Cocaine actions in a central noradrenergic circuit: enhancement of cerebellar Purkinje neuron responses to iontophoretically applied GABA

297

Brain Research, 546 (1991) 297-309 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116512T BRES 16512

Cocaine actions in a central noradrenergic circuit: enhancement of cerebellar Purkinje neuron responses to iontophoretically applied GABA Barry D. Waterhouse 1, Zachary N. Stowe 2, Carlos A. Jimenez-Rivera 1, Francis M. Sessler 1 and Donald J. Woodward 3 l Department of Physiology and Biophysics, Hahnemann University, Philadelphia, PA 19102-1192, 2Department of Psychiatry, Duke University Medical Center, Durham, NC 27710 and 3Department of Cell Biology and Anatomy, University of Texas Health Science Center, Dallas, TX 75235 (U.S.A.) (Accepted 6 November 1990) Key words: Cocaine; F-Aminobutyric acid; Cerebellum; Purkinje neuron; Microiontophoresis

Many recent studies have implicated the mesolimbic dopaminergic pathway as the central neurotransmitter system which is most likely responsible for the euphoria and abuse potential associated with cocaine self-administration. Nevertheless, cocaine also has well established interactions with the norepinephrine- and serotonin-containing pathways of the brain. In order to begin assessing potential nondopamine-mediated actions of cocaine in central circuits, we have initiated a series of experiments using the cerebellar Purkinje neuron as an electrophysiological test system. The strategy was to use the same experimental protocols employed in previous investigations of noradrenergic influences on putative amino acid transmitter action to examine the effects of exogenously applied cocaine on },-aminobutyric acid (GABA)-induced depressant responses of Purkinje cells. Accordingly, the inhibitory responses of Purkinje neurons to microiontophoretically applied GABA were examined before and after systemic or local iontophoretic administration of cocaine. Drug-induced changes in the spontaneous firing rate and GABA responsiveness of individual cells were assessed by quantitative analysis of perievent histograms. The results indicate that, like norepinephrine, cocaine at parenteral or iontophoretic doses subthreshold for producing direct suppression of spontaneous discharge can augment Purkinje neuron responses to GABA. Such potentiating effects of cocaine on GABA-mediated inhibition were not evident in animals pretreated with the selective noradrenergic toxin DSP-4. These findings indicate that cocaine can enhance central neuronal responsiveness to GABA in a manner identical to that shown previously for norepinephrine. Such actions in noradrenergic target circuits throughout the brain could contribute to the net behavioral response observed following cocaine administration.

INTRODUCTION Many previous biochemical studies have shown that cocaine is a potent inhibitor of norepinephrine (NE) reuptake mechanisms 3A7'ls'23'37. The net effect of this action in the CNS would be to elevate synaptic levels of tonically or phasically released NE. Because of the widespread distribution of central NE-containing fibers the above described action should produce an enhancement of noradrenergic function throughout the CNS. However, only a limited number of studies 33'35 have examined the physiological consequences of cocaine administration in noradrenergically innervated regions of the mammalian brain. Over a period of many years a facilitating effect of N E on neuronal responsiveness to afferent synaptic inputs and putative transmitter substances has been demon-

strated in the rat cerebellum 16"3°, cerebral cortex 15'45'46, facial motor nucleus 25, hippocampus 39, lateral geniculate nucleus 36 and lateral hypothalamus 6'4°. The results of these studies suggest that the synaptic release of endogenous N E may facilitate the transfer of information through central circuits by enhancing excitatory and inhibitory components of cellular responses to nonmonoamine synaptic inputs. In the present study, we have used the same experimental strategy employed in previous investigations of noradrenergic influences on putative transmitter action to examine the effects of exogenously administered cocaine on G A B A - i n d u c e d depressant responses of cerebellar Purkinje cells. The results indicate that parenterally and microiontophoretically applied cocaine can potentiate cerebellar neuronal responses to G A B A in a manner identical to that observed with NE. Such facilitating

Correspondence: B.D. Waterhouse, Department of Physiology and Biophysics, Mail Stop 409, Hahnemann University, Broad and Vine, Philadelphia, PA 19102, U.S.A.

298 effects o n G A B A - i n d u c e d inhibition w e r e not o b s e r v e d in n e u r o n s r e c o r d e d f r o m animals p r e t r e a t e d with the n o r a d r e n e r g i c - s p e c i f i c toxin D S P - 4 . A p r e l i m i n a r y r e p o r t o f t h e s e findings has b e e n p r e s e n t e d p r e v i o u s l y 47.

