Effects of microiontophoretically-applied morphine on the purkinje cell in the cerebellum of the cat

Neuropharmacology Vol. 28, No. 3, pp. 235-242, 1989 Printed in Great Britain

0028~3908/89 $3.00 + 0.00 Pergamon Press plc

EFFECTS OF MICROIONTOPHORETICALLY-APPLIED MORPHINE ON THE PURKINJE CELL IN THE CEREBELLUM OF THE CAT K. Department

of Pharmacology,

TACUCHI

and Y.

SUZUKI

Showa College of Pharmaceutical Tokyo 154, Japan

Sciences, 5-I-8, Tsurumaki,

Setagaya-ku,

(Accepted 17 August 1988) Summary-The effects of microiontophoretically-applied and pneumatically-applied morphine on the spontaneous discharge of Purkinje cells in the cerebellum of the anesthetized cat $ere examined. Microiontophoretic application of morphine produced both inhibitory and excitatory responses of the Purkinje cells. Pneumatic application of morphine produced similar effects to those of microiontophoresis. Both types of application of morphine induced dose-dependent responses. Excitatory responses were antagonized by naloxone (opiate antagonist), but inhibitory responses were not affected by naloxone, propranolol (beta-receptor antagonist) or methysergide (serotonin antagonist). Bicuculline and picrotoxin, GABA antagonists, abolished completely the morphine-induced inhibitory response. These results suggest that morphine-induced excitation is connected with the opiate system and that inhibition is related to the GABAergic system, in the cerebellum of the cat. Key words-morphine,

maloxone,

GABA,

microiontophoresis,

The functions of the periphery are influenced by the cerebellum. This area has a significant role in the modulation of physiological functions, as a regulator of the motor system and also as a coordinator of the autonomic system. It has previously been reported that the cerebellum plays an important role, not only in the motor system, but also in relation to the sensation of pain (Suzuki and Taguchi, 1983a). The existence of multiple opiate receptors in the central nervous system (CNS) is well known (Martin, Eades, Thompson, Huppler and Gilbert, 1976; Lord, Waterfield, Hughes and Kosterlitz, 1977; Wood, 1982). Concerning the cerebellum, there have been many studies on opiate binding sites in the mammalian brain. Cerebellum membrane preparations of rabbit and guinea-pig have been found to contain large proportions of mu- and kappa-opioid binding sites, respectively (Meunier, Kouahou, Puget and Moisand, 1983; Itzhak, Hiller and Simon, 1984; Frances, Moisand and Meunier, 1985). Ho, IHammonds and Li (1985) reported that fi-endorphin displaced 88% and 73% of tritiated diprenorphine from cerebellum and brain membranes respectively, suggesting multiple sites for the binding of I?-endorphin. Other reports demonstrated that the cerebellum has a low level of enkephalin and density of stereospecific opiate binding sites (Kuhar, Pert and Snyder, 1973; Simantov, Kuhar, Pasternak and Snyder, 1976). Sar, Stumpf, Miller, Chang and Cuatrecasas (1978) observed cell bodies in the cerebellum of the rat which contained enkephalin-like immunoreactivity. They identified these cells as Golgi type II cells. In the cat, most of the enkephalin-like immunoreactivity cells lie in the outer one-third of

Purkinje

cell, cerebellum.

