Indirect nucleus accumbens input to the prefrontal cortex via the substantia nigra pars reticulata: A combined anatomical and electrophysiological study in the rat

Neuroscience Vol. 61, No. 3, pp. 533-545, 1994

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Pergamon

0306-4522(94)E0138-T

Elsevier ScienceLtd Copyright © 1994 IBRO Printed in Great Britain. All rights reserved 0306-4522/94 $7.00 + 0.00

I N D I R E C T N U C L E U S ACCUMBENS INPUT TO THE P R E F R O N T A L CORTEX VIA THE SUBSTANTIA NIGRA PARS RETICULATA: A COMBINED ANATOMICAL AND ELECTROPHYSIOLOGICAL STUDY IN THE RAT J. M. DENIAU,*~"A. MENETREYtand A. M. THIERRY~ tUniversit6 Pierre et Marie Curie, D~partement de Neurochimie-Anatomie, Institut des Neurosciences U.A. 1199, 4 Place Jussieu, 75230 Paris Cedex 05, France :~INSERM U 114, Chaire de Neuropharmacologie, Colldge de France, 11 Place M. Berthelot, 75231 Paris Cedex 05, France Abstract--The nucleus accumbens is a major component of the ventral striatum through which most of the limbic affiliated cortical areas gain access to the basal ganglia circuitry. In this study, the organization of the pathways linking the nucleus accumbens to the thalamus, via the substantia nigra pars reticulata, was examined in the rat using anatomical and electrophysiological methods. Use of anterograde and retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase has established that the core of the nucleus accumbens innervates a dorsal region of the substantia nigra pars reticulata which projects to subfields of the mediodorsal and ventral medial thalamic nuclei. These subfields consist of the rostral pole of the mediodorsal nucleus with the exception of its central segment and a region of the ventral medial nucleus, medial to the mammillothalamic tract. Confirming the existence of a nucleus aceumbens nigrothalamic link, we have observed that electrical or chemical stimulation of the nucleus accumbens induces an inhibition of the spontaneous discharges of the nigral cells which project to the mediodorsal and ventral medial thalamic nuclei. Finally, the cortical projections of the thalamic subfields involved in the nucleus accumbens nigrothalamic circuit were determined using the anterograde and retrograde axonal transport of wheatgerm agglutinin conjugated with horseradish peroxidase. These subfields innervate mainly the prelimbic and to a lesser degree the orbital areas of the prefrontal cortex. The present data show that the substantia nigra pars reticulata is a major link beteen the core of the nucleus accumbens and the prefrontal cortex and provide further evidence for the concept of a parallel architecture in the basal ganglia thalamocortical circuits of the ventral striatum.

The basal ganglia are functionally related to the cerebral cortex through multisynaptic loop circuits. 2s The striatum, which corresponds to the major input structure of the basal ganglia, receives projections from all areas of the cerebral cortex. In turn, via the nigro- and pallidothalamic pathways, the striatum indirectly influences the cortical mantle. There is growing evidence that the functional architecture of these circuits is essentially parallel in nature} ,2,',m7,~9 The organization of the corticostriatal projections is such that information from functionally distinct cortical areas is processed in separate striatal subterritories. Moreover, according to the topographic organization of the striatal efferent pathways to the pallidum, to the substantia nigra and secondarily to the thalamus, very likely the segregation of corticostriatal inputs is further maintained through-

*To whom correspondence should be addressed. Abbreviations: MD, mediodorsal thalamic nucleus; NAcc,

nucleus accumbens; SN, substantia nigra; SNR, substantia nigra pars reticulata; VM, ventral medial thalamic nucleus; WGA-HRP, wheatgerm agglutininhorseradish peroxidase. 533

out the subsequent parts of the circuit. The parallel architecture of the trans-striatal circuits is well established for the dorsal striatum, but such an organization has not yet been completely demonstrated for the ventral striatum. The nucleus accumbens (NAcc), a main component of the ventral striatum, processes information from limbic affiliated cortical areas (medial prefrontal, insular, entorhinal and hippocampus; for review see Ref. 37) and, as a typical striatal structure, sends prominent projections to both the pallidal complex (including the entopeduncular nucelus and the ventral pallidal area) and the substantia nigra (SN). 7'20'26'36-38The NAcc comprises two main subdivisions, the core and the shell, which have been defined on the basis of histochemical and cytoarchitectural characteristics (for review see Ref. 37). Although the projections of the N A c c to the ventral pallidum originate from these two subdivisions, the projections to the SN arise mainly from the core. 7'37'3s The existence of a pathway from the N A c c to the ventral pallidum, and from the ventral pallidum to the mediodorsal thalamic nucleus, is consistent with the participation of the N A c c in a prefrontal

