Evidence that an accumbens to subpallidal GABAergic projection contributes to locomotor activity

Brain Research

Bulkritz, Vol. 11, pp. 309-314, 1983. 0 Ankho International

Inc. Printed in the U.S.A.

Evidence that an Accumbens to Subpallidal GABAergic Projection Contributes to Locomotor Activity GORDON

Department

of Physiology,

J. MOGENSON

AND MARK A. NIELSEN

University of Western Ontario, London, Ontario, Canada N6A 5Cl Received

23 May 1983

MOGENSON, G. J. AND M. A. NIELSEN. Evidence that an accurnbens to subpallidal GABAergic projection contributes to locomotor activity. BRAIN RES BULL ll(3) 309-314, 1983.-Neural projections from nucleus accumbens to

subpallidal region, which contains a major GABAergic component, have been demonstrated with anatomical and electrophysiological techniques. The possible contribution of this GABA projection to the initiation of locomotor activity was investigated using neuropharmacological techniques. Injecting picrotoxin, a GABA antagonist, into the ventral globus pallidus increased locomotor activity measured in an open-field test, confirming previous findings. Locomotor activity was also increased when picrotoxin was injected into the lateral preoptic area, the sublenticular part of the substantia innominata and bed nucleus of the stria terminalis. In another series of experiments locomotor activity initiated by injecting dopamine into the nucleus accumbens was attenuated by pretreating the lateral preoptic area, the substantia innominata and ventral globus pallidus with GABA. These observations provide evidence that GABAergic projections from accumbens to subpallidal region contribute to locomotor activity and raise the possibility that they have a role in exploratory locomotion and in certain goal-directed behaviors. Accumbens

Subpallidal region

GABA

Locomotor

activity

efferent projections from the accumbens. If this is so then, from our early experiments [8,14], it may be expected that injecting picrotoxin, a GABA antagonist, into the lateral preoptic region and the sublenticular substantia innominata should increase locomotion. Furthermore, injecting GABA into these subpallidal regions should attenuate locomotor activity initiated by injecting dopamine into the nucleus accumbens. The present study was undertaken to investigate these possibilities.

THERE has been considerable interest in recent years in the mechanisms of limbic-motor integration. This has come about mainly because of neuroanatomical observations, using newer tracer methods, demonstrating neural projections from limbic forebrain structures to the motor system, and particularly to the basal ganglia [3, 16,211. Since there is experimental evidence implicating limbic structures in motivational-emotional processes, associated with adaptive behavioral responses, these neural projections may provide a functional link between the limbic and motor systems. The nucleus accumbens has been shown to project to the ventral globus pallidus [20]. Since the nucleus accumbens also receives strong afferents from the amygdala and’ hippocampus, two prominent limbic forebrain structures, it appeared to be positioned strategically for limbic-motor integration [ 11,161. The accumbens to ventral globus pallidus projection was subsequently shown to contain a GABAergic component [2, 7, 8, 231, which was implicated in locomotor activity [13], and in angiotensin-elicited drinking behavior [9,131. The accumbens projection to the subpallidal region appear to be more extensive than suggested in the earlier experiments, and include the lateral preoptic region and the sublenticular substantia innominata as well as the ventral globus pallidus [1, 12, 17, 18, 231. Indeed it has been suggested by Heimer and coworkers [S] that this region of the classicallydesignated substantia innominata is .a rostroventral extension of the globus pallidus. Since neurons in these other subpallidal regions, like those in the ventral globus pallidus, are inhibited by single pulse stimulation of the nucleus accumbens [12], it seems likely that they receive GABAergic

METHOD

Male Wistar rats, weighing 215-265 g at the time of surgery, were anesthetized with sodium pentobarbital (50 mg/kg IP) and placed in a stereotaxic instrument with the incisor bar positioned 2.4 mm below the ear bars. Guide cannulas constructed from 23 gauge hypodermic needle tubing were inserted bilaterally 1.25 mm above the target site and were secured with dental acrylic to jewellers screws fixed in the skull. The target sites were: the sublenticular portion of the substantia innominata (7.0 mm anterior to the intraural line, 2.0 mm lateral to the midline, and 6.8 mm below the surface of the cortex), the ventral globus pallidus (7.0 mm anterior, 2.4 mm lateral and 6.4 mm ventral), the lateral preoptic area (7.0 mm anterior, 1.4-l .6 mm lateral and 6.8 mm ventral) and the nucleus accumbens (9.9 mm anterior, 1.3 mm lateral and 6.0 mm ventral). Steel wire insert pins were inserted into the guide cannulas to ensure that they were functional throughout the experimental period. The animals were given a one-week recovery period after surgery. They were then adapted to the test chamber for four