the granular layer, giving the appearance of a thin, regular layer close to the Purkinje cell layer. Thus, enkephalin may play a role in afferent and interneuronal cerebellar synaptic communication (Schulman, Finger, Brecha and Kaarten, 1981). From these findings, it may be suggested that endogenous opioid peptides have a role in physiological functions of the cerebellum. Many studies have reported that the actions of morphine in the CNS can lead to changes in the neurotransmitters such as serotonin (5-HT), norepinephrine (NE), gamma-aminobutyric acid (GABA) and opioid peptides, with respect to behaviour, analgesia, sedation and tolerance (Snelgar and Vogt, 1981; Sivam and Ho, 1983; Tanaka, Kohno, Tsuda, Nakagawa, Ida, Iimori, Hoaki and Nagasaki, 1983; Depaulis, Morgan and Liebeskind, 1987). Moreover, there have been many neuropharmacological studies of the effects of morphine in the CNS (Peieto-Gomez, Reyes-Vazquez and Dafny, 1984; Chaing and Pan, 1985; Frey and Huffman, 1985). In the cerebellum, it has been demonstrated that S-HT, NE, GABA and opioid peptides are involved in the regulation of the activities of the Purkinje cell (Nicoll, Siggins, Ling, Bloom and Guillemin, 1977; Waterhouse, Moises, Yeh and Woodward, 1982; Strahlendorf, Lee and Strahlendorf, 1984). The Purkinje cell of the cerebellum in rat has been observed to be inhibited or increased by iontophoretic administration of normorphine (Nicoll et al., 1977). Previously, this laboratory has observed excitatory and inhibitory effects of systemically-applied morphine on the spontaneous discharge of Purkinje cells (Suzuki and Taguchi, 1983b). However, the role 235

236

K.

TAGUCHI

of microiontophoretic administration of morphine on the serotonergic, noradrenergic or GABAergic systems in the spontaneous discharge of Purkinje cells has not been examined. The purpose of the experiments reported here was to investigate the effects of microiontophoreticallyapplied morphine on the Purkinje cell of the cerebellum of the cat. The present data shows that morphine, released iontophoretically onto cerebellar Purkinje cells, produced both excitatory and inhibitory effects. A mode of inhibition of Purkinje cells by morphine was antagonized by picrotoxin and bicucuiline, while excitation of the Purkinje cells was antagonized by naloxone. METHODS

Experiments were performed on 53 cats of both sexes, weighing 2.3-3.6 kg. Under ketamine (20 mg/kg, i.m.)-induced anesthesia, a tracheotomy was performed. The femoral vein and artery were cannulated and then the animal was placed in the stereotaxic instrument. Under further anesthesia with cr-chloralose (60mg!kg, iv.), lobules VI-VII of the vermis were exposed by craniotomy. The wound margin and pressure points were treated locally by infiltration of 8% xylocaine spray. The exposed tissues were immersed in paraffin liquid and the temperature of the paraffin pool was maintained at 37-38°C. The animal was paralyzed with pancuronium bromide (Mioblock; initial dose 20 mg/kg, iv.) and was artificially respired. Physiological conditions were maintained by monitoring the blood pressure and end-tidal CO, (3.545%). Body temperature was also maintained at 37-38’C by means of a heating pad. Five-barrelled glass micropipettes were used. The tip of the micropipette was broken back to a diameter of 5-12 pm, under microscopic control. The central barrel, used for the recording electrode, was filled with a 3 M-NaCl solution, dissolved with 10% fast green dye, to mark the recording sites (resistance 20-40 M). In the five-barrelled micropipettes, three of the four side barrels were filled with the following drugs: 0.1 M DL-noradrenaline hydrochloride, pH 4.5 (Nakarai Chem.), 0.5 M gamma-aminobutyric acid (GABA), pH 4.5 (Tokyo Chem. Ind.), 0.1 M morphine hydrochloride, pH 4.5 (Sankyo), 0.1 M naloxone hydrochloride, pH 4.5 (Endo Laboratories Inc.), 50mM bicuculline methobromide, pH 4.0 (Sigma), 20 mM picrotoxin, pH 4.0 (Sigma), 50 mM methysergide hydrogenmaleate, pH 4.0 (Sandoz) or 0.5 M DL-propranolol hydrochloride, pH 4.5 (Sigma). The remaining side barrel, the balance barrel, was filled with a solution of 2 M-NaCl. The substances were applied from the micropipettes adjacent to the recording site by microiontophoretic application or through pressure ejection. Microiontophoretic application was performed using a constant-current pump (Iontophoresis Pump Neuro Phore BH-2, Medical

SUZUKI

and Y.