MATERIALS AND METHODS The extracellular activity of individual cerebeilar Purkinje neurons was recorded from 35 female Long-Evans hooded rats, weight 200-300 g. Five of these animals were pretreated with the selective22 noradrenergic neurotoxin DSP-4 (CIBA-Geigy) according to the protocol outlined by Dooley et ai. 9. Surgical and electrophysiological recording procedures were the same for all animals.

DSP-4 treatment Ten animals were pretreated with CGP 6085 A (CIBA-Geigy) (2.7 mg/kg, i.p.), an inhibitor of 5-hydroxytryptamine uptake 43 to prevent DSP-4 toxicity toward serotonergic neurons. Thirty minutes later these animals received intraperitoneal injections of either NaCI vehicle alone (control) or DSP-4 (63 mg/kg, i.p.). The total volume of injections was maintained at 1.0 mi. Control and DSP-4 rats were then used in electrophysiology experiments, 10-12 days posttreatment. Dooley et al. 9 have reported substantial reductions of NE (<25% of control NE) in neocortex, hippocampus, cerebellum and spinal cord at 10 days following this treatment protocol. Following each DSP-4 experiment, the cerebellum was removed, placed in a preweighed vial and stored at -70 °C for later analysis of NE content by high pressure liquid chromatography (HPLC).

Surgical procedure Rats were initially anesthetized with halothane, intubated and allowed to breathe a mixture (0.5-0.75%) of halothane in oxygen for the duration of the experiment. Body temperature was monitored by a rectal probe and maintained between 36 and 37 *C with a heat lamp. An indwelling intraperitoneal catheter made of silastic (Dow Coming) with an inside diameter of 0.02 in. and a measured volume of 0.1-0.15 ml was inserted in the abdomen and secured with wound clips. Three animals were also implanted with femoral artery catheters to measure systolic blood pressure. Finally, the skull and dura over the posterior vermis of the cerebellum were removed and the exposed brain was covered with 2% agar in balanced saline solution.

Parenteral administration of cocaine Intraperitoneal injections of cocaine HCI (Merck & Co., Inc.) over a range of doses between 0.12 and 35.0 mg/kg were given in a NaC! vehicle at a volume of 0.2-0.8 ml. Each drug injection was preceded and followed by 0.3 ml of NaCl solution to determine control responses and ensure full delivery of drug, respectively.

maintained through a fourth side barrel filled with 3 M NaCI~ Action potentials of individual neurons were monitored on an oscilloscope and converted to standard digital pulses by a window discriminator. These pulses were integrated over 1-s intervals and displayed as a continuous ratemeter record. The window discriminator output was also led to a digital computer (S/20 Microeclipse, Data General) which summed unit activity in the form of perievent histograms. Such histograms were collected before, during and after parenteral or microiontophoretic administration of cocaine HCI. All of the results described in the present report were obtained from cells which were positively identified as Purkinje neurons by the presence of spontaneously occurring 'complex' spikes ~°. However, no attempt was made to determine the effect of cocaine on 'complex' spike activity. Once a single neuron was isolated, iontophoretic pulses (10 s duration) of GABA were applied at regularly spaced intervals (40 s). Microiontophoresis of GABA produced consistent reductions in Purkinje cell spontaneous 'simple' spike 8 discharge. Such inhibitory responses were quantitated by comparing the firing rate during GABA administration to the average discharge rate between drug pulses and by expressing the difference as a percentage of suppression of baseline firing frequency, accordingly. After cocaine injection or cessation of cocaine iontophoresis successive histograms were computed until recovery of the control pattern of response was observed. As described previously3°'46 differential effects of drug application on GABA-mediated inhibitory responses and background discharge of Purkinje neurons were assessed by comparing firing rates during identical response (R) epochs and periods of spontaneous activity (SA) in control (R1, SA1) and drug (R2, SA2) histograms. The response period was selected to begin at the particular bin in which counts deviated markedly from baseline and terminated at bins in which counts reapproaehed the baseline. To facilitate comparisons between histograms, equal numbers of GABA applications were used for each. This quantitative approach allowed the data to be more accurately evaluated in scatter diagrams (see Fig. 3). In addition, Student's t-tests were performed to assess the statistical significance of effects produced by cocaine 19. The following operational definitions were employed as before ~' 46 to describe the influence of cocaine on GABA-mediated responses. The descriptive term 'augmentation' (synonyms: facilitation, potentiation or enhancement) was used to denote cases in which the neuronal firing rate during GABA-induced inhibition was depressed at least 15% more by cocaine administration than was the baseline rate of background firing. Conversely, interactions were defined as 'antagonistic' when the background rate of firing was reduced proportionately more by cocaine application than was activity during GABA inhibition. Cocaine was assumed to have no significant effect when activity during periods of GABA-induced inhibition and spontaneous discharge were suppressed to an equivalent extent. This condition is represented by the dotted 45° equivalence line as shown in the graphs of Fig. 3.

