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Reciprocal pre- and postsynaptic actions of cocaine at a central noradrenergic synapse
EXPERIMENTAL
NEUROLOGY
Reciprocal
98,5 18-528 (1987)
Pre- and Postsynaptic Actions of Cocaine at a Central Noradrenergic Synapse DAVID K. PITTS AND JOEMARWAH’
Neuroscience Laboratory, Department ofPathology, University ofMedicine and Dentistry of New Jersey-School of Osteopathic Medicine, Camden, New Jersey 08103 Received December 4, 1986; revision received April 27, I987 Single-unit microelectrode studies were conducted to test the effects of systemic cocaine HCl on spontaneously firing single noradrenergic locus ceruleus (presynaptic) and cerebellar Purkinje (postsynaptic) neurons in rats in vivo. The spontaneous neuronal activity of all locus ceruleus neurons was inhibited by cocaine in a dose-dependent manner (0.5 to 2 mg/kg). These doses of cocaine elicited a predominant activation of postsynaptic Purkinje neurons. No effect of cocaine on neuronal action potential amplitude or slope was observed. Similar doses of the local anesthetic agent, procaine, did not affect action potential amplitudes or slopes of either locus ceruleus or Purkinje neurons. In addition, although cocaine elicited a significantly greater absolute change in the discharge rate of locus ceruleus neurons than of Purkinje neurons, the effects of procaine on those neurons were not significantly different from each other. The inhibition of locus ceruleus neurons by cocaine was significantly attenuated by pretreatment either with the cu2-adrenoceptor antagonist, yohimbine, or with reserpine. The activation of Purkinje neurons by cocaine was also significantly attenuated by reserpine pretreatment. Systemic cocaine administration (I m&kg, i.v.) did not potentiate the inhibitory effects of either locus ceruleus stimulation or local iontophoretic application of norepinephrine on Purkinje neuron discharge rate. We conclude that cocaine potently inhibits locus ceruleus neurons and this effect probably elicits Purkinje cell activation through disinhibition. o 1987 Academic preu, IX.
INTRODUCTION
Cocaine is both a potent local anesthetic agent and a psychotropic agent with significant central nervous system stimulant effects (19). Recreational use has increased dramatically in recent years and cocaine has emerged as a Abbreviations: LC-locus ceruleus, PC-Purkinje cell, tp-toe pinch, NE-norepinephrine. ’ This study was supported in part by a National Institute of Drug Abuse grant, ROl-DA04158, and an American Heart Association grant-in-aid, 83-757. 518 00144886/87
$3.00
Copyright @ 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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519
major drug of abuse. Despite the current popularity of cocaine, little is known about its effects on discrete central neurons. This is an important consideration, since cocaine is known to block neuronal monoamine reuptake (7,9) and many of the psychotropic effects of cocaine are generally attributed to its interactions with specific central monoaminergic neuronal systems (6, 13). To date the only reports of the effects of cocaine on the electrophysiologic activity of central neurons in vivo are those of Pitts and Marwah (15, 16, 18). They reported that cocaine potently inhibits central monoaminergic neurons. In addition, their preliminary finding (16) suggests that cocaine can also modulate postsynaptic neuronal activity. Cerebellar Purkinje neurons receive well defined inhibitory noradrenergic afferent fibers from the nucleus locus ceruleus (2, 14). In this study, we investigated the effects of cocaine on neurotransmission at the locus ceruleus-Purkinje neuron synapse. Utilizing single-unit electrophysiologic techniques to delineate the potential central sites/mechanisms of action, we examined the effects of cocaine on the pre- (locus ceruleus neurons) and postsynaptic (Purkinje neurons) neuronal components. We now report that cocaine potently modulates synaptic transmission at the above synapse and this modulation is most likely a “disinhibition” of spontaneously discharging Purkinje neurons due to the inhibitory effects of cocaine on locus ceruleus neurons. METHODS Sprague-Dawley rats (Harlan-Indianapolis, 180 to 290 g) were anesthetized with urethane ( 1.25 g/kg, i.p.), intubated following a tracheotomy, and continued to breathe spontaneously. Core temperature was monitored by a rectal thermistor probe and maintained at 37 & 1°C with a heating pad. A catheter was inserted into the lateral tail vein for the administration of drugs (administered during a period of approximately 30 s). The animals were then placed in a stereotaxic instrument for extracellular recordings from single neurons. The eletrical activity of single neurons was recorded with glass micropipets with tip diameters of about 1 pm. The micropipets were filled with 2 MNaCl saturated with fast green. The in vitro impedance for the micropipets was 2.5 to 5 MS2 measured at 135 Hz. The stereotaxic coordinates for recording from locus ceruleus (LC) neurons were: 1.1 mm posterior to lambda and 1.1 mm lateral to midline. The electrical activity from LC neurons was usually encountered at 5.0 to 6.0 mm below the skull surface. Single noradrenergic LC neurons were identified by the following criteria (4); (i) a characteristic longduration (“2 ms), positive-negative action potential, usually with a notch on the asending limb; (ii) a characteristic acceleration/inhibition of firing
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rate when pressure was applied to the contralateral hind paw (toe pinch, tp); (iii) the presence of neurons of the mesencephalic nucleus of cranial nerve V (trigeminal), which are readily activated by moving the lower mandible of the rat, just lateral to LC neurons; (iv) a zone of electrical silence just above the site of the LC neurons corresponding to the fourth ventricle; (v) a firing rate of 0.5 to 5 Hz; and (vi) a consistent, rapid, and reversible inhibition produced by small (10 &kg) systemic doses of clonidine. Further details regarding our recording procedure for LC neurons can be obtained from previous reports (10, 11). For Purkinje neuron (PC) recording, the skull and dura overlying the cerebellum were removed, and the cerebellar surface covered with 2% agar. The cisterna was opened at the foramen magnum to reduce brain pulsations. The recordings from single cerebellar PC neurons were made in the vermis, lobules VI and VII within a region with maximal depth approximately 1 mm from the brain surface (12). The neurons were identified by their characteristic discharge of complex and simple spikes (12). Neuronal activity was amplified, filtered, and monitored on an oscilloscope and then converted to constant-voltage pulses using a window discriminator. The