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substantia innominata: Biochemical and electrophysiological studies
0028-3908/89 $3.00+0.00 Pergamon Press plc
Neuropharmacology Vo1.28, no.7, pp.757-760, 1989 Printed in Great Britain
DOPAMINE IN THE RAT VENTRAL PALLIDUM/SUBSTANTIA INNOMINATA: BIOCHEMICAL AND ELECTROPHYSIOLOGICAL STUDIES
T. Celeste Napier' and Pamela E. Potter' 'Department of Pharmacology and Experimental Therapeutics, Loyola University of Chicago, Stritch School of Medicine, May-wood, IL 60153, U.S.A. *Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Hospital and Medical Center, Bronx, NY 70467 U.S.A. (Accep.ted24 Apti
19893
Recent anatomical literature suggests that dopaminergic projections ascending from the midbrain terminate within the ventral pallidum/ substantia innominata (VP/SI). The present investigation evaluated this possibility using standard biochemical and electrophysiologic approaches. Biochemical studies revealed that dopamine and its major metabolites are present within the rat VP/SI. Concentrations of these compounds were diminished greatly when dopaminergic neurons of the substantia nigra were destroyed. Electrophysiologic studies demonstrated that VP/S1 neurons often respond to local applications of dopamine with a decrease in firing rate. These observations support the contention that dopamine regulates neuronal activity within the VP/S1 and that cells of origin for at least a portion of this projection lie within the substantia nigra. KEY WORDS: dopamine, ventral pallidurn, substantia innominata, microiontophoresis
Dopaminergic projections from the midbrain terminate in various forebrain regions, including the striatum, nucleus accumbens (Fuxe, 1965) and globus pallidus (Lindvall and Bjorklund, 1979). Recent studies demonstrate that the ventral pallidum/substantia innominata (VP/SI) region of the basal forebrain also receives inputs from the midbrain in rats ((Haring and Wang, 1986; Fuller, Russchen and Price, 1987; Martinez-Murillo, Semenenko and Cuello, 1988) and in monkeys (Russchen, Amaral and Price, 1985). Using antibodies against dopamine, Voorn, Jorritsma-Byham, Van Dijk and Buijs (1986) observed immunoreactive fibers with varicosities within the rat ventral pallidurn. Furthermore, ligand binding studies suggest that dopamine receptors exist within the VP/S1 (Gehlert and Wamsley, 1985; Beckstead, Wooten and Trugman, 1988). Based on these reports, the present study was devised to address the following questions: (1) Is dopamina present within the VP/S1 and if so does this neurotransmitter (2) Does dopamine influence neuronal activity within the originate within the midbrain? VP/SIT
METHODS For identification of monoamine concentration, male Sprague Dawley rats (300-35Dpg) were decapitated and striatal tissue rostra1 to the crossing of the anterior commissure was dissected. The rest of the brain was freeze-mounted onto a microtome chuck, and the globus pallidus and VP/S1 were removed by micropunching from the appropriate tissue sections (500 microns thick), according to the atlas of Paxinos and Watson (1986). Monoamine and indoleamine concentrations were determined by high performance liquid chromatography with electrochemical detection as previously described (Hortnagl, Potter and Hanin, 1987). To verify that the dopamine originated within a midbrain region known to contain dopaminergic cell bodies, chemical destruction of the substantia nigra was performed. This was accomplished by injecting 6-hydroxydopamine (B/bg/4pl/Bmin/side) bilaterally into the substantia nigra (2.Omm Lat., 3.2mm Ant. to earbar zero, 7.7mm Vent. from skull surface) in rats which were pretreated with desmethylimipramine (20mg/kg i.p.) and anesthetized with pentobarbital (45mg/kg i.p.). Controls received intranigral injections of the 0.1% ascorbic acid vehicle instead of the neurotoxin. All rats were sacrificed one week after surgery and the aforementioned brain regions removed for assay.
