaxically in the ventromedial hypothalamus (VMH) [A 4.380 pm, L 0.5 mm, H -3.6 mm) (27)]. A reference electrode was placed in the frontal sinus. Multiunit activity was monitored during placement of the electrodes. When the signal-to-noise ratio was at least 3:1, the electrodes were fixed to the skull with dental cement and attached to terminals on an Amphenol plug. Several days later, the rats were placed in plastic cages within a sound-attenuated electrophysiologic test chamber. The electrodes were connected to Grass P511 preamplifiers by means of low-noise leads attached to terminals on the Amphenol plug. A commutator and counterbalanced arm allowed the an-
68
NACHUM
DAFNY
TABLE
1
Injection Schedule for the Development of Physical Morphine Dependence and Tolerance”
Day
Morning dose
Challenge doseb or midday dose
1 2 3 4 5
20 30 40 50
lob 20 30 40 lob
Evening dose 20 30 40 50
a All doses are mgfkg, i.p. b Recordings were for 30 min before and during the challenge dose and after naloxone, I .O w/kg.
imals to move freely. Spontaneous multiunit activity recorded from one or both electrodes in each animal was monitored with a multibeam oscilloscope and simultaneously was fed to window discriminators. The amplitude and fall time of the spikes were used to define discriminator settings for spike counting by the computer (Interdata 70). Simultaneously, the outputs from the discriminators were connected with an integrator and fed to a Beckman Model 411 polygraph to plot the frequency activity (spikes per second) on-line (40). Ten rats were used as a noninjected time control group, another 10 as a saline (injection) control group, and 69 animals as the morphine-treated experimental group. Each rat participated in two recording sessions 4 days apart; each session consisted of three periods of 30 min each. After 1 h of equilibration within the test chamber, recordings were taken for 30 min before and 30 min after an injection of morphine (10 mg/kg, i.p.) or saline (1 ml/kg) and for an additional 30 min after injection of naloxone (1 mg/ kg, i.p.) or saline. Drugs used were morphine sulfate (Merck) and naloxone hydrochloride (Endo Laboratories). The drugs were dissolved in 0.9% sodium chloride. The drug doses indicated refer to the salt form. All injections were administered in a volume of 1 ml/kg. The time control group also participated in three consecutive 30-min recording sessions, but was not injected with morphine or saline. After the recordings were obtained on day 1, animals in the morphine group were injected with incremental multiple doses of additional morphine (Table 1) (morning, noon, and evening) for 5 days to induce tolerance and physical dependence ( 13,29). Recording procedures identical to those on day 1 were resumed at noon on day 5, 4 h after the last morphine treatment, which is approximately 1 h before rats will exhibit initial behavioral withdrawal symptoms (unpublished observations). The mean and standard error were calculated for the frequency
HYPOTHALAMUS
69
AND MORPHINE
TABLE
2
Summary of Changes in Averaged Spontaneous Firing Rate Recorded from the Medial Basal Hypothalamus” Day 1
Day 5
O-30 Min Multi-unit no.
d
2SE
Time elfectb 1 2 3 4 5 6 7 8 9 10
2.8 3.4 5.7 8.2 10.5 15.4 17.2 17.5 24.3 21.6
0.8 1.3 0.6 3.1 1.2 2.3 2.1 4.6 3.8 4.4
O-30 Min 30-60 Min
-
T
-
1.2 0.4 0.2 1.6 2.1 1.7 2.5 2.2 3.7 5.7
-
-
First Saline effect’ 1 1.2 2 3.1 3 3.4 4 3.7 5 5.2, 6 8.3 7 13.5 8 14.9 9 19.6 10 35.4
60-90 Min 2SE
2.3 3.8 5.9 5.4 9.5 18.7 15.8 14.4 26.3 25.1
1.2 1.1 1.5 2.6 3.2 4.1 2.6 3.2 2.8 2.9
Injection Second
-
30-60 Min x
-
-
T
-
-
First 2.0 2.8 4.1 3.7 4.8 7.3 12.8 14.4 21.3 30.0
0.8 1.3 2.6 0.5 1.1 2.4 1.8 3.6 2.5 4.2
60-90 Min
Injection Second
T -
’ Each line represents the direction of responses of a particular recording compared with its own control recording. T, significant increase in firing; -, no change; SE, standard error from the mean (2 SE = P 5 0.05). * Time effect represents noninjected time control group. ’ Saline effect represents saline (injection) control group.