pulses were integrated over 1 or 2 s (PC) or 10 s (LC) epochs by a ratemeter and were then displayed on a chart recorder. Only one neuron and one dose of cocaine was studied in each animal. Experiments employing either antagonist or reserpine pretreatment were accomplished as follows: reserpine (10 mg/kg, i.p.) was administered 5 h and yohimbine (5 mg/kg, i.p.) 20 min before electrophysiologic recording (3, 18). The ceruleus-cerebellar noradrenergic pathway was stimulated with a concentric bipolar electrode (0.4-mm diameter) inserted stereotaxically into the brain through the ipsilateral hemisphere. Ten-second trains of 0.2-ms pulses at 10 Hz were applied at 3-min intervals. During this stimulation cocaine was administered systemically. Comparison of the rate of discharge before stimulus to the rate poststimulus in control and drug-treated rats provided quantification of the modulatory effects of cocaine on the noradrenergic pathway. Multibarrel (five barrels) micropipets were used to record extracellular action potentials of spontaneously active PC neurons and to apply norepinephrine at the site of recording by microiontophoresis. This paradigm was used for drug interaction studies involving central norepinephrine administration and recording after systemic cocaine administration. For multibarrel pipets the center recording barrel was filled with 2 M NaCl. Tip potentials from iontophoretic currents which might directly affect cell discharge were minimized by a current-balancing circuit which automatically passed an equal current of opposite polarity to drug ejection or holding currents through a peripheral barrel containing 4 A4 NaCl. Positive and negative currents were also passed through this barrel independently to check for possible current
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’ SAL
0.5
1
2
DOSE (YG/KG) FIG. 1. Bar graph illustrating the dose-related effects of cocaine (0.5 to 2.0 mg/kg, iv.) and saline (S) on Purkinje (PC, N = 19 activated neurons; see also Table 1) and locus ceruleus (LC) neurons (N = 29).
artifacts. Drugs were applied from the remaining side barreIs. Pulses of uniform current and duration were applied at equal intervals. Such a paradigm permits precise timing of ejection currents and interejection times so that artifacts due to variation of these parameters are minimized. Iontophoretic artifacts of current, drug pH, local anesthesia, or failure to release drugs were carefully controlled. Data from cells which did not show complete recovery of the agonist response after cessation of drug application were discarded. Data analysis was accomplished as follows: Bonferroni t test for multiple comparisons (5) of PC/LC dose-response data, a two-sample t test for antagonist data, and a paired t test for pre- vs. posttreatment data. The effects of reserpine pretreatment on cocaine-induced changes in PC discharge rate were ascertained by a chi-square test based on the classification of responses as depicted in Table 1 for all animals receiving the I-mg/kg dose of cocaine. Mean values used in statistical tests were calculated as mean percentage change in neuronal firing rate 2 min after cocaine administration as a function of a 2-min baseline firing rate just prior to drug administration. A P value of less than 5% was considered statistically significant. The following drugs (purchased from Sigma) were used in the present study: norepinephrine bin&rate, cocaine HCl, clonidine HCl, yohimbine HCl, and reserpine.
RESULTS Cocaine (0.5 to 2 mg/kg, iv.) invariably inhibited LC neurons and usually activated PC neurons (Fig. 1, Table 1). These effects were evident within 40
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PITTS AND MARWAH TABLE 1 Responses of Purkinje (PC) and Locus Ceruleus (LC) Neurons to Intravenous Cocaine Response”
Neuron type
Dose b-m&.4
PC
LC
Activated
Inhibited
0.5 1.0 1.0 (R)’ 2.0
4 I1
-
0.5 1.0 1.O(R)’ 2.0
-
1 8
3 -
Biphasic
Noeffect
Nb
1 1
5 15
6 2
7 11
-
10 13 10 6
-
2 -
1 -
1
-
10 13 4 6
-
3 -
’ Responses to intravenous cocaine (2 min after administration) were categorized from ratemeter records as follows: activation, >5% increase; inhibition, >5% decrease; no effect, f5%; biphasic, inhibition/excitation. b The number of animals reported for a particular cocaine dose in Table 1 occasionally exceeds the number of animals reported in Figs. 1 and 2, because the figures represent data expressed only for particular time points and types of responses, and Table 1 represents observations made in all animals studied. ’ Animals pretreated with reserpine, 10 m&kg, i.p., 5 h before electrophysiologic recording.
s of drug injection and recovery toward baseline commenced about 5 min after drug administration. Repeated systemic injections of physiologic saline vehicle (vehicle for cocaine) in equivalent volume did not affect either LC or PC neuron firing. The three doses of cocaine tested elicited a significantly greater absolute change (P < 0.005) in the tiring rate of LC as opposed to PC neurons. The effects of procaine (0.5 to 2.0 mg/kg, i.v.) on LC and PC neurons were not significantly different from that of saline controls (data not shown). For both LC and PC neurons cocaine exerted a significantly greater effect on neuronal firing than similar doses of procaine (0.5 to 2.0 mg/kg; LC:P < 0.005, PC? < 0.05). Table 1 summarizes the effects of various doses of cocaine on LC and PC neurons. Over the dose range tested, cocaine consistently inhibited LC neuronal firing (N = 29). At these same doses the effect of cocaine on PC neurons was activation (74%) inhibition (lo%), no effect ( 13%), or biphasic (3%). The values in parentheses represent the percentage of 3 1 neurons tested showing the particular response. Clearly, the predominant effect in 3 1 PC neurons tested from 3 1 separate animals was an activation of neuronal firing. The inhibitory effects of cocaine (1 mg/kg) on LC neurons were significantly attenuated by pretreatment with reserpine (a monoamine-depleting
COCAINE ACTION
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ON NE SYNAPSE
+-PC -o- PCXR) -0 --•-
LC(R) LC(Y)
-a- LC
MINUTES FIG. 2. Time course for the in viva effects of intravenous cocaine (1 mg/kg) on spontaneously firing single locus ceruleus (LC) and Purkinje (PC) neurons in intact, reserpine (R, 10 mg/kg, i.p.)- and yohimhine (Y, 5 mg/kg, i.p.)-pretreated rats. Each point represents a 10-s period (PC mean of five 2-s bins; LC: one 10-s bin) plotted every 20 s. The circles and squares represent percentage change in LC and PC activity, respectively. Filled symbols represent control animals (LC: N = 13, PC N = 8 activated neurons) and blank symbols represent reserpine-depleted (LC: N = 5; PC: N = 6 neurons with no effect; see also Table I) animals. Yohimbine-pretreated animals (N = 5) are depicted by the dashed line.
