Brain Research, 94 (1975) 45-55
45
@~ Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
D O P A M I N E A N D N O R E P I N E P H R I N E LEVELS IN T H E N U C L E U S ACCUMBENS, O L F A C T O R Y T U B E R C L E A N D CORPUS STRIATUM FOLLOWING LESIONS 1N T H E V E N T R A L T E G M E N T A L AREA
G E O R G E F. K O O B , G. J E A N B A L C O M AND J A M E S L. M E Y E R H O F F
Division of Neuropsychiatry, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D.C. 20012 (U.S.A.) (Accepted M a r c h 9th, 1975)
SUMMARY
Dopamine and norepinephrine levels were examined in 3 forebrain regions following unilateral lesions either in the ventral medial tegmental area (VMT) or in the substantia nigra. The dopamine and norepinephrine content of the nucleus accumbens, olfactory tubercle and corpus striatum were assayed ipsilaterally and contralaterally in unilaterally lesioned rats sacrificed 2, 5, 10, and 20 days after the placement of the lesions. In the nucleus accumbens and olfactory tubercle ipsilateral dopamine levels were significantly reduced below the contralateral levels at 2 days, and were decreased by 56 % and 65 %, respectively, 10 days after the lesion. A 30 % reduction of dopamine levels occurred in corpus striatum as well, following lesions in the VMT. Lesions in the substantia nigra decreased ipsilateral dopamine levels by 68% in the corpus striatum, without affecting dopamine levels in the olfactory tubercle or nucleus accumbens. Norepinephrine levels on the side ipsilateral to the lesion did not significantly differ from contralateral levels in any of the 3 regions following lesions either in the VMT or in the substantia nigra. These results demonstrate the specificity of projection in the mesolimbic dopamine system as suggested by the original histofluorescence studies.
1NTRODUCTION
Two dopaminergic pathways ascending from cell bodies in the midbrain and projecting to forebrain areas have been identified in the rat. A pathway from the pars compacta of the substantia nigra to the corpus striatum has been identified in studies utilizing brain lesions and both histofluorescence histochemistry z,la,2a and biochemical assay 11,19. A more medial pathway from a cell group located dorsolateral to the inter-
46 peduncular nucleus has been suggested utilizing only the histofluorescence technique 4, 10,23. Dopamine-rich ventral tegmental cells may project rostrally to the ventral striatum (i.e., the nucleus accumbens and the olfactory tubercle) via the pathway currently referred to as the 'mesolimbic dopamine system'. We define the ventral tegmental area (VMT) as the region surrounded ventromedially by the interpeduncular nucleus, dorsolateralty by the medial lemniscus and laterally by the substantia nigra. The purpose of this study was to confirm the neurochemical specificity of this pathway utilizing a combined technique of electrolytic lesion, discrete dissection, and sensitive enzymatic-isotopic assay for dopamine and norepinephrine. This procedure has several advantages over the histofluorescence technique. The present techniques allow a quantitative description of changes occurring in specific brain regions and thus can provide information about the time course necessary for depletion due to denervation. Further, the enzymatic-isotopic assay does not require additional pharmacological manipulation to dissociate dopamine from norepinephrine as does the histofluorescence technique. MATERIALS AND METHODS
Male, 300 g albino Wistar-derived rats from the Walter Reed colony were anesthetized with sodium pentobarbital and secured in a Kopf stereotaxic instrument. Electrolytic lesions were made in 30 rats, A current of 1 mA (D.C., rectal anode) was passed for 5 sec through a stainless steel electrode (0.254 mm in diameter) that was insulated to the tip. In 24 of the rats, unilateral lesions were made with electrodes aimed at the ventral tegmental area dorsolateral to the interpeduncular nucleus. The lesion side was assigned in a random manner. Four groups of 6 rats were decapitated 2, 5, 10 or 20 days following the operation. A sham operated group of 6 rats received electrode penetration to the lesion location but without passage of current. Six additional rats were given unilateral lesions in the substantia nigra, pars compacta utilizing two ipsilateral penetrations, with 1 mA of current for 5 sec. The rats with nigral lesions and the sham operated rats were sacrificed 10 days following the operation.
Region dissection After decapitation, the brains were quickly removed from the cranial cavity, and a coronal cut was made at the level of the infundibulum, and the portion of the brain caudal to tuberal hypothalamus was saved for histological verification of the lesions. After removal of the olfactory bulbs, the rostral portion of the brain was sectioned in the mid-sagittal plane. Two further coronal cuts were made: the first cut bisected the anterior commissure and optic chiasm; the second cut was 3 mm anterior to the first cut. This ensured inclusion of olfactory tubercle and nucleus accumbens in their entirety in the brain slice formed by the two cuts. The olfactory tubercle and nucleus accumbens were freed from the rostral side of the second slice (Fig. 1). The corpus striatum was obtained by freehand dissection.
47
TO
CAA
~
CAA
TO
i
Fig. 1. Three-dimensional drawing of the dissection of the forebrain redrawn from the illustrations in the atlas of K6nig and Klippe117. a, nucleus accumbens; cp, nucleus caudatus putamen; CAA, commissura anterior, pars anterior; TO, tuberculum olfactorium.
