The progressive changes of neuronal activities of the nigral dopaminergic neurons upon withdrawal from continuous infusion of cocaine

Brain Research, 594 (1992) 315-318 © 1992 Elsevier Science Pubhshers B.V. All rights reserved 0006-8993/92/$05.00

315

BRES 25403

The progressive changes of neuronal activities of the nigral dopaminergic neurons upon withdrawal from continuous infusion of cocaine H o n g Z h a n g , T o n g H. Lee a n d E v e r e t t H. Ellinwood Jr. Department of Psychiatry, Duke UniversityMedical Center, Durham, NC 27710 (USA) (Accepted 28 July 1992)

Key words: Cocaine; Dopaminergic; Substantia nigra pars compacta; Apomorphine; Single-unit recording; Electrophysiology

Sensitivity of the nigral dopamine neurons to intravenous apomorphine was exantined in rats pretreated with continuous cocaine or saline for two weeks. One day after withdrawal from pretreatment, a subsensitivity was observed while a supersensitivity was found on day 7, along with a significant increase in the baseline firing rates. In humans, an increased sensitivity of dopamine soma/dendritic autoreceptors may partly account for the symptomatology associated with the intermediate phase of withdrawal f om cocaine binges.

Psychomotor stimulants, such as cocaine (COC) and amphetamine (AMP), have been widely abused in this country ~, and the intermediate withdrawal period following compulsive abuse is associated with a high rate of recidivisms'~. Our previous studies 4,n have shown that, following an early phase of subsensitivity to low doses of apomorphine (APO, 18 h after withdrawal), dopamine (DA) neurons in the substantia nigra pars compacta (SNC) become supersensitive 7 days after a withdrawal from continuous AMP pretreatment. It was proposed that these changes are partly due to those in somatodendritic DA autoreceptors and may contribute to the behavioral changes observed in both human and animals during withdrawal4-6't2. Although AMP and COC produce a similar functional effect on the central DA system (i.e., increased DA transmission), they possess different modes of action Is, whi:h might affect the outcome of chronic pretreatment. Therefore, we have directly examined the time-dependent changes in APO sensitivity of the nigral DA neurons following continuous COC pretreatment. Our results indicate that continuous COC and AMP pretreatments lead to similar changes in the sensitivity of the nigral DA neurons. Following a minimum of 7 days of acclimatization, male, Sprague-Dawley rats (170-225 g) were pre-

treated with continuous COC or saline for 2 weeks. As we described previously 12, rats were anesthetized with methoxyflurane and a small incision was made on the back of the rats (incision site infiltrated with lidocaine). Single osmotic minipumps (Alzet Model 2ML2, Palo Alto, CA) filled with normal saline or COC solution (concentration adjusted to yield an approximate infusion rate of 40 mg/kg/day) were then implanted subcutaneously. The minipumps were removed two weeks later using a similar surgical procedure. 1, 7 or 14 days after the removal of the minipumps, sensitivity of single nigrai DA neurons to APO was examined using an extracellular recording technique. Separate saline control groups were used for the three COC withdrawal groups. For extracellular recordings from single nigral DA neurons, rats were mounted on a stereotaxic apparatus under chloral hydrate anesthesia (400 mg/kg, i.p.). A 3 mm burr hole was drilled in the area overlying the SNC (1.7 mm lateral and 3.2 mm anterior to lambdal4), and a glass-coated tungsten electrode (impedance I0 MD at 1,000 Hz) lowered 6.5-8.0 mm below the dura. The adequacy of anesthesia was determined by an absence of the foot-withdrawal response to a toe pinch and maintained throughout the experiment by a constant

Correspondence: Hong Zhang, Department of Psychiatry, Box 3870, Duke University Medical Center, Durham, NC 27710, USA.