agent; P < 0.00 1, N = 10) or yohimbine (an qadrenoceptor antagonist; P < 0.02, N = 5) compared with control animals (N = 13; Figure 2 and Table 1). The activating effects of cocaine on PC neurons were also significantly (P < 0.005) attenuated by pretreatment with reserpine (N = 7) compared with control animals (n = 15; Fig. 2 and Table 1). Figure 3A depicts the effects of cocaine on a spontaneously firing LC neuron. Cocaine (C) rapidly and reversibly inhibited neuron firing. This inhibition of neuronal firing was significantly attenuated by pretreatment with the a2-adrenoceptor antagonist, yohimbine (5 mg/kg, i.p., 20 min before recording, Fig. 3B), or reserpine (Fig. 3E). LC neurons from reserpine-treated animals were resistant (often initially excited; see also Fig. 2) to the inhibitory effects of cocaine. However, these same neurons were potently inhibited by clonidine and activated by yohimbine. Figure 3C shows the activating effects of cocaine on a spontaneously firing Purkinje neuron. The recovery from this activation is shown in Fig. 3D. Figure 3F depicts the lack of effects of cocaine on a spontaneously firing PC neuron from an animal administered reserpine. Stimulation ofthe LC decreased the activity of postsynaptic spontaneously discharging PC neurons (Fig. 4). Cocaine significantly attenuated the LCelicited inhibition of PC neuron activity (P-c 0.05, N = 5).
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FIG. 3. Ratemeter histograms comparing the effects of cocaine (I mg/kg, i.v.) on single LC and PC neurons in control animals or animals pretreated with either yohimbine (5 mg/kg, i.p.) or reserpine (10 mgjkg, i.p.). C-cocaine, CL-clonidine (20 pg/kg, i.v.), tp-toe pinch, a nociceptive stimulus, Y-yohimbine (200 &kg, i.v. X2). (A)-single LC neuron from control animal. (B)-single LC neuron from a yohimbine-pretreated animal. (C)-single PC neuron from a control animal. (D)-recovery of the PC neuron depicted in (C) 26 min after cocaine administration. (E)-single LC neuron from a reserpine-pretreated animal. Note the excitatory effect of cocaine on this neuron and the ability of the a,-adrenoceptor agonist, clonidine, to completely silence this neuron. The nociceptive stimulus (tp X5) accelerated the firing rate of the otherwise silent cell indicating its continued presence at the electrode tip. Subsequent administration of yohimbine reversed the effects of clonidine on this neuron. (F)-single PC neuron from a reserpine-pretreated animal. The calibration bars under each panel represent a 2-min period.
The typical effects of iontophoretically applied norepinephrine are depicted in Fig. 5A. Norepinephrine inhibited PC neuronal firing (first four panels in figure). The systemic administration of cocaine failed to alter this norepinephrine-mediated inhibition of PC neuron discharge. The cumulative (four animals) inhibition of PC neuron firing is depicted in Fig. 5B. The mean percent inhibition before cocaine (32.0 f 2.8%) was not significantly different from that after cocaine (29.9 f 3.1%; P > 0.10). DISCUSSION Previous studies (2, 14) established that the projection from the pontine nucleus locus ceruleus to cerebellar Purkinje neurons is noradrenergic. Activation of this pathway results in the release of norepinephrine from presynaptic LC neuron terminals, which subsequently interacts with P-adrenocep-
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AFTER COCAINE S I
FIG. 4. Two ratemeter histograms generated from a single PC neuron. The histogram on the left represents mean counts per I-s bin for five different trials of LC stimulation. During the stimulation period (S, 10 s) the LC was stimulated with a bipolar electrode (10 Hz, 0.2 ms, 20 V). Mean percentage inhibition of spontaneous activity (I, averaged for 15 s) of this neuron for five trials was 56%. The histogram on the right shows the mean effects of five LC stimulation trials on the same neuron after cocaine administration (1 mg/kg, i.v.) Stimulation parameters were identical to those used during the drug-free condition. Mean percentage inhibition of spontaneous activity of this neuron for five trials was 2790.
tors situated on postsynaptic Purkinje soma/dendrites (2). Activation of these /3-adrenoceptors elicits inhibition of PC firing (2). Our results suggest that the activation of PC cells by cocaine observed in the current study is most likely due to indirect effects. This appears to be a plausible explanation for the lack of effects of cocaine on PC neurons in animals after reserpine administration (monoamine depleted). In animals without reserpine inhibition by cocaine of the tonically discharging inhibitory presynaptic LC neurons (2) would release the postsynaptic PC neurons from noradrenergic inhibitory restraint. This would result in an activation of neuronal firing. The mechanism(s) responsible for inhibition of LC neurons are also indirect, as LC neurons in animals after reserpine pretreatment were resistant to the inhibitory effects of cocaine. The mechanism responsible for the transient “paradoxical” excitation of LC neurons in animals after reserpine by systemic cocaine administration is unknown. Central monoamine depletion may unmask excitatory influences of cocaine (direct or indirect) on spontaneously firing LC neurons. The reserpine regimen used in the present study was previously shown not to completely attenuate the brief increase in mean arterial pressure induced by systemic cocaine administration ( 17). Therefore, systemically induced cardiovascular effects cannot be ruled out as possibly contributing to this “paradoxical” excitation. There currently exists no evidence regarding the ability of cocaine to interact directly with adrenoceptors. Thus it appears unlikely that cocaine elicits neuron inhibition by directly activating inhibitory a2-adrenoceptors situated on LC neurons (4). The most plausible explanation appears to be that cocaine potentiates the effects of an inhibitory “neurotransmitter” that is tonically released onto LC soma/dendrites. This transmitter may conceivably be
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PITTS AND MARWAH
A)
5
20-
H i! f E oBEFORE
AFTER
FIG. 5. Panel A represents eight sequential I .5-min segments of a ratemeter histogram from a single PC neuron which received norepinephrine (NE) by iontophoretic application (trial; 30 nA, 30 s) every 3 min. Each trial was separated by a 2.5-min recovery period (not shown in entirety). Iontophoretic application of NE elicited an inhibitory response of similar magnitude in each trial even though cocaine was administered after the first four trials. Panel B illustrates the mean percentage inhibition from iontophoretic NE application for four different neurons (from four different animals) before and afier intravenous cocaine administration (1 mg/kg). These means were constructed from four pre- and four postcocaine trials as described above for each of the PC neurons. The control mean percent inhibition from iontophoretic NE trials was not significantly different (P > 0.10) from the mean after cocaine administration.