Biochemical analysis The dissected regions were placed in 100-300 vol. of cold 0.1 N perchloric acid and sonicated for approximately 1 rain in a Heat Systems Model 185 sonicator. After centrifugation at 25,000 × g at 4 °C for 15 rain, the supernatant fluid was stored in glass vials at --70 °C until assayed for norepinephrine and dopamine by the sensitive enzymatic-isotopic method as described by Coyle and Henry 8. For each region, control and lesion samples for a given animal were always measured in the same assay. Interassay variability was found to be 13.5 % for norepinephrine and 17 ~ for dopamine. Anatomical evaluation oJ lesions Each caudal half of the brain was fixed in 10 % formalin. Serial sections (52/~m thick) were cut on a freezing microtome and every other section through the lesion was stained with cresylecht violet. Statistical procedures The norepinephrine and dopamine levels (in #g/g brain tissue) of the VMTlesioned groups were analyzed within a two-factor repeated-measures analysis of variance with the levels from the lesioned and non-lesioned sides forming the repeated factor. The paired t-test was used for within group comparisons following the analysis of variance and for comparing catecholamine levels in the lesioned and non-lesioned sides of the brains of the substantia nigra and sham-lesioned groups sacrificed at l0 days. Significance was taken at P < 0.05. RESULTS
Area ventralis tegmenti lesions The lesions in the ventromedial tegmental area were approximately l m m in
48
Fig. 2. T h e upper p h o t o m i c r o g r a p h is t a k e n t h r o u g h the midbrain at the level o f the superior colliculus. A unilateral lesion in the ventromedial tegmental area is present o n the left side. Magnification, ",: 10. T h e lower p h o t o m i c r o g r a p h is t a k e n t h r o u g h the s a m e level o f the m i d b r a i n a n d illustrates the presence o f a unilateral lesion in the substantia nigra, pars c o m p a c t a .
49
A2,2
A2.0
AI.8
AI.6
AI.3
Fig. 3. Drawings of serial sections through the midbrain of the rat, redrawn from illustrations in the atlas of KOnig and Klippe117 showing the extent of the lesions in the VMT. Drawings in the left-hand column illustrate our smallest VMT lesion, while the drawings in the right-hand column represent our largest VMT lesion.
length a n d 0.5-0.6 m m in w i d t h a n d characteristically extended fi'om j u s t c a u d a l to the m a m m i l l a r y bodies to just a n t e r i o r to the rostral p o n s (Fig. 3). A l l the lesions were unilateral with no visible tissue destruction on the c o n t r a l a t e r a l side. Fig. 2, upper p a r t , shows a p h o t o m i c r o g r a p h o f a typical lesion in the V M T . The lesion destroyed the t e g m e n t u m i m m e d i a t e l y d o r s o l a t e r a l to the i n t e r p e d u n c u l a r nucleus.
Regional weights T a b l e I shows the tissue weights o f the 3 dissected regions when they were comp a r e d by sides. The lesions were r a n d o m l y assigned by sides and there was no signific a n t difference between the weights for a n y o f the regions. The coefficient o f variation in weight for each side was a p p r o x i m a t e l y 20°J~ for the corpus s t r i a t u m and 25-30 for the o l f a c t o r y tubercle and nucleus accumbens. TABLE I WEIGHT
OF BRAIN REGIONS
Right side Left side
Corpus striatum*
Nucleus accumbens Olfactory tubercle
62.1 :] 12.7 59.2:1 8.4
16.1 :} 3.4 14.8 :F 4.0
* Values represent means ~cS.D. in mg. n ~ =30.
9.9 7t: 2.5 10.2 ] 3.4
50
CORPUS STRIATUM /,gigI
Oopomine
ooD
20.0
--
/¢g/g--I0.40
Norepinelohrine
S
2
5 I0 20 [~ays Post-lesion
Fig. 4. Levels of norepinephrine and dopamine in the corpus striatum following VMT lesions contralateral (stippled bar) and ipsilateral (clear bar) to lesion. Histogram represents mean ± S.E,M. for 6 rats in each group. S represents sham operated animals sacrificed 10 days after surgery, * denoted P < 0.05.
Corpus striatum The dopamine levels on the lesion side in the corpus striatum decreased by 30 when compared to the contralateral levels on days 2, 5, 10 and 20 (Fig. 4). Analysis of the data revealed a significant treatment effect (F = 57.8, df = 1/20, P < 0.05) and a significant days effect (F ~-- 12.5, df~-- 3/20, P < 0.05). The ipsilateral levels were significantly lower than the contratateral levels in each group except for the sham group and day 5 post-lesion group (P < 0.05, paired t-test). The V M T lesions produced no significant change in norepinephrine in the striatum when the lesion side was compared with the contralateral side or between any of the post-lesion groups.