309

MOGENSON

3 IO

AND NIELSEN

FIG. 1. Sites of unilateral injections of picrotoxin in the region of the ventral globus pallidus, the lateral preoptic area and sublenticular substantia innominata. Doses of picrotoxin required to produce a four-fold increase in locomotor activity as compared to locomotion after injections of 09% NaCl solution are desig-

nated by the following symbols: (0.1 &O. 1 ~1) (0.2 pg10.2 PI), (0.4 &0.2 PI), (>0.4 &0.2 ~1). Brain drawings are adapted from Fig. 9 of Mogenson, Swanson and Wu [ 121 and to facilitate comparison of the behavioral observations of the present study with the earlier anatomical and electrophysiological observations, the same designations are used for the subpallidal region. Abbreviations: AHA, Anterior hypothalamus; AAA, amygdaloid area; AC, anterior commissure; BST, bed nucleus of the stria terminalis; CP, caudate putamen; GP, globus pallidus; IC, internal capsule; LPO, lateral preoptic area; MPO, medial preoptic area; OT, optic tract; PVH, paraventricular nucleus; SFO, subfomical area; SI, sublenticular substantia innominata; SO, supraoptic nucleus; VL, lateral ventricle; V,, third ventricle.

days before the effects of intracranial drug injection on IOcomotor activity were investigaied. Locomotor activity was recorded in an open-field test chamber (69X69x46 cm) by an electronic counter which detected interruptions of four independent light beams. Light beam sources were placed 23 cm apart and 5 cm above the floor on two adjoining walls. The rat was placed in this box for a 20 min pretest interval before injecting drugs. Unilateral and bilateral injections were made by inserting, to a depth of 1.25 mm beyond the guide cannula, a 30 gauge stainless steel cannula, connected by PE-10 tubing to a Hamilton microliter syringe. Injections of 0.2, 0.4, 0.5, and 1.0 ~1 were carried out over 30,60,70 and 120 set periods, respectively. Following an injection, the animal was returned to the box for 20 min and, in addition to recording the number of light beams broken each five min, its general behavior was observed. The first series of 30 rats received unilateral injections of three doses of picrotoxin (0.1 pg/O. 1 p&O.2 &0.2 ~1 and 0.4 pglO.2 ~1) in counterbalanced order. Each injection of picrotoxin was followed 24 hr later by a control injection of 0.9% NaCl of equal volume into the same cannulae. Following these tests the animals were divided into three sub-

groups for the following treatments. The effects of unilateral injections of picrotoxin (0.2 pdO.2 ~1) were compared with the effects of combined injections of PTXiGABA (0.2, 4 pg/O.2 ~1 and 0.2, 8 pg/O.2 ~1) and GABA alone (see Fig. 3, total of 25 injection sites). The effects of bilateral injections of picrotoxin (0.1 pgil ~1,0.2 pg/2 ~1, and 0.4 pg/O.2 ~1) were investigated (14 sites). These injections were made in a counterbalanced sequence and alternated with bilateral injections of O.% NaCI. The effects of unilateral injections of dopamine (20 /.&O.S ~1) were investigated (7 sites) with O.% NaCl injections as controls. A second series of animals was divided into two groups. In both groups, dopamine injections to the nucleus accumbens were held constant throughout the test (20 p&O.5 ~1) while GABA injections to the subpallidal region varied (10 &0.5 ~1 to 40 pg11.0 ~1). One group (n=6) received injections of GABA ipsilateral to the unilateral injection of dopamine. During control tests these rats received GABA (40 &l.O ~1) to the subpallidal region contralateral to the site of the dopamine injection to the accumbens. The other group (n=6) received bilateral injections of dopamine and GABA. During control tests these rats received bilateral in-