757
Preliminary Notes
758
For neurophysiologic evaluation of VP/S1 dopamine, extracellular recordings of single VP/S1 neurons were evaluated during local applications of the neurotransmitter. Using chloral hydrate anesthetized rats, action potentials were detected with a glass pipette filled with a 2M NaCl solution. The signals were isolated, amplified and monitored using standard electrophysiologic procedures. A five barrel pipette was glued in parallel to the recording pipette with each of the four side barrels filled with one of the following compounds: dopamine hydrochloride (0.2M), haloperidol (O.lM), GABA (0.2M). monosodium glutamate (0.2M) or norepinephrine (0.2M). The center barrel was filled with 2M NaCl solution saturated with fast green. This barrel served as an automatic electrical balance to offset net charges at the pipette tip and ejection of fast green was used to mark the recording site.
RESULTS Biochemical experiments revealed that dopamine and its major acid metabolites are contained within VP/S1 tissue (Table 1). Lesions of dopaminergic cells within the substantia nigra produced significant depletions of dopamine in the striatum, globus pallidus and the VP/SI. Content of serotonin and its metabolite, 5-hydroxyindoleacetic acid was not affected by nigral lesions (Table 1).
TABLE 1: EFFECT OF 6-HYDROXYDOPAMINE TREATMENT ON HONOANINE AND INDOLAMINE CONTENTS
TISSUE
DA
DOPAC
5HT
SHIM
11.6 f 0.7 9.7 f 0.8
15.8 + 1.1 15.5 + 1.0
STRIATUM Control C-OHDA
357.0 f 19 28.1 rf 11.4***
57.4 f 3.6 3.1 + 1.4***
GLOBUS PALLIDUS Control 6-OHDA
127.0 + 16 8.6 * 2.2***
28.7 + 5.1 4.0 f 0.9***
39.7 f 4.3 45.9 * 2.0
67.9 + 5.7 60.6 f 4.9
83.6 It:5.2 78.7 _+ 6.5
74.4 * 10.0 60.5 + 6.4
VENTRAL PALLIDUM Control 6-OHDA
44.2 f 10.2 12.8 + 1.8**
12.4 f 2.8 4.9 f 0.8*
* p
Electrophysiologic recordings of 30 neurons (from 11 rats) were verified histologically to be within the VP/S1 region assayed in the biochemical experiments. Action potential wave forms, firing patterns and firing rates (9.5 + 1.1 [SEMI Hz) are similar to those observed previously for neurons in this brain region (e.g. Aston-Jones, Shaver and Dinan, 1985: Lamour, Dutar, Rascal and Jobert, 1986). Forty-three percent of the encountered VP/S1 neurons demonstrated altered firing rates upon local application of dopamine; 10% increased firing while 33% responded with a rate suppression (Figures 1 and 2). Microiontophoresis of haloperidol attenuated the dopamine-induced suppression in 4 of 7 cells tested (Figure 1~). Nineteen of the neurons which responded to dopamine also were evaluated for sensitivity to local applications of norepinephrine. Rate suppression was again the most observed response. However, 75% of the cells tested for responses to both norepinephrine and dopamine were affected differentially by the two monoamines (see Figure la). Activity of VP/S1 neurons was decreased by local application of GABA and, although the neurons were not as responsive to glutamate as neurons in other forebrain regions, all cells which responded to the excitatory amino acid did so with an increase in firing (data not shown). DISCUSSION This study demonstrates that dopamine is present within the VP/SI. Levels of dopamine are low in comparison to the striatum, but this observation agrees with anatomical evidence of sparse innervation of the VP/S1 by dopaminergic neurons (MartinezMurillo et al. 1988) The possibility that the midbrain is a site of origin for VP/S1
Preliminary Notes
759
E!?
!!? Dopamine
c.
6 NorepinephrineHaloper
idol
1111*
Fieure 1, Rate histograms generated for three (A, B and C) spontaneously-active VP/S1 neurons. The vertical axis is spikes per set and the horizontal calibration is 2 min. Numbers above the bars illustrate the amount of current (in nA) applied to the pipette. Duration of current application is indicated by the length of each bar. The neuron illustrated 'A' increased firing rate after the ejecting current was turned off to the pipette containing norepinephrine, whereas the neuron shown in 'B' responded to norepinephrine applications with a decrease in activity. Note that 20 nA was below threshold for a response to dopamine for the neuron depicted in 'B'. Haloperidol alone slightly increase firing in 'C' and was able to attenuate dopamine-induced rate suppression when co-iontophoresed with dopamine.
