BRAIN RESEARCH

ELSEVIER Brain Research 740 (1996) 151 - 161

Research report

Subpallidal outputs to the nucleus accumbens and the ventral tegmental area: anatomical and electrophysiological studies

M i c h a e l W u ~, A l a n W . H r y c y s h y n a S t e f a n M. B r u d z y n s k i b,

" Department (~['Anatomy and Cell Biology, Unifiers'it3" of Western Ontario. London, Ont. N6A 5CI, Cana~hl h Deparmwnt ~?[ Clinical Neurological Sciences. London Health Sciences Centre. London. Ont. N6A 5A5, Canada

Accepted 2 July 1996

Abstract

The goal of this study was to investigate the functional organization of the subpallidal ~ accumbens direct and indirect feedback loops by both anatomical and electrophysiological methods. The results of the dextran-conjugated rhodamine injections into the subpallidal area has shown three distinct projections: (1) a substantial pathway from the subpallidal area to the ventral tegmental area, (2) a more diffuse rostral projection from the subpallidal area to the core area of the nucleus accumbens, and (3) a sparse pathway projecting rostrodorsally from the subpallidal area toward the thalamic regions. Electrical or chemical stimulation of the subpallidal region, which was studied by the axonal tracer, evoked inhibitory responses in the majority (60 and 80%, respectively) of the accumbens and ventral tegmental area neurons in a standard extracellular recording study. Less than 1 / 3 of the accumbens or ventral tegmental area cells showed an increase in the mean firing rate. The majority (77.5%) of all responded neurons had a latency of less than l/) ms. Furthermore, injection of glutamate into the subpallidal area not only altered the firing pattern of the accumbens neurons, but also attenuated their excitatory responses elicited by the electrical stimulation of the ventral subiculum. Our results indicate that the subpallidal area plays a predominantly inhibitory role in the ventral tegmental area-accumbens-subpallidal circuitry, presumably by its GABAergic projections, and may also modulate subicular input into the nucleus accumbens.

Keywords." Nucleu~ accumbens: Subpallidal area: Ventral tegmental area: Single unit recording: Dextran-conjugated-rhodamine lracer: Limbic-mtm)r integration

1. Introduction

The rapid advancement of neuronal tracing and trans- mitter immunocytochemistry techniques in recent years has provided the anatomical framework for investigation o f the l i m b i c - m o t o r i n t eg ra t i ve m e c h a n i s m s [2,8,10,16.35,44]. The anatomical findings have indicated that the nucleus accumbens, a major component of the ventral striatum, is strategically located in the brain. It receives both cortical and allocortical glutamate excitatory input from the limbic system and a major dopamine input from the mesolimbic dopamine projection originating from the ventral tegmental area (VTA) [9,49,50,54]. Numerous studies have shown that the nucleus accumbens projects to the rostral division of the ventral globus pallidus, termed ventral pallidum (VP), and to its caudal division desig-

' Corresponding author. Department of Clinical Neurological, London Health Sciences Centre, University Hospital, P.O. Box 5339, Postal Stn. A, London, Ont N(~A 5A5, Canada. Fax: (1) (519) 663-3753.

nated as the subpallidal area (SP). which includes the substantia innominata and the lateral aspect of the lateral hypothalamic-preoptic region [48,51,52]. In turn, neurons of the SP, project to the pedunculopontine nucleus (PPN), a major part of the mesencephalic locomotor region (MLR) which is located in the dorsolateral mesencephalic reticular formation [12,13,16,17,24-28,36,45,46,48,51,52]. This output pathway from the basal ganglia has been shown to play a critical role in conveying the limbically-initiated signals to the motor system [24-28]. The limbic and mesolimbic signals reaching the nucleus accumbens are translated into command signals for the locomotor system. enabling adaptive behavioural responses [22,27,28]. There is evidence from previous behavioural studies that this limbic -~ accumbens ---, SP -~ MLR pathway essen- tially contributes to exploratory locomotor activity [26-28].