316 infusion of chloral hydrate through a lateral tail vein ~s. The body temperature of the rats was monitored and maintained at 36-38°C with a temperature-control pad. Single DA neurons in the SNC were identified by their locations (6.5-8.0 mm below the brain surface) and the following characteristics of their spontaneous activities: (1) a long action potential (> 2.5 ms) with a distinct initial segment spike; (2) slow, bursting or regular, firing pattern (10-90 spikes/10 s); and (3) low-pitched sound on an audio monitor t.s. Following a minimum of 10-min baseline collections, increasing doses of A P e (Sigma, St. Louis) were administered every 2 min through a tail vein until the spontaneous activity ceased or a maximum dose of 500 /~g/kg was reached. Haloperidol ( 5 0 / t g / k g i.v.) was given in most of the experiments to reverse the A P e effect. At the end of each experiment, the recording site was lesioned with a 10 ttA current for 30 s, and was subsequently confirmed by histological processing of the brain. Only one cell was recorded from each animal. The effect of A P e was expressed as the percentage inhibition of the baseline firing rate. The neuronal sensitivity to A P e was represented as EDs0, which was obtained by using the third-order polynomial regression on the dose-response curves 17. The ED~0's, as well as the baseline firing rates, of DA neurons were evaluated with Student's t-test. In all cases, P < 0.05 was considered statistically significant. The effect of continuous COC pretreatment on the baseline firing of the nigral DA neurons during withdrawal is shown in Table 1 In control animals the firing rates ranged from 10 to 90 spikes/10 s, and were positively correlated with their sensitivity to A P e (rcp-

TABLE !

Nettronal acrid'tries of the SNC DA neurons upon withdra'~val Treatment group Day I a saline COC

Baseline firing

l~'Dso

~spikes/ lO ~,~

~ttg / kg, i,t'.)

31 ± I! (13) 34± 12 (12)

12± 8(13) 26±20(12) h

38± 12(12) 49± 15(19) b

I! ± 7(12) 6± 4(18) c

39± 17(10) 38±11 (9)

9± 5 (9) 12± 2 (8)

Day 7 '~

saline COC Day 14 '~ saline COC

The ED~,'s were obtained from the dose-response curves by fitting them with the third-order polynomial I~, All results are expressed as mean ± S.D. The number of animals tested in each group is indicated m parentheses. " The number of days from saline or COC withdrawal: b p < 0.05: " P < 004,

resented as EDs0's, r = 0.35, n = 34, P < 0.04, the three saline groups combined). Although no significant change (t25 = 0.6, P > 0.5) in baseline firing rates was found on day 1 of COC withdrawal, a significant inc r e a s e (t31 = 2.1, P < 0.05) was observed 7 days after withdrawal. The baseline firing rate returned to the control level on day 14 of withdrawal (t~9 = 0.3, P > 0.7). In contrast to the control group, no significant correlation was found between the baseline firing rates and EDs0'S among any of the three COC pretreatment groups (r < 0.5, P > 0.1, three groups combined). Continuous COC infusion also induced timedependent changes in the sensitivity of the nigrai DA neurons to A P e during withdrawal (Table I). Thus, a subsensitivity to A P e was observed on the first day of withdrawal ( t 2 5 - 2.1, P < 0.05) followed by a supersensitivity on day 7 (t29 = 2.1, P<0.04). The ED5o returned to the control level on day 14 of COC withdrawal (tts = 1.6, P > 0.1). The APe-induced inhibition of firing rates could be reversed with haloperidol in all DA neurons tested (> 80% of total samples in each group). In most cases (> 90% of those tested), the firing rates return to the baseline levels (+ 10%); in addition, no difference in the magnitude of haloperidol-induced reversal was observed between any of the COC and control groups. In summary, continuous COC infusion for 14 days leads w' (1) significantly increased baseline firing rates on day 7 of COC withdrawal; (2) a loss of correlation between the baseline firing rate and the ED.~0 at each withdrawal period tested; and (3) time-dependent changes in the neuronal sensitivity to low doses of A P e (a subsensitivity on day 1 and a supersensitivity on day 7 of withdrawal), Fig, 1 summarizes the doseresponses of each withdrawal group and combined control groups (no significant difference was found among control groups, data not shown). The most significant finding ";n the present study is that continuous COC infusion induces time-dependent changes in the sensitivity of the nigral DA neurons to low doses of A P e during withdrawal. A subsensitivity of the nigral DA neurons to A P e was present on day 1 of withdrawal followed by a supersensitivity on day 7. These time-dependent changes are comparable to our previous observations following continuous AMP protreatment 4'~2, Since low doses of A P e appear to inhibit the firing rates of single DA neurons via its action on soma/dendritic DA autoreceptors IO'20, the changes observed in this study may represent sensitivity changes in the impulse-regulating soma/dendritic DA autorecopters. Thus, chronic elevation in dendritic DA release during continuous COC pretreatment may lead to a down-regulation of DA autoreceptors, which is