20
1
I 20
APPLIED
44
60
M
CURRENT (nA) TO PIPmE CONTAINING
loo
120
DOPAMINE
&zure 2, Current-response curves illustrating the inhibitory effects of iontophoretic dopamine on VP/S1 neurons (n-10). The curve demonstrated a significant regression (df-1,30; F-11.17; p<.OOS).
dopamine is supported by the demonstration that destruction of nigral dopaminergic cells depleted VP/S1 dopamine. Anatomists report that the substantia nigra pars compacta and pars lateralis, as well as the ventral tegmental area, project to the VP/S1 (Haring and Wang, 1986; Fuller et al. 1987; Martinez-Murillo et al. 1988). In the present study, the injector tips were verified histologically to be within the substantia nigra zona compacta, but the injected solution might have spread to other midbrain areas known to contain dopaminergic soma. However, the observation that VP/S1 dopamine remained at about 30% of control levels while near total depletion was obtained in striatal and dorsal globus pallidus tissue, suggests that the nigral lesions were relatively complete and VP/S1 projections originating from the ventral tegmentum and/or pars lateralis remained at least in part, intact.
760
Preliminary Notes
Neurons of the VP/S1 responded to microiontophoretically-applied dopamine, suggesting a physiological role for this transmitter within the VP/SI. A portion of encountered neurons ware insensitive to dopamine, thus it appears that dopaminergic inputs project to a subpopulation of VP/S1 cells. Since the VP/S1 regulates both motor behaviors and cognition, it would be of interest to identify the dopaminoceptive cells and determine if they are involved with one or both of these functions.
ACKNOWLEDGEMENTS The authors thank Ms. Dowon An for her excellent technical assistance. Work supported in part by BRSG, Loyola University of Chicago, Stritch School of Medicine.
REFERENCES
Aston-Jones G. Shaver R. and Dinan T.G. (1985) Nucleus basalis neurons exhibit axonal branching with decreased impulse conduction velocity in rat cerebrocortex. Brain Res. 325: 271-205. Beckstead R.M. Wooten G.F. and Trugman J.M. (1988) Distribution of Dl and D2 dopamine receptors in the basal ganglia of the cat determined by quantitative autoradiography. J. Comp. Neural. 268: 131-145. Fuller T.A. Russchen F.T. and Price J.L. (1987) Sources of presumptive glutamergic/aspartergic afferents to the rat ventral striatopallidal region. J. Comp. Neural. 258: 317-338. Fuxe K. (1965) Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system. Acta Physiol. Stand. 64 (Suppl. 247): 39-85. Gehlert D.R. and Wamsley J.K. (1985) Dopamine receptors in the rat brain: Quantitative autoradiographic localization using [3H]sulpiride. Neurochem. Int. 7: 717-723. Haring J.H. and Wang R.Y. (1986) The identification of some sources of afferent input to the rat nucleus basalis magnocellularis by retrograde transport of horseradish peroxidase. Brain Res. 366: 152-158. Hortnagl H. Potter P.E. and Hanin I. (1987) Effect of cholinergic deficit induced by ethylcholine aziridinium (AF64A) on noradrenergic and dopaminergic parameters in rat brain. Brain Res. 421: 75-84. Lamour Y. Dutar P. Rascal 0. and Jobert A. (1986) Basal forebrain neurons projecting to the rat frontoparietal cortex: Electrophysiological and pharmacological properties. Brain Res. 362: 122-131. Lindvall 0. and Bjorklund A. (1979) Dopaminergic innervation of the globus pallidus by collaterals from the nigrostriatal pathway. Brain Res. 172: 169-173. Martinez-Murillo R., Semenenko F. and Cue110 A.C. (1988) The origin of tyrosine hydroxylase-immunoreactive fibers in the regions of the nucleus basalis magnocellularis of the rat. Brain Res. 451: 227-236. Paxinos G. and Watson C. The rat brain in stereotaxic coordinates. (1986) Academic Press Inc., Orlando, Florida. Russchen F.T., Amaral D.G. and Price J.L. (1985) The afferent connections of the substantia innominata in the monkey, Macaca fascicularis. J. Camp. Neurol. 242: l-27. Voorn P. Jorritsma-Byham B. Van Dijk C. and Buijs R.M. (1986) The dopaminergic innervation of the ventral striatum in the rat: A light- and electron-microscopical study with antibodies against dopamine. J. Camp. Neurol. 251: 84-99.