stored in LC axon terminals (collaterals) or in terminals of other catecholamine inputs that impinge on LC soma/dendrites (1). The identity of this transmitter is most likely catecholaminergic, as our data indicate that the inhibitory effects of cocaine on LC neurons are significantly attenuated by pretreatment with yohimbine (an 5zz-adrenoceptor antagonist) but not by naloxone (a mu receptor antagonist).
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Baseline LC neuron firing rate has been reported to be significantly elevated in reserpine and yohimbine-pretreated animals ( 18). It is unlikely that this elevated baseline firing rate is responsible for the attenuation of the inhibitory effects of cocaine on LC neurons because a*-adrenoceptor antagonists (piperoxane and yohimbine) can also reverse such inhibition (data not shown). Results from experiments using the electrical stimulation of the LC source of noradrenergic Purkinje neuron agerent fibers suggest that the modulatory effect/s of cocaine on monoaminergic synapses is greater at the level of the LC soma than at LC terminals. If the effect on LC terminals predominated (i.e., inhibition of reuptake of tonically released noradrenaline) then the resultant effect on PC neurons should be an inhibition of firing. Instead an attenuation of the inhibitory effects of LC stimulation was observed. Similar results have been obtained with amphetamine at another central noradrenergic synapse (8). Systemically administered cocaine did not potentiate the inhibitory effects of iontophoretically applied norepinephrine on Purkinje neurons. This finding is also consistent with a predominant effect of cocaine on LC soma resulting in a decrease in impulse flow. Three aspects suggest that the effects of cocaine on PC and LC neurons cannot be attributed to the local anesthetic properties of this compound. First, the effects of cocaine on both LC and PC neurons were qualitatively and quantitatively different from the effects of the structurally related local anesthetic agent, procaine. Second, cocaine did not elicit a decrease in neuronal spike height or slope of either LC or PC neurons. Such a decrease is characteristic of a local anesthetic effect (20). Third, cocaine was less effective in altering neuronal firing in reserpine-treated animals. Finally, the inhibitory effects of cocaine on the central neurons studied are not directly related to its effects on blood pressure, as the neuronal effects are of much greater duration than the relatively brief increase in mean arterial pressure (17, 18). The data reported here delineate for the first time significant central effects of cocaine on all the components of an identified central monoaminergic synapse. Such data are not meant to imply that this is the only central synapse that is exquisitively sensitive to cocaine. What our data do demonstrate is that cocaine elicits a multitude of pre and postsynaptic effects, which collectively contribute to the global psychotropic state seen after cocaine administration. Additional studies on the effects of cocaine on well defined central monoaminergic neurons/synapses is likely to yield significant mechanistic information. REFERENCES G. K. 1978. Feedback regulation of central monoaminergic neurons evidence from single cell recording studies. Pages l-32 in M. B. H. YOUDIM, W. LOVEN-
I. AGHAJANIAN,
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2. 3. 4. 5. 6.
PITTS AND MARWAH BERG, D. R. SHARMAN, AND J. R. LAGANDO, Eds., Essays in Neurochemistry on Neuropharmacology. Wiley, New York. BLOOM, F. E. 1978. Central noradrenergic systems: physiology and pharmacology. Pages 13 l-l 64 in M. E. KILLAM AND A. DIMASCIO, Eds., Psychopharmacology-A Generation OfProgress. Raven Press, New York. ENGBERG, G., AND T. H. SVENSSON. 1979. Amphetamine-induced inhibition of central noradrenergic neurons: a pharmacological analysis. Life&i. 24,2245-2254. CEDARBAUM, J. M., AND G. K. AGHAIANIAN. 1976. Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine and activation by the a-antagonist piperoxane. Brain Res. 112: 413-419. GILL, J. L. 1978. Completely randomized designs and analysis of variance. Design and Analysis of Experiments in the Animal and Medical Sciences, pp. 176- 177 Iowa State Univ. Press, Ames. GOLD, M. S., AND . VEREBY. 1984. The psychopharmacology of cocaine. Psychiat. Ann.
