Role of GABAergic systems in the modulation of intracranial self-stimulation in the rat

Role of GABAergic Systems in the Modulation of Intracranial Self-Stimulation in the Rat JEFFREY

M. GOLDSTEIN,

KRYSTYNA

Biomedical Research Department,

LESZCZYNSKA

AND JEFFREY

ICI Americas Inc., Wilmington,

B. MALICK

DE 19897

GOLDSTEIN, J. M., K. LESZCZYNSKA AND J. B. MALICK. Role of GABArrgic s.wtems in the rn~~~ul~~tj~~~ qf ititrft~ru~i~ti s~~~.~~~rnula~~ttn in the rot. BRAIN RES. BULL. 5: Suppf. 2,275-278, 1980.-The effects of GABA agonists and antagonists on intracranial self-stimulation (ICSS) were studied in rats utilizing an electrode-cannula which permitted infusion of drugs directly into the brain at the same site at which ICSS was elicited (medial forebrain bundle). Muscimol (0.001-0.03 pg) and GABA (3-10 pg) produced a dose-related suppression of ICSS. Following infusion of the same behavio~lly active doses used in the ICSS experiments, no signi~cant depression of motor-activity was observed; thus, a selective effect on the reward system was suggested as a mechanism for muscimol- and GABA-induced suppression of ICSS. The GABA antagonist bicuculline blocked the effects of GABA on ICSS but failed to block the effects of muscimol on ICSS. Since muscimol has a higher affinity for the GABA receptor than GABA itself, higher doses of antagonist would be required to block the effects of muscimol at such sites. U~o~unately, higher doses of bicuculline could not be used in this behavioral model. The antagonism of GABA effects on ICSS by bicuculline still implicates GABA receptors as being the site of this activity. Thus, GABAergic systems appear to modulate reinforcement pathways at the level of the medial forebrain bundle. Self-stimulation Electrode-cannula

GABA

Muscimol

Bicuculline

PHARMACOLOGY of intracranial self-stimulation (ICSS) has been extensively investigated (for a review see [IO]). Representative agents from each of the major classes of psychoactive drugs including hypnotics, neuroleptics, anxiolytics, stimulants, antidepressants, psychedelics, analgesics, anticonvulsants and anticholinergics have been tested in ICSS. In addition, research on the possible neurotransmitters and neuromodulators that may mediate ICSS has revealed that noradrenergic, dopaminergic, serotonergic, and cholinergic systems participate in this behavior [9]. It is therefore surprising that the role of the gammaaminobuty~c acid (GABA) system in ICSS has received little attention, especially in view of the considerable evidence that GABA is an important inhibitory neurotransmitter in the CNS [IS]. Kent and Fedinets [7] investigated the effects of GABA blockade on ICSS and found that the GABA antagonists picrotoxin and bicuculline, at doses that did not impair shock-escape reinforced lever pressing, produced a dosedependent depression of lateral hypothalamic selfstimulation. The effects of GABA agonists or of GABA itself were not studied. On the other hand, Hasegawa [4] reported that the intraventricuIar (ICV) administration of GABA did not produce a consistent effect on substantia nigra selfstimulation, whereas ICV administration of norepinephrine facilitated it. No other studies were found in the current literature that could resolve the role of GABAergic processes in ICSS. THE

Copyright

c‘ 1980 ANKHO

Inte~ational

Motor activity

lntracerebral

infusion

Accordingly, the present studies were designed to determine the role of GABA agonists and antagonists on medial forebrain bundle ICSS. Since GABA does not readily penetrate the blood-b~in barrier, an electrode-cannula was used to inject GABA and muscimol, a reported GABA agonist [6], directly into the brain at the same site at which ICSS was elicited. The electrode-cannula technique was previousfy used to determine the effects of Substance P in ICSS [3]; this technique was found to be very sensitive to the effects of intracerebrally administered drugs. In addition, the specificity of the effect of GABA and muscimol in ICSS was tested by dete~ining whether these drugs, at doses that were effective in ICSS, produced non-specific decrements in motor behavior that might have interfered with the rats’ ability to lever press for ICSS.

