Alteration of the neuronal response to amphetamine in the neostriatum by pretreatment with a centrally acting anticholinergic

Neuropharmacolog~~. 1977, 16. 455.461. Pergamon Press,‘Prmted in Gt. Britain

ALTERATION OF THE NEURONAL RESPONSE TO AMPHETAMINE IN THE NEOSTRIATUM BY

PRETREATMENT WITH A CENTRALLY ACTING ANTICHOLINERGIC

C. J. WILSON, JANICE M. JURASKA and P. M. GROVES

Department of Psychology, University of Colorado, Boulder, Colorado 80309, U.S.A.

(Accepted 12 Maq’ 1976)

Summary-Intraperitoneal injection of 1.0 mg/kg (-)-scopolamine hydrobromide had no effect upon the firing rate of caudate-putamen neurones recorded in immobilized, locally anaesthetized rats, although the pattern of their spike trains over time was somewhat altered. Pretreatment of animals with this same dose of scopolamine, but not with 1.0 mg/kg (-)-scopolamine methyl bromide, trans- formed the normally biphasic response of caudate-putamen neurones to 2.5 mg/kg (+)-amphetamine into a response consisting of only a depression of firing rate. A less common response of caudate- putamen neurones to amphetamine, a prolonged increase in firing rate, was unaffected by scopolamine pretreatment. The transient initial potentiation of activity seen in most caudate-putamen neurones to precede the amphetamine-induced depression of unit activity therefore appears to require the activity of a central muscarinic receptor. The results are discussed in relation to the interaction of dopaminergic and cholinergic systems in the striatum.

Following acute amphetamine administration, changes in the spontaneous activity of neostriatal neurones which follow a course similar to that of behaviour induced by amphetamine have been observed in rats. Intraperitoneal injection of amphetamine in

doses ranging from 0.5 to 4.0 mg/kg leads to a marked depression of the firing rate of most caudate-putamen neurones in immobilized, locally anaesthetized rats, which parallels the appearance and time course of behaviours seen in unrestrained rats gi-ven compar-

able doses (Groves and Rebec, 1976; Groves, Rebec and Segal, 1974). Like amphetamine-induced stereotyped behaviour (e.g. Creese and Iversen, 1972), the depression of activity of these neostriatal neurones is attenuated or abolished by lesions of the nigro- striatal bundle which deplete neostriatal dopamine

(Groves, Rebec and Harvey, 1975), and may be reversed by subsequent treatment with haloperidol (Groves et al., 1974), suggesting that both may result, in part, from an amphetamine-induced facilitation of dopaminergic transmission in the nigro-neostriatal system.

Neither the depression of neostriatal unit activity nor stereotyped behaviour follows immediately upon amphetamine administration, however. In a number of behavioural studies (e.g. Segal, 1975), amphetamine-induced stereotyped behaviour in rats has been reported to follow a transient period of increased locomotor activity, the intensity of which increases with increasing doses of amphetamine. Costa11 and Naylor (1972) have suggested that the delay in the onset of stereotyped behaviour after amphetamine administration may be due in part to the compensatory activation of a hypothetical cholinergic neural system set into motion by the catecholamine-releasing effects

of amphetamine. Evidence for a cholinergic role in the production of stereotyped behaviour is derived from the ability of anti-muscarinic agents to exagger- ate the effects of amphetamines (Arnfred and Ran- drup, 1968; Mennear, 1965; Naylor and Costall, 1971; Klawans, Rubovits, Pate1 and Weiner, 1972) and apomorphine (Scheel-Kriiger, 1970), and the antagonism of these effects by cholinergic agonists (e.g. Proctor, Potts, Ashley and Denefield, 1967). The existence of

an antagonistic relationship between dopaminergic and cholinergic systems is supported by a large body of related clinical (e.g. Duvoisin, 1967), pharmacologi- cal (e.g. And&n and Bkdard, 1971, Costa11 and Olley, 1971; Neil], 1976), and biochemical (Racagni, Cheney, Trabucchi and Costa, 1976) evidence.