14: 7 14-723. 7. HADFIELD, M.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
G., D. E. W. MOH, AND J. A. ISMAY. 1980. Cocaine: effect of in vivo administration on synaptosomal uptake of norepinephrine. Biochem. Pharmacol. 29: 18611863. HUANG, Y. H., AND J. W. MAAS. 198 1. d-Amphetamine at low doses suppresses noradrenergic functions. Eur. J. Pharmacol. 75: 187-l 95. KOE, B. K. 1976. Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain. J. Pharmacol. Exp. Ther. 199: 649661. MARWAH, J., AND G. K. AGHAJANIAN. 1982. Relative potencies of alpha-l and alpha-2 antagonists in the locus coeruleus, dorsal raphe and dorsal lateral geniculate nuclei: an electrophysiological study. J. Pharmacol. Exp. Ther. 222: 287-293. MARWAH, J., J. M. KEHNE, R. L. COMMISSARIS, J. LAKOSKI, W. SHAW, AND M. DAVIS. 1983. Spinal clonidine inhibits neural firing in locus coeruleus. Brain Res. 276: 379-382. MARWAH, J., B. J. HOFFER,H. M. GELLER, AND R. FREEDMAN. 1981. Electrophysiological interactions of antipsychotic drugs with central noradrenergic pathways. Psychopharmacology73: 126-133. MULE, S. J. 1984. The pharmacodynamics of cocaine abuse. Psychiatr. Ann. 14: 724-727. OLSON, L., AND K. FUXE. 197 1. On the projections from the locus coeruleus noradrenaline neurons: the ccrebellar innervation. Brain Res. 28: 165-l 7 1. PITS, D. K., AND J. MARWAH. 1986. Effects of cocaine on the electrical activity of single noradrenergic neurons from locus coeruleus. Life Sci. 38: 1229- 1234. Pxns, D. K., AND J. MARWAH. 1986. Electrophysiological effects of cocaine on central monoaminergic neurons. Eur. J. Pharmacol. 131: 95-98. PITTS, D. K., AND J. MARWAH. 1987. Cardiovascular effectsof cocaine in anesthetized and conscious rats. Life Sci. 40: 1099- 1111. Purrs, D. K., AND J. MARWAH. 1987. Electrophysiological actions of cocaine on noradrenergic neurons in rat locus ceruleus. J. Pharmacol. Exp. Ther. 240: 345-35 1. RITCHIE, J. M., AND N. M. GREENE. 1980. Local anesthetics. Pages 300-320 in A. GOODMAN GILMAN, S. GOODMAN, AND A. GILMAN, Eds., The Pharmacological Basis ofTherapeutics. Macmillan, New York. SHANES, A. M. 1958. Electrochemical aspects of physiological and pharmacological action in excitable cells. Pharmacol. Rev. 10: 59-273.
NEUROLOGY
Reciprocal
98,5 18-528 (1987)
Pre- and Postsynaptic Actions of Cocaine at a Central Noradrenergic Synapse DAVID K. PITTS AND JOEMARWAH’
Neuroscience Laboratory, Department ofPathology, University ofMedicine and Dentistry of New Jersey-School of Osteopathic Medicine, Camden, New Jersey 08103 Received December 4, 1986; revision received April 27, I987 Single-unit microelectrode studies were conducted to test the effects of systemic cocaine HCl on spontaneously firing single noradrenergic locus ceruleus (presynaptic) and cerebellar Purkinje (postsynaptic) neurons in rats in vivo. The spontaneous neuronal activity of all locus ceruleus neurons was inhibited by cocaine in a dose-dependent manner (0.5 to 2 mg/kg). These doses of cocaine elicited a predominant activation of postsynaptic Purkinje neurons. No effect of cocaine on neuronal action potential amplitude or slope was observed. Similar doses of the local anesthetic agent, procaine, did not affect action potential amplitudes or slopes of either locus ceruleus or Purkinje neurons. In addition, although cocaine elicited a significantly greater absolute change in the discharge rate of locus ceruleus neurons than of Purkinje neurons, the effects of procaine on those neurons were not significantly different from each other. The inhibition of locus ceruleus neurons by cocaine was significantly attenuated by pretreatment either with the cu2-adrenoceptor antagonist, yohimbine, or with reserpine. The activation of Purkinje neurons by cocaine was also significantly attenuated by reserpine pretreatment. Systemic cocaine administration (I m&kg, i.v.) did not potentiate the inhibitory effects of either locus ceruleus stimulation or local iontophoretic application of norepinephrine on Purkinje neuron discharge rate. We conclude that cocaine potently inhibits locus ceruleus neurons and this effect probably elicits Purkinje cell activation through disinhibition. o 1987 Academic preu, IX.
INTRODUCTION
Cocaine is both a potent local anesthetic agent and a psychotropic agent with significant central nervous system stimulant effects (19). Recreational use has increased dramatically in recent years and cocaine has emerged as a Abbreviations: LC-locus ceruleus, PC-Purkinje cell, tp-toe pinch, NE-norepinephrine. ’ This study was supported in part by a National Institute of Drug Abuse grant, ROl-DA04158, and an American Heart Association grant-in-aid, 83-757. 518 00144886/87
$3.00
Copyright @ 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
COCAINE ACTION
ON NE SYNAPSE
519
major drug of abuse. Despite the current popularity of cocaine, little is known about its effects on discrete central neurons. This is an important consideration, since cocaine is known to block neuronal monoamine reuptake (7,9) and many of the psychotropic effects of cocaine are generally attributed to its interactions with specific central monoaminergic neuronal systems (6, 13). To date the only reports of the effects of cocaine on the electrophysiologic activity of central neurons in vivo are those of Pitts and Marwah (15, 16, 18). They reported that cocaine potently inhibits central monoaminergic neurons. In addition, their preliminary finding (16) suggests that cocaine can also modulate postsynaptic neuronal activity. Cerebellar Purkinje neurons receive well defined inhibitory noradrenergic afferent fibers from the nucleus locus ceruleus (2, 14). In this study, we investigated the effects of cocaine on neurotransmission at the locus ceruleus-Purkinje neuron synapse. Utilizing single-unit electrophysiologic techniques to delineate the potential central sites/mechanisms of action, we examined the effects of cocaine on the pre- (locus ceruleus neurons) and postsynaptic (Purkinje neurons) neuronal components. We now report that cocaine potently modulates synaptic transmission at the above synapse and this modulation is most likely a “disinhibition” of spontaneously discharging Purkinje neurons due to the inhibitory effects of cocaine on locus ceruleus neurons. METHODS Sprague-Dawley rats (Harlan-Indianapolis, 180 to 290 g) were anesthetized with urethane ( 1.25 g/kg, i.p.), intubated following a tracheotomy, and continued to breathe spontaneously. Core temperature was monitored by a rectal thermistor probe and maintained at 37 & 1°C with a heating pad. A catheter was inserted into the lateral tail vein for the administration of drugs (administered during a period of approximately 30 s). The animals were then placed in a stereotaxic instrument for extracellular recordings from single neurons. The eletrical activity of single neurons was recorded with glass micropipets with tip diameters of about 1 pm. The micropipets were filled with 2 MNaCl saturated with fast green. The in vitro impedance for the micropipets was 2.5 to 5 MS2 measured at 135 Hz. The stereotaxic coordinates for recording from locus ceruleus (LC) neurons were: 1.1 mm posterior to lambda and 1.1 mm lateral to midline. The electrical activity from LC neurons was usually encountered at 5.0 to 6.0 mm below the skull surface. Single noradrenergic LC neurons were identified by the following criteria (4); (i) a characteristic longduration (“2 ms), positive-negative action potential, usually with a notch on the asending limb; (ii) a characteristic acceleration/inhibition of firing