METHOD

Male Wistar rats, 200-250 g, were anesthetized with sodium pentobarbital (50 mg/kg, IP) and stereotaxically implanted with electrode-cannulae (Plastic Products Co., Roanoke, VA) into the medial forebrain bundle (coordinates measured from bregma: posterior 0.6 mm, lateral 1.7 mm, and 9.5 mm below the skull). The coordinates used were taken from the atlas of Pellegrino and Cushman [S]. One week following surgery, rats were trained for ICSS in a

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GOLDSTEIN.

standard operant box (stimulus parameters: 60 Hz sine wave, 250 msec duration, current range 30-100 PA). Current was adjusted for individual rats to maintain responding between 2000 and 4000 responses/30 min. The experimental design for intracerebral drug administration consisted of a 10 min preinfusion period immediately followed by drug infusion and a 20 min postinfusion period. Drug infusion was accomplished by insertion of an internal cannula that extended 1 mm beyond the tip of the guide cannula to coincide with the tips of the stimulating electrode. The internal cannula was connected via a length of PE tubing to a Sage infusion pump, set to deliver 1 ~1 over 24 set; the internal cannula was allowed to remain in place an additional 30 set to permit drug absorption. In the drug antagonism studies, bicuculline was injected subcutaneously 10 min prior to infusion of GABA or muscimol. In preliminary testing the dose of bicuculline selected for the antagonism studies (0.25 mg/kg) did not significantly affect ICSS. GABA and muscimol were dissolved in a sterile water vehicle immediately before infusion, and doses for each drug were calculated on a microgram per rat basis. Bicuculline was dissolved in HCl, neutralized to pH 5.0 with NaOH, saline added for a final concentration of 0.15 M, and injected in a volume of 1 ml/kg. Drug effects were statistically evaluated using a paired Student’s t-test comparing mean preinfusion responses to mean postinfusion responses. Each 30 min session was broken down into two 5 min preinfusion periods and four 5 min postinfusion periods. Statistical comparisons using the mean of the two 5 min preinfusion time periods were made with each of the postinfusion time periods.

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FIG. 1. Self-stimulation rates, expressed as a percent of pretreatment baseline, following the infusion of muscimol (O=O.OOl wg, N=6; V=O.O03 pg, N=6; q=0.03 pg, N=6) or sterile water (0, N=6). *p
Motor Activity

Male Wistar rats, 200-250 g, were anesthetized with sodium pentobarbital (50 mglkg, IP) and stereotaxically implanted with guide cannulae (Plastic Products Co., Roanoke, VA) into the medial forebrain bundle at the coordinates given previously. One week following surgery, rats were divided into three groups which received either sterile water (vehicle), GABA, or muscimol at doses that were effective in ICSS. The infusion procedure was the same as described previously. Motor activity was recorded in an Animex activity monitor (Columbus Instruments, Columbus, OH) for 30 min after drug infusion. Data were statistically evaluated by comparing the vehicle-treated group to the drug-treated groups using the Student’s t-test.

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At the conclusion of the studies, rats were sacrificed with an overdose of sodium pentobarbital, and electrode-cannula and cannula placements histologically verified. Examination of several representative brains indicated that placements were within the medial forebrain bundle at the level of the lateral hypothalamus. RESULTS

Figure 1 shows the results of intracerebral infusion of muscimol on ICSS. The lowest dose of muscimol infused (0.001 pg) failed to produce a significant alteration in ICSS at any of the time periods tested (5, 10, 15, and 20 min postinfusion). The next higher dose of muscimol (0.003 pg) produced a significant decrease in ICSS only at the 15 min