Although this pathway likely plays a key role in the translation of limbic signals into locomotor responses, it can no longer be assumed that the serially linked limbic -~ accumbens -~ SP ~ MLR circuit represents a

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0006-89c)3196)00859-1

152 M. Wu et al. / Brain Research 740 (1996) 151-161

unidirectional flow of information which reflects this inte- gration. It is now known that almost every major limbic region, as well as the nucleus accumbens and the SP, receive modulatory input from dopamine neurons which arise from the VTA and, in turn, the VTA receives teed- back from almost all of these structures [22,38]. There is additional electrophysiological data suggesting that the SP output fibres not only project to the MLR but also project back, directly to the nucleus accumbens [15,19]. Earlier electrophysiological evidence has suggested that a feed- back loop may exist from the nucleus accumbens to the SP, to the VTA, and back to the nucleus accumbens [19,22,23,32,51]. This loop could affect the release of dopamine in the nucleus accumbens and could ultimately be responsible for changes in locomotor activity. Neurons projecting from the V P / S P to the nucleus accumbens are likely GABAergic [21] and they may connect with GABAergic neurons in the nucleus accumbens which pro- ject to the VTA [53]. Although this GABAergic- GABAergic connection has not yet been confirmed [41], it could decrease GABAergic input to the VTA and, thus, produce a disinhibition of dopaminergic neurons, an in- crease in dopamine release, and consequently, amplifica- tion of locomotor activity [53]. Recent anatomical studies have also shown that a reciprocal GABAergic projection exists between the VP and the nucleus accumbens [8].

These cumulated results suggest that, in addition to activating neurons in the MLR [5,33,52], the V P / S P out- put may also influence, directly or indirectly, the neuronal activity of the nucleus accumbens. These loops may have important implications in the control of the output signals for exploratory locomotor activity and other behaviours [1,22]. The goal of this study was to investigate the functional organization of the SP ~ accumbens direct and indirect feedback connections by combined anatomical and electrophysiological methods. To achieve this goal, neuroanatomical tracer techniques were used to determine the extent and topographic organization of fibers originat- ing from the SP and projecting to the nucleus accumbens and to the VTA. In subsequent experiments, electrophysio- logical extracellular recordings were carried out to study the neuronal activity in the nucleus accumbens and in the VTA in response to electrical or chemical stimulation of the relevant SP region which received injections of the tracer.

2. Materials and methods

2.1. Anatomical tracing technique and microscopy

Twelve male Wistar rats weighing 260-280 g were used. The rats were anaesthetized with sodium pento- barbital (Somnotol, 60 mg/kg , i.p., M.T.C. Pharmaceuti- cals, Cambridge, Ont.) and placed in a Kopf stereotaxic frame. Dextran-conjugated rhodamine (DCR) (Molecular

Probes, Inc., Eugene, OR) was used as the anterograde tracer and was stereotaxically injected into the SP. The stereotaxic coordinates (in ram) were measured from the zero interaural line (A), midline (L) and the surface of the cortex (V) according to the atlas of Paxinos and Watson [39] and were A = 8.0-8.6, k = 1.8-2.8, V - 6 . 8 - 8 . 4 . Initially, DCR (5% solution w / v in double distilled water, 0.5 /xl at 0.25 /xl/min) was bilaterally pressure-injected into the SP. In later experiments, however, the DCR (2.5% solution in 0.45% NaCI) was applied iontophoretically into the SP. The DCR contained in glass micropipettes (40-60 /xm inner tip diameter) was ejected by 5 /xA of anodal current (Constant Current Generator, Grass Instrument Co., Quincy, MA), in 7-s pulses every 14 s over 30 rain of the ejection period (Electromatic Timing Recycler, Electro- matic Inc.). Following the delivery of the tracer, the cannula remained in place for at least 10 rain betore being withdrawn while the polarity of the current was reversed to prevent leakage of the tracer along the cannula tract. Before and following the surgery, the rats received the analgesic, buprenorphine (Temgesic, Reckitt Colman, Great Britain).

After 3 to 6 days of survival time, rats were sacrificed with an overdose of sodium pentobarbital (i.p.) and per- fused transcardially with 50 ml of 0.9% NaC1 solution followed by the fixative (500 ml of 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde). The brains were removed and immersed overnight in 15% buffered sucrose solution at 4°C. Frozen, 32 p~m thick, transverse sections were cut and placed in a phosphate buffer solu- tion. Subsequently, the sections were mounted on chrome- alum gelatin coated slides, air dried, and coverslipped with mounting medium for f luorescence microscopy (Vectashield, Vector Laboratories Inc., Burlingame, CA).

The sections were examined using a Zeiss Axioskop microscope (Carl Zeiss, Germany) equipped with a Sony DXC-930 three chip CCD camera (Sony Corporation, Japan). Images were collected into the IBM compatible Personal Computer through Northern Exposure image inte- gration software on line (Empix Imaging, Toronto, Ont.) via the Sony DXC camera. Images collected on Northern Exposure were spliced using Mocha software off line (Jandel Corporation, San Rafael, CA). The spliced images were then brought into the Adobe Photoshop program (Adobe System Incorporated, Mountain View, CA) and enhanced by setting the pixel value of all background noise ( < 40 grey level) to 0 (black) and by adjusting the contrast. Lettering of the images was added using Corel- Draw software (Corel Corporation, Ottawa, Ont.). The final images were printed out with the Tektronix Phaser 440 printer (Tektronix Inc., Wilsonville, OR).