Local application of cocaine Cocaine HCI, 1 mg/ml 0.003 M adjusted to pH 4.5, was directly applied to cells through multibarrel micropipettes by microiontophoresis (1-70 nA).

Data acquisition Five-barrel micropipettes, tip diameter 4-6 /~m, were used to record the spontaneous 'simple' spike discharge of Purkinje neurons 1° and to apply chemical substances at the recording site by microiontophoresis. The center barrel, filled with 4 M NaCl, was used for recording (3-5 MD impedance). Three side barrels, filled by capillary diffusion for 12-24 h, contained drug solution as follows: 0.25 M GABA, pH 3.8 (Sigma); and cocaine HCI 0.003 M, pH 4.8 (Merck & Co., Inc.). A crystal dock regulated timer (WPI-Digipuise 1800) controlled the iontophoresis unit (BH-2, Medical Systems) so that constant pulses of uniform duration and magnitude could be passed at equal intervals through the drug barrels (40-100 MK) impedance). Automatic current balancing was

RESULTS

Effects of systemic cocaine on G A B A responses and spontaneous discharge o f cerebellar Purkinje neurons T h e effects o f i n t r a p e r i t o n e a l l y a d m i n i s t e r e d c o c a i n e (0.12-35.0 spontaneous

mg/kg)

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w e r e e x a m i n e d in 25 cells r e c o r d e d f r o m 19 animals. In t h e m a j o r i t y o f cases (n = 14), a single d o s e of c o c a i n e was t e s t e d o n o n e n e u r o n p e r animal. T h e effects of increasing d o s a g e s of c o c a i n e w e r e s t u d i e d in 6 n e u r o n s and in 3 e x p e r i m e n t s m o r e t h a n o n e n e u r o n p e r rat was

299 studied. In those experiments where multiple units or cumulative doses of cocaine were tested, each recording session or subsequent dose increment was separated by 20 min or longer. We were unable to distinguish any differences in spontaneous firing rate or G A B A responsiveness (i.e. G A B A ejection currents necessary for evoking inhibition) in cells recorded from naive versus previously drug-tested animals. Slight (5%) to moderate (25-35%) suppression of Purkinje cell spontaneous firing rate was observed at all doses of cocaine studied; 0.12, 0.25, 0.5, 1.0, 2.0, 10.0 and 35.0 mg/kg. Overall, a depressant effect of the drug on spontaneous discharge was observed in 22 of 25 cases. At doses between 1.0 and 10.0 mg/kg, cocaine consistently (10 of 15 cases) enhanced Purkinje cell responses to G A B A in a manner similar to the example shown in Fig. 1. In this case a control injection of 0.9% saline (NaCI) produced little change in firing rate during the G A B A response epochs and periods of spontaneous

activity. However, within 2 min after a 1.0 mg/kg injection of cocaine, discharge rate during the period of amino acid-induced inhibition was decreased 69% from 32.55 (R1) to 9.98 spikes/s (R2) while spontaneous firing was only reduced 25% from 40.35 (SA1) to 30.16 (SA2) spikes/s. The net effect of this differential action of cocaine on GABA-mediated inhibition and spontaneous discharge was that the cell's response (R/SA) to amino acid application was augmented from control levels (19-67%). It is also important to note in Fig. 1 that potentiation of the G A B A response was evident prior (1-3 min postdrug injection) to the onset of the direct depressant effect of cocaine on spontaneous firing rate. Recovery to the control level of G A B A inhibitory response began around 13 rain postdrug injection and was complete by 16 min postinjection. Such net potentiating effects of cocaine on G A B A were not routinely observed at doses below 1.0 mg/kg. At doses above 10.0 mg/kg (see Fig. 2) cocaine produced a

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Fig. 1. Low dose cocaine augments Purkinje cell response to GABA. Continuous ratemeter (left) and corresponding perievent histogram (right) records illustrate the response of a Purkinje neuron to iontophoretic pulses of GABA 7 nA (solid bars) before and after i.p. injections of NaC1 and cocaine HCI (1.0 mg/kg). Each perievent histogram sums cell activity during 5 consecutive GABA administrations beginning with the last 5 before NaCI administration and then at 4 min post-NaCl and 2, 8 and 16 rain postcocaine injection. Quantitative anaiysis of these histograms indicated that GABA-induced depression of spontaneous discharge was minimally affected by NaCI, but was markedly enhanced over control levels within 2 min after cocaine administration, from 19 to 67% inhibition of firing rate. Gradual recovery to the control condition was observed over a period of 16 min postdrug injection.