520
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AND
MARWAH
rate when pressure was applied to the contralateral hind paw (toe pinch, tp); (iii) the presence of neurons of the mesencephalic nucleus of cranial nerve V (trigeminal), which are readily activated by moving the lower mandible of the rat, just lateral to LC neurons; (iv) a zone of electrical silence just above the site of the LC neurons corresponding to the fourth ventricle; (v) a firing rate of 0.5 to 5 Hz; and (vi) a consistent, rapid, and reversible inhibition produced by small (10 &kg) systemic doses of clonidine. Further details regarding our recording procedure for LC neurons can be obtained from previous reports (10, 11). For Purkinje neuron (PC) recording, the skull and dura overlying the cerebellum were removed, and the cerebellar surface covered with 2% agar. The cisterna was opened at the foramen magnum to reduce brain pulsations. The recordings from single cerebellar PC neurons were made in the vermis, lobules VI and VII within a region with maximal depth approximately 1 mm from the brain surface (12). The neurons were identified by their characteristic discharge of complex and simple spikes (12). Neuronal activity was amplified, filtered, and monitored on an oscilloscope and then converted to constant-voltage pulses using a window discriminator. The pulses were integrated over 1 or 2 s (PC) or 10 s (LC) epochs by a ratemeter and were then displayed on a chart recorder. Only one neuron and one dose of cocaine was studied in each animal. Experiments employing either antagonist or reserpine pretreatment were accomplished as follows: reserpine (10 mg/kg, i.p.) was administered 5 h and yohimbine (5 mg/kg, i.p.) 20 min before electrophysiologic recording (3, 18). The ceruleus-cerebellar noradrenergic pathway was stimulated with a concentric bipolar electrode (0.4-mm diameter) inserted stereotaxically into the brain through the ipsilateral hemisphere. Ten-second trains of 0.2-ms pulses at 10 Hz were applied at 3-min intervals. During this stimulation cocaine was administered systemically. Comparison of the rate of discharge before stimulus to the rate poststimulus in control and drug-treated rats provided quantification of the modulatory effects of cocaine on the noradrenergic pathway. Multibarrel (five barrels) micropipets were used to record extracellular action potentials of spontaneously active PC neurons and to apply norepinephrine at the site of recording by microiontophoresis. This paradigm was used for drug interaction studies involving central norepinephrine administration and recording after systemic cocaine administration. For multibarrel pipets the center recording barrel was filled with 2 M NaCl. Tip potentials from iontophoretic currents which might directly affect cell discharge were minimized by a current-balancing circuit which automatically passed an equal current of opposite polarity to drug ejection or holding currents through a peripheral barrel containing 4 A4 NaCl. Positive and negative currents were also passed through this barrel independently to check for possible current
COCAINE
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ACTION
ON NE SYNAPSE
521
’ SAL
0.5
1
2
DOSE (YG/KG) FIG. 1. Bar graph illustrating the dose-related effects of cocaine (0.5 to 2.0 mg/kg, iv.) and saline (S) on Purkinje (PC, N = 19 activated neurons; see also Table 1) and locus ceruleus (LC) neurons (N = 29).
artifacts. Drugs were applied from the remaining side barreIs. Pulses of uniform current and duration were applied at equal intervals. Such a paradigm permits precise timing of ejection currents and interejection times so that artifacts due to variation of these parameters are minimized. Iontophoretic artifacts of current, drug pH, local anesthesia, or failure to release drugs were carefully controlled. Data from cells which did not show complete recovery of the agonist response after cessation of drug application were discarded. Data analysis was accomplished as follows: Bonferroni t test for multiple comparisons (5) of PC/LC dose-response data, a two-sample t test for antagonist data, and a paired t test for pre- vs. posttreatment data. The effects of reserpine pretreatment on cocaine-induced changes in PC discharge rate were ascertained by a chi-square test based on the classification of responses as depicted in Table 1 for all animals receiving the I-mg/kg dose of cocaine. Mean values used in statistical tests were calculated as mean percentage change in neuronal firing rate 2 min after cocaine administration as a function of a 2-min baseline firing rate just prior to drug administration. A P value of less than 5% was considered statistically significant. The following drugs (purchased from Sigma) were used in the present study: norepinephrine bin&rate, cocaine HCl, clonidine HCl, yohimbine HCl, and reserpine.
RESULTS Cocaine (0.5 to 2 mg/kg, iv.) invariably inhibited LC neurons and usually activated PC neurons (Fig. 1, Table 1). These effects were evident within 40
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PITTS AND MARWAH TABLE 1 Responses of Purkinje (PC) and Locus Ceruleus (LC) Neurons to Intravenous Cocaine Response”
Neuron type
Dose b-m&.4
PC
LC
Activated
Inhibited
0.5 1.0 1.0 (R)’ 2.0
4 I1
-
0.5 1.0 1.O(R)’ 2.0
-
1 8
3 -
Biphasic
Noeffect
Nb
1 1
5 15
6 2
7 11
-
10 13 10 6
-
2 -
1 -
1
-
10 13 4 6
-
3 -
’ Responses to intravenous cocaine (2 min after administration) were categorized from ratemeter records as follows: activation, >5% increase; inhibition, >5% decrease; no effect, f5%; biphasic, inhibition/excitation. b The number of animals reported for a particular cocaine dose in Table 1 occasionally exceeds the number of animals reported in Figs. 1 and 2, because the figures represent data expressed only for particular time points and types of responses, and Table 1 represents observations made in all animals studied. ’ Animals pretreated with reserpine, 10 m&kg, i.p., 5 h before electrophysiologic recording.