System Co.); the retaining current was l&12 nA. All substances were injected as cations. The pressure was applied through tubing fixed to each drug barrel of the micropipette and the magnitude of pressure delivered was controlled by a pneumatic pump (Medical System Co.). Single unit spontaneous activity of Purkinje cells was recorded from the vermis (lobules VI-VII) in the cerebellar cortex. Extracellular recording was performed through the electrode, which was connected to a preamplifier and the spike potentials of the Purkinje cells were measured by means of a window discriminator. Electrical activity was displayed on a medical oscilloscope with an audiomonitor. A signal processor (Model 7T07, Nihondenki San-ei Instrument Co., Ltd) was used for compiling the data in the form of pulse density variation histograms. The spontaneous activity of each cell was monitored in the first instance for l&l5 min to ensure a stable baseline before recording began. The effects of the previous drug on the spontaneous discharge were observed after ejection for 60 sec. The next drug was ejected l-3 min after recovery of the responses. The actions of morphine were scored when the spontaneous activity changed the pre-drug firing rate by at least 30%. Antagonistic actions to the morphineinduced response was scored as a significant action when the response was reduced at least 50% of control values. After the recording, a negative current of 20 nA was applied for IO-l.5 min through the central barrel, filled with fast green dye. The cerebellum was removed and fixed in 10% formalin. Several days later, the cerebellum was cut with a freezing-microtome at 50 pm sections to determine the recording site. Statistical analysis was performed using the Student’s t-test for significant differences. All quantitative measurements were calculated, taking account of mean & standard error. RESULTS 1. Identtjication and characterization of the Purkinje ceil

The spontaneous discharge of the Purkinje cell consisted of simple and complex spikes (Eccles, Ito and Szendgothai, 1967) and displayed a regular firing rate (20-50 spikejsec) with an average firing

Table

1. Neuronal responses pneumat~~lly-applied

Ejection currents or pressure 50” 100” ;:

to microiontophoreticalfy-applied morphine on Purkinje cells No. of neurons morphine-induced

Total no. of neurons 20 48 15 14

Excitation

Inhibition

5 (25) 13 (27) 2~3) 3(21)

7 (35) 17 (35) 5 (33) 6 (43)

The numbers in parentheses represent nMicroiontophoresis currents (aA). bPressures (psi).

or

exhibiting responses

the percentages

No effect 8 (4) 18 (38) 8 (54) J (36) of neuroos.

Morphine and the Purkinje rate of 35 f 13.5 spike/set. The complex spike was further identified through observation of the climbing fiber response elicited by stimulation of the inferior olive nucleus. The spontaneous discharge was inhibited (decrease of firing rate) by microiontophoretic application of NE (30 nA) and GABA (30 nA) which are regarded as inhibitory neurotransmitters of the Purkinje cell. The Purkinje cell was therefore identified through observation of these characteristic patterns of the spontaneous discharge. i! Effects of morphine on the spontaneous discharge of the Purkinje cell The responses of 68 Purkinje cells (50nA-20 units, 100 nAA8 units) to microiontophoretic application of two incremental currents (50 and 100nA) c’f morphine were investigated. Table 1 shows the neuronal responses to ejected morphine. Both excitatory responses (Fig. 1A) and inhibitory responses (Fig. 1B) were observed. The low level

GABA

(A)

MOR

Szl&

100

E3

2 min

60 -

40 -

20 -

o2 min

Fig. I. Effects of microiontophoretic application of morphine on the spontaneous discharge of a Purkinje cell in the ;,erebellum. (A) Frequency histogram of the firing rate for morphine (MOR)-induced excitatory response. Note that the effects of each dose of morphine (30, 50 and lOOnA) were a weaker inhibition, followed by a dose-dependent excitation of the spontaneous activity. (B) Frequency histogram of the firing rate for the morphine-induced inhibitory response. Note that these effects of morphine (30, 50 and 100 nA) resulted in a dose-dependent inhibition of the spontaneous activity of the Purkinje cell. In this and all subsequent Figures showing a frequency histogram, the duration of ejection of drug is indicated by the horizontal bars above each record; numbers directly above each bar refer to the microiontophoretic current in nanoamperes used for the ejection of drugs. The ordinate scale is the firing rate in spikes per second. The time bar is 2min.