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Fig. 2. High dose cocaine antagonizes Purkinje cell responses to GABA. Continuous ratemeter (left) and corresponding perievent histograms (right) show the response of a single Purkinje cell to iontophoretic pulses of GABA 40 nA (solid bars) before and after intraperitoneal injection of cocaine hydrochioride (35 mg/kg). Each perievent histogram sums cell activity during 5 GABA applications. Quantitative analysis of these histograms revealed that in comparison to control conditions G A B A was less effective in suppressing Purkinje cell spontaneous firing rate following cocaine injection. For example, during the first 7 min after cocaine administration discharge rate during the GABA responSe epoch was increased 23% from 15.82 (R1) to 19.45 (R2) spikes/s, whereas spontaneous firing rate was decreased 16% from 38,37 (SA1)to 32.05 (SA2) spikes/s; thus yielding a reduction in the magnitude of the GABA inhibitory response (R/SA) from 59% to 39%. This antagonizing effect of cocaine on GABA responses persisted for 28 min postinjection, after which time a gradual return to the control level of GABA inhibition was observed.

net antagonism of G A B A inhibition in all but one case tested (n = 5). The graphs presented in Fig. 3A and B. contrast the effects of parenterally administered cocaine on G A B A induced inhibition and spontaneous firing rate for all cells tested. For purposes of correlating the present findings with data from previous studies, the experiments were grouped in two categories; those where cells were tested with doses (10-35 mg/kg, i.p.) of cocaine (Fig. 3B) known to produce alerting effects and increased locomotion 38 in laboratory animals, i.e. 'high' doses; and those where the dose of the drug was in the range of 0.12-2.0 mg/kg, i.p., i.e. 'low' doses. Points lying well above the 45 ° equivalence line indicate cases where the

rate of firing during the G A B A r e s p o n s e was depressed to a greater extent following cocaine administration than was the rate of spontaneous discharge. Data points plotted close to or below the equivalence line indicate cells where the efficacy of G A B A inhibition was unchanged or reduced by cocaine relative to changes in spontaneous firing rate. Overall, this analysis indicated (see also Table I) that augmentation of G A B A inhibition of Purkinje cell firing was most consistently observed following systemic administration of cocaine at doses between 1.0 and 10.0 mg/kg (i.p.). This drug4nduced enhancement of G A B A efficacy relative to changes in spontaneous discharge was statistically significant (paired t-test, t = 4.695, P<0.004, df = 13) for the entire sample

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% Suppression of Spontaneous Discharge Fig. 3. Comparison of the effects of systemically and locally administered cocaine on spontaneous discharge and G A B A inhibitory responses of cerebellar Purkinje cells recorded from normal and NE-depleted rats. Shown here are summaries of results from individual experiments where varying routes of administration and doses of cocaine were interacted with Purkinje neuron activity. Each symbol (filled and open circles) represents data obtained from one Purkinje cell and compares the effect of cocaine on discharge frequency during periods of spontaneous activity and G A B A application. The dotted 45° lines represent the prediction of an equivalent depressant effect of cocaine on spontaneous discharge and firing rate during the GABA-induced response. Data points above the dotted 45° line indicate cases where G A B A inhibition was augmented relative to changes in background firing. Those points lying below the dotted line represent cells where the G A B A response was antagonized relative to changes in spontaneous discharge.

302 TABLE I Net effect o f cocaine on Purkinje cell responsiveness to GA BA * Cocaine (mg/kg)

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Additional independent analyses of cocaine's effects on spontaneous discharge and G A B A - i n d u c e d inhibition further defined the relationship between drug dose and enhancement of G A B A efficacy and distinguished this effect from cocaine's direct depressant effects. The

graphs in Figs. 4 and 5 illustrate the individual and averaged percent changes in spontaneous firing and G A B A - i n d u c e d inhibition that were observed following intraperitoneal injection of saline or cocaine at doses ranging from 0.12 to 35.0 mg/kg. Although variance in the magnitude of effect for individual cases was observed, the trend toward facilitation of G A B A - m e d i a t e d inhibition of firing rate was most apparent at cocaine doses between 0.5 and 10.0 mg/kg whereas higher doses of the