Olfactory tubercle The dopamine levels in the olfactory tubercle decreased more substantially than in the striatum following the lesions (Fig. 5). There was a 40 700decrease in dopamine ipsilateral to the lesion by day 2, and a 6 5 ~ decrease by day 10. The data reveal a significant treatment effect (F = 83.8, df= 1/20, P < 0.05) and a significant days effect (F z 7.4, df-- 4/50, P < 0.05). The ipsilateral levels were significantly lower
51 OLFACTORY TUBERCULE
p.g/g
Dopomine
20.0-
t~g/g
Norepinephrine
2,0-
I.O-
S
2
5
I0
20
Days Post-lesion
Fig. 5. Levels of norepinephrine and dopamine in olfactory tubercle. Legend as in Fig. 4. than the contralateral levels in each group, except for the sham group (P < 0.05, paired t-test). There was no significant difference in norepinephrine in the olfactory tubercle within or between any of the groups (Fig. 5). Nucleus accumbens
The dopamine levels in the nucleus accumbens closely parallel the changes observed in the olfactory tubercle (Fig. 6). There was a 39 ~ decrease in dopamine on the lesion side on day 2 and a 5 6 ~ decrease by day i0. The data reveal a significant treatment effect (F = 60.9, d r = 1/20, P < 0.05). The ipsilateral levels were significantly lower than the contralateral levels in each group, except for the sham group (P < 0.05, paired t-test). The lesions produced no significant change in norepinephrine in the nucleus accumbens when the lesion side was compared to the unoperated side. An analysis of the data revealed a significant variation in N E levels (Fig. 6) between days (F = 3.6, d f = 3/20, P < 0.05). These differences in the nucleus accumbens are too large to be attributable solely to interassay variability, and may be explained by the possible inclusion of the nucleus interstitialis stria terminalis in the nucleus accumbens sample in some of the groups.
52
NUCLEUSACCUMBENS ~'glgi Dopamine 20.0--]
I0.0-~
~O/O Norepieel0hrine 4.02.0S
2
5 10 DaysPost-lesion
20
Fig. 6. Levels of norepinephrine and dopamine in nucleus accumbens. Legend as in Fig. 4. Substantia nigra lesions T h e lesions a i m e d at the s u b s t a n t i a nigra, p a r s c o m p a c t a , characteristically ex-
t e n d e d as far as the V M T lesions in the r o s t r a l - c a u d a l p l a n e b u t e x t e n d e d a greater distance in the m e d i a l - l a t e r a l p l a n e because o f the d o u b l e p e n e t r a t i o n (Fig. 2, lower part). The d o p a m i n e levels in the c o r p u s s t r i a t u m decreased 68 ~ on the lesion side (P < 0.05, p a i r e d t-test), b u t were n o t significantly c h a n g e d in the o l f a c t o r y tubercle o r nucleus a c c u m b e n s (Table II). N o r e p i n e p h r i n e levels d i d n o t differ in any o f these b r a i n regions. TABLE II DOPAMINEAND NOREPINEPHRINELEVELSAFTERUNILATERALLESIONSIN THE SUBSTANTIANIGRA Values represent means ± S.E,M. for unoperated (contralateral) and lesion sides, n = 6. Unoperated control corpus striatum: NE levels, 0.38 ~ 0.03 #g/g; DA levels, 16.0 ~ 0.5/~g/g.
Corpus striatum Olfactory tubercle Nucleus accumbens
Dopamine (izg/g wet weight)
Norepinephrine (#gig wet weight)
Contralateral side
Lesion s i d e
Contralateral side
Lesion side
18.3 :k 2.0 9.1 d-: 1.1 10.0 -~ 0.7
5.9 ± 0.8* 9.0 ± 1.4 8.7 _~: 1.0
0.33 4- 0.06 0.60 4- 0.10 1.47 _-k_0.22
0.23 ~ 0.02 0.66 ~ 0.!0 1.08 :k_ 0.20
* P < 0.05, paired t-test.
53 DISCUSSION
These data demonstrate that lesions in the ventral medial tegmental area of the midbrain result in a significant decrease in levels of dopamine in the nucleus accumbens, olfactory tubercle, and corpus striatum. Lesions of the pars compacta of the substantia nigra result in a significant decrease in levels of dopamine in the corpus striatum only. The decreases in dopamine in the corpus striatum following lesions of the substantia nigra were more than twice as great as the decreases following VMT lesions. The nigro-striatal pathway was discovered by the histofluorescence technique 1 and previous studies have shown that lesions in the pars compacta of the substantia nigra significantly decrease dopamine levels in the corpus striatum 11. Studies using silver degeneration ts and axonal transport 12 have confirmed the existence of the nigrostriatal dopamine pathway. Early histofluorescence studies described a great collection of catecholamine cells located in the ventromedial midbrain tegmentum, an area adjacent to the interpeduncular nucleus 4. These medial cells were labeled A10, but no distinct borderline was indicated between the medial A10 and the more lateral A9 of the substantia nigra. Subsequent studies described a marked bilateral disappearance of dopamine nerve terminals in the nucleus accumbens and olfactory tubercle following bilateral lesions in the interpeduncular area 2a. Thus, the results of the present study provide additional evidence for the existence of a medial dopaminergic pathway from the ventral tegmentum to the olfactory tubercle and nucleus accumbens (i.e., ventral striatum). The changes in dopamine in the corpus striatum caused by lesions in the VMT may be interpreted in two ways. One explanation could be that cell bodies in the VMT as well as the substantia nigra project to the neostriatum. An alternate explanation would be that only the substantia nigra projects to the neostriatum and the VMT lesions simply destroy some nigro-striatal fibers of passage or more medial cell bodies of the substantia nigra. In the present study, VMT lesions produced a 30 ~o decrease in dopamine in the corpus striatum, while substantia nigra lesions produced a 60 ~;~ decrease. Faull and Laverty 11 demonstrated that lesions restricted to the pars compacta of the substantia nigra produced a 60 .%~decrease in dopamine in the striatum whereas lesions which extended into the ventromedial tegmentum produced a more complete loss of striatal dopamine. These data are not inconsistent with the hypothesis that some component of the neostriatal projection arises from the VMT. A more likely possibility, however, would seem to be that the VMT lesions destroyed some pars compacta cells in their more medial aspect or may have interrupted fibers of passage from the pars compacta as they turn medially before coursing rostrally 2a. These catecholamine changes resulting from lesions in the tegmental area appear to be specific for dopamine. Norepinephrine levels were not altered significantly by the lesions in any of the regions at any time. Also, the actual endogenous levels for norepinephrine in these 3 areas were very small when compared to the dopamine values. Dopamine-norepinephrine ratios were 20/1 in the olfactory tubercle, 10/l in the nucleus accumbens and 50/1 in the corpus striatum. It is noteworthy that levels of