cell

237

(50nA) application of morphine altered significantly the spontaneous discharge of 12 out of 20 (60%) Purkinje cells tested. Seven out of 12 cells showed inhibitory responses, but 5 out of 12 cells were excited (increase of firing rate) by morphine. The high level current (100 nA) application of morphine altered significantly the spontaneous discharge of 30 out of 48 (62%) Purkinje cells tested. Seventeen out of 30 Purkinje cells showed inhibitory responses, while 13 out of 30 cells were excited by morphine. The excitatory effects of 50 and 100 nA of morphine on the spontaneous discharge of Purkinje cells were observed in terms of average firing rates of 63.8 + 24.5 spike/set (n = 5) and 80.0 f 30.0 spike/set (n = 13) respectively. It is interesting to note that the morphine-induced excitatory response consisted of an initial weaker inhibition followed by excitation. Figure 1A is a typical record illustrating the dose-dependent excitation of the spontaneous discharge of the Purkinje cell by morphine applied at 30, 50 and 100nA current. This excitatory effect of morphine usually reached its maximum just before termination of the iontophoresis. The inhibitory effects of 50 and 100 nA of morphine were observed in terms of average firing rates of 26.6 f 7.5 spikes/set and 10.6 f 5.3 spikes/set, respectively. Figure 1B illustrates this dose-dependent inhibition of the spontaneous discharge of the Purkinje cell by morphine, applied at 50 and 100 nA current. In order to determine whether the microiontophoretic-induced responses were elicited directly by the substance concerned or by the electric current, the effects of pneumatically-applied morphine were also examined. Effects of pneumatically-applied morphine were studied in 29 Purkinje cells. The pressures used were 3 pounds per square inch (psi; 15 units) and 5 psi (14 units). Pneumatic application of the small dose of morphine significantly altered the spontaneous discharge of 7 out of 15 (46%) of the Purkinje cells tested: inhibition occurred in 5 out of 7, and excitation in 2 out of 7. The large dose of morphine significantly altered the spontaneous discharge of 9 out of 14 (64%) of the Purkinje cells tested; inhibition was observed in 6 out of 9 and excitation, in 3 out of 9 (see Table 1). In addition, incremental effects of pneumatically-applied morphine at both pressures were similar to those for microiontophoresis, showing a dose-dependent response. current

3. Antagonistic effects of naloxone induced response

on morphine-

To determine the specificity of the effects of morphine, with respect to mediation by the opiate system, naloxone was applied concurrently with morphine on the Purkinje cell. Naloxone (30 nA) antagonized morphine-induced excitation in all cells (8 units). On the other hand, the inhibitory effects of morphine (100 nA) on the Purkinje cells (8 units) were in no case antagonized by naloxone (Table 2). Figure 2

238

K.

TAGUCHI and

illustrates the antagonism by naloxone of the excitatory effects of morphine and shows that the inhibitory effects of morphine were not affected. Naloxone also had no effect on the Purkinje cells and the inhibitory response to GABA (30 nA) of the Purkinje cells was not affected by microiontophoretically-applied naloxone. Thus, it is suggested that the excitatory effect of morphine are perhaps mediated directly by the opiate system and that the inhibitory effect of morphine is mediated by another system. 4. Interaction antagonists

between

morphine

und

Table 2. Effects of microiontophoretically-applied naloxone, propranolol, methysergide, bicuculline and picrotoxin, on morphine (100 nA)-induced excitatory and inhibitory responses of Purkinje C&S