2.2. Elec'trophysiological technique

A group of 20 male Wistar rats weighing 250-350 g were used. The animals were anaesthetized with urethane

M. Wu et al. / Brain Research 740 ( 1996~ 151 161 15

(1,15-1.2 g /kg, i.p.) and placed in a Kopf stereotaxic frame. Their body temperature was monitored by a rectal probe and the YSI-402 model telethermometer (Yellow Spring Instrument Co.) and maintained at 36-38°C by a radiant heat lamp during the recording session. The skin, skull and dura above the ventral subiculum, SP and VTA were removed to position the stimulating electrodes or injection cannulae, and burr holes of 3 -4 mm in diameter were drilled in the skull above the nucleus accumbens and/or VTA to lower the recording microelectrode. The stereotaxic coordinates for the ventral subiculum, mea- sured from the interaural zero line (A), midline (L) and the surface of the cortex (V), were (in ram) A = 2.1-2.7, L = 4.6-5.4, and V = 6.5-7.5. The coordinates for the nucleus accumbens were A = 9.5-11.7, L = 0.5-2.0, and V = 6.0-7.4, for the SP were A = 8.0-8.6, L = 1.8-2.8, and V = 6.8-8.4, and for the VTA were A -- 3.0-4.5, L = 0.2-1.2, and V = 7.8-8.6. The stimulating sites and drug injection sites were ipsilateral to the recording site. Stainless-steel, concentric, bipolar electrodes (NE-100, tip separations of 1).5 ram, Rhodes Medical Instruments, Inc., Woodland Hill. CA) were used to deliver the electrical stimulation to the SP or ventral subiculum sites. The stimulating pulses were generated by a Grass S-44 stimula- tor coupled to a Grass Stimulus Isolation Unit (SIU-5). For stimulation, monophasic square-wave pulses were used with 0.15 ms duration and current intensities of 100-1000 /.tA.

Glass micropipettes were used for extracellular record- ings of single units fi'om the nucleus accumbens and the VTA. The single barrel microelectrodes were pulled by a Narishige vertical microelectrode puller (model PE-2) to a tip diameter of 1-5 /,*m. The microelectrodes were filled with 0.5 M sodium acetate containing 2% pontamine sky blue for marking of the recording sites. Recorded poten- tials were fed into a DC differential preamplifier (Axoprobe, Axon Instruments, Forter City, CA), which was connected to a signal processing unit (Intronix, Rex- dale, Ont.) for high-pass filtering and further amplification, and then displayed on an oscilloscope (Tektronix, Beaver- ton OR). Unit activity was sampled on-line by an IBM AT computer and peristimulus time histograms were compiled from data files off line. Significant changes in the activity of neurons following stimulation were identified and quan- tified by comparing the height of each 1 ms post-stimulus bin of the peristimulus time histogram with the average height of the bin during the 100 ms period before the stimulus. The onset and termination of a significant change in the mean firing rate was defined by the occurrence of five consecutive bins with the height being within one standard deviation of the baseline mean firing rate.

Since electrical stimulation activates both the cell bod- ies and fibres of passage, chemical stimulation with gluta- mate solution was also used in order to selectively activate cell bodies. For chemical stimulation, glutamate (0.1 /xM/0.1 /xl) was microinjected into the SP over a period

of 20 s during the recording session. A Hamilton CR-700 microsyringe connected with PE-10 polyethylene tubing (Clay Adams, Parsippany, NJ) to a 30 gauge stainless-steel injection cannula was used for the injections.

Recording sites in the accumbens or the VTA were marked for later histological identification by passing a cathodal current of 6-8 /xA lk~r 10-15 min to eject pontamine sky blue into the brain. Injection sites of gluta- mate were marked with 0.1 /.d of India ink. Stimulation sites were marked with an iron deposit by passing 10 #A anodal current through the stimulating electrodes for I rain. The animal was then sacrificed by an overdose of urethane and perfused through the heart with 50 ml ol + 0.9% NaC1 solution followed by 50 ml of buffered formal- dehyde containing 2% potassium ferricyanide. The ferri- cyanide reacted with the iron deposit to form Prussian blue which labelled the position of the stimulating electrode tip. The brain was then removed and fixed in formalin for 24 h. Transverse, 70 /xm thick histological sections were cut on a freezing microtome and were stained with thionine for microscopic examination to verify the recording and stimulating sites.