If a compensatory cholinergic activation does occur during the initial stages of amphetamine intoxication,

it could account for the biphasic nature of the response of neostriatal neurones to amphetamine. In the present experiments, changes are described in the neuronal response to amphetamine administration in animals pretreated with scopolamine or its quatern- ary analog, methscopolamine.

METHODS

Male albino rats, weighing from 250 to 450g were anaesthetized by ether inhalation, and placed in a stereotaxic instrument using atraumatic ear bars (Kopf Instruments) covered with a local anaesthetic ointment (Lidocaine). Ether anaesthesia was maintained for a period of 30 to 45 min, during which all surgery was performed. A short, midsaggital scalp incision exposed the calvarium, and a small burr hole was drilled in the skull over the neostriatum on each

455

N.P Ih 7;R A

356 C. J. WILSON. JANICE M. JURASKA and P. M. GROVES

side of the midline at a point determined using a previously calibrated electrode. The areas in and sur- rounding the wound were infiltrated thoroughly with

procaine hydrochloride (Novocaine) by subcutaneous injections on all sides of the wound and local application to all cut edges. Commercial eyedrops (Visine) were applied to prevent cornea1 drying, and a small opening was made in the dura on each side. The animal was then immobilized with 2.0 mg/kg tubocurar-

ine hydrochloride (Abbott), ether anaesthesia was dis- continued. and the subject was artificially respired using a Harvard Instruments rodent respirator attached to a rubber cone which fits snugly over the snout. Heart rate, body temperature. and breath-by- breath expired carbon dioxide were monitored con-

tinuously. Local anaesthesia of the scalp and contact points was maintained by repeated application of Novocaine at 1 hr intervals for the remainder of the experiment.

Glass-coated tungsten microelectrodes with tip dia- meters of 1 to 5 microns and impedances of 1 to 3 m0 (measured at 500 Hz) were lowered into the caudate-putamen on each side and moved slowly until a single unit was isolated (3: I signal to noise ratio or better). Typically, action potentials so isolated were monitored for a period of 20 to 60 min to insure stability of the recording. Neuronal firing rates were moni-

tored on line by means of a Schmitt-trigger circuit and high speed printer-counter on a minute by minute basis. and action potentials were diplayed at

high sweep speed on the face of a Tektronix 565 oscilloscope and at low speed on a Federal 18” screen monitor scope.

Unit activity accepted on the basis of constant signal to noise ratio and relatively stable firing frequency was monitored for an additional 10 min prior to drug administration in order to establish a control firing rate. Immediately afterward. animals in the experimental group (n = 8) received an intraperitoneal in-

jection of 1 mg/kg scopolamine hydrobromide (Sigma) followed 15 min later by an intraperitoneal injection of 2.5 mg/kg (+) amphetamine sulphate. In one control group (n = 5). 1 mg/kg scopolamine hydrobromide was injected but no amphetamine was given. In another control group (11 = 8) 1 mg/kg scopolamine methyl bromide (Sigma) was injected followed 15 min later by 2.5 mg/kg (t ) amphetamine sulphate. In animals given scopolamine alone, neuronal firing rates were monitored for periods’of from 55 to 150min following drug administration. In animals given scopolamine or methscopolamine followed by amphetamine, unit activity was monitored for approximately 300min or until firing frequency approached or stabilized at or near predrug firing rate following drug-induced changes. Data obtained from units failing to recover to within 60 percent of predrug baseline firing rate within this time, or failing to maintain a 3:l signal-to-noise ratio, are not included in this report.

Upon completion of the experiment. subjects were

given a lethal dose of pentobarbital and the electrode tip marked with a small lesion. Animals were then sacrificed by intracardial perfusion with normal saline

followed by formalin, and the recording position veri- fied in 60 micron sections stained with cresyl violet.