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channel which parallels the sensorimotor circuits of the dorsal striatum) 7 ~9.39In contrast, the projections of the core of the N A c c to the substantia nigra pars reticulata (SNR) has been considered as a possible exception to the parallel architecture of the striatal circuits. 37 Indeed, through its efferents to the SNR, this output of the NAcc has been suggested to be directed towards motor thalamic nuclei, indicating that the N A c c could play a role of interface between limbic and m o t o r systems. However, besides its major projection to the m o t o r thalamic nuclei, there is now evidence that the S N R also provides a substantial innervation to thalamic areas in relation with the prefrontal cortex. ~°'15'23 Thus, the possibility exists that, via the SNR, the output of the N A c c remains within the prefrontal circuitry and is not oriented towards the motor system. Therefore, the present anatomical and electrophysiological study was undertaken in the rat to further characterize the nigro-thalamo-cortical output circuits of the NAcc. F o r this purpose, using the axonal transport of wheatgerm agglutininhorseradish peroxidase complex ( W G A - H R P ) , we endeavoured to delineate the nigral regions which receive the terminal field of N A c c efferents and determine the thalamic areas innervated by this nigral region. In addition, the cortical projections of these thalamic areas were also investigated. Finally, to ascertain the existence of a functional connection

between the NAcc and nigrothalamic neurons, the effect of N A c c stimulation on nigrothalamic cells (identified by antidromic activation) was analysed. EXPERIMENTAL PROCEDURES

Anatomical analysis

Experiments were performed on 15 adult male Sprague-Dawley rats (Charles River, France). For all surgical procedures, animals were anaesthetized with an initial injection of pentobarbitone (40 mg/kg i.p.; Pentobarbital sodique, Sanofi) supplemented by administration of ketamine (30 mg/kg i.m., Imalg~ne, Rh6ne-M~rieux). Single unilateral injections of WGA-HRP (Sigma product, 2.5% in 0.9% saline) were stereotaxically placed in the NAcc, the SN and the thalamus using glass micropipettes (internal tip diameter 15 #m) and iontopboretic delivery methods (positive current pulses of 5~A for 5-15min). Following a survival period of 36-48 h, the animals were deeply anesthetized with pentobarbitone and intracardiaUy perfused with 0.9% saline (100ml), 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4; 500 ml) and a 10% sucrose solution in phosphate buffer (0.1M; pH7.4; 200ml). The brains were removed and stored overnight in this buffer before 50 #m frontal plane sections were cut on a freezing microtome and processed for histochemistry using the tetramethyl benzidine method of Mesulamfl~ Sections were then mounted onto chrome-alum-coated slides, counterstained with Safranin and dehydrated through alcohol to xylene for light microscopic examination. The local diffusion of the tracer at the injection sites and the patterns of anterograde and retrograde labelling were analysed under both dark-field and bright-field illumination, reconstructed

Fig. 1. Projection of the core of NAcc to the SN (I) and distribution of nigral cells projecting to the rostral MD (II) and the medial VM (III). The injection sites of WGA-HRP in the NAcc and the thalamus are shown in the left part of the figure. In each case, the distribution of anterograde and retrograde labelling is illustrated on frontal sections of the SN. Caudal left, rostral right, cc, corpus callosum; CPu, caudate-putamen; G, gelatinosus nucleus; LD, lateral dorsal thalamic nucleus; MD, mediodorsal thalamic nucleus; SNC, substantia nigra pars compacta; VL, ventral lateral thalamic nucleus; VM, ventral medial thalamic nucleus; VP, ventral posterior thalamic nucleus.

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Nucleus accumbens nigrothalamic circuit with the aid of a drawing tube at high power magnification ( x 400) and photographed.