Neuropharmacology Vo1.28, no.7, pp.757-760, 1989 Printed in Great Britain
DOPAMINE IN THE RAT VENTRAL PALLIDUM/SUBSTANTIA INNOMINATA: BIOCHEMICAL AND ELECTROPHYSIOLOGICAL STUDIES
T. Celeste Napier' and Pamela E. Potter' 'Department of Pharmacology and Experimental Therapeutics, Loyola University of Chicago, Stritch School of Medicine, May-wood, IL 60153, U.S.A. *Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Hospital and Medical Center, Bronx, NY 70467 U.S.A. (Accep.ted24 Apti
19893
Recent anatomical literature suggests that dopaminergic projections ascending from the midbrain terminate within the ventral pallidum/ substantia innominata (VP/SI). The present investigation evaluated this possibility using standard biochemical and electrophysiologic approaches. Biochemical studies revealed that dopamine and its major metabolites are present within the rat VP/SI. Concentrations of these compounds were diminished greatly when dopaminergic neurons of the substantia nigra were destroyed. Electrophysiologic studies demonstrated that VP/S1 neurons often respond to local applications of dopamine with a decrease in firing rate. These observations support the contention that dopamine regulates neuronal activity within the VP/S1 and that cells of origin for at least a portion of this projection lie within the substantia nigra. KEY WORDS: dopamine, ventral pallidurn, substantia innominata, microiontophoresis
Dopaminergic projections from the midbrain terminate in various forebrain regions, including the striatum, nucleus accumbens (Fuxe, 1965) and globus pallidus (Lindvall and Bjorklund, 1979). Recent studies demonstrate that the ventral pallidum/substantia innominata (VP/SI) region of the basal forebrain also receives inputs from the midbrain in rats ((Haring and Wang, 1986; Fuller, Russchen and Price, 1987; Martinez-Murillo, Semenenko and Cuello, 1988) and in monkeys (Russchen, Amaral and Price, 1985). Using antibodies against dopamine, Voorn, Jorritsma-Byham, Van Dijk and Buijs (1986) observed immunoreactive fibers with varicosities within the rat ventral pallidurn. Furthermore, ligand binding studies suggest that dopamine receptors exist within the VP/S1 (Gehlert and Wamsley, 1985; Beckstead, Wooten and Trugman, 1988). Based on these reports, the present study was devised to address the following questions: (1) Is dopamina present within the VP/S1 and if so does this neurotransmitter (2) Does dopamine influence neuronal activity within the originate within the midbrain? VP/SIT
METHODS For identification of monoamine concentration, male Sprague Dawley rats (300-35Dpg) were decapitated and striatal tissue rostra1 to the crossing of the anterior commissure was dissected. The rest of the brain was freeze-mounted onto a microtome chuck, and the globus pallidus and VP/S1 were removed by micropunching from the appropriate tissue sections (500 microns thick), according to the atlas of Paxinos and Watson (1986). Monoamine and indoleamine concentrations were determined by high performance liquid chromatography with electrochemical detection as previously described (Hortnagl, Potter and Hanin, 1987). To verify that the dopamine originated within a midbrain region known to contain dopaminergic cell bodies, chemical destruction of the substantia nigra was performed. This was accomplished by injecting 6-hydroxydopamine (B/bg/4pl/Bmin/side) bilaterally into the substantia nigra (2.Omm Lat., 3.2mm Ant. to earbar zero, 7.7mm Vent. from skull surface) in rats which were pretreated with desmethylimipramine (20mg/kg i.p.) and anesthetized with pentobarbital (45mg/kg i.p.). Controls received intranigral injections of the 0.1% ascorbic acid vehicle instead of the neurotoxin. All rats were sacrificed one week after surgery and the aforementioned brain regions removed for assay.
757
Preliminary Notes
758
For neurophysiologic evaluation of VP/S1 dopamine, extracellular recordings of single VP/S1 neurons were evaluated during local applications of the neurotransmitter. Using chloral hydrate anesthetized rats, action potentials were detected with a glass pipette filled with a 2M NaCl solution. The signals were isolated, amplified and monitored using standard electrophysiologic procedures. A five barrel pipette was glued in parallel to the recording pipette with each of the four side barrels filled with one of the following compounds: dopamine hydrochloride (0.2M), haloperidol (O.lM), GABA (0.2M). monosodium glutamate (0.2M) or norepinephrine (0.2M). The center barrel was filled with 2M NaCl solution saturated with fast green. This barrel served as an automatic electrical balance to offset net charges at the pipette tip and ejection of fast green was used to mark the recording site.