RESULTS

Changes in neuronal activity in the control group

given 1 mg/kg methscopolamine followed 15 min later by 2.5 mg/kg (+) amphetamine sulphate are illus-

trated in Figure 1. Mean firing rate in Figures I-4 is expressed as a percentage of the mean rate during the 10min predrug control period. Firing rate is

shown on a minute by minute basis for the 15 min period between injections, and for the I5 min period following amphetamine administration. Thereafter, data are the means for 5 min observation intervals.

Slashes represent the time required for each of the neurones in the sample to recover to 60 percent of its predrug rate. The activity of each neurone subsequent to this recovery criterion (usually a “rebound” increase in firing rate) has been deleted.

Changes in neuronal activity in this group were

similar to those obtained from non-pretreated animals given comparable doses of (+) amphetamine (e.g., Groves rt al., 1974; Rebec and Groves, 1975). While methscopolamine produced little change in neuronal firing rates during the 15 min after intraperitoneal injection, (+) amphetamine produced an initial

brief increase in firing rate followed by a prolonged depression of neuronal activity which in this sample of 8 neurones, lasted from approximately 85 to 325 min from the time of amphetamine injection. The combination of methscopolamine and amphetamine

had profound peripheral effects, including heart rate increases of approximately 35?,. For three other neurones not shown in Figure 1. the combination of methscopolamine pretreatment and amphetamine administration led to an increase in firing rate lasting

60 to 130 min. Spontaneous firing rates in the caudate-putamen

were generally slow, but showed considerable variability across cells. In the present sample of 28 neurones recorded in 21 subjects, the mean predrug firing rate ranged from 5 to 836 spikes/min, with a mean of 146. The wide range of spontaneous firing rates for neurones encountered in the caudate-putamen could not be related either to the positions of the neurones within the nucleus or to variability between subjects. Neurones with widely differing rates may be recorded in the same area of the nucleus, and even simultaneously from the same electrode. Furthermore, neurones recorded under conditions meeting our cri- teria for signal to noise ratio and stability of spike height generally had very stable firing rates, which varied little during the 30 to 70min period of observation preceding drug administration. Most neurones fired in a bursty pattern, sporadically issuing a sequence of action potentials in rapid succession, fol-

Amphetamine and neuronal activity 457

LOmg /kg methscop. + 2.5 mg/kg d-omph.

mscop. omph.

Fig. 1. The effects of pretreatment with l.Omg/kg methscopolamine and subsequent injection of 2.5 mg/kg (+)-amphetamine sulphate on the activity of eight caudate-putamen neurones recorded in 6 rats. Mean firing rate is expressed as a percent of the mean rate for the 10min preceding the methscopolamine injection. Single neurone activity is shown on a minute by minute basis for the 15 min between injections and for the 15 min period after amphetamine administration. Subsequent data are means of 5 min intervals. The slashed lines indicate the time at which each neurone in the sample recovered to 60% of its control firing rate, and the time at which data from that neurone are deleted from the averaged values. The microelectrode tip placements for the eight neurones are

shown to the right of the graph.

1.0 mg/ kg stop. n=5

50 -

1 stop.

Time, min

Fig. 2. The effects of intraperitoneal injection of 1 mg/kg (-)-scopolamine hydrobromide on the activity of five caudate-putamen neurones, and the position at which each neurone was recorded. Slashes

in this case indicate the length of time each neurone was held for observation.

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C. J. WILSON, JANICE M. JURASKA and P. M. GROVES

1.0 mg / kg atop. + 2.5 mg f kg d-omph. n=8

Fig. 3. The effects of pretreatment with l.Omg/kg scopolamine and the subsequent injection of 2.5 mg/kg (+)-amphetamine sulphate on the activity of eight caudate-putamen neurones. and the position at which each neurone was recorded. Firing rate is expressed as a percent of the pretreatment

mean rate as in Figure 1.

1.0 mg/kg stop. + 2.5 mg/kg d-amph.

v ’ +jo ’ -30 ’ Lo ’ z3ib )O LiiO ’ Jo ’ 110 ’ 110 ’ A

stop. omph.