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FIG. 2. Self-stimulation rates, expressed as a percent of pretreatment baseline, following the infusion of GABA (0=3 pg, N=6; V=6 fig, N=7; Cl=10 pg, N=8) or sterile water (@, N=8). *p
time period whereas the highest dose tested (0.03 pg) produced a significant effect at each of the time periods tested. The effect of intracerebral infusion of GABA on ICSS is shown in Fig. 2. GABA at 3 and 6 pg failed to significantly

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GABA AND SELF-STIMULATION

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FIG. 3. Effects of bicuculline pretreatment on muscimol-induced suppression of ICSS at 5, IO, 15, and 20 min post drug. ***p
TABLE THE

EFFECTS

Treatment

alter ICSS whereas GABA at 10 pg produced a significant suppression of ICSS at 10, 1.5, and 20 min post drug; no significant effect was observed at the 5 min time period. The results of the antagonism studies are shown in Fig. 3 and 4. Bicuculline, at a dose that failed to affect ICSS itself, did not prevent the suppression of ICSS produced by muscimol at 0.03 Kg (see Fig. 3). However, bicuculline significantly blocked the effects of GABA on ICSS (see Fig. 4). In order to test for the specificity of the effects of muscimol and GABA in ICSS, both drugs were tested for their effects on motor activity following infusion of the same behaviorally active doses that produced suppression of ICSS (muscimol 0.03 Fg, GABA 10 fig). The results of this study are presented in Table 1. Muscimol and GABA did not produce a significant decrease in motor activity as compared to the vehicle control group after infusion into the brain; a moderate increase in activity with both drugs was observed that was not statistically significant. DISCUSSION

Muscimol was about 330 times more potent than GABA in producing an equi-effective suppression of ICSS. These findings appear highly compatible with the recent findings of Yarbrough [l I] that on the basis of iontophoretic ejection currents muscimol was considerably more potent than GABA in inhibiting spontaneously active cortical neurons in rats. However, the present data do not agree with that of Hasegawa [4]. In the present study GABA produced a doserelated suppression of ICSS; Hasegawa [4] reported that GABA did not produce a consistent effect on ICSS. Closer examination of the data presented by Hasegawa [4] revealed that GABA did produce an inhibitory effect but it was not dose-related. In these studies GABA was infused intracerebroventricularly and ICSS was elicited from the substantia nigra. In the present study, GABA was infused directly into the same site at which ICSS was elicited, which was the

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OF INTRACEREBRAL INFUSION MUSCIMOL ON MOTOR ACTIVITY

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SN.S.=Not significant.

medial forebrain bundle. The dose of GABA required to produce an effect on ICSS following infusion into the ventricle was considerably higher than that used in the present studies. An effect on ICSS that was not dose-related may have been a result of the higher doses employed by Hasegawa [41 which may have produced a nonseIective disruptive effect on ongoing behavior. It is possible that the infusion of GABA into the substantia nigra using an eiectrode-cannula may have produced suppression of ICSS comparable to the present results. The specificity of the GABA and muscimol effects on ICSS in the present studies is further supported by the results of the motor activity studies. GABA and muscimol, infused into the same site at which ICSS was suppressed, failed to suppress motor acitvity. In fact, there was a moderate though nonsignificant increase in activity with both drugs compared to the vehicle control group. These results demonstrate that suppression of ICSS by GABA and muscimol is not simply due to a depression of motor activity per se or to nonspecific CNS depression. Rather, it appears that GABA and muscimoi may be interfering with the reward system and making the stimulation less reinforcing since the rats continue to press the lever for ICSS but at lower rates. Kent and Fedinets [7] suggest that the suppression of ICSS