3. Results

3.1. Axonal tracer experiments

Initially, either pressure injection or iontophoretic pro- cedures were used in order to determine which method would give results that labelled only efferent pathways from the SP, with little or no retrograde labelling, lon- tophoretic ejections of DCR were found to give the best results with clear anterograde labelling of axons and with very limited retrograde labelling of the somata outside the injection site. Contrary to that which was observed follow- ing pressure injection, only a few cells close to the injec- tion site were labelled retrogradely by iontophoresis.

Iontophoretic applications of DCR were characterized by an ejection site with better defined boundaries than that given by pressure injection and were limited to the SP. All ejections were placed within the rostral half of the dor- socentral SP, that is, the ejection was made within that portion of the SP which lies underneath the caudal end of the anterior commissure and the bed nucleus of the stria terminalis. These ejection sites contained abundant fluores- cent labelling of neuronal cell bodies, their dendrites and the surrounding neuropil.

Ejections of DCR into the SP produced extensive an- terograde axonal labelling which allowed for good visual- ization of the SP efferent pathways and terminal fields of interest, even at low magnification. At higher magnifica- tions, axons ran parallel to one another within the labelled pathways, while within the terminal fields, labelled preter- minal axons and terminals appeared as irregularly lying beaded fibres with terminal puncta. The distribution pat-

154 M. Wu et a l . /Brain Research 740 (1996) 151 If)l

tern of the SP efferents was similar in all cases. Fig. 1 illustrates the results from a representative animal. La- belled axons were observed to leave the SP and course either rostrally or caudally. Those that ran rostrally were moderate in number and produced a diffusely labelled pathway that swept dorsally to terminate ipsilaterally as a light terminal field within the dorsal aspect of the rostral core area of the nucleus accumhens. Only a few retro- gradely labelled cells were seen, mainly in the dorsocaudal area of the nucleus accumbens, just dorsal to the anterior commissure.

From the caudal border of the ejection site within the SP, a heavy projection of labelled axons was always seen coursing through the medial forebrain bundle to terminate within the ipsilateral VTA. This terminal field consisted of extensive terminal arborization and was much heavier than that seen in the nucleus accumbens. A third pathway was also visualized. It appeared that these fibres first travelled within the medial forebrain bundle, and then turned dor- sally, just caudal to the bed nucleus of the stria terminalis presumably to terminate within the habenulae or the mediodorsal thalamic nucleus.

3.2. Electrophysiological recording experiments

A total of 85 neurons were studied in the nucleus accumbens and VTA in response to electrical or chemical stimulation of the SP.

3.2.1. Responses o[" the accumbens neurons to electrical stimulation of the SP

Extracellular recordings were made from 33 neurons in the nucleus accumbens in nine rats. Thirty-two out of 33 neurons (97%) responded to the SP stimulation. Among them, three neurons (9.1%) were antidmmically activated. About 61% of the tested accumbens neurons were inhib- ited by SP stimulation whereas only 27.3% of the accum- bens neurons were excited by the SP stimulation. Exam- pies of both those responses are shown in Fig. 2. Most of the onset latencies of the responses (86.2%) were less than 10 ms. The duration of the excitatory or inhibitory re- sponses were less than 30 ms lbr more than hall of the neurons tested (19 out of 33, 57.6%). Eight neurons (24.2%) had response durations between 30 and 50 ms, while six neurons (18.2%) had response durations longer than 50 ms. The detailed list of responses obtained from the accumbens neurons to the electrical stimulation of the SP is presented in Table IA.

3.2.2. Responses o/ the VTA neurons to electrical stimula- tion of the SP

There were 32 neurons recorded from the VTA in six rats. Twenty-one out of these neurons (65.6%) had a long spike duration and a slow discharge rate (2.6 Hz on average) which resembled the responses of dopamine neu- rons, and therefore, were classified as type A neurons. Eleven out of 32 neurons (34.4%) had a fast discharge rate

Fig. 1. A: a photomontage of a series of fluorescence photomicrographs shown on a sagittal brain section taken about 1.4 mm lateral to the midline. The montage shows the subpallidal region (SP) projections following DCR placement in the dorsal subpallidal urea, just behind the caudal bnrdcr of the anterior commissura (ac). Anterogradely labelled fibres, sweeping both caudally and mstrally from subpallidal area and terminating m the nucleus aecumbens (NA) and the ventral tegmental area (VTA), are readily distinguished. B: a high magnification fluorescence photomicrograph of anterogradely labelled fibres within the ipsilateral nucleus accumbens (rectangle in A). Notice the punctate labelling and the anterogradely labelled axons containing varicosities (arrows). Scales: 400 /~m in A and 135 /xnl in B.