Electrophysiological approach Adult male Spragne-Dawley rats (N = 20) anaesthetized with ketamine (100 mg/kg i.p. supplemented by 50 mg/kg i.m. injections) were fixed in a conventional stereotaxic apparatus. Their body temperature was monitored and kept at 37.5°C with a homeothermic warming blanket (Harvard Apparatus, Kent, U.K.). Activities of single nigral ceils were recorded extracellularly using glass micropipettes (7-10 Mfl) filled with 4% Pontamine Sky Blue dissolved in a 0.6 M sodium chloride solution. Amplified spike potentials were A/D converted through voltage discriminators and peristimulus time histograms of unit firing were constructed on-line using a computer connected to the 1401 Cambridge Electronic Design Interface (Cambridge Electronic Design Limited, Cambridge, U.K.). Typically, 100 stimuli were used for each histogram. Nigrothalamic neurons were identified by their antidromic activation following stimulation of the ipsilateral ventral medial and mediodorsal thalamic nuclei. The antidromic potentials were characterized by their fixed latency at threshold and their collision with spontaneous discharges within an appropriate time interval. Electrical stimulations of the NAcc (A 10.6; H 3.2; L 1.8), and of the mediodorsal (A 6.7; L 0.5; H 4.3) and the ventral medial (A 6.8; L 0.9; H 2.6) thalamic nuclei, ipsilateral to the recording site, were performed through co-axial stainless steel electrodes (diameter 400#m, tip barrel distance 300/~m), positioned stereotaxically according to the atlas of Paxinos and Watson. 3° Electrical stimuli consisted of monopolar pulses of 0.2 ms width and 100-300/~A intensity. In some cases, a chemical stimulation of the NAcc has been achieved by local application of glutamate.

Monosidum glutamate (0.1 M in 0.15 M saline) was administered via a glass micropipette (internal tip diameter 40 # m) fitted to a pneumatic picopump (PV 820, World Precision Instruments, U.K.) allowing the delivery of calibrated pulses of pressure (4 p.s.i., 2 s). The effectiveness of glutamate application was controlled by local unit recording performed with a tungsten microelectrode (2-5 Mf~). In some experiments in which the conduction time of action potentials of the NAcc-SN pathway was determined, a bipolar co-axial stimulating electrode was implanted in the SN (A 3.7; L i.8; H 1.6) and antidromic activation of cells in the ipsilateral NAcc was extracellularly recorded using a glass micropipette (as above). At the end of each recording session, the tips of the stimulating electrodes were marked by electrical deposit of iron (15/~A anodal, 15s) and observed in histological sections following a ferri-ferrocyanide reaction. The tip of the recording microelectrode was marked by iontophoretic ejection of Pontamine Sky Blue (8 #A cathodal, 20 min), which allowed determination of the positions of the recorded cells. Brains were fixed with a 10% formalin solution and the positions of electrodes identified microscopically in serial frozen sections (100/~m) stained with Cresyl Violet. RESULTS

Nucleus accumbens efferents to the substantia nigra The distribution p a t t e r n o f the projections from the N A c c to the S N was investigated in two animals. Unilateral injections of W G A - H R P were m a d e into the core o f the N A c c t h r o u g h o u t its extent (Fig. 1, I). These injections resulted in a dense terminal labelling

~VPL II Fig. 2. Concurrent visualization of retrogradely labelled neurons in the striatum and anterogradely labelled fibres in the thalamus following an injection of W G A - H R P into the dorsomedial SNR. (I) W G A - H R P injection site. (II) Distribution of retrogradely labelled ceils in the striatum. Note that most of the labelled cells are present in the core of the NAcc. Numbers indicate the approximate distance in mm from the interaural line. (III) Anterograde labelling within the MD and the VM. AV, anteroventral thalamic nucleus; CL, central lateral thalamic nucleus; CM, central medial thalamic nucleus; Hb, habenular nucleus; IAM, interanteromedial thalamic nucleus; mt, mammillothalamic tract; VPL, ventral posterolateral thalamic nucleus. Other abbreviations as in Fig. 1.

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in the ipsilateral SN. Confirming previous observations, 7'26'36 the labelled terminal field occupied a mediolaterally oriented strip in the dorsal edge of the pars reticulata and the adjacent portion of the over-

leying pars compacta (Figs 1, I and 3A, B). The projections of the N A c c were strictly confined medially in the rostral part of the SN and progressively extended more laterally at caudal levels.

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Fig. 3. (A, B) Dark-field photomicrographs of anterograde labelling in the SN following an injection of WGA-HRP into the core of the NAcc (injection site illustrated in Fig. I). (C-E) Anterograde labelling in the MD (D) and the VM (E) after injection of WGA-HRP into the dorsomedial SNR (injection site shown in Fig. 2). Rectangles in C delineate the regions of the MD and VM shown in the photomicrographs. Arrows in D and E point to the anterograde labelling. (F, G) Retrogradely labelled cells in the SNR following injection of WGA-HRP into the rostromedial part of the MD (F) or VM (G). Injection sites are depicted in Fig. 2. ZI, zona incerta. For other abbreviations see Figs 1 and 2. Scale bar = 350/~m (A); 150~m (B-D): 50~m (F, G).