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Fig. 5. Dose-response relationship of cocaine effects on OABA-induced suppression of Purkinje cell firing. Plotted here are the percentage changes in Purkinje cell responses to GABA followingintraperitoneal injections of 0.9% saline (NaCI) and varying doses of cocaine HCI. Each point (filled circle) represents quantitative analysis of data from a single experiment where Purkinje cell activity was monitored before and after drug administration. Asterisks connected by the dashed line indicate the average cell response at each drug dose tested. Dotted horizontal line represents the control response to GABA prior to cocaine or NaCI injection. drug tended to antagonize G A B A responses (see Fig. 5). By contrast, cocaine exerted a predominantly depressant effect on spontaneous firing rate (see Fig. 4) which increased in magnitude with increasing drug dose.

Time course of cocaine's effects The latency and duration of cocaine's effect on GABA-induced inhibition was determined from 13 cases where Purkinje neuron responsiveness to G A B A was assessed by analysis of perievent histograms collected every 3-4 min for a period of up to 80 min postdrug injection. The graphs in Fig. 6A, B illustrate cocaineinduced changes in neuronal responsiveness to iontophoretic pulses of G A B A as a function of time. Drug effects observed at low (0.25-2.0 mg/kg) doses were generally rapid in onset (<1 min), peaked between 3 and 12 min postdrug injection and diminished back to control levels by 15-20 rain postinjection. At higher doses (10-35 mg/kg), the onset of cocaine's effect was just as rapid, but the duration of effect was longer in proportion (i.e. 30-45 rain) to the increased dosage. A similar time course of

action was noted for cocaine's direct depressant effect on background firing (data not shown),

Effect of iontophoretic cocaine on GABA-induced inhibition Since any effects observed following systemically administered cocaine can result from actions at sites afferent to recorded neurons, a series of experiments were conducted in which direct iontophoretic application of cocaine was interacted with cerebellar Purkinje neuron responses to GABA microiontophoresis. As with parenteral administration, iontophoretic cocaine (5-60 hA) was capable of suppressing the spontaneous firing rate of all Purkinje cells tested (n = 16). Cocaine was also capable of producing a mild-tomoderate enhancement of GABA-induced inhibitory responses relative to direct depression or no change of background firing in 10 of 16 cases tested (see Table I). In the example shown in Fig. 7 cocaine augmented the cell's inhibitory response to G A B A while causing little suppression of spontaneous discharge. In 6 remaining

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cases, cocaine produced roughly equivalent changes (less than 15% difference) in cell firing rate during G A B A application and spontaneous firing yielding no enhancement or in one case a net antagonism of the G A B A inhibitory response. A graphic summary of the effects of iontophoretic cocaine on GABA-induced responses versus spontaneous discharge for all cells tested is shown in Fig. 3C. The net facilitating effect of iontophoretic cocaine on Purkinje cell responsiveness to G A B A was statistically significant (paired t-test, t = 4.263, P<0.0007, df = 14) for the entire sample of cells tested. Cocaine actions in DSP-4-treated animals

The effects of iontophoreticaily or systemically administered cocaine on G A B A - i n d u c e d responses and spontaneous discharge were further evaluated in 11 Purkinje neurons recorded from 3 DSP-4-treated animals. High performance liquid chromatography (HPLC) determina-

tion of cerebellar monoamine content revealed a 71.5% reduction of norepinephrine and a 20,0% reduction of serotonin in these animals relative to weight-matched controls (see Table II). In 4 cells recorded from two DSP-4-treated animals, parenteral cocaine (1.0-10 mg/ kg, i.p.) was ineffective in augmenting GABA-induced suppression of activity despite producing a direct depressant effect on spontaneous firing rate. The records shown in Fig. 8A illustrate one such case where net responses to G A B A were not enhanced following cocaine injection (2.0 mg/kg i.p.) despite a mildly depressant effect (-12.3%) on spontaneous firing, from 19.86 spikes/s before drug to 17.41 spikes/s after drug. Quantitative analysis of histogram records for this cell indicated that iontophoretic pulses of G A B A produced a 21% suppression of spontaneous discharge d u r i n g t h e predrug control period, 23% inhibition following NaCI administration and 24, 19 and 18% suppression of firing at 0--4, 5-8 and 9-12 min postcocaine injection. A subsequent injection