M. Wu et al. / Brain Research 740 (1996) 151 161 155

no. of spikes

20q

1o~

O, ~0

A

,t I,]lllllllll~l ~llll~th~liJ~llllLl~l~'llIJIJL[llllilLll~ll~[,li,liil, ~l~lllJL~ll,lll 100 1200 1300 1400 ~500 ms r

no. of spikes

20-

tO ~

~0

A

[ ~ , , _

It00 1200 1300 400 1500 m~

B

no. of spikes

20 l

r '°l I 0 IlO 0 1200 1300 1400 r500 ms i

Fig. 2. Peri-stmmlus histograms showing the typical responses of the spontaneously active accumbens neurons to stimulation of the subpallidal area. A: inhibitory response (onset latency of 4 ms) to single-pulse stimulation of the subpallidal area. B: excitatory response (onset latency of 7 ms) to single-pulse stimulation of the subpallidal area. Electrical stimulations were 500 p,A in intensity, 0.15 ms in duration at 1 Hz in both A and B. Hi-,tograms were compiled from 156 sweeps for A, and 800 sweeps fol B.

(12.6 Hz on average) and a short spike duration and were classified as type B neurons. As indicated in Table 1B, single-pulse electrical stimulation of the SP inhibited 12

B

no. of spikes

201

l 0

' ]

l0 It00 ~200 1300 [400 tS00 n |~

Fig. 3. The typical responses of the type A neuron in the ventral tegmental area to stimulation of the subpallidal area. A: inhibitory response with onset latency of 2 ms produced by stimulation of the subpallidal area at 500 /xA. B: excitatory response with onset latency o1"6 ms produced by stimulation of the subpallidal area a! 5()0 /,tA. HN- tograms were compiled of 259 and 924 sweeps, respectively.

(57.2%) and facilitated 7 (33.3%) of the type A neurons, and inhibited 7 (63.6%) and facilitated 3 (27.3%) of the type B neurons. The onset latency for both the excitatory and the inhibitory responses were similar. Sixty percent of

Table I Responses of tile accumbens and the VTA neurons to electrical stimulation of the subpallidal area

Type of response Onset latency (ms)

< 5 5 10 > 10

Number of neurons

(A) Accumbens neurons ill nine rats: Inhibition 10 Excitation 3 Antidromic actt val i()ll 1 No responses Total

(B) VTA ncurons m six rats: TVIIC A nelllY)ll~(

Inhibition 3 Excitation 2 No responses Total 7)7~e B neHvolts

Inhibition 6 Excitat ion 2

No responses Total

8 2 4 2 2

4 5 3 2

0 I

0 1

20 9 3 I

33

12

7

2

21

7

3

I

II

60.6

27.3

~). l }.()

I00.()

57.2 33.3

~').5 IOO.O

63.6

27.3

O. l

1 0 0 . 0

156 M. Wu et al./Brain Research 740 (1996) 151 161

all responses had onset latency less than l0 ms, while the remaining responses (31%) had onset latency longer than 10 ms. Examples of excitatory and inhibitory responses of type A neurons is shown in Fig. 3.

A

3.2.3. Effects of glutamate injection into the SP on neu- ronal actit,i~ in the nucleus accumbens

Extracellular recordings of neuronal activity were ob- tained from 20 neurons in the nucleus accumbens in six rats. The mean firing rate of the spontaneously firing neurons was reduced in 16 neurons (80%) when glutamate (0.1 /~1) was injected into the SP, while the mean firing rate of the remaining four (20%) spontaneously firing neurons was increased after the intra-SP glutamate injec- tion. Examples of these responses are presented in Fig. 4. Five of the 20 neurons were also tested in response to electrical stimulation of the ventral subiculum. All neurons responded to the electrical stimulation of the ventral subiculum: four neurons were excited and one was inhib- ited by the subicular stimulation. An example of these responses is shown in Fig. 5.