54 norepinephrine in the nucleus accumbens are 5 times as great as levels in the corpus striatum. This may be due to inclusion along with the nucleus accumbens ot' the ventral part of the nucleus interstitialis stria terminalis. The latter structure has a very high concentration of norepinephrine '~ and the inclusion of the ventral part could have a significant effect. Although the levels of dopamine decreased significantly on day 2, the maximal depletion in both the nucleus accumbens and olfactory tubercle was found on day 10. Moreover, the same degree of depletion persists on day 20. Thus, the minimal postlesion time for obtaining maximal depletion of dopamine in these regions would appear to be between 5-10 days. The neurochemical, neuroanatomical and neuropharmacological characteristics of the nigro-striatal system have been extensively investigated3,16 and more attention recently has begun to be focused on the mesolimbic dopamine system. Helmet and Wilson 14 have observed that the cytoarchitecture of the 'ventral striatum' (nucleus accumbens and the olfactory tubercle) is similar to that of the caudate nucleus. They suggest that the nucleus accumbens and olfactory tubercle together form a continuous projection field for both allocortex and the ventral tegmentum14. Moreover, just as neocortex projects to the caudo-putamen, so allocortex projects to the ventral striatum. Specifically, the hippocampus projects via the fornix to the nucleus accumbens and the piriform cortex projects to the olfactory tubercle la. Neuropharmacological and neurochemical similarities have been described between neostriatum and 'ventral striatum'. A dopamine-sensitive adenylate cyclase is found in homogenates of both regions and various phenothiazines6,1~ and butyrophenones inhibit this enzyme in both regions to a similar degree a. Both the antipsychotic effects and parkinsonian side effects of these drugs have been attributed to this inhibition. Evidence correlating therapeutic efficacy of antipsychotic medication with dopamine-blccking potency, as well as other evidence, has led to speculation that brain dopamine systems~t and specifically the ventral striatum 2~ may be sites of the pathophysiology underlying beth amphetamine and schizophreniform psychoses. In the rat, the mesolimbic dopamine system has been implicated in apomorphine-induced stereotypy7. Recent evidenceg, 20, however, has made this conclusion controversial and has implicated the mesolimbic system in locomotor behavior. Although the functions of this system are not completely understood, the ability to selectively deplete it of dopamine over a predictable time course promises to be useful in further analysis of its role in behavior. ACKNOWLEDGEMENTS
The authors wish to acknowledge the excellent technical assistance of Mr. Clifton Johnson, Mr. Curtis King, and Mr. Michael Kelly.
REFERENCES 1 ANDI~N,N.-E., CARLSSON,A., DAHLSTR6M~A., FUXE, K., HILLARP, N.-A., AND LARSSON,K.,
55
2 3 4
5 6
7 8 9 10
I1 12 13
14
15
16 17 18 19 20 21 22 23
Demonstration and mapping out of nigro-neostriatal dopamine neurons, Life Sei., 3 (1964) 523530. AND~N, N.-E., DAtiLSXR6M, A., FtJXE, K., AND LARSSON, K., Further evidence for the presence of nigro-neostriatal dopamine neurons in the rat, Amer. J. Anat., 116 (1965) 329 333. AND/~N, N.-E., DAHLSTROM, A., FUXE, K., AND LARSSON, K., Functional role of the nigro-neostriatal dopamine neurons, Acta pharmacol. (Kbh.), 24 (1966) 263 274. ANDI~N,N.-E., DAHLSTROM, A., FUX~, K., LARSSON, K., OLSON, L., AND UNGERSTEDT, U., Ascending monoamine neurons to the telencephalon and diencephalon, Aeta physiol, scand., 67 0966) 313-326. BROWNSTEIN, M., SAAVEDRA, J. M., AND PALKOVITS, M., Norepinephrine and dopamine in the limbic system of the rat, Brain Research, 79 (1974) 431-436. CLEMENT-CORMIER, Y. C., KEBABIAN, J. W., PETZOLD, G. L., AND GREENGARD, P., Dopaminesensitive adenylate cyclase in mammalian brain: a possible site of action of antipsychotic drugs, Proe. nat. Aead. Sci. (Wash.), 71 (1974) 1113-1117. COSTALL, B., AND NAYLOR, R. J., The role of telencephalic dopaminergic systems in the mediation of apomorphine-stereotyped behavior, Europ. J. Pharmacol., 24 (1973) 8 24. COYLE, J. T., AND HENRY, D., Catecholamines in fetal and new born rat brain, J. Neurochem., 21 (1973) 61 67. CREESE, I., AND IVERSEN, S. D., The role of forebrain dopamine systems in amphetamine induced stereotyped behavior in the rat, Psychopharmaeologia (Bed.), 39 (1974) 345-357. DAHLSTROM, A., AND FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1 55. FAULL, R. L. M., AND LAVERTY, R., Changes in dopamine levels in the corpus sttiatum following lesions in the substantia nigra, Exp. Neurol., 23 (1969) 332-340. FIBIGER, H. C., MCGEER, E. G., AND ATMAD, A. S., Axoplasmic transport of dopamine in nigrostriatal neurons, J. Neurochem., 21 (1973) 383-385. FtJxE, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine terminals in the central nervous system, Acta physiol, stand., 64, Suppl. 