In order to test the relationship between the inhibitory effects of morphine and inhibitory interneurons (i.e. basket cells) the neurotransmitter of which is considered to be regarded as GABA, the authors studied the effects of the interaction between morphine and bicuculline or picrotoxin (GABA antagonists) on the spontaneous discharge of the Purkinje cells. Figure 3 illustrates the antagonism by bicucu~line of morphine-induced responses. Bicuculline (30 nA) antagonized the inhibitory effect of morphine (100 nA) in 5 out of 6 (83%) Purkinje cells and only one was excited by bicuculline. The excitatory effects of morphine (100 nA) on the Purkinje cells (5 units) were in no case antagonized by bicuculhne (Table 2). Picrotoxin (30 nA) antagonized morphine-induced inhibition in all cells (5 units); the excitatory effects of morphine on Purkinje cells (6 units) were not antagonized by picrotoxin (Fig. 4,

GABA

30

(A)

MOR 100

I

Naloxone Propranolol Methysergide Bicuculline Picrotoxin

8 4 6 5 5

Naloxone Propranolol Methysergide Bicuculline Picrotoxin

8 5 7 6 6

No. of neurons where mo~hine-indu~d excitatory response was affected Augmentation

Antagonism

0 0 0 l(17) 0

0 0 0 5(83) 61100)

5. interaction antagonists

between

morphine

and

a 3

(Bf 6Of-

r?arious

Purkinje cells receive two inhibitory inputs from the noradrenergic and serotonergic pathways. To

NALOXONE 30nA I

a

MOR 100 Es

8(10@ S(lO0) 71100) 0 0

Table 2). Bicuculline and picrotoxin alone were tested on 22 cells and a slight increase of spontaneous firing was observed in a11 cells. The GABA-induced inhibitory response was completety antagonized by microiontophoretically-applied bicuculline or picrotoxin (Figs 3 and 4). From these results, it is possible to conclude that the inhibitory effects of morphine were mediated by the GABAergic system.

100

e

100

i%

30 (nA)

I

NALOXONE 30nA GABA z

No effect

0 8(100) 0 0 0 4(100) 0 0 6(100) 0 0 5(lOO) 0 0 5(1Mf) No. of neurons where morphine-induced inhibitory response was affected

The numbers in parentheses represent the percentages of neurons.

100

t%

Total no. of neurons

Drugs

GABA

t

Y. Suzuto

100 e

100 e

30 -

100 30 (nA;

8m

I

2 min

Fig. 2. Frequency histograms illustrating the effects of naloxone on responses of a Purkinje cell to morphine (I 00 nA). Naloxone (30 nA) antagonized completely morphine (MOR)-induced excitation (A), but failed to antagonize mo~hine-induced inhibition (3).

Morphine and the Purkinje cell BICUCULLINE3OnA G4BA t%lR

p

MBA

K)R

60

is BICUCULLINE 3Oti

Q:

l;t

(B

j

-

2 min

Fig. 3. Frequency histograms illustrating the effects of b&cull&e, a GABA antagonist, on responses of a Purkinje cell to morphine (50 nA). Bicuculline (30nA) failed to antagonize the morphine (MQR)-induced excitation (A), but antagonized completely the morphine- and GABA-induced inhibitory responses (B). test the interaction between these inhibitory inputs ztnd the morphine-induced responses, morphine was applied concurrently with propranolol (B-receptor antagonist) and methysergide (serotonin antagonist) on Purkinje cells. Neither the excitatory (4 units) nor the inhibitory (5 units) effects of morphine (100 nA) on the Purkinje cells were significantly changed by ~~on&urrently-applied propranoiol (30 nA)_ Methy!;ergide (30 nA) also failed to alter significantly both l.he excitatory (t; units) and inhibitory (7 units) eflects of morphine (Table 2). Therefore, it is suggested that there was no interaction between the effects of morphine and the noradrenergic system or the zierotonergic system on the Purkinje cells.