Fig. 4. The response of a single neurone to amphetamine injection illustrating a less common response in the caudate-putamen. Although this neurone was recorded in an animal pretreated with scopolamine, its response is atso typical of neurones responding in this way in methscopoIamine-pretreated animals. Four such neurones were recorded in subjects pretreated with scopolamine; three were recorded in

methscopoiamine-pretreated subjects.


Methscopolomine pretreatment Scopolamine pretreatment

Control

IO min after amphetamine

30 min after amphetamine

Recovery

Fig. 5. Examples of neuronal activity of neurones included in the samples of Figure 1 and Figure 3, photographed directly from data film. Samples from the predrug control period, the period during which the initial potentiation is usually seen in response to amphetamine, the period during which most cells show a depression of activity, and the recovery period are shown. The calibration marks

represent 1 set (horizontal) and 300 pV (vertical).

lowed by a variable interval of relative quietude (see e.g. Fig. 5). Intraperitoneal injection of 1 mg/kg scopolamine had no significant effect on the firing rate of any of 5 caudate-putamen neurones monitored for up to 150 min after intraperitoneal administration. The data from this group of neurones are shown in

Figure 2. In 4 of the 5 cells, however, a change in the pattern of action potentials over time could be detected. This consisted of a tendency toward a

tighter grouping of spikes within a burst, and a de- creased burst duration. This change, which tended to give the spike trains a more bursty quality, occurred within 5 to 20min after scopolamine administration. It was not seen in one neurone, which showed very little tendency toward bursty firing previous to drug injection.

Figure 3 illustrates the effects of (+)-amphetamine for eight caudate-putamen neurones in subjects given the drug 15 min after pretreatment with 1 mg/kg scopolamine. Again, scopolamine appeared to have no consistent effect on the firing rate during the 15 min period between injections. In contrast to the results obtained in control subjects pretreated with methscopolamine, however, the depression of activity which followed amphetamine administration was almost im- mediate, occurring within 3 to 10 min after the injec-

tion. No initial increase in activity was evident. In an analysis of variance performed on 1 min mean rates for the groups shown in Figures 1 and 3 over the first 30 min following amphetamine administration, this difference between groups appeared as a sig-

nificant pretreatment group X time interaction (F = 4.4, df = 29/406, P < 0.05). A significant time

effect was also seen (F = 2.0, df = 29/406, P < 0.05). Overall mean firing rates for the first 30 min following amphetamine did not differ significantly for the two

groups (F = 1.1, df = l/14, P > 0.05). In four additional neurones, not included in the

group data shown in Figure 3, intraperitoneal

amphetamine administration was followed by a prolonged increase in neuronal firing rate lasting from 60 to 130min, similar to those seen in methscopolamine pretreated animals as well as non-pretreated animals or animals subjected to other experimental treatments as previously reported (e.g., Rebec and Groves, 1975; Groves and Rebec, 1977).

Examples of single unit activity as photographed directly from the face of the oscilloscope are provided in Figure 5. On the left are examples of extracellularly recorded action potentials at various times during the experiment in an animal given methscopolamine pre-

treatment followed by amphetamine. On the right are similar examples from an animal given scopolamine pretreatment followed by amphetamine.

DISCUSSION

For most caudate-putamen neurones, low doses of amphetamine lead to a depression of firing rate which develops 10 to 40tiin after intraperitoneal injection. At higher doses, an initial, brief potentiation of neuronal activity develops prior to the depression of firing

460 C. J. WILSON, JANICE M. JURASKA and P. M. GROVES

rate (Groves et cd., 1974). Unlike the amphetamine- induced depression, the initial transient potentiation of unit firing appears to be unaffected by lesions of the nigro-striatal pathway, suggesting that it results from an effect of amphetamine on non-dopaminergic systems intrinsic to the caudate-putamen. or the activation of some system affecting the neostriatum by way of its cortical or brainstem afferents (Groves et al., 1975). The present results suggest that the system or systems responsible for the initial potentiation seen during this period require the integrity of central cholinergic neuronal transmission.