247 (1965) 37-84. HEIMER, L., AND WILSON, R. U., The subcortical projections of the allocortex: similarities in the neural associations of the hippocampus, the piriform cortex, and the neocortex. In M. M. SANTINI (Ed.), The Golgi Centennial Symposium: Perspectives in Nenrobiology, Raven Press, New York, 1975, in press. HORN, A. S., CUELLO, A. C., AND MILLER, R. J., Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effects of drugs on the uptake mechanism and stimulation of adenylate cyclase activity, J. Neurochem., 22 (1974) 265-270. HORNYKIEWIEZ, O., Dopamine (3-hydroxytyramine) and brain function, Pharmaeol. Rev., 18 (1966) 925-964. KOMG, J. F. R., AND KLIPPEL, R. A., The Rat Brain: a Stereotaxie Atlas of the Forebrain attd Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963. MOORE, R. Y., AND H~LLER, A., Monoamine levels and neuronal degeneration in rat brain following lateral hypothalamic lesions, J. Pharmaeol. exp. Ther., 156 (1967) 12-22. OLTMANS, G. A., AND HARVEY, J. A., LH syndrome and brain catecholamine levels after lesions of the nigrostriatal bundle, Physiol. Behav., 8 (1972) 69-78. PIJNENBURG, A. J. J., AND VAN ROSSUM, J. M., Stimulation of locomotor activity following injection of dopamine into the nucleus accumbens, J. Pharm. Pharrnacol., 25 (1973) 1003 1005. SNYDER, S. H., Amphetamine psychosis: a 'model' schizophrenia mediated by catecholamines, Amer. J. P,sychiat., 130 (1973) 61-67. STEVENS, J. R., An anatomy of schizophrenia, Arch. gen. Psychiat. (Chic.), 29 (1973) 117 189. UNGERS'rED'r,U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand., 82, Suppl. 307 (1971) 1 ~ 8 .
45
@~ Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
D O P A M I N E A N D N O R E P I N E P H R I N E LEVELS IN T H E N U C L E U S ACCUMBENS, O L F A C T O R Y T U B E R C L E A N D CORPUS STRIATUM FOLLOWING LESIONS 1N T H E V E N T R A L T E G M E N T A L AREA
G E O R G E F. K O O B , G. J E A N B A L C O M AND J A M E S L. M E Y E R H O F F
Division of Neuropsychiatry, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D.C. 20012 (U.S.A.) (Accepted M a r c h 9th, 1975)
SUMMARY
Dopamine and norepinephrine levels were examined in 3 forebrain regions following unilateral lesions either in the ventral medial tegmental area (VMT) or in the substantia nigra. The dopamine and norepinephrine content of the nucleus accumbens, olfactory tubercle and corpus striatum were assayed ipsilaterally and contralaterally in unilaterally lesioned rats sacrificed 2, 5, 10, and 20 days after the placement of the lesions. In the nucleus accumbens and olfactory tubercle ipsilateral dopamine levels were significantly reduced below the contralateral levels at 2 days, and were decreased by 56 % and 65 %, respectively, 10 days after the lesion. A 30 % reduction of dopamine levels occurred in corpus striatum as well, following lesions in the VMT. Lesions in the substantia nigra decreased ipsilateral dopamine levels by 68% in the corpus striatum, without affecting dopamine levels in the olfactory tubercle or nucleus accumbens. Norepinephrine levels on the side ipsilateral to the lesion did not significantly differ from contralateral levels in any of the 3 regions following lesions either in the VMT or in the substantia nigra. These results demonstrate the specificity of projection in the mesolimbic dopamine system as suggested by the original histofluorescence studies.
1NTRODUCTION
Two dopaminergic pathways ascending from cell bodies in the midbrain and projecting to forebrain areas have been identified in the rat. A pathway from the pars compacta of the substantia nigra to the corpus striatum has been identified in studies utilizing brain lesions and both histofluorescence histochemistry z,la,2a and biochemical assay 11,19. A more medial pathway from a cell group located dorsolateral to the inter-
46 peduncular nucleus has been suggested utilizing only the histofluorescence technique 4, 10,23. Dopamine-rich ventral tegmental cells may project rostrally to the ventral striatum (i.e., the nucleus accumbens and the olfactory tubercle) via the pathway currently referred to as the 'mesolimbic dopamine system'. We define the ventral tegmental area (VMT) as the region surrounded ventromedially by the interpeduncular nucleus, dorsolateralty by the medial lemniscus and laterally by the substantia nigra. The purpose of this study was to confirm the neurochemical specificity of this pathway utilizing a combined technique of electrolytic lesion, discrete dissection, and sensitive enzymatic-isotopic assay for dopamine and norepinephrine. This procedure has several advantages over the histofluorescence technique. The present techniques allow a quantitative description of changes occurring in specific brain regions and thus can provide information about the time course necessary for depletion due to denervation. Further, the enzymatic-isotopic assay does not require additional pharmacological manipulation to dissociate dopamine from norepinephrine as does the histofluorescence technique. MATERIALS AND METHODS
Male, 300 g albino Wistar-derived rats from the Walter Reed colony were anesthetized with sodium pentobarbital and secured in a Kopf stereotaxic instrument. Electrolytic lesions were made in 30 rats, A current of 1 mA (D.C., rectal anode) was passed for 5 sec through a stainless steel electrode (0.254 mm in diameter) that was insulated to the tip. In 24 of the rats, unilateral lesions were made with electrodes aimed at the ventral tegmental area dorsolateral to the interpeduncular nucleus. The lesion side was assigned in a random manner. Four groups of 6 rats were decapitated 2, 5, 10 or 20 days following the operation. A sham operated group of 6 rats received electrode penetration to the lesion location but without passage of current. Six additional rats were given unilateral lesions in the substantia nigra, pars compacta utilizing two ipsilateral penetrations, with 1 mA of current for 5 sec. The rats with nigral lesions and the sham operated rats were sacrificed 10 days following the operation.