s of drug injection and recovery toward baseline commenced about 5 min after drug administration. Repeated systemic injections of physiologic saline vehicle (vehicle for cocaine) in equivalent volume did not affect either LC or PC neuron firing. The three doses of cocaine tested elicited a significantly greater absolute change (P < 0.005) in the tiring rate of LC as opposed to PC neurons. The effects of procaine (0.5 to 2.0 mg/kg, i.v.) on LC and PC neurons were not significantly different from that of saline controls (data not shown). For both LC and PC neurons cocaine exerted a significantly greater effect on neuronal firing than similar doses of procaine (0.5 to 2.0 mg/kg; LC:P < 0.005, PC? < 0.05). Table 1 summarizes the effects of various doses of cocaine on LC and PC neurons. Over the dose range tested, cocaine consistently inhibited LC neuronal firing (N = 29). At these same doses the effect of cocaine on PC neurons was activation (74%) inhibition (lo%), no effect ( 13%), or biphasic (3%). The values in parentheses represent the percentage of 3 1 neurons tested showing the particular response. Clearly, the predominant effect in 3 1 PC neurons tested from 3 1 separate animals was an activation of neuronal firing. The inhibitory effects of cocaine (1 mg/kg) on LC neurons were significantly attenuated by pretreatment with reserpine (a monoamine-depleting
COCAINE ACTION
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ON NE SYNAPSE
+-PC -o- PCXR) -0 --•-
LC(R) LC(Y)
-a- LC
MINUTES FIG. 2. Time course for the in viva effects of intravenous cocaine (1 mg/kg) on spontaneously firing single locus ceruleus (LC) and Purkinje (PC) neurons in intact, reserpine (R, 10 mg/kg, i.p.)- and yohimhine (Y, 5 mg/kg, i.p.)-pretreated rats. Each point represents a 10-s period (PC mean of five 2-s bins; LC: one 10-s bin) plotted every 20 s. The circles and squares represent percentage change in LC and PC activity, respectively. Filled symbols represent control animals (LC: N = 13, PC N = 8 activated neurons) and blank symbols represent reserpine-depleted (LC: N = 5; PC: N = 6 neurons with no effect; see also Table I) animals. Yohimbine-pretreated animals (N = 5) are depicted by the dashed line.
agent; P < 0.00 1, N = 10) or yohimbine (an qadrenoceptor antagonist; P < 0.02, N = 5) compared with control animals (N = 13; Figure 2 and Table 1). The activating effects of cocaine on PC neurons were also significantly (P < 0.005) attenuated by pretreatment with reserpine (N = 7) compared with control animals (n = 15; Fig. 2 and Table 1). Figure 3A depicts the effects of cocaine on a spontaneously firing LC neuron. Cocaine (C) rapidly and reversibly inhibited neuron firing. This inhibition of neuronal firing was significantly attenuated by pretreatment with the a2-adrenoceptor antagonist, yohimbine (5 mg/kg, i.p., 20 min before recording, Fig. 3B), or reserpine (Fig. 3E). LC neurons from reserpine-treated animals were resistant (often initially excited; see also Fig. 2) to the inhibitory effects of cocaine. However, these same neurons were potently inhibited by clonidine and activated by yohimbine. Figure 3C shows the activating effects of cocaine on a spontaneously firing Purkinje neuron. The recovery from this activation is shown in Fig. 3D. Figure 3F depicts the lack of effects of cocaine on a spontaneously firing PC neuron from an animal administered reserpine. Stimulation ofthe LC decreased the activity of postsynaptic spontaneously discharging PC neurons (Fig. 4). Cocaine significantly attenuated the LCelicited inhibition of PC neuron activity (P-c 0.05, N = 5).
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FIG. 3. Ratemeter histograms comparing the effects of cocaine (I mg/kg, i.v.) on single LC and PC neurons in control animals or animals pretreated with either yohimbine (5 mg/kg, i.p.) or reserpine (10 mgjkg, i.p.). C-cocaine, CL-clonidine (20 pg/kg, i.v.), tp-toe pinch, a nociceptive stimulus, Y-yohimbine (200 &kg, i.v. X2). (A)-single LC neuron from control animal. (B)-single LC neuron from a yohimbine-pretreated animal. (C)-single PC neuron from a control animal. (D)-recovery of the PC neuron depicted in (C) 26 min after cocaine administration. (E)-single LC neuron from a reserpine-pretreated animal. Note the excitatory effect of cocaine on this neuron and the ability of the a,-adrenoceptor agonist, clonidine, to completely silence this neuron. The nociceptive stimulus (tp X5) accelerated the firing rate of the otherwise silent cell indicating its continued presence at the electrode tip. Subsequent administration of yohimbine reversed the effects of clonidine on this neuron. (F)-single PC neuron from a reserpine-pretreated animal. The calibration bars under each panel represent a 2-min period.