SUBPALLIDAL

GABA PROJECTIONS

311

AND LOCOMOTION

jections of 0.9% NaCl to the subpallidal region following bilateral pretreatment of the accumbens with dopamine. After completion of the experiments, the animals were sacrificed by an overdose of urethane. Their brains were perfused with 50 ml of 0.9% NaCl followed by 50 ml of buffered formalin, removed from the skull, and fmed in formalin. They were then sectioned with a freezing microtome at 80 p and stained with thionin for histological determination of injection sites. Locomotor activity was expressed as the five min average of the total number of photobeams broken during the twenty min test period. The effects of unilateral injections of picrotoxin to the subpallidal region were analysed with a repeated measures analysis of variance [25]. All possible dose-dependent responses were tested for significance using simple linear regression analysis [27]. Differences between regions in the magnitude of dose-dependency was analyses using the Newman-Keuls multiple range test on the regression slopes [27]. All pair-wise analyses were completed using the t-test for paired comparisons [27]. SALINE

RESULTS

Unilateral injections of picrotoxin into different sites in the subpallidal region increased locomotor activity, F( 1,53)=679.5, p
.2/.2

PICROTOXIN

.4/.2

DOSE tug/Ml)

FIG. 2. Dose-dependent effects of unilateral injections of increasing doses of picrotoxin into the dorsal portion of the lateral preoptic area. Each of the rats (n=9) was injected with each of the doses of picrotoxin. Injections of picrotoxin were counterbalanced with injections of 0.9% NaCl of equal volume. Results are expressed as the mean+SEM (vertical bars). The numbers at the bottom of each bar indicate the number of animals injected.

earliest experiments (compare Fig. 3 with Fig. 2). The addition of 4 and 8 pg of GABA to the 0.2 pg injection of picrotoxin to subpallidal sites reduced locomotor activity in a dose-dependent manner, F(1,49)=14.54, pcO.01. Also shown in Fig. 3 is locomotor activity after injecting 4 pg of GABA alone into the subpallidal region, which resulted in a small, but statistically significant, reduction in locomotor activity compared to the control of 0.9% NaCl, t(8)=3.65, pco.01. GABA (10,20,40 pg) was injected into subpallidal sites to see whether or not there was a reduction in locomotor activity initiated by injections of dopamine into the nucleus accumbens. The injections of GABA and dopamine were initially unilateral and then bilateral. The results are presented in Fig. 4. When dopamine was injected into the accumbens, locomotor activity was increased to approximately 45 responses/5 min for unilateral injections and to approximately 100 responses/5 min for bilateral injections. Treating the subpallidal region with GABA reduced the dopamineinitiated locomotion in a dose-dependent manner, unilateral: F(1,58)=5.62,p<0.05; bilateral: F(1,29)=14.33,p<0.01. The results of control tests are presented in the bars at the right in Fig. 4. GABA (40 fig) injected into the subpallidal region contralateral to the unilateral injection of dopamine into the nucleus accumbens resulted in a decrease in locomotion that was less than the decrease from GABA injected ipsilaterally, t(10)=4.25,p<0.01. Bilateral injections of O.% NaCl did not reduce locomotor activity initiated by dopamine injected bilaterally into the nucleus accumbens. DISCUSSION

Injections

of picrotoxin

into the ventral globus pallidus

MOGENSON

AND NIELSEN

UNILATERAL 60

5 fj

u ‘TX .2

PTX.;&ABA

P;;F6AEiA

150rBILATERAL I

GABA 4

tNJECTtON (pq) FIG. 3. Locomotor activity elicited by unilateral injections of picrotoxin f0.2 118) was attenuated in a dose-dependent manner when GABA was added to the injection solution. Nine rats were injected with a solution containing 4 gg of GABA and 2 pg of picrotoxin. Seven rats were injected with a solution containing 8 fig of GABA and 2 pg of picrotoxin. Nine rats were injected with GABA (4 fig) alone. For each of the 25 rats these iniections with GABA were counterbalanced ivith injections of picroroxin alone and 0.9% NaCl solution. The results are expressed as mean-+SEM (vertical bars). The numbers at the bottom of each bar refer to the number of animals injected.