317

SENSITIVITYOF SNC DA NEURONS TO APOMORPHINE 100;~

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1 10 100 1000 APO CUMULATIVE DOSE (ug/kg,l.v.) Fig. 1. Inhibitory effect of i.e. apomorphine on the spontaneous firing of nigral DA neurons. Since no significant difference was found among control groups they have been combined for the plotting (filled squares, CONTROL). A subsensitivity was found on day I of cocaine withdrawal (open squares, COC DAY 1) followed by a supersensitivity on day 7 (hourglasses, COC DAY 7). The sensitivity returned to the control level on day 14 of withdrawal (asterisks, COC DAY 14).

manifested on day 1 of withdrawal by the subsensitivity to APO. The supersensitivity, on the other hand, may be a compensatory response of the nigral DA neurons following the removal of COC. Local application of more specific D 2 agonists within the SNC and/or zona reticulata (which contain extensive DA dendritic network), as well as in vitro recordings, would shed light on this hypothesis. In addition to DA ~ut¢rcceptors, the role of other mechanisms in the time-dependent changes in APO sensitivity would have to be investigated. For example, Pitts et al. I~ have suggested that changes in Dm modulation of autoreceptor sensitivity, and not those in autoreceptors themselves, may be responsible for the APO subsensitivity shortly after daily injections of DAMPH (APO is a mixed D~/D 2 agonist). Thus, they reported that, following 24 h of withdrawal, the N-ST DA neurons were normoscnsitive to intravenous quinpirole, a selective D2 (autoreceptor) agonist. Only when the selective D~ agonist, SKF 38393, was simultaneously administered were the DA neurons subsensitive to quinpirole (i.e. a shift of the dose-response curve to the right). A similar mechanism may also be involved in the subsensitivity observed on day 1 of withdrawal from continuous COC. Likewise, the D I mechanism cannot be ruled out as a factor contributing to the APO supersensitivity on day 7. Furthermore, continuous COC infusion might also indirectly change the APO sensitivity of DA neurons via other neurotransmitter systems that interact with DA neurons either in the