Two neurons which were activated by the stimulation of the ventral subiculum were tested for the effect of gluta- mate injection into the SP. As shown in Fig. 6, the excitatory responses resulting from subicular stimulation were attenuated immediately after the injection of gluta- mate (0.1 /~1) into the SP. The excitatory responses from

A nO. of spikes

40 GLU - - GLU - -

20

0 i100 1200 1300 '400 1500 s '

no. of spikes

I 0

l0 ~100 1200 1300 1400 500 ms I

B no. of spikes

40q GLU - -

f0 150 I100 1150 1200 r250 s'

Fig. 5. An example of an accumDens neuron which responded to both

electrical stimulation of the ventral subiculum and the glutamate injection

into the subpallidal area. A: peri-stimulus histogram showing the in- hibitory responses of a accumbens neuron to the stimulation of the ventral subieulum (500 /~A, 1 Hz, 207 sweeps). B: t ime frequency histogram

showing the neuronal activity of the same accumbens neuron which was

reduced by the injection of glutamate into the subpallidal area. In this cell

glutamine injection time was 27 s.

the subicular stimulation started to recover 9 min after the glutamate injection and were fully recovered after 45 min.

A summary of all electrical and chemical stimulation sites in the SP is illustrated in Fig. 7.

B no. of spikes

40 GLU - -

20

01, .,, b., , , , . 0 1100 1200 1300 ~400 '500 s I

Fig. 4. Time frequency histograms showing the responses of the accum- t e n s neurons to the injection of glutamate (0.1 /xM/0 .1 p.l) into the subpallidal area. A: results of the injection of glutamate into the subpalli- dal area which reduced the neuronal activity of the accumbens neurons

(80% of the accumbens neurons tested). B: result of the injection of glutamate into the subpallidal area which increased the neuronal activity (20% of the accumbens neurons).

4. Discussion

4.1. Anatomical considerations

Examination of the present results of the axonal tracer experiment has shown three distinct projections from the VP: (1) a substantial pathway to the VTA, (2) a more diffuse rostral projection to the core area of the nucleus accumbens, and (3) a less prominent pathway projecting rostrodorsally from the VP toward the thalamic regions.

The anterograde labelling obtained by iontophoretic ejection of DCR is consistent with results of recent studies showing that the DCR is a very sensitive anterograde neuronal tracer [34,43]. Although, both the iontophoretic and pressure injections of DCR produced excellent antero- grade labelling with various degrees of retrograde labelling [34], the iontophoretic method appeared to be more selec-

M. Wu et al. / Brain Research 740 ~1996) 151-161 157

tive in obtaining only anterograde labelling. The retrograde labelling of a few neurons obtained after iontophoretic application of DCR might be explained in the following way. A DCR-labelled cell was most probably labelled (1) because its axon was damaged by the ejecting cannula, then the tracer was incorporated into the axon at the damaged site, and then transported back to the soma of the cell, or (2) because of leakage of the tracer which could occur during placement or withdrawal of the cannula, even though the holding current of reverse polarity was applied to prevent such leakage along the cannula tract.

The results of this anatomical study are in agreement with recent studies describing ventral pallidal connections [2,8, l 1,30]. The most conspicuous projection was the palli- dotegmental pathway, however, the less conspicuous recip- rocal connections between the nucleus accumbens and the V P / S P may represent part of an important feedback loop

in the ventral striatopallidal and pallidostriatal relation- ships. A dense projection from the nucleus accumbens to the VP has been described [16.36]. Of particular interest is the pallidostriatal projection from the VP to the accumbens [2,3,11,40]. This projection was reported to be topographi- cally organized and, to a certain extenk to parallel the pattern of reciprocal innervation from the nucleus accum- bens to the VP [8,11]. Results of the present study showed a projection from the rostral half of the dorsocentral SP which terminates in a diffuse pattern mostly within the core region of the nucleus accumbens. A similar pattern of projection has been also obtained in another study after iontophoretic application of fluoro-gold into the medial core of the nucleus accumbens which resulted in retro- gradely labelled neurons in both the subcommissural VP and in the dorsomedial portion of the VP and rostral SP [8]. An important functional consideration is that the

no. or spikes

10

0

A no . o f s p i k e s

2o 1

,01 i

D

a , _ _ . 0J~s_ i , , j , ,j , L : F100 1200 1300 1400 500 ms 0 100 200 '300 r400 500 rns

no . o f s p i k e s

2° 1 i i

i1 f0

B E n o . o f $ p l l ~ s

,0 i

0Jr0' L i I100 1200 r300 r400 sO0 ms IlO0 ~200 '300 '400 ;500 ms

n o . o f s p i k e s

20!