GOLDSTEIN,

278

produced by picrotoxin and bicuculline is due to an effect on reinforcement mechanisms rather than by disrupting motor activity; our results with GABA antagonists support their observations. The ability of bicuculline to significantly block the effects of GABA on ICSS suggests that the effects of GABA are mediated by an interaction with the GABA receptor. The failure of bicuculline to block the effects of muscimol on ICSS does not necessarily rule out the possibility that muscimol is producing its effects on ICSS via the GABA receptor also since Enna et ~1. [2] have shown that muscimol is about 10 times more potent than GABA in displacing radioactive GABA bound to membranes from rat brain. This would suggest that more antagonist may be needed to prevent the action of muscimol on the GABA receptor than of GABA on its receptor. In the present studies, the dose of bicuculline could not be increased since it produces effects by itself on ICSS at higher doses. An alternative explanation for the lack of antagonism by bicuculline of the effects of muscimol on ICSS may be that muscimol is producing nonspecific CNS effects not related to the GABA system. This is unlikely since the motor activity studies did not re-

LESZCZYNSKA

AND MALAICK

veal a general depressive effect of muscimol at the doses which suppress ICSS. In conclusion, the present studies have shown that GABA and muscimol, when infused into the same site in the brain at which ICSS was elicited, produced a dose-related suppression of ICSS. The failure of GABA and muscimol to affect motor activity at the same doses that suppressed 1CSS suggests a specific effect of these drugs on the reward system. The apparent lack of antagonism by bicuculline of the inhibitory effects of muscimol may be related to the higher affinity of muscimol for the GABA receptor: more antagonist

may be necessary to block the effects of muscimol than of GABA and higher doses of bicuculline could not be used in this behavioral model. Thus, GABAergic systems appear to modulate activity in reinforcement pathways at the level of the medial forebrain bundle in rats. Although it is not easy to resolve why GABA agonists and antagonists both appear to produce selective suppression of ICSS, it may be that if GABAergic neurons play a modulatory role, any alteration in their activity may upset a homeostatic balance resulting in disruption of activity in reward pathways.

REFERENCES

1. Curtis, D. R. and G. A. R. Johnson. Amino acid transmitters in 2.

3.

4.

5.

the mammalian central nervous system. Erg&n. Ph.vsio/. 69: 97-188, 1974. Enna, S. J., J. F. Collins and S. H. Snyder. Stereospecificity and structure-activity required of GABA receptor binding in rat brain. Bruin Res. 124: 185-190, 1977. Goldstein, J. M. and J. B. Malick. Effect of Substance P on medial forebrain bundle self-stimulation in rats following intracerebral administration. Pharmac. Biochem. Brhav. 7: 475-478, 1977. Hasegawa, K. Changes in the substantia nigral self-stimulation behavior by intraventricular injection of norepinephrine, dopamine, and GABA. Folia Pharmac. jup. 72: 985-990, 1976. Iversen, L. L. and F. E. Bloom. Studies on the uptake of 3HGABA and 3HGlycine in slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography. Brain RPS. 41: 131-143, 1972.

6. Johnston, G. A. R., D. R. Curtis, W. C. DeGroat and A. W. Duggan.

Central

Biochem.

Pharmuc.

actions

of ibotenic

17: 2488-2489,

acid and

muscimol.

1%8.

7. Kent, E. W. and P. Fedinets. Effects of GABA blockade on lateral hypothalamic self-stimulation. Brain Res. 107: 628-632, 1976.

8. Pellegrino, L. J. and A. J. Cushman. A Sfrrrotcrxic At/us of the Rat Brain. New York: Meredith, 1967. 9. Rolls, E. T. TOP Brain end Re~~~trd. Oxford: Pergamon Press, 1975. 10. Wauquier, A. The influence of psychoactive drugs on brain self-stimulation in rats: a reVieW. In: Bruin-Srimukation ReI~‘ard. edited by A. Wauquier and E. T. Rolls. Amsterdam: NorthHolland, 1976, pp. 123-170. 11. Yarbrough, G. G. Nipecotic acid and 2,4-diaminobutyric acid enhance the actions of muscimol on cerebral cortical neurons. Corn.

.I.

Physiol. Pharmac.

56: 443-446, 1978.