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Fig. 7. Effects of iontophoreticaily applied cocaine on Purkinje neuron responses to GABA. Continuous ratemeter (top) and corresponding perievent histograms (bottom) show the responses of a single Purkinje cell to iontophoretic pulses of GABA 7 nA (solid bars) before, during and after continuous microiontophoresis of cocaine HCI 1 nA (broken bar). Each histogram sums unit activity during 5 consecutive GABA applications. Solid lines and numbers above histograms indicate the duration of the GABA ejection pulse and percentage decrease in spontaneous discharge induced by GABA application, respectively. During cocaine iontophoresis, background firing rate remained constant while activity during the GABA response period was reduced, thus yielding an enhancement (from 45% to 56%) of GABA's inhibitory action. Recovery to the control pattern of response was observed following termination of the cocaine ejection current.

of cocaine at 10 mg/kg i.p. also failed to enhance responses of this n e u r o n to G A B A application (data not shown). I o n t o p h o r e t i c a l l y a p p l i e d cocaine was also ineffective in a u g m e n t i n g Purkinje cell responses to G A B A in each of 7 cells tested in D S P - 4 - t r e a t e d animals. F o r the cell shown in Fig. 8B i o n t o p h o r e t i c pulses of G A B A prod u c e d a 25% suppression of spontaneous discharge during the control p e r i o d , 17, 18 and 20% suppression during cocaine iontophoresis at 10, 20 and 30 n A

respectively a n d 20% suppression after t e r m i n a t i o n of the cocaine ejection current. F u r t h e r m o r e , direct inhibitory actions of i o n t o p h o r e t i c cocaine on s p o n t a n e o u s firing were w e a k e r in D S P - 4 - t r e a t e d animals relative to controls as evidenced by the generally higher (20-70 n A ) ejection currents r e q u i r e d to depress P u r k i n j e cell discharge. The graph in Fig. 3D summarizes the effects of systemic and i o n t o p h o r e t i c cocaine on G A B A responsiveness and s p o n t a n e o u s firing rate in cells r e c o r d e d

TABLE II Cerebellar monoamine levels (ng/mg protein) in control and DSP-4-treated rats

NE, norepinephrine; 5-HT, serotonin; C, control case; X, experimental case. Individual cases

NE Control DSP-4-treated 5-HT Control DSP-4-treated

Mean

% Depletion

C 1 = 2.8 X1 = 0.7

C2 = 2.6 X: = 0.8

Ca = 2.9 X 3 = 0.9

2.8 + 0.12 0.8 + 0.08

71.5%

C I = 1.8 XI = 1.1

C2 = 1.2 X 2 = 1.2

1.6

-

1.5 _+0.25 1.2 + 0.05

20.0%

C 3 =

306

DSP4 All

NaCI .3ml ip

Cocaine 2mg/kg ip

w

20

a

nun

am

an

n

in

; BII

in

oi

aln

m

10

12

Cocaine 10nA GABA 38nA

an

nm

ill

am

14

mm

m

ian

mm

1(~ rain

20nA

I I I I I I I I I l I l l l l 1OllllllllllllllllllIIIllllll IIIlIlIIIIOlllll I l l l l l I I I l I I I l I l I I I I l l l l l l l l l l l l I I

30nA ~ooel ioaoloo oaonlooOlllOlllllallgll

l i l l l l g l O Ilnalu

6 0 sec Fig. 8. Cocaine actions in NE-depleted animals. Ratemeter records illustrate the effects of systemically (A) and iontophoretically (B) applied cocaine on spontaneous firing rate and GABA-induced inhibitory responses of two Purkinje neurons recorded from animals pretreat¢d with the noradrenergic neurotoxin, DSP-4. In each case cell responses to iontophoretic pulses of GABA (solid bars) were monitored before, during and after cocaine administration. In A cocaine injection (2 mg/kg, i,p.) was preceded by a volumetric equivalent control injection (Lp.) of 0.9% saline (NaCI). In B cocaine was ejected from the pipette with currents of 10, 20 and 30 nA. GABA responsiveness was not significantly affected by either route of cocaine administration.

from D S P - 4 - t r e a t e d animals. In contrast to similar e x p e r i m e n t s c o n d u c t e d in control animals (Fig. 3 A - C ) , there was no evidence of a cocaine-induced selective e n h a n c e m e n t of G A B A inhibition in these N E - d e p l e t e d rats ( p a i r e d t-test, t = 0.697, P < 0 . 5 0 2 , df = 9). DISCUSSION T h e p r i m a r y goal of the present study was to investi-

gate the possibility that cocaine could mimic previously o b s e r v e d 3° facilitating actions of N E on G A B A - m e d i a t e d inhibitory responses of Purkinje neurons. Such an outcome might be expected based on the results of b i o c h e m ical studies 17"18 which indicate that a m a j o r net effect o f cocaine within the CNS is to elevate synaptic levels of endogenously r e l e a s e d N E . The b r o a d e r question raised by these studies is w h e t h e r certain of the physiological actions of cocaine can account for or contribute to the