,0] i i

0J: 0

C F no . o f s p i k e s

20~

i

1.1

o. ~. (I ~ i~ l o ) 400 5ol l t i t , 1 flo 211o 3(H) 41)(I 5qlql in~

Fig. 6. Peri-stimulus histograms showing the excitatory responses of an accumbens neuron to the electrical stimulation of the ventral subiculum and its subsequent attenuation by the glutamate injection into the subpallidal area. A: excitatory responses to single-pulse stimulation of the ventral subiculum. B,C: attenuation of the excitatory responses of the same neuron to electrical stimulation of the ventral subiculum right after and 6 rain after the injection of the glutamate into the subpallidal area, respectively. D,E: slow recovery of the excitatory responses of the neuron to the ventral subiculum stimulation 9 and 12 min after the glutamate injection, respectively. F: a fully recovered excitatory response of the neuron to the ventral subiculum stimulation 45 rain after the glutamate injection. All histograms were compiled of 206 sweeps.

158 M. Wu et a l . /Brain Research 740 (1996) 151-161

Fig. 7. A composite diagram of all stimulation sites (circles) shown on the frontal sections of the rat brain 8.2 and 8.08 mm anterior from the interaural plane according to the stereotaxic atlas [39]. Circles on the left hand side of both the brain sections (n = 6) indicate deposition sites for India ink which correspond to injection sites for 0.1 #1 of glutamate. Circles on the right hand side (n = 13) indicate Prussian blue deposits which correspond to sites for electrical stimulation. The scale bar is 1 ram. Abbreviations: AA, anterior amygdaloid area; BN, bed nucleus of stria terminalis; cc, corpus callosum: CP, caudate putamen; DB, horizon- tal limb of diagonal band; fx, fornix; GP, globus pallidus; ic, internal capsule; MC, magnocellular preoptic nucleus; MPA, medial preoptic area; ox, optic chiasm; SF, septofimbrial nucleus; SH, striohypothalamic nucleus; sm, stria medullaris; SP, subpallidal area; TS, triangular septal nucleus; VP, caudal end of the ventral pallidum.

VP/SP and accumbens is reciprocally connected by GABAergic neurons [8,55].

4.2. Electrophysiological considerations

The most important finding of this study is that electri- cal or chemical stimulation to the region of the SP, into which DCR was ejected, evoked mostly inhibitory re- sponses (60 and 80% of responding cells, respectively) of the spontaneously firing accumbens and VTA neurons in a standard extracellular recording procedure. Less than 1/3 of the accumbens or VTA cells showed an increase in the mean firing rate. The majority (78.5%) of all the respond- ing neurons had a latency of less than 10 ms.

The present results remain in agreement with earlier electrophysiological findings that demonstrated a substan- tial monosynaptic VP + accumbens feedback. The spon- taneously active accumbens neurons responded in a vari- able way to VP/SP stimulation (26% of cells were inhib- ited, 24% excited, 12% 'driven', - i.e. activated silent cells, and 16% affected in a complex way) [15]. In the present study, inhibition of the mean firing rate represented the predominant type of response (62.5% of responding

neurons). The difference between these results may be explained by the difference of location of the stimulation site in the VP/SP. While in the study by Hakan et al. [15], the stimulating electrode was placed in a broadly defined region including the lateral hypothalamus, the substantia innominata, and the VP, electrodes in the present study were located within the SP proper, mostly in the centro- dorsal aspect of the SP. It is interesting to note that some of the inhibitory responses of the accumbens neurons lasted more than 80 ms in our study, and up to 450 ms in the other study [15]. The predominance of inhibitory con- nections between the VP/SP and the nucleus accumbens is further corroborated by the finding that the projection from the ventral VP/SP to the accumbens is primarily GABAergic. In some instances, over 80% of the retro- gradely labelled cells in the VP/SP-accumbens projection were also double labelled for glutamic acid decarboxylase mRNA [8]. It seems therefore, that the reciprocal connec- tions between the VP/SP and the nucleus accumbens are mutually GABAergic. There are, however, other results suggesting that the GABAergic terminals from the VP/SP cells may not terminate directly on GABAergic neurons in the nucleus accumbens [41].

Furthermore, the present results suggest that injection of glutamate into the SP not only altered the firing pattern of the accumbens neurons but also reversibly attenuated exci- tatory responses elicited by electrical stimulation of the ventral subiculum. These results imply that SP neurons may have a modulatory influence on the hippocampal input to the nucleus accumbens. These observations are also consistent with the findings that neurons in the nu- cleus accumbens have monosynaptic convergence of in- puts from fimbria and the VP/SP [15].