Region dissection After decapitation, the brains were quickly removed from the cranial cavity, and a coronal cut was made at the level of the infundibulum, and the portion of the brain caudal to tuberal hypothalamus was saved for histological verification of the lesions. After removal of the olfactory bulbs, the rostral portion of the brain was sectioned in the mid-sagittal plane. Two further coronal cuts were made: the first cut bisected the anterior commissure and optic chiasm; the second cut was 3 mm anterior to the first cut. This ensured inclusion of olfactory tubercle and nucleus accumbens in their entirety in the brain slice formed by the two cuts. The olfactory tubercle and nucleus accumbens were freed from the rostral side of the second slice (Fig. 1). The corpus striatum was obtained by freehand dissection.
47
TO
CAA
~
CAA
TO
i
Fig. 1. Three-dimensional drawing of the dissection of the forebrain redrawn from the illustrations in the atlas of K6nig and Klippe117. a, nucleus accumbens; cp, nucleus caudatus putamen; CAA, commissura anterior, pars anterior; TO, tuberculum olfactorium.
Biochemical analysis The dissected regions were placed in 100-300 vol. of cold 0.1 N perchloric acid and sonicated for approximately 1 rain in a Heat Systems Model 185 sonicator. After centrifugation at 25,000 × g at 4 °C for 15 rain, the supernatant fluid was stored in glass vials at --70 °C until assayed for norepinephrine and dopamine by the sensitive enzymatic-isotopic method as described by Coyle and Henry 8. For each region, control and lesion samples for a given animal were always measured in the same assay. Interassay variability was found to be 13.5 % for norepinephrine and 17 ~ for dopamine. Anatomical evaluation oJ lesions Each caudal half of the brain was fixed in 10 % formalin. Serial sections (52/~m thick) were cut on a freezing microtome and every other section through the lesion was stained with cresylecht violet. Statistical procedures The norepinephrine and dopamine levels (in #g/g brain tissue) of the VMTlesioned groups were analyzed within a two-factor repeated-measures analysis of variance with the levels from the lesioned and non-lesioned sides forming the repeated factor. The paired t-test was used for within group comparisons following the analysis of variance and for comparing catecholamine levels in the lesioned and non-lesioned sides of the brains of the substantia nigra and sham-lesioned groups sacrificed at l0 days. Significance was taken at P < 0.05. RESULTS
Area ventralis tegmenti lesions The lesions in the ventromedial tegmental area were approximately l m m in
48
Fig. 2. T h e upper p h o t o m i c r o g r a p h is t a k e n t h r o u g h the midbrain at the level o f the superior colliculus. A unilateral lesion in the ventromedial tegmental area is present o n the left side. Magnification, ",: 10. T h e lower p h o t o m i c r o g r a p h is t a k e n t h r o u g h the s a m e level o f the m i d b r a i n a n d illustrates the presence o f a unilateral lesion in the substantia nigra, pars c o m p a c t a .
49
A2,2
A2.0
AI.8
AI.6
AI.3
Fig. 3. Drawings of serial sections through the midbrain of the rat, redrawn from illustrations in the atlas of KOnig and Klippe117 showing the extent of the lesions in the VMT. Drawings in the left-hand column illustrate our smallest VMT lesion, while the drawings in the right-hand column represent our largest VMT lesion.
length a n d 0.5-0.6 m m in w i d t h a n d characteristically extended fi'om j u s t c a u d a l to the m a m m i l l a r y bodies to just a n t e r i o r to the rostral p o n s (Fig. 3). A l l the lesions were unilateral with no visible tissue destruction on the c o n t r a l a t e r a l side. Fig. 2, upper p a r t , shows a p h o t o m i c r o g r a p h o f a typical lesion in the V M T . The lesion destroyed the t e g m e n t u m i m m e d i a t e l y d o r s o l a t e r a l to the i n t e r p e d u n c u l a r nucleus.
Regional weights T a b l e I shows the tissue weights o f the 3 dissected regions when they were comp a r e d by sides. The lesions were r a n d o m l y assigned by sides and there was no signific a n t difference between the weights for a n y o f the regions. The coefficient o f variation in weight for each side was a p p r o x i m a t e l y 20°J~ for the corpus s t r i a t u m and 25-30 for the o l f a c t o r y tubercle and nucleus accumbens. TABLE I WEIGHT
OF BRAIN REGIONS
Right side Left side
Corpus striatum*
Nucleus accumbens Olfactory tubercle
62.1 :] 12.7 59.2:1 8.4
16.1 :} 3.4 14.8 :F 4.0
* Values represent means ~cS.D. in mg. n ~ =30.