Taguchi and Hagiwara, 1987), These discrepancies have been accounted for, at least in part, by the opiate-receptor subtypes, the metabolite of morphine, animal strain or anesthesia used. Moreover, the reports that microiontophoretic morphineinduced inhibition was not antagonized by naloxone were also observed in the nucleus caudate, nucleus reticularis paramedianus, nucleus reticularis and olfactory ~uber~le-nucleus accumbens region (Bradley

PICROTOXIN 30nA MOR &WA 100 30 100 100 30

30 100

= =

DISCUSSION

Microiontophoretically-applied morphine was found to increase, as well as decrease, the firing rate ,sf Purkinje cells in the cerebellum of the cat. It had been previously suggested that systemically-applied morphine may have shown two types of action on Purkinje cells (Suzuki and Taguchi, 1983a). The Purkinje cell of the cerebellum in rat has been observed to be inhibited or increased by normorphine and both effects were blocked by Ihe narcotic antag onist, naloxone (Nicoll et & 1977). In the present study, naloxone failed to reverse the depressant effects of morphine, but the excitatory effects were antagonized by microiontophoretically-applied naloxone. Similar results for the iontophoretic application of leucine enkephalin (6-receptor agonist) on the Purkinje cell have been reported (Suzuki, Ishida,

Fig. 4. Frequency histograms illustrating the effects of picrotoxin, a GABA antagonist, on responses of a Purkinje cell to morphine (100 nA). Picrotoxin (30 nA) antagonized completely the morphine- and GAB&induced inhibitory responses.

240

K. TAGUCHI and Y. SUZUKI

and Bramwell, 1977; Dingledine, Iversen and Breuker, 1978). These results showed that morphine may not affect the opiate receptor directly but there is interaction between other receptors. In the present experiments, the two different (excitatory and inhibitory) effects of morphine on Purkinje cells may be mediated by different kinds of opiate of other receptors. The excitatory effects of morphine were not antagonized by microiontophoresis of picrotoxin, bicuculline, methysergide or propranolol but excitatory effects were antagonized by naloxone, suggesting that they may be mediated by opiate receptors in the cerebellum. It has been demonstrated that morphine acts on two different receptors in the periaqueductal gray matter (Jacquet and Lajtha, 1976). Similar observations have been reported for the reticular formation (Haigler and Spring, 1978), the amygdaloid complex (Rodges and File, 1979) and the ventromedial hypothalamus (Peieto-Gomz, Reyes-Vazquez and Dafny, 1984). These reports showed that a morphine-induced decrease could be blocked by naloxone. However the morphineinduced increase in the firing rate was not blocked by naloxone. On the other hand, different observations have also been reported, whereby naloxone was able to block a morphine-induced increase in firing in the hippocampus (Nicoll et al., 1977; DeFrance, Stanley, Taberk, Marchand and Dafny, 1980). These reports are similar to those the present authors obtained for the action of morphine on Purkinje cell discharge in the cerebellum. It has been suggested that these findings did not exclude the possibility of the existence of a disinhibitory mechanism. Previous studies have reported predominantly excitatory effects of opioid peptides and of an opiate on hippocampal pyramidal cell activity, effects mediated presumably through a disinhibitory mechanism (Zieglgansberger, French, Siggins and Bloom, 1979; Siggins and Zieglgansberger, 1981). Previous reports of the iontophoretic application of Mg2+, which blocks release of transmitter, demonstrated that the excitatory effect of leucine enkephalin may contribute to presynaptic effects (Suzuki et al., 1987). Therefore, it is possible that the excitatory effects of morphine may be related to disinhibition of interneurons through an opiate receptor in the cerebellum. It is well known that Purkinje cells receive an input from the basket cell, which contains GABA, acting as an inhibitory transmitter. It has been reported that iontophoretic application of GABA induces inhibition in cerebellar Purkinje cells and this effect can be blocked by bicuculline, as a GABA antagonist (Kawamura and Provini, 1970). Regarding the relationship between the mechanisms of morphine and the GABAergic system, several studies have indicated a role for GABA in analgesia, behaviour and tolerance to opiates (Yoneda, Takashima and Kuriyama, 1976; Depaulis, Morgan and Liebeskind, 1987) and also in the antagonism of opiate-induced convulsions