The inhibitory effects resulting from SP stimulation could also be induced by direct or indirect activation of dopamine neurons in the VTA. As shown in the present as well as previous studies, the effects of VP stimulation can reach VTA neurons directly [1 I] or indirectly via the PPN [14,37]. Thirty-three percent of electrophysiologically dis- tinguished dopaminergic neurons in the VTA showed exci- tatory responses with short latencies to stimulation of the SP. Activation of VTA neurons can release dopamine in the nucleus accumbens with its resultant inhibitory effects. Pharmacological disinhibition of the VP/SP neurons has been postulated to involve an increase of accumbens dopamine via the feedback projection from the mesolimbic dopamine system [1,21]. Involvement of this VTA feed- back loop, however, would require a considerably longer latency than that predominantly observed in our study. Thus, the results of the present study suggest that a direct SP ~ accumbens pathway is most likely responsible for the inhibitory effects in the nucleus accumbens. If so, the GABAergic projection from the SP to the nucleus accum- bens may also influence the limbic input to the nucleus accumbens. On the other hand, some of the accumbens neurons showed latencies longer than 10 ms ( 12.5%, Table

M. W , et al. / Brain R,'vearch 740 ~ 1996 J /"7 / /(J I [ 50

I A) in response to SP stimulation, and thus, these long latency responses could have resulted from an indirect VTA relay. Dopamine release in the nucleus accumbens may also be controlled by an inhibitory influence of V P / S P neurons on dopaminergic cells in the VTA. Pre- dominance of inhibitory responses (57.2%, Table 1B) of electrophysiologically distinguished dopaminergic neurons in the VTA to SP stimulation would suggest existence of such a con t ro l mechanism.

4.3. l~u#u'tiomtt con.~'iderations

A substantial amoun t of experimental data implicates the pathway l'ronl limbic structures to the nucleus accum- bens --, VP /SP --, MLR in regulation of locomotor activity [22.25 29]. The GABAergic projection from the nucleus accumbens to the VP /SP appears to be a critical component in regulating behavioural responses. It is be- lieved that inhibition of this GABAergic projection by endogenous dopamine in the accumbens initiates locomo- tor activity. This conclusion is based on a number of observations. Pharmacological agents which antagonize the dopamine action in the nucleus accumbens [32], or which block dopamine release or synaptic activity in the accum- bens [6]. were reported to cause a decrease in exploratory locomotor activity. Systemic application of amphetamine, which restilted in an increase in locomotor activity, was reported to significantly reduce extracellular concentration of GABA in the VD in an in r ive microdialysis study [4]. Injection of GABA or its agonist, muscimol, into the VP inhibited locomotor activity induced from the nucleus ac- cumbens [20,31] or inhibited activity caused by apomor- phine following 6-OHDA lesion of the nucleus accumbens [47]. Contrary, injection of the GABA antagonist, picro- toxin into the VP resulted i l l all increase of locomotor activity [7.3 11.

In light (11 these findings, reciprocal inhibitory connec- tions between the VP//SP and accumbens add new infer marion about functions of this circuitry. Furthermore, the present findings also show a predominantly inhibitory effect of SP stimulation on the mean firing rate of VTA neurons. This result is in agreement with the results of studies using tracer techniques combined with in situ hybridization for glutamate decarboxylase mRNA which showed a GABAergic projection from the VP to the VTA [21]. Recent hehavioural data has further emphasized that the integrity el lhe circuit that contains the VTA, the nucleus accumbens and the VP, is required for the mani- (estation of noxelty-induced exploratory activity in rats 11 8]. Application of the dopamine antagonist, fluphenazine, rote the accnmbens, muscimol, a GABAa agonist into the VP, or baclofen, a GABAu agonist, into the VTA pre- vented novehs.-induced locomotor activation without sup- pressing the activity of habituated animals [18]. It is be- lieved thai this circuit, mediating novelty-induced ex- ploratory aciixit-, is limbically-driven and the input from

the ventral subiculum to the nucleus accumbens initiates the response via the nucleus accumbens ~ VP /SP --, MLR pathway [22,25,28,29J. Thus, during exploratory ac- tivity associated with novelty, excitatory responses from the ventral subiculum would be relayed to the accumhens neurons. The GABAergic projection fnnn the VP /SP to the nucleus accumhens would cause release el: GABA in the nucleus accumbens and a subsequent inhibition of the novelty-driven behaviour. Our present findings ha'~e shown that, indeed, stimulation of the SP with glutamate attenu- ated excitatory responses of the accumbens neurons caused by electrical stimulation of the ventral subiculunL This linc of discussion is also supported by beha\ioural findings that injection of GABA into the nucleus accumhens inhibited dopamine-induced hyperactivity [42]. The present results and the results of others indicate that the VP and its rostral extension. SP, play a predominantly inhibitory role in this circuitry, prestimably by its GABAergic projections both to the nncleus accumbens and to the VTA.

Acknowledgements

The study was supported by an operating grant to S.M. Brudzynski from the Medical Research Council of Canada.

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