307 behavioral effects of the drug. Many studies have linked cocaine's actions at dopaminergic synapses to the behavioral reinforcing and addictive properties of the drug 49, however few studies to date have focused on the potential for cocaine to interact with central noradrenergic synapses and bring about other cocaine-related effects. While no direct evidence links the cerebellum with cocaine-induced behaviors, the cerebellar Purkinje neuron provides considerable advantages as a test system for examining cocaine actions because of its well established synaptic connections 1°, the absence of a substantial dopaminergic innervation 42, and the well-characterized nature of NE's actions at this synaptic site 4'28-31. The results of the present study indicate that both systemically and locally applied cocaine can augment cerebellar Purkinje cell responses to G A B A in a manner identical to that observed during iontophoretic application 3° or synaptic release 29 of norepinephrine. Moreover, these effects are weak or absent in animals where NE has been partially depleted by the selective neurotoxin DSP-4.

Electrophysiological effects of cocaine In the present study, two actions of cocaine were noted following either systemic or local application of the drug. At doses between 1.0 to 10.0 mg/kg i.p. cocaine enhanced inhibitory neuronal responses to GABA. Depressant effects on background firing were also observed at these doses as well as at doses up to 35 mg/kg. Neither of these actions was consistently observed following injections of NaCI. Overall, the trend which emerged suggested that cocaine-induced augmentation of G A B A responsiveness was a lower threshold action of the drug relative to suppression of spontaneous firing. Indeed, in several cases cocaine administration caused marked enhancement of G A B A responses accompanied by little or no change in background firing rate. Similar direct depression of spontaneous discharge and modulation of G A B A responses were observed with iontophoretic administration of the drug at 10-30 nA. We were unable to confirm a previous report 33'35 of cocaine-induced activation of rat cerebellar Purkinje cells, despite the fact that only the anesthetic (halothane vs urethane) and route of drug administration (i.v. versus i.p.) were different between the two studies.

Site of cocaine action The changes in G A B A efficacy and spontaneous firing rate of Purkinje cells observed following systemic administration of cocaine could have resulted from drug interactions locally or at sites afferent to the cerebellum. For example, Pitts and M a r w a h 32'34 have demonstrated a consistent depressant effect of cocaine (0.0625-2.0 mg/kg i.v.) on locus coeruleus neurons which are the major

source of noradrenergic input to Purkinje cells. Likewise, Cunningham and Lakoski have shown that cocaine (0.25-2.0 mg/kg i.v.) can suppress the activity of serotonin-containing dorsal raphe neurons, an effect which may reasonably extend to other brainstem raphe neurons that provide the serotonergic innervation of the cerebellar cortex3. However, in the present study augmentation of GABA-mediated inhibition and suppression of Purkinje cell spontaneous discharge were not only observed after systemic administration of the drug, but also during direct iontophoretic application of cocaine. This result provides strong evidence that at least a component of cocaine's effects in the cerebellar cortex occur as a result of interactions with local neuronal processes.

Mode of cocaine action A further issue for consideration is the means through which cocaine produced changes in Purkinje cell responsiveness to G A B A and spontaneous discharge. One likely possibility is that these effects were indirectly mediated by increased synaptic levels of endogenous NE, a condition which could result from cocaine blockade of monamine reuptake 17'18. In previous studies, results similar to those observed here were obtained following electrical stimulation of the coeruleo-cerebellar pathway 21'29 or during iontophoretic administration of NE 2°' 3o,31. Since the cerebeUar cortex lacks substantial dopaminergic innervation 42 and since iontophoretic dopamine does not augment Purkinje neuron responsiveness to G A B A and has only weak depressant effects on Purkinje cell spontaneous discharge 3°, it is unlikely that cocaineinduced increases in synaptic levels of this monoamine could underlie the locally mediated effects reported here. Furthermore, the results of experiments in NE-depleted animals indicate a reduced capacity for cocaine to enhance G A B A action. Despite these arguments the possibility must still be considered that some aspects of cocaine's action on Purkinje neurons may be due to its ability to block the reuptake of locally released serotonin 37. There have been reports that iontophoretically applied serotonin can directly inhibit Purkinje cell firing24 as well as enhance GABA-induced suppression of Purkinje cell discharge 41. Moreover, although cocaine did not augment Purkinje cell responses to G A B A in DSP-4-treated animals, it was still capable of inhibiting Purkinje cell spontaneous discharge. This residual effect of cocaine on spontaneous firing might also be explained by an action of cocaine or a metabolite on non-monoamine presynaptic terminals or Purkinje cell membranes. The local anesthetic action of cocaine could, for example, cause a reduction in membrane excitability and depress spontaneous cell activity. Despite these additional possibilities, the findings re-