Nucleus accumbens nigrothalamic circuit

Nigrothalamic projections from the nigral area innervated by the nucleus accumbens Thalamic areas receiving projections from cells located in the region of the SN innervated by the NAcc were studied using the anterograde and retrograde transport of W G A - H R P .

Distribution of anterogradely labelled fibres in the thalamus following wheatgerm agglutinin-horseradish peroxidase injection into the substantia nigra. In three animals, W G A - H R P injections were placed within the dorsal SN. The tracer remained limited to this region and infiltrated the dorsal pars reticulata, the pars compacta and the adjacent part of the ventral tegmental area (Fig. 2, I). The localization of the injection site in the terminal field of afferents from the NAcc was attested by the resulting distribution of retrogradely labelled neurons within the core of the NAcc and the immediate adjacent region of the dorsal striatum (Fig. 2, II). As a result of this injection, anterogradely labelled axons were found mainly in the ipsilateral mediodor-

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sal (MD) and ventral medial (VM) thalamic nuclei (Figs 2, III and 3D, E). Within the MD, labelled fibres occupied mostly the medial and ventral edges of the nucleus in its rostral pole but avoided the central segment. Labelled fibres were not observed in the caudal part of the MD. Within the VM, the labelled terminals were distributed within a narrow band localized at the level of the mammillothalamic tract. Although labelled fibres were present throughout the rostrocaudal extent of the nucleus, their density was more prominent rostrally. In addition to these two main projection fields in the VM and the MD, a few labelled fibres were also observed in the region of the centralis medialis adjacent to the MD and in the rostral parafascicular nucleus, along the fasciculus retroflexus.

Distribution of retrogradely labelled cells in the substantia nigra following wheatgerm agglutininhorseradish peroxidase injection into the ventral medial and the mediodorsal thalamic nuclei. W G A - H R P injections were placed into the subfields of the MD

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Fig. 4. Anterogradely labelled fibres in the cerebral cortex following injection of WGA-HRP centred in the regions of the MD (A) and of the VM (B) innervated by the dorsomedial SNR. The injection sites are shown in Fig. 1. The labelling of thalamocortical axons is illustrated on frontal sections. Caudal left, rostral right. The dotted lines indicate the boundary of cortical layer I. ACd, anterior cingulate cortex; aci, anterior commissure, intrabulbar; AG, agranular cortex; AGI, agranular cortex lateral; AGm, agranular cortex medial; Aid, agranular insular cortex dorsal; fmi, forceps minor corpus callosum; IL, infralimbic area of the medial frontal cortex; LO, lateral orbital area; MO/VO, medial/ventral orbital area; PL, prelimbic area of the medial frontal cortex; VLO, ventrolateral orbital area. Other abbreviations as in previous figures.

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and the VM as described above (Fig. 1, II, III) to investigate the distribution of retrogradely labelled neurons in the SN. One successful injection located in the MD subfield infiltrated the ventral and medial regions of the nucleus as well as the adjacent part of the central segment. Most of the retrogradely labelled neurons were confined to the dorsal part of the pars reticulata and sparse labelled cells were also observed within the pars compacta (Figs 1, II and 3F). Interestingly, the distribution pattern of these neurons was strikingly similar to that of the NAcc axon terminals within the SN. These neurons occupied a medial position in the rostral SN and progressively shifted more laterally at caudal levels. In addition, a few labelled cells were also observed ventrolaterally along the cerebral peduncle. In two rats, the injections were centred into the medial part of the VM (Fig. 1, III). The injection sites extended to the lateral edge of the mammillothalamic

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tract and also involved the ventral part of the nucleus gelatinosus. The distribution of the retrogradely labelled neurons in the SN was reminiscent of that obtained after the MD injection (Figs 1, III and 3G). Most of the labelled cells were present in the dorsomedial part of the SNR, in the region where the NAcc efferents were shown to terminate. A few other labelled cells were scattered along the ventrolateral edge of the SN. Projections to the cerebral cortex o f the thalamic areas implicated in the nucleus accumbens nigrothalamic circuit

Following the identification of the rostral MD and the medial VM as the main targets of the NAcc-nigrothalamic circuit, the cortical areas innervated by these two thalamic regions were delineated by anterograde and retrograde axonal transport of WGA-HRP,

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Fig. 5. Charts of retrogradely labelled cells in the thalamus following injections of WGA-HRP into the medial/ventral orbital (A), the lateral/ventrolateral orbital(B), the prelimbic (C) and the medial agranular (D) cortical areas. For each case, the maximal extent of the injection site is shown in the left part of the figure and the distribution of retrogradely labelled cells is represented on frontal sections from caudal (left) to rostral (right). Re, reuniens thalamic nucleus; Zl, zona incerta. Other abbreviations as in previous figures.