The typical effects of iontophoretically applied norepinephrine are depicted in Fig. 5A. Norepinephrine inhibited PC neuronal firing (first four panels in figure). The systemic administration of cocaine failed to alter this norepinephrine-mediated inhibition of PC neuron discharge. The cumulative (four animals) inhibition of PC neuron firing is depicted in Fig. 5B. The mean percent inhibition before cocaine (32.0 f 2.8%) was not significantly different from that after cocaine (29.9 f 3.1%; P > 0.10). DISCUSSION Previous studies (2, 14) established that the projection from the pontine nucleus locus ceruleus to cerebellar Purkinje neurons is noradrenergic. Activation of this pathway results in the release of norepinephrine from presynaptic LC neuron terminals, which subsequently interacts with P-adrenocep-
COCAINE
ACTION
BEFORE COCAINE l-5’
ON NE SYNAPSE
525
AFTER COCAINE S I
FIG. 4. Two ratemeter histograms generated from a single PC neuron. The histogram on the left represents mean counts per I-s bin for five different trials of LC stimulation. During the stimulation period (S, 10 s) the LC was stimulated with a bipolar electrode (10 Hz, 0.2 ms, 20 V). Mean percentage inhibition of spontaneous activity (I, averaged for 15 s) of this neuron for five trials was 56%. The histogram on the right shows the mean effects of five LC stimulation trials on the same neuron after cocaine administration (1 mg/kg, i.v.) Stimulation parameters were identical to those used during the drug-free condition. Mean percentage inhibition of spontaneous activity of this neuron for five trials was 2790.
tors situated on postsynaptic Purkinje soma/dendrites (2). Activation of these /3-adrenoceptors elicits inhibition of PC firing (2). Our results suggest that the activation of PC cells by cocaine observed in the current study is most likely due to indirect effects. This appears to be a plausible explanation for the lack of effects of cocaine on PC neurons in animals after reserpine administration (monoamine depleted). In animals without reserpine inhibition by cocaine of the tonically discharging inhibitory presynaptic LC neurons (2) would release the postsynaptic PC neurons from noradrenergic inhibitory restraint. This would result in an activation of neuronal firing. The mechanism(s) responsible for inhibition of LC neurons are also indirect, as LC neurons in animals after reserpine pretreatment were resistant to the inhibitory effects of cocaine. The mechanism responsible for the transient “paradoxical” excitation of LC neurons in animals after reserpine by systemic cocaine administration is unknown. Central monoamine depletion may unmask excitatory influences of cocaine (direct or indirect) on spontaneously firing LC neurons. The reserpine regimen used in the present study was previously shown not to completely attenuate the brief increase in mean arterial pressure induced by systemic cocaine administration ( 17). Therefore, systemically induced cardiovascular effects cannot be ruled out as possibly contributing to this “paradoxical” excitation. There currently exists no evidence regarding the ability of cocaine to interact directly with adrenoceptors. Thus it appears unlikely that cocaine elicits neuron inhibition by directly activating inhibitory a2-adrenoceptors situated on LC neurons (4). The most plausible explanation appears to be that cocaine potentiates the effects of an inhibitory “neurotransmitter” that is tonically released onto LC soma/dendrites. This transmitter may conceivably be
526
PITTS AND MARWAH
A)
5
20-
H i! f E oBEFORE
AFTER
FIG. 5. Panel A represents eight sequential I .5-min segments of a ratemeter histogram from a single PC neuron which received norepinephrine (NE) by iontophoretic application (trial; 30 nA, 30 s) every 3 min. Each trial was separated by a 2.5-min recovery period (not shown in entirety). Iontophoretic application of NE elicited an inhibitory response of similar magnitude in each trial even though cocaine was administered after the first four trials. Panel B illustrates the mean percentage inhibition from iontophoretic NE application for four different neurons (from four different animals) before and afier intravenous cocaine administration (1 mg/kg). These means were constructed from four pre- and four postcocaine trials as described above for each of the PC neurons. The control mean percent inhibition from iontophoretic NE trials was not significantly different (P > 0.10) from the mean after cocaine administration.
stored in LC axon terminals (collaterals) or in terminals of other catecholamine inputs that impinge on LC soma/dendrites (1). The identity of this transmitter is most likely catecholaminergic, as our data indicate that the inhibitory effects of cocaine on LC neurons are significantly attenuated by pretreatment with yohimbine (an 5zz-adrenoceptor antagonist) but not by naloxone (a mu receptor antagonist).
COCAINE ACTION
ON NE SYNAPSE
527
Baseline LC neuron firing rate has been reported to be significantly elevated in reserpine and yohimbine-pretreated animals ( 18). It is unlikely that this elevated baseline firing rate is responsible for the attenuation of the inhibitory effects of cocaine on LC neurons because a*-adrenoceptor antagonists (piperoxane and yohimbine) can also reverse such inhibition (data not shown). Results from experiments using the electrical stimulation of the LC source of noradrenergic Purkinje neuron agerent fibers suggest that the modulatory effect/s of cocaine on monoaminergic synapses is greater at the level of the LC soma than at LC terminals. If the effect on LC terminals predominated (i.e., inhibition of reuptake of tonically released noradrenaline) then the resultant effect on PC neurons should be an inhibition of firing. Instead an attenuation of the inhibitory effects of LC stimulation was observed. Similar results have been obtained with amphetamine at another central noradrenergic synapse (8). Systemically administered cocaine did not potentiate the inhibitory effects of iontophoretically applied norepinephrine on Purkinje neurons. This finding is also consistent with a predominant effect of cocaine on LC soma resulting in a decrease in impulse flow. Three aspects suggest that the effects of cocaine on PC and LC neurons cannot be attributed to the local anesthetic properties of this compound. First, the effects of cocaine on both LC and PC neurons were qualitatively and quantitatively different from the effects of the structurally related local anesthetic agent, procaine. Second, cocaine did not elicit a decrease in neuronal spike height or slope of either LC or PC neurons. Such a decrease is characteristic of a local anesthetic effect (20). Third, cocaine was less effective in altering neuronal firing in reserpine-treated animals. Finally, the inhibitory effects of cocaine on the central neurons studied are not directly related to its effects on blood pressure, as the neuronal effects are of much greater duration than the relatively brief increase in mean arterial pressure (17, 18). The data reported here delineate for the first time significant central effects of cocaine on all the components of an identified central monoaminergic synapse. Such data are not meant to imply that this is the only central synapse that is exquisitively sensitive to cocaine. What our data do demonstrate is that cocaine elicits a multitude of pre and postsynaptic effects, which collectively contribute to the global psychotropic state seen after cocaine administration. Additional studies on the effects of cocaine on well defined central monoaminergic neurons/synapses is likely to yield significant mechanistic information. REFERENCES G. K. 1978. Feedback regulation of central monoaminergic neurons evidence from single cell recording studies. Pages l-32 in M. B. H. YOUDIM, W. LOVEN-
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