cell body and/or terminal regions (e.g. serotonin projection from the raphe nucleus) 9'~5. In this study, the significant changes in the baseline activity of the nigral DA neurons were also observed following chronic COC infusion. Unexpectedly, the APO supersensitivity on day 7 was associated with an increased baseline firing rate. This finding is in contrast to no significant changes during withdrawal from continuous AMP infusion 4J2, and, more importantly, is opposite to what would be expected from the increased negative feedback. Several explanations could be offered for the apparent discrepancy between the APO supersensitivity and baseline firing rates. First, it is possible that the increased firing rates on day 7 is a random finding. As we have shown previously ~2, baseline firing rates of DA neurons do not show consistent changes following chronic AMP or COC pretreatment, however, the fairly large number of neurons tested in this study has made this explanation less convincing. Secondly, changes in autoreceptor sensitivity may have a more direct effect on the number of spontaneously active DA neurons (e.g. supersensitivity associated with a decrease in number), and possibly the remaining active neurons adjust their baseline firing rates to compensate for the changes in the number of active neurons. It has been shown that not all nigral DA neurons are spontaneously active s'9, and, therefore, withdrawal from chronic COC pretreatment might lead to an increased percentage of quiescent DA neurons in the SNC. Although we did not observe an increased difficulty in finding DA neurons on day 7, more systematic examination using population sampling method (by passing recording electrodes a fixed number of times through predetermined coordinates II) may reveal a decreased number of spontaneously active neurons. Thirdly, the increased baseline firing could also be an indirect result of the COC-induced changes in other brain sites that regulate DA activity levels (e.g. striatum, pars reticulata of substantia nigra and raphe nucleusg.~5). Finally, instead of changing the synaptic mechanisms (i.e. regulation by autoreceptors or afferents), chronic COC infusion might also lead to direct changes in various membrane conductances. Although this hypothesis is speculative, it is well documented that the spontaneous activity of DA neurons is, in large part, determined by intrinsic membrane properties rather than synaptic excitatory or inhibitory inputs !°. In addition, other studies have clearly shown that various experimental manipulations (e.g. conditioning) can lead to long-term changes in membrane conductances (cae+-dependent after-hyperpolarization) 3. Another interesting finding in this study is a loss of the correlation between the baseline firing and the

318 EDso'S at all withdrawal time periods tested. Since the exact mechanism(s) underlying the correlation between the two measures is not known, the significance of this finding is not clear, however, since the nigral afferent pathways seem to be an important factor in the rate/ sensitivity correlation 2, our finding may reflect changes in these afferent pathway(s). The intermediate withdrawal phase (3-10 days) following •COC (or AMP) binge episodes in humans is characterized by fatigue, loss of physical/mental ener~, and aahcdonia, which, in turn, may be largely responsible for the high rate of recidivism during this period s'6. We hypothesize that the inability to compensate during increased demand on the D A system (due to increased negative feedback) is one of the neurobiological changes underlying the intermediate withdrawal syndrome. In this study, we have examined the changes observed in the nigral D A neurons; we are now in the process of determining the sensitivity of the DA neurons in the ventral tegmental area to APO and quinpirole administered either intravenously or micro-iontophoretically. These studies would complete a database on the effects of continuous COC infusion on the impulse regulation of both nigrostriatal and mesolimbic DA neurons. It is emphasized that clinical manifestation of COC withdrawal is a syndrome, often with both 'motoric', and 'mental/mood' components s,~'. In chronic stimulant regimens, most researchers have focused on the meso-accumbens DA pathway because of its relation to reinforcement and the proposed role of the reinforcement mechanism in mediating effects of chronic COC I'~. in addition to motor functions, however, the nigrostriatal DA pathway is thought to subserve a function in the behavioral selection/activation process during behavioral activation in response to sensory stimuli 7,~'~. Considering that compulsive stimulant abusers in the intermediate withdrawal phase exhibit a limited behavioral repertoire and an inability to initiate/sustain activity or attention s,e, dysfunction of the nigrostriatal DA neurons may significantly contribute to the expression of behavioral deficits noted during withdrawal. Therefore, examination of both the: "figrostriatal and mesolimbic DA pathways may provide a more complete picture of changes that occur during withdrawal from chronic COC pretreatment. Results from a more comprehensive examination may provide for an animal model of human COC withdrawal, which, in turn, may be utilized to screen for potential therapeutic modes for maintaining COC abstinence. The authors wish to thank Dr. George King for his helpful discussions, and Zheng-Yu Xue, Cackle M. Joyner, Rosemary Hunter-