539

Nucleus accumbens nigrothalamic circuit

Distribution of anterogradely labelled fibres in the cerebral cortex after wheatgerm agglutinin-horseradish peroxidase injections into the ventromedial parts of the mediodorsal and the ventral medial thalamic nuclei. The cortical projections of M D and VM subfields innervated by the dorsal SNR were also examined in the animals injected with W G A - H R P into these thalamic regions (Fig. 1, II, III). When injections were centred into the rostromedial part of the MD, anterogradely labelled fibres were observed in the anterior cingulate, the prelimbic, the lateral orbital and the adjacent part of the ventrolateral orbital cortical areas (Fig. 4A). In all these cortical areas, the bulk of thalamic fibres was directed to layer III and the upper part of layer V. A more modest labelling was also observed in layer I. When the injection was placed in the medial VM, labelled fibres were found in the prelimbic, the orbital, the anterior cingulate and the adjacent part of the medial agranular cortical area (Fig. 4B). In all these areas labelled fibres were restricted to the upper part of layer I. Distribution of retrogradely labelled cells in the ventral medial and the mediodorsal thalamic nuclei following injections of wheatgerm agglutininhorseradish peroxidase into different areas of the frontal cortex. To further identify the cortical areas innervated by the thalamic subfields of MD and VM related to the NAcc nigrothalamic circuit, W G A - H R P injections were made into different areas of the frontal cortex. Retrograde labelling in the MD and VM subfields was only observed after injections located in either the medial/ventral orbital, the prelimbic or the lateral/ventrolateral orbital areas (Fig. 5A-C). In contrast, when the injection was placed in the medial agranular motor area, even though labelled neurons were found in the VM and MD, they were localized outside the regions innervated by the dorsomedial sector of the SNR (compare Figs 5D and 2, III). In this case (Fig. 5D), labelled cells were located in the lateral segment of the MD, the portion of the VM adjacent laterally to the mammillothalamic tract and in the ventral lateral nucleus and the adjacent intralaminar nuclei centralis lateralis and paracentralis. Response of identified nigrothalamic neurons to stimulation of the nucleus accumbens Electrophysiological characterization of nigral cells projecting to the ventral medial and the mediodorsal thalamic nuclei. Nigrothalamic cells were electrophysiologically identified on the basis of their antidromic activation from medial regions of either the MD (N = 31) or the VM (N = 23). The mean latency of the antidromic potentials was 6.1 + 0.6 ms for the nigral cells projecting to the M D and 4.3 __+0.4 ms for those innervating the VM. As well as a relatively slow conduction time, these nigrothalamic neurons displayed the typical electrophysiological features of SNR cells. Their spike potentials were brief when compared to those of the dopaminergic cells of the

SN pars compacta ( > 2 m s ) and they presented a relatively high rate of spontaneous discharge (10-40Hz). Consistent with the anatomical observations, most of the neurons antidromically activated from the M D or the VM were found in the dorsomedial part of the SNR. Conduction time of the nucleus accumbenssubstantia nigra pathway. The conduction time of the NAcc-SN pathway was determined by the antidromic activation method (Fig. 6). Following stimulation of the dorsomedial SNR, 63 of the 98 NAcc cells tested were found to be antidromically driven. These cells were localized within the core of the NAcc and the adjacent ventral part of the caudateputamen. The latencies of the antidromic responses were in the range of 12-25 ms (mean 16.5 + 0.3 ms). Effect of nucleus accumbens stimulation on nigrothalamic neurons. Sixteen of the 23 cells identified as projecting to the VM, and 24 of the 25 antidromically identified from the M D responded to electrical stimulation of the NAcc. The response consisted of a brief and complete inhibition of spontaneous discharge lasting for 23.7_ 1.7ms for nigro-MD cells and 21.2 + 1.5 ms for nigro-VM cells (Fig. 7). The latency of the inhibitory responses, which could be reliably

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Fig. 6. Conduction time of the NAcc-SN pathway as determined by the antidromic activation method. (Top) Antidromic activation of an NAcc cell following stimulation of the SN. The antidromic response is characterized by its fixed latency and collisions with spontaneous spikes (arrowhead). (Bottom) Histogram of latencies of the antidromic spikes.