9.9 7t: 2.5 10.2 ] 3.4
50
CORPUS STRIATUM /,gigI
Oopomine
ooD
20.0
--
/¢g/g--I0.40
Norepinelohrine
S
2
5 I0 20 [~ays Post-lesion
Fig. 4. Levels of norepinephrine and dopamine in the corpus striatum following VMT lesions contralateral (stippled bar) and ipsilateral (clear bar) to lesion. Histogram represents mean ± S.E,M. for 6 rats in each group. S represents sham operated animals sacrificed 10 days after surgery, * denoted P < 0.05.
Corpus striatum The dopamine levels on the lesion side in the corpus striatum decreased by 30 when compared to the contralateral levels on days 2, 5, 10 and 20 (Fig. 4). Analysis of the data revealed a significant treatment effect (F = 57.8, df = 1/20, P < 0.05) and a significant days effect (F ~-- 12.5, df~-- 3/20, P < 0.05). The ipsilateral levels were significantly lower than the contratateral levels in each group except for the sham group and day 5 post-lesion group (P < 0.05, paired t-test). The V M T lesions produced no significant change in norepinephrine in the striatum when the lesion side was compared with the contralateral side or between any of the post-lesion groups.
Olfactory tubercle The dopamine levels in the olfactory tubercle decreased more substantially than in the striatum following the lesions (Fig. 5). There was a 40 700decrease in dopamine ipsilateral to the lesion by day 2, and a 6 5 ~ decrease by day 10. The data reveal a significant treatment effect (F = 83.8, df= 1/20, P < 0.05) and a significant days effect (F z 7.4, df-- 4/50, P < 0.05). The ipsilateral levels were significantly lower
51 OLFACTORY TUBERCULE
p.g/g
Dopomine
20.0-
t~g/g
Norepinephrine
2,0-
I.O-
S
2
5
I0
20
Days Post-lesion
Fig. 5. Levels of norepinephrine and dopamine in olfactory tubercle. Legend as in Fig. 4. than the contralateral levels in each group, except for the sham group (P < 0.05, paired t-test). There was no significant difference in norepinephrine in the olfactory tubercle within or between any of the groups (Fig. 5). Nucleus accumbens
The dopamine levels in the nucleus accumbens closely parallel the changes observed in the olfactory tubercle (Fig. 6). There was a 39 ~ decrease in dopamine on the lesion side on day 2 and a 5 6 ~ decrease by day i0. The data reveal a significant treatment effect (F = 60.9, d r = 1/20, P < 0.05). The ipsilateral levels were significantly lower than the contralateral levels in each group, except for the sham group (P < 0.05, paired t-test). The lesions produced no significant change in norepinephrine in the nucleus accumbens when the lesion side was compared to the unoperated side. An analysis of the data revealed a significant variation in N E levels (Fig. 6) between days (F = 3.6, d f = 3/20, P < 0.05). These differences in the nucleus accumbens are too large to be attributable solely to interassay variability, and may be explained by the possible inclusion of the nucleus interstitialis stria terminalis in the nucleus accumbens sample in some of the groups.
52
NUCLEUSACCUMBENS ~'glgi Dopamine 20.0--]
I0.0-~
~O/O Norepieel0hrine 4.02.0S
2
5 10 DaysPost-lesion
20
Fig. 6. Levels of norepinephrine and dopamine in nucleus accumbens. Legend as in Fig. 4. Substantia nigra lesions T h e lesions a i m e d at the s u b s t a n t i a nigra, p a r s c o m p a c t a , characteristically ex-
t e n d e d as far as the V M T lesions in the r o s t r a l - c a u d a l p l a n e b u t e x t e n d e d a greater distance in the m e d i a l - l a t e r a l p l a n e because o f the d o u b l e p e n e t r a t i o n (Fig. 2, lower part). The d o p a m i n e levels in the c o r p u s s t r i a t u m decreased 68 ~ on the lesion side (P < 0.05, p a i r e d t-test), b u t were n o t significantly c h a n g e d in the o l f a c t o r y tubercle o r nucleus a c c u m b e n s (Table II). N o r e p i n e p h r i n e levels d i d n o t differ in any o f these b r a i n regions. TABLE II DOPAMINEAND NOREPINEPHRINELEVELSAFTERUNILATERALLESIONSIN THE SUBSTANTIANIGRA Values represent means ± S.E,M. for unoperated (contralateral) and lesion sides, n = 6. Unoperated control corpus striatum: NE levels, 0.38 ~ 0.03 #g/g; DA levels, 16.0 ~ 0.5/~g/g.
Corpus striatum Olfactory tubercle Nucleus accumbens
Dopamine (izg/g wet weight)
Norepinephrine (#gig wet weight)
Contralateral side
Lesion s i d e
Contralateral side
Lesion side
18.3 :k 2.0 9.1 d-: 1.1 10.0 -~ 0.7
5.9 ± 0.8* 9.0 ± 1.4 8.7 _~: 1.0
0.33 4- 0.06 0.60 4- 0.10 1.47 _-k_0.22
0.23 ~ 0.02 0.66 ~ 0.!0 1.08 :k_ 0.20
* P < 0.05, paired t-test.