Aaron and Michael P. Bates for their excellent help with the chronic animal treatment and part of the histological confirmation work. This study was supported by NIDA Grants DA06519 and SRCD[5PS0-DA05303]. 1 Bunney, B.S., Aghajanian, G.K. and Roth, R.H., Comparison of effects of t.-dopa, amphetamine and apomorphine on firing rate of rat dopaminergic neurons, Nature, 245 (1973) 123-125. 2 Chiodo, L.A., Dopamine-containing neurons in the mammalian central nervous system: electrophysiology and pharmacology, Neuroscl. Biobehav. Rev., 12 (1988) 49-91. 3 Coulter, D.A., Lo Turco, J.J., Kubota, M., Disterhoft, J.F., Moore, J.W., and Alkon, D.L., Classical conditioning reduces amplitude and duration of calcium-dependent after hyperpolarization in rabbit hippocampal pyramidal cells, J. Neurophysiol., 61 (1989) 971-981. 4 Ellinwood Jr., E.H. and Lee, T.H., Effect of continuous systemic infusion of D-amphetamine on the sensitivity of nigral dopamine cells to apomorphine inhibition of firing rates, Brain Res., 273 (1983) 379-383. 5 Gawin, F.H. and Ellinwood Jr., E.H., Cocaine and other stimulants: action, abuse and treatment, N. Eng. J. Med., 318 (1988) 1173-1182. 6 Gawin, F.H. and Ellinwood Jr., E.H., Cocaine dependence, Annu. Rev. Med., 40 (1989) 149-161. 7 Goldman-Rakic, P.S. and Selemon, L.D., New frontiers in basal ganglia research, Trends Neurosci., 13 (1990) 241-244. 8 Grace, A.A. and Bunney, B.S., Intracellular and extracellular electrophysiology of nigral dopaminergic neurons. I. Identification and characterization, Neurosci., I0 (1983) 317-331. 9 Grace, A.A. and Bunney, B.S., Opposing effects of striatonigral feedback pathways on midbrain dopamine cell activity, Brain Res., 333 (1985) 271-284. I0 Grace, A.A., The regulation of dopamine neuron activity as determined by in vivo and in vitro intracellular recordings. In L.A. Chiodo and A.S. Freeman (Eds,), The Neurophysiolo~ of Dopamine Systemv, Lake Shore Publications, Detroit, 1987, pp, 1-67, 11 Hollerman, J.R, and Grace, A,A,, The effects of dopamine-depleting brain lesion on the electrophysiological activity of rat substantia nigra dopamine neurons, Brain Res., 533 (1990) 203212. 12 Lee, I.H. and Ellinwood Jr., E.H., Time-dependent changes in the sensitivity of dopamine neurons to low doses of apomorphine following amphetamine infusion, Brain Res., 483 (1989) 17-29. 13 Markou, A. and Koob, G.F., Postcocaine anhedonia: an animal model of cocaine withdrawal, Neuropsychopharmacology,4 (1991) 17-26. 14 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 15 Pitts, D.K. and Marwah, J., Cocaine and central monoaminergic neurotransmission: a review of electrophysiological studies to amphetamine and antidepressants, Life $ci., 42 (1988) 949-968. 16 Pins, D,K,, Freeman, A.S. Kelland, M,D. and Chiodo, L.A., Repeated amphetamine: reduced dopamine neuronal responsiveness to apomorphine but not quinpirole, Fur. J. PkarmacoL, 162 (1989) 167-171. 17 Pitts, D,K,, K(:lland, M,D,, Shen, R.Y,, Freeman, A.S, and Chiodo, L,A,, Statistical analysis of dose-response curves in extracellular electrophysiologicai studies of single neurons, Synapse, 5 (1990) 281-293, 18 Rosenber8, H,C,, Tietz, EJ., Zhang, H. and Chiu, T.H., Tolerance to diazepam and methyl-g-carboline-3-carboxylate measured in substantia nigra of benzodiazepine tolerant rats, Life Sci., 46 (1989) 519-525. 19 Stevens, J.R., An anatomy of schizophrenia? Arch. Gen. Psychiatry, 29 (1973) 177-189.20. 20 Waddington, J.L. and O'Boyle, K.M., Drugs acting on brain dopamine receptors: a conceptual re-evaluation five years after the first selective D l antagonist, Pharmacol. Ther., 43 (1989) ! -52.