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Fig. 7. Inhibitory effect of NAcc stimulation on nigrothalamic neurons projecting to the MD (A) or VM (B). (Left) Experimental design. (Middle) Antidromic activation of SNR ceils followingstimulation of the MD (top) or the VM (bottom). The antidromic response is characterized by its fixed latency and collisions with spontaneous spikes (arrowhead). (Righ) Inhibitory response of identified nigrothalamic cells to stimulation of the core of NAec (poststimulus time histograms: I00 sweeps; oscilloscope traces: 15 superimposed sweeps).

determined thanks to the high spontaneous activity of the neurons and the clear-cut onset of the response, was 17.2 +__1.3 ms for the VM-projecting cells and 18 + 0.7 ms in the case of neurons projecting to the MD. Complementary experiments were undertaken to ascertain if the responses evoked by electrical stimulation of the NAcc were or were not related to activation of axons passing through the structure. For this purpose, responses of single identified nigrothalamic cells to electrical or chemical stimulation (achieved by local micropressure injections of glutamate) applied at different depths within the NAcc and the overlying dorsal striatum were examined. The results obtained with electrical stimulation are illustrated in Fig, 8. The inhibitory response of the nigrothalamic cells evoked when the stimulating electrode was located in the core of the NAcc gradually decreased in more dorsal stimulation sites. Similar observations were made under chemical stimulations with glutamate. The chemical activation of the NAcc neurons resulted in a clear-cut and long-lasting inhibition of discharge of the nigrothalamic cells (Fig. 9). This inhibitory response was progressively lost when the glutamate injections was placed more dorsally in the striatum. At this level, the chemical stimulation generated a sustained discharge of the nigral cells.

DISCUSSION

The present anatomical and electrophysiological study investigated the functional organization of the NAcc nigrothalamic circuits in the rat. The results demonstrate that the NAcc exerts and inhibitory synaptic influence on a subset of nigrothalamic cells localized in the dorsornedial part of the SNR. These nigral cells innervate subfields of the VM and the MD specifically connected to the prelimbic and orbital cortical areas. These observations suggest that the NAcc-nigral pathway is part of a ventral striatal prefrontal loop circuit which parallels the sensorimotor circuits of the dorsal striatum. The substantia nigra pars reticulata as an output station of the nucleus accumbens

The SNR has been classically considered as a major output station of the basal ganglia involved in the expression of dorsal striatal functions. This concept stems from several studies which established that the SNR provides a major link between the caudate-putamen and structures extrinsic to the basal ganglia,s'~3'2s'35Although the existence of a projection from the NAcc to the SN has long been reported, 7'26'36 the contribution of the SNR in the expression of the ventral striatal functions has not yet been investigated. Our study further demonstrates that the NAcc

541

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Fig. 8. Comparison of the responses evoked on an identified nigro-MD cell by electrical stimulation of the NAcc and the overlying striatum. (A) Antidromic activation from the MD (top: fixed latency; bottom: collision with a spontaneous spike). (B) Localization of stimulation sites in the dorsal striatum and the NAcc. (C) Poststimulus time histogram showing the response of the nigro-MD cell to stimulations (arrow) applied at each of the stimulation sites (1-4).

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Fig. 9. Inhibition of the spontaneous firing rate of a nigroMD neuron to local application of glutamate in the NAcc. (Left) Schematic representation of the different sites of glutamate application (1-3). (Right) Changes in spontaneous discharge rate of the cell induced by glutamate injection (11). Note the decreased efficiencyof the evoked inhibition for dorsal injection sites.