(Dingledine et al., 1978). Furthermore, a recent study in the rat has shown that one of dual actions of morphine in the CNS may consist of blockade of GABA A receptors (Jacquet, Saederup and Squires, 1987). The reports of these experiments suggest that morphine probably interacts with GABAergic drugs. In receptor binding studies, opiates, in large concentrations displace the binding of GABA receptors (Sivam, Nabeshima and Ho, 1982; Jacquet er al., 1987). Acute administration of morphine produces a decrease in the binding of GABA in the cerebellar cortex and striatum. Thus, morphine may produce some of effects by modulating the GABAergic system (Ticku and Huffman, 1980). These reports show that the effects of morphine are, in part, mediated by the GABAergic system in the cerebellum. In the present experiments, the inhibitory effects of morphine were not antagonized by microiontophoresis of naloxone, methysergide or propranolol. However, the inhibitory effects of iontophoretically-applied morphine on Purkinje cells were blocked by bicuculline and picrotoxin, acting as GABA-receptor antagonists. Morphine-induced inhibition may occur through an interaction with GABA-receptors in the cerebellum. From these results, it is clear that the depressant actions of morphine on the cerebellar Purkinje cells were affected by the GABA receptor. A previous study has shown that iontophoretically applied naloxone antagonized the depression of the firing rate produced by GABA (Dingledine et al., 1978). Naloxone, in large doses, causes convulsions in mice and potentiates the convulsant activity of bicuculline. In addition, naloxone displaces [3H]GABA from receptor sites in the forebrain and cerebellum of the rat, with similar low potency. Considering the relationship between GABA and naloxone, the data presented here showed that naloxone did not reverse GABA-induced depression of the activity of Purkinje cells. Thus, there was no interaction between the effects of GABA and naloxone on the activity of Purkinje cells. It is known that the Purkinje cell is regulated, in part, by serotonin or norepinephrine located on the cerebellum (Hoffer, Siggins, Oliver and Bloom, 1973; Chan-Palay, 1976). Cerebellar Purkinje cells in the rat, for example, are uniformly inhibited by microiontophoretic application of NE and this inhibitory effect of NE is blocked by the P-blocker, sotalol (Waterhouse, Moises, Yeh and Woodward, 1982). The present data demonstrates that the excitation of inhibition of Purkinje cells after iontophoretic application of morphine was not antagonized by propranolol, a P-receptor antagonist. Thus, the action of morphine was not influenced by the noradrenergic system in producing excitation or inhibition of the activity of Purkinje cells. Regarding serotonin, an immunocytochemical study has revealed the presence of serotonin-positive fibers in the Purkinje cell layer (Bishop and Ho, 1985). Microiontophoretically applied serotonin pro-

Morphine and the Purkinje cell

duced slow depression in cerebellar Purkinje cells and is presumed to exert a direct effect on the postsynaptic receptors of Purkinje ceils (Strahlendorf, Lee and Strahlendorf, 1984). It is considered that morphine may influence the activity of serotonergic neurons of the cerebellum. Iontophoretically applied methysergide did not block morphine-induced excrtation or inhibition and the data revealed that the erects of morphine were not mediated by the activity of serotoninergic neurons in the cerebellum. In conclusion, these results suggest that excitation of the activity of Purkinje cells in the cerebellum induced by microiontophoretically applied morphine is related to the opiate system and that inhibition is related to the GABAergic system. ~&~~~~~e~ge~e~f~-We are very grateful to Dr Yuji Maruyama for helpful comments on the manuscript. We thank Sandoz, and Endo Laboratories, respectively, for their generous gifts of methysergide and naloxone.

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