increased locomotor activity, confirming previous observations [8,14]. Furthermore, as predicted in the Introduction from a recent anatomical and electrophysiolo~cal study, locomotor activity was also increased when picrotoxin was injected into the sublenticular substantia innominata and lateral preoptic region. The responsive sites were throughout much of the subpallidal region, as shown in Fig. 1, corresponding to the projection fields of the nucleus accumbens, as demonstrated by anatomical and electrophysiological techniques [ 121. As the dose of picrotoxin was increased the magnitude of the locomotor response increased. On the other hand, when GABA was added to the picrotoxin and injected into these subpallidal sites the picrotoxin-initiated locomotor activity was attenuated in a dose-dependent manner, Previously, it was reported that injecting GABA into the ventral globus pallidus attenuated locomotor activity initiated by injecting dopamine into the nucleus accumbens 181, suggesting that this dopamine-initiated locomotor activity is mediated by GABAergic projections to the ventral globus pallidus from the accumbens 12,191. This finding was confirmed in the present study and, in addition, similar attenuating effects occurred in a dose-dependent manner when GABA was injected into the sublenticular substantia innominata and lateral preoptic area. It appears, therefore, that locomotion initiated by injecting dopamine into the nucleus accumbens is mediated by GABAergic projections to the sub-pailidal region. Other neuropharmacologicalbehavioral experiments have implicated these GABAergic

TRE,

blo MENT

““G&yf IO/.5

SUBPALLIDAL

INJECTION

(pg/pI)

FIG. 4. Locomotor activity elicited by injecting dopamine into the nucleus accumbens was attenuated in a dose-dependent marmel when GABA was injected into the subpallidal region. At top are results for 12 unilateral injections in 6 rats in which GABA was injected into the sub~liidal region ipsilateral to the dopamine injection into the accumbens. As a controi, these rats also received a GABA 140 &I ~1) injection into the subp~lidal region contralateral to the site of the injection of dopamine into the accumbens. At bottom are results for 6 rats in which dopamine and GABA were injected bilaterally. As a control, these rats received a bilateral injection of 0.9% NaCl to the subpallidal region following bilateral pretreatment of the accumbens. Results are expressed as the meantSEM (vertical bars). The numbers at the bottom of each bar indicate the number of injections.

projections in drinking responses initiated by central injections of Angiotensin II 19,131. Locomotion was selected for investigation in the present study because it is a fundament~ component of a number of adaptive behaviors such as food procurement, escape from predators or the pursuit by the male animal of the female coming into estrous. Since iimbic integrative processes contribute to the initiation of these adaptive behaviors it has been suggested that signals for behavioral response initiation reach the motor system from the limbic system [ 11,211. There is some evidence that signals from the hippocampus may initiate exploratory locomotion [6] and that signals from the amygdala may contribute to taste-associated, food procurement [ 151.The results of electrophysiological recording experiments have suggested a model and a gating mech-

SUBPALLIDAL

GABA PROJECTIONS

313

AND LOCOMOTION

anism for limbic-motor integration which emphasizes the contributions of the nucleus accumbens and its mesolimbic dopamine afferents [15,26]. This model, when applied to behavioral response initiation should include a role for the GABAergic projection from the nucleus accumbens to the subpallidal region investigated in the present study. The efferent neural pathways from the subpallidal region that contribute to the initiation of locomotor activity have not been studied. One possibility is neural projections that have been demonst~ted from the magnoc~llul~ nucleus to the cerebral cortex [IO]. Another possibility is neural connections from the subpallidal region to mediodorsal thalamus, which in turn project to the anterior cingulate cortex and supplementary motor cortex [5]. If these neural pathways contribute to initiating locomotion they may not be essential in the rat which, after removal of the cerebral cortex, retains a high level of locomotor activity [24]. However, with encephalization of function, as in the primate, these neural connections involving subpallidal region and mediodorsal thalamus may have a more important role in the initiation of locomotion. A third possibility is that signals for the initiation of locomotion follow the recently demonstrated projections from the subpallidal area (which includes the

sublenticular substantia innominata, lateral preoptic area, bed nucleus of the stria terminalis and ventral globus pallidus) to the vicinity of the nucleus pedunculopontis [221, the site of a functional system that has been designated the mesencephalic locomotor region (MLR) [4]. The MLR in turn sends neural projections to spinal cord “generators” for rhythmical control of limb movements 141. It will now be of considerable interst to undertake experiments to see whether or not disrupting these neural pathways from the subp~lid~ region to the mediodors~ thalamus or the the MLR attenuate locomotor activity initiated by injecting picrotoxin into the subpallidal region or by injecting dopamine into the nucleus accumbens. These proposed experiments relate to the question of whether the forebrain mechanisms for behavioral response initiation (e.g., accumbens, subpallidal region) are associated functionally with brainstem mechanisms (MLR) concerned with locomotion. ACKNOWLEDGEMENTS

The authors thank Becky Woodside and Vince Nicol for assistance in preparing the illustrations. Supported by the National Sciences and Engineering Research Council of Canada.

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AND NIELSEN

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