RESULTS Biochemical experiments revealed that dopamine and its major acid metabolites are contained within VP/S1 tissue (Table 1). Lesions of dopaminergic cells within the substantia nigra produced significant depletions of dopamine in the striatum, globus pallidus and the VP/SI. Content of serotonin and its metabolite, 5-hydroxyindoleacetic acid was not affected by nigral lesions (Table 1).
TABLE 1: EFFECT OF 6-HYDROXYDOPAMINE TREATMENT ON HONOANINE AND INDOLAMINE CONTENTS
TISSUE
DA
DOPAC
5HT
SHIM
11.6 f 0.7 9.7 f 0.8
15.8 + 1.1 15.5 + 1.0
STRIATUM Control C-OHDA
357.0 f 19 28.1 rf 11.4***
57.4 f 3.6 3.1 + 1.4***
GLOBUS PALLIDUS Control 6-OHDA
127.0 + 16 8.6 * 2.2***
28.7 + 5.1 4.0 f 0.9***
39.7 f 4.3 45.9 * 2.0
67.9 + 5.7 60.6 f 4.9
83.6 It:5.2 78.7 _+ 6.5
74.4 * 10.0 60.5 + 6.4
VENTRAL PALLIDUM Control 6-OHDA
44.2 f 10.2 12.8 + 1.8**
12.4 f 2.8 4.9 f 0.8*
* p
Electrophysiologic recordings of 30 neurons (from 11 rats) were verified histologically to be within the VP/S1 region assayed in the biochemical experiments. Action potential wave forms, firing patterns and firing rates (9.5 + 1.1 [SEMI Hz) are similar to those observed previously for neurons in this brain region (e.g. Aston-Jones, Shaver and Dinan, 1985: Lamour, Dutar, Rascal and Jobert, 1986). Forty-three percent of the encountered VP/S1 neurons demonstrated altered firing rates upon local application of dopamine; 10% increased firing while 33% responded with a rate suppression (Figures 1 and 2). Microiontophoresis of haloperidol attenuated the dopamine-induced suppression in 4 of 7 cells tested (Figure 1~). Nineteen of the neurons which responded to dopamine also were evaluated for sensitivity to local applications of norepinephrine. Rate suppression was again the most observed response. However, 75% of the cells tested for responses to both norepinephrine and dopamine were affected differentially by the two monoamines (see Figure la). Activity of VP/S1 neurons was decreased by local application of GABA and, although the neurons were not as responsive to glutamate as neurons in other forebrain regions, all cells which responded to the excitatory amino acid did so with an increase in firing (data not shown). DISCUSSION This study demonstrates that dopamine is present within the VP/SI. Levels of dopamine are low in comparison to the striatum, but this observation agrees with anatomical evidence of sparse innervation of the VP/S1 by dopaminergic neurons (MartinezMurillo et al. 1988) The possibility that the midbrain is a site of origin for VP/S1
Preliminary Notes
759
E!?
!!? Dopamine
c.
6 NorepinephrineHaloper
idol
1111*
Fieure 1, Rate histograms generated for three (A, B and C) spontaneously-active VP/S1 neurons. The vertical axis is spikes per set and the horizontal calibration is 2 min. Numbers above the bars illustrate the amount of current (in nA) applied to the pipette. Duration of current application is indicated by the length of each bar. The neuron illustrated 'A' increased firing rate after the ejecting current was turned off to the pipette containing norepinephrine, whereas the neuron shown in 'B' responded to norepinephrine applications with a decrease in activity. Note that 20 nA was below threshold for a response to dopamine for the neuron depicted in 'B'. Haloperidol alone slightly increase firing in 'C' and was able to attenuate dopamine-induced rate suppression when co-iontophoresed with dopamine.