provides a substantial input to SNR cells. This projection originates mainly from a region of the core of the NAcc, adjacent dorsally to the anterior commissure, and terminates in the dorsal part of the SN within a longitudinal lamina extending throughout the rostrocaudal extent of the structure. Confined medially in the rostral half of the SNR, this lamina extends laterally at more caudal levels. This circumscribed domain of axon terminals of the NAcc suggests that the NAcc selectively controls a particular subpopulation of nigral output neurons. In fact, the intranuclear distribution of the parent cells of the various nigral efferent systems (i.e. nigrothalamic, tectal and tegmental) obey a rigorous topographic arrangement. 5'1°'14'15'29'32As recently established for the rat SNR, the basic principle of this topographic arrangement is an "onion-like" organizational plan such that neurons which project to functionally distinct regions of the thalamus, the tectum and/or the tegmentum are segregated spatially along a series of curved laminae enveloping a longitudinal core located dorsolaterally in the SNR.~° In accordance with the existence of such a compartmentalization of nigral efferents, the present study shows that the dorsal nigral cells localized in the projection field

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of the NAcc efferents innervate restricted subfields of the MD and the VM. Indeed, after injection of W G A - H R P into the dorsal SNR, anterogradely labelled fibres were observed specifically within the rostral pole of the MD, with the exception of its central subdivision, and in a rostral part of the VM medial to the mammillothalamic tract. Furthermore, when W G A - H R P was injected into these two thalamic subfields, retrogradely labelled cells were found in the region of the dorsal SNR innervated by the NAcc. In addition, a few labelled neurons were also observed in the ventrolateral part of the SNR, outside of the terminal field of the NAcc efferents. This labelling was likely due to the extension of the injection site to more caudal aspects of the M D and to the VM region adjacent laterally to the mammillothalamic tract, since these thalamic areas have been shown to be innervated by cells of the ventrolateral SNR) ° The existence of a functional link between the NAcc and the nigral cells innervating the rostral MD and the medial VM is further established by our electrophysiological findings, which showed that the NAcc exerts a synaptic influence on these nigrothalamic cells. Indeed, stimulation of the core of the NAcc induced a clear-cut and short duration (20 ms) inhibition of spontaneous activities in most of the dorsal nigral cells projecting to the MD or the VM. In agreement with the observations of Scarnati et al., s4 the inhibitory effect evoked in SNR cells by stimulating the NAcc does not result from a spurious co-activation of axons originating from the overlying dorsal striatum. Indeed: (i) electrical stimulation of the dorsal striatum did not affect the activity of the nigrothalamic cells located dorsally in the SNR; (ii) the latency of the inhibitory responses evoked by stimulation of the NAcc was consistent with the relatively long conduction time of the NAcc-SNR pathway (mean latency: 16.5 ms) but not with the conduction time of the projections from the dorsal striatum (mean latency: 9.8 ms), as determined previously by Ryan et al. 33 on the basis of the antidromic activation method; (iii) local application of glutamate into the NAcc, a procedure which activates cell bodies without affecting fibres, decreased the firing rate of the SNR cells projecting to the MD and the VM. By contrast, when the glutamate injection was made in the dorsal striatum, these nigrothalamic cells displayed a sustained increase of firing. As discussed in our previous electrophysiological analyses,9 these evoked activations may result from the activation of synaptic circuits that subserve a contrasting process which focuses the striatal inhibitory signals on particular sets of nigrothalamic neurons. Cortical extension nigrothalamic circuit

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accumbens

A precise topographical organization of the thalamocortical projections arising from the M D in the rat has been described by Groenewegen) 6 In accordance

with this organization, the present anatomical data indicate that the region of the rostral MD affiliated to the NAcc-nigrothalamic circuit mainly projects to the prelimbic area and to a lesser extent to the medial/ventral and to the lateral orbital areas. It is worth noting that in the present anterograde tracing experiments, the important labelling observed in the lateral orbital area after injection of W G A - H R P into the rostral MD was partly due to a diffusion of the tracer into the central segment of the MD. Indeed, as indicated by retrograde axonal transport from injections of W G A - H R P into different regions of the prefrontal cortex, the M D projection to the lateral orbital area mainly originates from neurons located within the central subdivision of the M D just beside the subfield innervated by the NAcc-nigral circuit. In the rat, the projections from the VM are known to innervate the whole frontal pole of the cerebral cortex, including the motor areas. 3'2t The innervation is organized topographically and forms an extensive plexus in layer I. In our experiments, when the injection of W G A - H R P was performed into the rostromedial part of the VM, labelled fibres were observed in layer I of the medial and lateral prefrontal areas and of the medial agranular area. The regions of the VM which innervate these different cortical areas were defined more precisely following injection of W G A - H R P into the prelimbic and orbital areas. Indeed, retrogradely labelled cells were found mainly in a part of the VM medial to the mammillothalamic tract. In contrast, when injections were placed in the medial agranular area, labelled cells were found in VM regions lateral to this fibre tract. These observations indicate that the VM region related to the NAcc-nigrothalamic circuit innervates the prelimbic and orbital areas but not the medial agranular cortex. The nucleus accumbens nigrothalamic pathway as a component o f the family of parallel basal ganglion circuits