The locus of the antagonistic interaction of dopaminergic and cholinergic influences on amphetamine-

induced behaviour has not yet been established. It has seemed attractive to attribute this interaction in part to the intrinsic organization of the striatum. The neostriatum contains one of the highest concen- trations of acetylcholine in the central nervous system (e.g. Fonnum, 1973). This neurotransmitter appears to be synthesized in striatal interneurones, which appear to be contacted by dopaminergic axon vari-

cosities (Hattori. Singh, McGeer, E. and McGeer. P., 1976). Many studies have shown consistent relation- ships between striatal dopaminergic transmission and striatal acetylcholine (e.g. Stadler, Lloyd. Gadea-Ciria and Bartholini, 1973; Racagni it al., 1976). These observations and others have suggested to numerous

investigators the possibility that the effects of dopaminergic activity in the neostriatum may attain their expression in the neostriatal output in part by way of cholinergic interneurones (e.g. McGeer. E..

McGeer, P., Grewaal and Singh, 1975; Groves, Wil- son, Young and Rebec, 1975). Furthermore, these in-

terneurones may very well be interconnected synapti- cally, providing the basis for a possible direct antagonism between dopaminergic and cholinergic influences upon such elements (Racagni c’t uI., 1976).

While those models do provide a substrate for in-

teracting dopaminergic and cholinergic influences

upon behaviour, other evidence suggests that non-

striatal mechanisms are also relevant. For example.

while anticholinergic drugs enhance stereotypic be-

haviour in response to amphetamine or dopaminergic

agonists. they do not by themselves induce stereotypy

(&heel-Kriiger. 1970), and local application of anticholinergics into the striatum does not enhance the stereotypy induced by amphetamine (Costall and

Naylor, 1972). Also, the apparent ability of anticholinergics to antagonize the behavioural effects of dopaminergic blockade with haloperidol varies according to which of the several effects of haloperidol is chosen for study (Setler. Sarau and McKenzie, 1976).

Scopolamine, administered at a dose that enhances amphetamine-induced stereotyped behaviour (1 mg/kg), had, by itself, no consistent effect on the activity of a sample of spontaneously active striatal neurones. Thus, the evidence does not reveal a tonic antagonistic dopaminergic-cholinergic interaction on

neostriatal neurones. Nevertheless the existence of an antagonism of a different sort between dopaminergic and cholinergic influences upon neostriatal unit activity is indicated by the effect of scopolamine pretreatment upon the neuronal response to amphetamine. The initial acceleration of firing rate observed to precede the depression of activity which, for most neostriatal neurones follows amphetamine administration, was abolished by prior treatment with scopolamine. Thus it would appear that amphetamine acts

upon neostriatal neurones not only by means of its facilitation of dopaminergic transmission, which leads to a depression of activity in most cells, but also by way of some other system involving a cholinergic synapse, whose effects upon neostriatal activity are antagonistic to those of dopamine. The result of sco-

polamine pretreatment upon the neuronal response to amphetamine appears as an abolition of the delay between the administration of amphetamine and the depression of neuronal activity which is its most per- sistent effect, and so is in agreement with the sugges- tion of Costall and Naylor (1972) that pretreatment with anticholinergics may act in part to reduce or abolish effects of amphetamine other than those due

to facilitation of dopaminergic transmission.

AcknoMledyements---The authors would like to thank Stephen J. Young for his help and advice at several stages of the preparation of this manuscript, and Pat Wilson for her expert technical assistance. We also thank Smith, Kline and French for supplying the (+)-amphetamine sulphate. This work was supported in part by grant MH 19515 and Research Scientist Development Award K02 MH 70706 from the National Institute of Mental Health, and grant DA01467 from the National Institute of Drug Abuse to P.M.G. and J.M.J. acknowledges support from NIH grant EY 01500 to Eva Fifkova.

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Alteration of the neuronal response to amphetamine in the neostriatum by pretreatment with a centrally acting anticholinergic