20
1
I 20
APPLIED
44
60
M
CURRENT (nA) TO PIPmE CONTAINING
loo
120
DOPAMINE
&zure 2, Current-response curves illustrating the inhibitory effects of iontophoretic dopamine on VP/S1 neurons (n-10). The curve demonstrated a significant regression (df-1,30; F-11.17; p<.OOS).
dopamine is supported by the demonstration that destruction of nigral dopaminergic cells depleted VP/S1 dopamine. Anatomists report that the substantia nigra pars compacta and pars lateralis, as well as the ventral tegmental area, project to the VP/S1 (Haring and Wang, 1986; Fuller et al. 1987; Martinez-Murillo et al. 1988). In the present study, the injector tips were verified histologically to be within the substantia nigra zona compacta, but the injected solution might have spread to other midbrain areas known to contain dopaminergic soma. However, the observation that VP/S1 dopamine remained at about 30% of control levels while near total depletion was obtained in striatal and dorsal globus pallidus tissue, suggests that the nigral lesions were relatively complete and VP/S1 projections originating from the ventral tegmentum and/or pars lateralis remained at least in part, intact.
760
Preliminary Notes
Neurons of the VP/S1 responded to microiontophoretically-applied dopamine, suggesting a physiological role for this transmitter within the VP/SI. A portion of encountered neurons ware insensitive to dopamine, thus it appears that dopaminergic inputs project to a subpopulation of VP/S1 cells. Since the VP/S1 regulates both motor behaviors and cognition, it would be of interest to identify the dopaminoceptive cells and determine if they are involved with one or both of these functions.
ACKNOWLEDGEMENTS The authors thank Ms. Dowon An for her excellent technical assistance. Work supported in part by BRSG, Loyola University of Chicago, Stritch School of Medicine.
REFERENCES
Aston-Jones G. Shaver R. and Dinan T.G. (1985) Nucleus basalis neurons exhibit axonal branching with decreased impulse conduction velocity in rat cerebrocortex. Brain Res. 325: 271-205. Beckstead R.M. Wooten G.F. and Trugman J.M. (1988) Distribution of Dl and D2 dopamine receptors in the basal ganglia of the cat determined by quantitative autoradiography. J. Comp. Neural. 268: 131-145. Fuller T.A. Russchen F.T. and Price J.L. (1987) Sources of presumptive glutamergic/aspartergic afferents to the rat ventral striatopallidal region. J. Comp. Neural. 258: 317-338. Fuxe K. (1965) Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system. Acta Physiol. Stand. 64 (Suppl. 247): 39-85. Gehlert D.R. and Wamsley J.K. (1985) Dopamine receptors in the rat brain: Quantitative autoradiographic localization using [3H]sulpiride. Neurochem. Int. 7: 717-723. Haring J.H. and Wang R.Y. (1986) The identification of some sources of afferent input to the rat nucleus basalis magnocellularis by retrograde transport of horseradish peroxidase. Brain Res. 366: 152-158. Hortnagl H. Potter P.E. and Hanin I. (1987) Effect of cholinergic deficit induced by ethylcholine aziridinium (AF64A) on noradrenergic and dopaminergic parameters in rat brain. Brain Res. 421: 75-84. Lamour Y. Dutar P. Rascal 0. and Jobert A. (1986) Basal forebrain neurons projecting to the rat frontoparietal cortex: Electrophysiological and pharmacological properties. Brain Res. 362: 122-131. Lindvall 0. and Bjorklund A. (1979) Dopaminergic innervation of the globus pallidus by collaterals from the nigrostriatal pathway. Brain Res. 172: 169-173. Martinez-Murillo R., Semenenko F. and Cue110 A.C. (1988) The origin of tyrosine hydroxylase-immunoreactive fibers in the regions of the nucleus basalis magnocellularis of the rat. Brain Res. 451: 227-236. Paxinos G. and Watson C. The rat brain in stereotaxic coordinates. (1986) Academic Press Inc., Orlando, Florida. Russchen F.T., Amaral D.G. and Price J.L. (1985) The afferent connections of the substantia innominata in the monkey, Macaca fascicularis. J. Camp. Neurol. 242: l-27. Voorn P. Jorritsma-Byham B. Van Dijk C. and Buijs R.M. (1986) The dopaminergic innervation of the ventral striatum in the rat: A light- and electron-microscopical study with antibodies against dopamine. J. Camp. Neurol. 251: 84-99.