of electrical discharges (spikes per second) for each of the six 30-min recording periods in the two experimental days (days 1 and 5). Several comparisons were made. On day 1 of the experiment, the discharges after morphine, naloxone, or saline were compared with those of the previous period; differences equal to or greater than two standard errors were con-
70
NACHUM
DAFNY
sidered significant changes induced by the treatment and were classified as an increase or decrease in firing rate. Identical calculations and comparisons were made for the second recording session on day 5 of the experiment. Comparisons were made between the two recording sessions (day 1 compared with day 5) using the mean + 2 SE as a criterion to classify differences between the recording sessions induced by the treatments (Table 3). The first 30 min of the recording for each multiunit was used as a control for the statistical comparisons. Similar procedures (recording and calculation) were carried out for the two control groups (Table 2). In the time control group, three periods of 30-min recording were obtained on days 1 and 5, but no injections were administered. In the saline control group, additional saline injections (1 ml/kg each) were given morning, noon, and evening, as in the morphine group (Table 1). On day 5, the recordings were taken before and after two saline injections as with the morphine treatment group (Table 2). At the end of each experiment, the rats were killed for histological verification of electrode sites (9-12). Twenty-five percent of the electrode placements were scattered around the VMH, e.g., in the arcuate nucleus, and no significant differences in the recording could be identified from those placed in the target site (VMH). We grouped all our data and classified our experimental neuronal population recordings as obtained from the medial basal hypothalamus (MBH). RESULTS The activity from 93 multiunits was recorded twice: once on day 1 of the experiment and again on day 5. These recordings included 10 multiunits from the untreated time control group, 10 from the saline-injected control , TABLE
3
Summary of the Significant Changes Elicited by the Drugs in Averaged Spontaneous Firing Rate Recorded from the Medial Basal Hypothalamus” Day 1 Group
N
M
I
8 10 20 22
t t 1 1
II
IIIa IIIb IV V
6 I
-
Day 5 Nal
M
Nal
T T
1 T T 1 T
t t T T
-
-
Spon day 5 Sport day 1 T t T T
-
a M, morphine, 10 mg/kg, i.p.: Nal, naloxone, I .O mg/kg, i.p.; T, increase in firing; 1, decrease in firing; -, no.change; N, number of recording multiunits. Further explanation in text.
HYPOTHALAMUS
N=23,1
N=1613
0
128 rnY
2%
71
AND MORPHINE
0
128 ran
25%
FIG. I. Time interval histograms of spontaneous (control) multiunit activity before and after drug treatments. N, total number of spikes/5 min. The 5-min data were taken 15 to 20 min after each treatment. Left--control, middle-morphine (10 mg/kg), right-naloxone (1 .O w/b).
group, and 73 from the morphine-treated group. In the two control groups only a few random changes (4/80) in firing rates were observed. These changes occurred on day 1 or 5 of the recording sessions (Table 2). Morphine and Naloxone Efects in Naive Rats (Day I). On day 1 of the experiment, 73 multiunits were recorded from 69 animals before and after morphine (10 mg/kg) and naloxone (1.0 mg/kg) treatment. The spontaneous activity of 60/73 multiunits was modified significantly by the morphine challenge dose (Table 3, day 1). In the majority of the responsive multiunits (42/60), morphine induced a decrease in electrical discharges (Fig. 1); in the remaining multiunits (18/60), morphine caused a significant increase in firing rate (Table 3). Naloxone antagonized morphine effects only in those multiunits which exhibited a decrease in firing rate after
72
NACHUM
DAFNY
morphine administration (Table 3, IIIa,b and Fig. l), and induced further increases (agonist effect) in about half the multiunits in which the morphine challenge dose caused an increase in activity (Table 3, II); in the other half of this neuronal population, naloxone failed to modify the morphine effects (Table 3, I). Six multiunits failed to modify their electrical discharges after morphine injection, but changed their activity after naloxone injection (Table 3, IV). Morphine and Naloxone Efects in Morphine Tolerant-Dependent Rats (Day 5). Thirty-eight of the 60 previously responsive multiunits failed to
respond to the morphine challenge dose on day 5 of the experiments (Table 3, I, II, and IIIa, day 5). In approximately half the multiunits from group III, however, morphine caused reversal effects (Fig. 1 and Table 3, IIIb, day 5), i.e., on day 1 morphine caused a decrease in activity, whereas in tolerant-dependent rats (day 5) the same dose of morphine induced an increase in electrical discharges. Moreover, those multiunits which altered their firing rates only after naloxone in naive rats (Table 3, IV, day 1) became sensitive to morphine in tolerant-dependent animals and altered their activities after the morphine challenge dose on day 5. Naloxone affected more neurons on day 5 (66/73) than on day 1 in naive animals (58/ 73). Naloxone on day 5 modified the activity of the recordings which were responsive to morphine on day 1, but which failed to alter their firing rates after the morphine challenge dose on day 5 (Table 3, I, II, and IIIa). Naloxone also antagonized morphine effects in the multiunits which were unaffected by morphine on day 5 (Table 3, IIIb) and exhibited similar effects in the six multiunits from group IV (Table 3, day 5). Seven multiunits did not change their electrical discharges after treatment with either drug (Table 3, group V). Comparison Between Activity Recorded in Premorphine Challenge Dose (Control) on Days 1 and 5 of the Experiment. Forty-four multiunits dis-