Mainly based on the connections of the dorsal striatum, Alexander et al. 1"2 have proposed that the basal ganglia and their affiliated cortical and tbalamic areas are composed of a family of circuits organized in parallel manner which remain largely segregated from one another, both structurally and functionally. There is evidence that this principle can be extended to the ventral striatal circuits. In the rat, Groenewegen and Berendse 6'~7 have characterized a family of ventral striato-thalamo-cortical channels related to different areas of the prefrontal cortex. Each ventral striatal channel is fed by a particular subdivision of the prefrontal cortex, involves anatomically distinct sectors of the ventral striatum and of the ventral pallidum and is focused via the M D to the prefrontal area of origin. The present findings have characterized an additional ventral striatal circuit which involves the SNR instead of the ventral pallidum. This circuit consists of the core of the NAcc

Nucleus accumbens nigrothalamic circuit AGm ACd

PL

~~~'~ _"~,~,

VM

Fig. 10. Schematic representation of the NAccSNR-thalamo-prefrontal loop suggested from the present study. Abbreviations as in previous figures.

and the dorsomedial part of the SNR, and is focused via the rostral M D and the medial VM onto the prefrontal cortex, mainly the prelimbic area (Fig. 10). In contrast to the ventral pallidal circuits which originate from the core and shell subdivisions of the NAcc, 17'2°'38'39 the nigral circuit is more specifically related to the core (Refs 7, 38 and present study). Thus, the two main compartments of the NAcc, the core and the shell, along with their respective projections to the SNR and ventral pallidum, could participate in distinct parallel channels with distinct functional specificities. This hypothesis is supported by the fact that the core and shell subdivisions of the NAcc receive different sets of cortical inputs. 17'37 Functional considerations

A basic process in the expression of dorsal striatal functions via the nigrothalamic pathway is demonstrated to be disinhibition.9 By virtue of their high rate of spontaneous activity, the GABAergic SNR

543

cells exert a tonic inhibitory influence on their target thalamic nuclei. Consequently, activation of the inhibitory striatal input to the SNR tends to suppress the tonic inhibitory influence of SNR cells, thereby resulting in a disinhibition of thalamic neurons. Very likely, this disinhibitory process also operates in the ventral striatal nigrothalamic circuit. Similarly to the dorsal striatum, the NAcc exerts a inhibitory influence on its target nigrothalamic cells. Furthermore, these nigrothalamic cells display a tonic discharge pattern and, as shown recently by Ray et al., 31 use G A B A as a neurotransmitter. Besides these major functional similarities, a distinctive feature consists of the relatively slow conduction time of the ventral as compared to the dorsal striato-nigro-thalamic circuit. 12.33 A corollary of the parallel organization of the basal ganglia thalamocortical circuits is their participation in different functions. The functional attribute of the circuit involving the NAcc and the SNR has not yet been precisely defined. Considering the massive innervation provided by the SNR to motor thalamic nuclei, it has been suggested that via the nigrothalamic pathway, the NAcc might participate in the cortical processing of motor activity) 7 However, the present study demonstrates that the SNR cells which receive afferents from the NAcc innervate subfields of the MD and the VM which are mainly affiliated to the prelimbic area of the prefrontal cortex. These observations indicate that via the SNR, the NAcc is likely to be involved in high integrative functions rather than in the execution of movement per se. Indeed, in the rat, the prefrontal cortex is implicated in the organization of complex behaviours and in the control of emotional and motivational processes. 24 With regard to this last aspect, the prelimbic area of the rat prefrontal cortex has been shown to control the autonomic nervous system. 27 This cortical area projects directly to autonomic motor centres in the brain stem and spinal cord. 4'27 Moreover, stimulation of the prelimbic area results in changes in blood pressure and gastric motility.4,22'27 Thus, by analogy with the "motor" channel of the dorsal striatum involved in skeletomotor control, the NAcc (core)-nigrothalamic prefrontal circuit may represent a "visceral" channel implicated in the control of autonomic function. Acknowledgements--This work was supported by CNRS

and INSERM and by a grant from the Human Frontier Program. We wish to thank Dr I. Glowinski for fruitful scientific discussion.

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