53 DISCUSSION
These data demonstrate that lesions in the ventral medial tegmental area of the midbrain result in a significant decrease in levels of dopamine in the nucleus accumbens, olfactory tubercle, and corpus striatum. Lesions of the pars compacta of the substantia nigra result in a significant decrease in levels of dopamine in the corpus striatum only. The decreases in dopamine in the corpus striatum following lesions of the substantia nigra were more than twice as great as the decreases following VMT lesions. The nigro-striatal pathway was discovered by the histofluorescence technique 1 and previous studies have shown that lesions in the pars compacta of the substantia nigra significantly decrease dopamine levels in the corpus striatum 11. Studies using silver degeneration ts and axonal transport 12 have confirmed the existence of the nigrostriatal dopamine pathway. Early histofluorescence studies described a great collection of catecholamine cells located in the ventromedial midbrain tegmentum, an area adjacent to the interpeduncular nucleus 4. These medial cells were labeled A10, but no distinct borderline was indicated between the medial A10 and the more lateral A9 of the substantia nigra. Subsequent studies described a marked bilateral disappearance of dopamine nerve terminals in the nucleus accumbens and olfactory tubercle following bilateral lesions in the interpeduncular area 2a. Thus, the results of the present study provide additional evidence for the existence of a medial dopaminergic pathway from the ventral tegmentum to the olfactory tubercle and nucleus accumbens (i.e., ventral striatum). The changes in dopamine in the corpus striatum caused by lesions in the VMT may be interpreted in two ways. One explanation could be that cell bodies in the VMT as well as the substantia nigra project to the neostriatum. An alternate explanation would be that only the substantia nigra projects to the neostriatum and the VMT lesions simply destroy some nigro-striatal fibers of passage or more medial cell bodies of the substantia nigra. In the present study, VMT lesions produced a 30 ~o decrease in dopamine in the corpus striatum, while substantia nigra lesions produced a 60 ~;~ decrease. Faull and Laverty 11 demonstrated that lesions restricted to the pars compacta of the substantia nigra produced a 60 .%~decrease in dopamine in the striatum whereas lesions which extended into the ventromedial tegmentum produced a more complete loss of striatal dopamine. These data are not inconsistent with the hypothesis that some component of the neostriatal projection arises from the VMT. A more likely possibility, however, would seem to be that the VMT lesions destroyed some pars compacta cells in their more medial aspect or may have interrupted fibers of passage from the pars compacta as they turn medially before coursing rostrally 2a. These catecholamine changes resulting from lesions in the tegmental area appear to be specific for dopamine. Norepinephrine levels were not altered significantly by the lesions in any of the regions at any time. Also, the actual endogenous levels for norepinephrine in these 3 areas were very small when compared to the dopamine values. Dopamine-norepinephrine ratios were 20/1 in the olfactory tubercle, 10/l in the nucleus accumbens and 50/1 in the corpus striatum. It is noteworthy that levels of
54 norepinephrine in the nucleus accumbens are 5 times as great as levels in the corpus striatum. This may be due to inclusion along with the nucleus accumbens ot' the ventral part of the nucleus interstitialis stria terminalis. The latter structure has a very high concentration of norepinephrine '~ and the inclusion of the ventral part could have a significant effect. Although the levels of dopamine decreased significantly on day 2, the maximal depletion in both the nucleus accumbens and olfactory tubercle was found on day 10. Moreover, the same degree of depletion persists on day 20. Thus, the minimal postlesion time for obtaining maximal depletion of dopamine in these regions would appear to be between 5-10 days. The neurochemical, neuroanatomical and neuropharmacological characteristics of the nigro-striatal system have been extensively investigated3,16 and more attention recently has begun to be focused on the mesolimbic dopamine system. Helmet and Wilson 14 have observed that the cytoarchitecture of the 'ventral striatum' (nucleus accumbens and the olfactory tubercle) is similar to that of the caudate nucleus. They suggest that the nucleus accumbens and olfactory tubercle together form a continuous projection field for both allocortex and the ventral tegmentum14. Moreover, just as neocortex projects to the caudo-putamen, so allocortex projects to the ventral striatum. Specifically, the hippocampus projects via the fornix to the nucleus accumbens and the piriform cortex projects to the olfactory tubercle la. Neuropharmacological and neurochemical similarities have been described between neostriatum and 'ventral striatum'. A dopamine-sensitive adenylate cyclase is found in homogenates of both regions and various phenothiazines6,1~ and butyrophenones inhibit this enzyme in both regions to a similar degree a. Both the antipsychotic effects and parkinsonian side effects of these drugs have been attributed to this inhibition. Evidence correlating therapeutic efficacy of antipsychotic medication with dopamine-blccking potency, as well as other evidence, has led to speculation that brain dopamine systems~t and specifically the ventral striatum 2~ may be sites of the pathophysiology underlying beth amphetamine and schizophreniform psychoses. In the rat, the mesolimbic dopamine system has been implicated in apomorphine-induced stereotypy7. Recent evidenceg, 20, however, has made this conclusion controversial and has implicated the mesolimbic system in locomotor behavior. Although the functions of this system are not completely understood, the ability to selectively deplete it of dopamine over a predictable time course promises to be useful in further analysis of its role in behavior. ACKNOWLEDGEMENTS
The authors wish to acknowledge the excellent technical assistance of Mr. Clifton Johnson, Mr. Curtis King, and Mr. Michael Kelly.
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