played significant modified predrug spike discharge rates on day 5 of the experiment compared with those obtained on day 1 (Table 3, Spon day 1 compared to Spon day 5). Behavioral Observations. In all animals, neither morphine (10 mg/kg) nor naloxone (1 mg/kg) treatments on day 1 of the experiment induced any gross behavioral changes. On days 3 to 5 the animals were waiting anxiously in their cages at the injection time (see Table 1) to get their morphine treatment, i.e., drug-seeking behavior. After they received the morphine injection, they returned to their “normal” behavior. On day 5, naloxone injections induced the expected behavioral withdrawal phenomena. All animals, whether they had responded electrophysioiogically or not to naloxone injections in day 5 of the experiment, behaviorally responded within 1 to 2 min by exhibiting withdrawal symptoms: wet-dog shakes,
HYPOTHALAMUS
AND
MORPHINE
13
frequent stretching of their bodies, diarrhea, excitability, running, jumping, and hyperactivity to environmental stimuli outside their cages. DISCUSSION The phenomenon of tolerance has been under investigation for almost 100 years. However, systematic neurophysiological investigations of the properties and parameters of this interesting physiologic and pharmacologic property of the opiates have been few (31). We used multiunit recording techniques in freely behaving animals to examine the electrical discharges from medial basal hypothalamic neurons from the same neuronal pool after a challenge dose of morphine when the animal was naive to morphine and later when the same animal became physically dependent on morphine. The objective of the present study was to determine if the neuronal population in this central site exhibited neurophysiologic evidence of tolerance or dependence to morphine. From the present observations we conclude that the observed changes in neuronal activity were induced mainly by the treatments with morphine. Neither time nor saline treatment in the two control groups elicited detectable systematic effects on MBH neurons, whereas morphine injections affected the vast majority of the neuronal population. Moreover, in the majority of the recordings, the morphine antagonist, naloxone, reversed the morphine-induced effects. In previous experiments (13, 29) and the present one, naloxone injection on day 5 induced behavioral signs of withdrawal (24) within 1 to 2 min. Based on the standard criterion, the animals in our study on day 5 of the experiment were morphine dependent. Moreover, the present study demonstrated that spontaneous discharges from MBH neurons exhibit six types (Table 3) of responses to morphine and naloxone treatments. These data suggest that the effects observed are direct and specific because nonspecific responses are less variable (8). Furthermore, these findings are consistent with the current theories suggesting that there are multiple sites of opiate receptors (22, 33, 39). Three types of physiologic-pharmacologic phenomena were observed in the present study. Changes in prechallenge spontaneous activity on day 5 compared with the activity obtained in the premorphine period on day 1 recorded from the same animal and the same locus, can be related to the physiologic alterations underlying dependence. The effects elicited by the morphine challenge dose in naive animals compared with the responses to morphine challenge in the same animals on day 5 can be related to tolerance, which is commonly defined as loss of responsiveness to a particular dose. Responses to naloxone in animals after chronic morphine treatment can be related to withdrawal. In the present study, a large fraction of the
14
NACHUM
DAFNY
neuronal population exhibited changes in premorphine challenge activity recorded on day 5 of the experiment compared with the recordings obtained in the premorphine period on day 1 of the experiment. It is possible to assume that the “new” baseline activity is related to the electrophysiologic properties underlying the development of dependence. The neurons may require additional morphine to maintain this “new” baseline activity, and this modified activity in the physically dependent animals may be the electrophysiologic correlate of the dependence phenomenon. There has been great interest in what happens to opiate receptors as a result of the development of a dependency on opiates. Theories have abounded for years (5-7, 37, 38) that changes in receptor numbers or receptor properties are involved