308 p o r t e d here do support the contention that cocaineinduced e n h a n c e m e n t of G A B A - e f f i c a c y is d e p e n d e n t upon release of e n d o g e n o u s NE.

stration of N E ' s ability to facilitate inhibitory and excitatory synaptic target neuronal circuits such as the and cerebellum 16'3° has been central

Behavioral correlates o f cocaine's electrophysiological actions A m p h e t a m i n e which produces a set of behavioral actions similar to cocaine's 11'12 and likewise shares

of this hypothesis. Accordingly, under a p p r o p r i a t e behavioral conditions, synaptically released N E may prime cells for optimal responsiveness to n o n - m o n o a m i n e synaptic inputs; thus improving their signal integrating properties. R e c e n t studies 44 have shown that locally applied N E can alter receptive field p r o p e r t i e s of primary sensory neurons through e n h a n c e m e n t of stimuluse v o k e d inhibition. B a s e d on the results of the present study cocaine a p p e a r s capable of activating such intrinsic physiological mechanisms and because of this ability may generate conditions like those o b s e r v e d during behavioral arousal where e n h a n c e d sensivity to sensory stimuli is a characteristic feature.

cocaine's ability to increase synaptic levels of N E , is also capable of producing noradrenergic-like facilitating actions on P u r k i n j e cell responses to iontophoretically a p p l i e d G A B A in both anesthetized 26 and awake, behaving 27 rats. T h e only differences n o t e d b e t w e e n the two drugs in these electrophysiological assays is the longer-lasting action of a m p h e t a m i n e which m a y be a reflection of its longer half-life in brain tissue 13'48. In addition to its behavior-reinforcing actions, cocaine is well known for its alerting effects and its ability to increase sensitivity to sensory stimuli 7. Within this context it should be n o t e d that the most current view (see refs., 1,14 for reviews) of the ascending n o r a d r e n e r g i c system's role in brain function is to maintain states of arousal o r vigilance as well as participate in orienting responses to novel environmental stimuli. The d e m o n REFERENCES 1 Aston-Jones, G., Behavioral functions of locus coeruleus derived from cellular attributes, Physiol. Psychol., 13 (1985) 118-126. 2 Azzaro, A.J., Ziance, R.J. and Rutledge, C.O., The importance of neuronal uptake of amines for amphetamine-induced release of 3H-norepinephrine from isolated brain tissue, J. Pharm. Exp. Ther., 189 (1974) 110-118. 3 Bishop, G.A. and Ho, R.H., The distribution and origin of serotonin immunoreaetivity in the rat cerebellum, Brain Research, 331 (1985) 195-207. 4 Bloom, EE., Holler, B.J. and Siggins, G.R., Norepinephrine mediated synapses: a model system for neuropharmacology, Biol. Psychiat., 4 (1972) 157-177. 5 Bozarth, M.A. and Wise, R.A., Toxicity associated with long term intravenous heroin and cocaine self-administration in the rat, J. Am. Med. Ass., 254 (1985) 81-83. 6 Cheng, J.-T., Sessler, EM., Azizi, S.A., Chapin, J.K. and Waterhouse, B.D., Electrophysiological actions of norepinephrine in rat lateral hypothalamus: II. An in vitro study of the effects of iontophoretically applied norepinephrine on LH neuronal responses to y-amino butyric acid (GABA), Brain Research, 446 (1988) 90-105. 7 Cohen, S., Cocaine: acute medical and psychiatric complications, Psychiat. Ann. 14 (1984) 747-749. 8 Cunningham, R.A. and Lakoski, J.M., The interactions of cocaine with serotonin dorsal raphe neurons: single unit extracellular recording studies, Neuropsychopharmacology, 3 (1990) 41-50. 9 Dooley, D.J., Bihiger, H., Hauser, K.L., Bischoff, S.E and Waldheimer, P.C., Alteration of central alpha 2- and betaadrenergie receptors in the rat after DSP4, a selective noradrenergic neurotoxin, Neuroscience, 9 (1983) 889-898. 10 Eccles, J.C., Ito, M. and Szentagothai, J., The cerebellum as a neuronal machine, Springer, New York, 1967, p.335. 11 Ellinwood, E.H. and Kilbey, M.M. (Eds.), Cocaine and Other

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