in these processes. Efforts to demonstrate such changes have been made (26, 34), but neither the number nor the binding affinity of receptors appeared to be altered. It was postulated by Collier (6, 7), however, that chronic opiate treatment can alter the concentration of neurotransmitter, providing two explanations or possible ways to interpret our data: chronic opiate treatment leads to (i) increases in activity of excitatory neurotransmitters that elicit the increase in firing discharges observed, or (ii) decreases in the inhibitory neurotransmitters (disinhibition) induce this excitation. It is interesting in this context that 38 recordings (Table 3; I, II, and IIIa) in which predrug firing rates were higher on day 5 than on day 1 of the experiment, failed to respond to morphine on day 5 of the experiment, whereas on day 1 morphine induced in these units significant alterations in their electrical discharges. This observation is in agreement with Collier’s statement (7) that physical dependence is always accompanied by tolerance. Thirty-eight of 60 multiunits failed to respond to the morphine challenge dose on day 5; whereas, on day 1 significant alterations were elicited by the same dose of morphine (Table 3; I, II, and IIIa). Because the tolerance phenomenon is regarded as a reduction in the pharmacologic response after repeated use of a drug (24), these neurons recorded from the MBH exhibited the classical sign of tolerance. The multiunits represented in Table 3 as group IIIb represent what we believe is another phenomenon, i.e., the direction of their firing rates induced by morphine on day 5 was opposite that observed on day 1 of the experiment. It has been reported ( 19, 3 1, 36) that in the absence of the original morphine response, tolerance may be expressed as a reversal in response direction due to the selective loss of a previous influence. Other experiments suggest that reversal of direction of morphine responses after development of tolerance may not be due solely to loss of one component of morphine action, but may be caused by supersensitivity to an opposing action. Frequently, high doses of morphine produce responses which are opposite those induced by low doses (9- 11,
HYPOTHALAMUS
AND
MORPHINE
75
13, 29). Often, low doses of morphine reduce and high doses increase activity. In the present experiments, reversal of responses after development of tolerance could be explained either by loss of responsiveness to inhibitory effects of morphine or by an increase in responsiveness to excitatory effects of morphine. One striking difference in responsiveness to morphine suggests the presence of two populations of functionally distinct neurons in the MBH. The two populations are presented as groups IIIa,b in Table 3. Both populations responded on day 1 with decreases in firing rates, but differed on day 5 in the following respects: group IIIa displayed increased predrug activity, increase in firing rates after morphine, and decreased activity after naloxone. The differences cannot be explained on the basis of a failure of group IIIb multiunits to develop tolerance, as the direction of response to morphine was reversed on day 5. The altered responses to morphine challenge injections in physically morphine-dependent animals are probably related to tolerance. Tolerance is generally regarded as a reduction in the pharmacologic response after repeated use of a drug. After the original morphine response is lost, tolerance may be expressed either as no response to a challenge dose of morphine on day 5 of the experiment, or as a reversal in direction of the response due to selective loss of previously dominant influences (24, 3 1). All animals exhibited withdrawal symptoms 1 to 2 min after naloxone injection. Responses to naloxone on day 5 can be used as an indication of withdrawal and the degree of dependence (20). In physically morphinedependent animals, naloxone affected more recording sites than was observed on day 1 of the experiment (Table 3). The improved efficacy of naloxone was suggested to be a sensitive indicator of the initiation and development of narcotic tolerance (20). Moreover, it was suggested that morphine tolerance-dependence is usually associated with an increase in the antagonist potency of naloxone (14,20). Using morphine and naloxone in morphine naive and physically dependent animals makes it possible to identify as many as six response patterns that can be correlated with different functional properties of neurons and it provides a model for the separation of tolerance from dependence in further investigations. REFERENCES 1. BORELL, J., I. LLORENS, AND S. BORRELL. 1974. Study of the effects of morphine on adrenal corticosteroids, ascorbic acid and catecholamines in unanesthetized and anesthetized cats. Horm. Res. 5: 351-358. 2. BRUNI, J. F., D. VAN VUGT, S. MARSHALL, AND J. MEITES. 1977. Effects of naloxone, morphine and methionine enkephalin on serum prolactin, luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone and growth hormone. Life Sci. 21: 461-466.
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for qualitative and quantitative unit