Psychoneuroendocrinology, 1977, Vol. 2, pp. 237-248. Pergamon Press. Primed in Great Britain
PEPTIDE HORMONES A N D CENTRAL DOPAMINE NEURON SYSTEMS WALTER LICHTENSTEIGER,RUTH LIENHART and HANS GEORG KOPP Department of Pharmacology, University of Ziirich, Ziirich, Switzerland (Received 2 August 1976) SUMMARY (1) In ovariectomized, estrogen-progesterone-pretreated rats, the dopamine (DA) neuron group of the arcuate nucleus of the hypothalamus which also projects to the intermediate lobe of the pituitary, and that of the substantia nigra, reacted markedly within 30 rain to single intraperitoneal injections of a-MSH. The response, an increase in cellular fluorescence intensity, was disclosed by histocbemical microfluorimetry. (2) ACTH 4-10 and prolactin had a similar action whereas ACTH 1-24 was ineffective in the dose used. (3) The response of both DA neuron groups was abolished by lesions of the area postrema and by more rostral brainstem lesions, indicating the participation of ascending systems. (4) Both neuron groups were further influenced by electrical stimulation in posteromediai thalami¢ areas known to be involved in central MSH effects. (5) Pretreatment with atropine blocked the stimulationinduced response of both DA neuron groups and the nigral reaction to a-MSH and reduced that of the arcuate DA neurons to the peptide, which suggests the participation of cholinergic systems. Key Words--a-melanocyte-stimulating hormone (a-MSH); dopamine neurons; substantia nigra; area postrema.
INTRODUCTION THE BEHAVIORALand other central effects of the pars intermedia hormone, a-melanocytestimulating hormone (a-MSH), and of related peptides, count among the most striking actions of these substances in mammals (de Wied, 1969; Kastin, Miller, Nockton, Sandman, Schally & Stratton, 1973). The intermediate lobe of the pituitary receives an abundant projection of dopaminergic (DA) fibers which belong to the tubero-infundibular or tuberohypophyseal D A neuron system (Bj6rkltmd, Falck, Hromek, O w m a n & West, 1970; Bj6rklund, Moore, Nobin & Stenevi, 1973). This D A neuron group readily responds to electrical stimulation in various extrahypothalamic areas thought to be involved in the integration of endocrine and behavioxal processes (Table I; Lichtensteiger, 1971; Lichtensteiger & Keller, 1974; Lichtensteiger & Lienhart, 1975b). Although the stimulatory responses were often accompanied by changes in serum levels of anterior pituitary hormones, it appeared to us that some of the extrahypothalamic influences on the tubero-hypophyseal D A system might also be related to the control of the intermediate lobe. In other words, this D A system might constitute a link between extrahypothalamic structures involved in the action of behaviorally active peptides and the intermediate lobe, a site of production o f such peptides. We approached this question first by investigating whether the tuberohypophyseal D A neurons reacted to systemically administered a - M S H and related peptides. 237
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WALTERLICHTENSTF~OER,RUTHLENHARTand HANSGEORGKOPP
TABLE I. EXTRAHYPOTHALAMIC STIMULATION SITES ELICITING AN ACUTE RISE IN FLUORESCENCE INTENSITY OF DA NERVE CELL GROUPS IN URETHANE-ANESTHETIZED RATS
Electrode position Medial preoptic area Diagonal tract nucleus Medial arnygdaloid nucleus Bed nucleus of stria terminalis Ventral tegmental area of midbrain (Tsai) Parafascicular thalamic nuc. Posteromedian and medial thalamic nuclei Med. habenular nucleus Dorsal hippoeampus
Parameters of stimulation (1)
DA neuron groups studied (reference)
100/sec, 100 #A, 5-30 min 100/sec, 100 #A, 10min 100[sec, 100 #A, 10-15 min 100[sec, 100 #A, 10min
Arcuate, nigral (2,3,4) Arcuate (3) Arcuate (3 and unpubl.) Arcuate (3)
100/sec, 100 #A, 10 min 30[sec, I00 t~A, 10min
Arcuate (3) Arcuate, nigral (4)
30/sec, 100/~A, 10 min 30/see, 100/~A, 10rain 30/sec, 100/~A, 10 min
Arcuate, nigral (4) Arcuate, nigral (4) Arcuate, nigral (4)
(1) Rats ovariectomized for 3 weeks, estrogen-progesterone-pretreated. Bipolar concentric electrode, 0.5 msec monophasic positive pulses, 15 see on/off. (2) Lichtensteiger, 1971 ; (3) Lichtensteiger & Keller, 1974; (4) Lichtensteiger & Lienhart, 1975b. In the same animals, an extrahypothalamic D A system, the nigrostriatal system, was also studied. Observations on similar reactions of the arcuate and nigral D A systems to certain environmental and other functional changes (e.g. acute exposure to cold, Lichtensteiger, 1969a; Lienhart, Lichtensteiger & Langemann, 1975) suggested that there might exist coordinate responses of the two systems despite differences in other situations such as the estrous cycle (Lichtensteiger, 1969b). The peptide effects were investigated in female Sprague-Dawley-Ivanovas rats ovariectomized for 3 weeks and pretreated for 1 day with 5 /~g estradiol-dipropionate and 2 mg progesterone (s.c.), since the extrahypothalamic influences had previously been studied on the same animal model. All experiments were performed in the afternoon, the rats being kept at a regular light-dark cycle (14 hr light period; Lichtensteiger & Lienhart, 1975b, 1977). NOTE ON THE MICROFLUORIMETRIC ANALYSIS OF DA NEURON GROUPS The functional state of the two D A neuron groups was assessed by a microfluorimetric technique based on the histochemical fluorescence method of Falck and Hillarp (Lichtensteiger, 1969b, 1970). Thereby, the intensity of the fluorescent light emitted by catecholamine-containing nerve cells is expressed as a fraction of a norepinephrine-containing gelatin standard processed together with the tissue. Account is taken of non-specific fluorescence of brain tissue (tissue blank) and fluorescence of the norepinephrine-free gelatin (standard blank). The distributions of relative fluorescence intensities of individual neurons are characteristic for a given functional state and neuron group. In studies on the nigral D A neuron group, we tlave observed a close non-linear correlation between mean fluorescence intensity and mean firing rate (Lichtensteiger, Felix, Lienhart & Hefti, 1976); in agreement with earlier less direct" evidence (Lichtensteiger, 1971), an increase in mean firing rate was accompanied by a rise in mean cellular fluorescence intensity. Because of certain variations! in fluorescence intensity throughout the DA neuron groups, measure-
239
M S H ON NIGRAL AND HYPOTHALAMIC DOPAMINE NEURONS
m e n t s were always t a k e n at regular intervals between defined antero-posterior levels. I n the arcuate D A n e u r o n group, n e u r o n s of the whole area were studied, while in the s u b s t a n t i a nigra m e a s u r e m e n t s were restricted to the lateral half to two-thirds o f the z o n a c o m p a c t a (for details, see Lichtensteiger & Keller, 1974; L i e n h a r t et aL, 1975). RESPONSE O F T H E A R C U A T E A N D N I G R A L D A N E U R O N G R O U P S TO a-MSH, A C T H 1-24, ACTH 4-10 A N D P R O L A C T I N W h e n a d m i n i s t e r e d i.p. i n doses used i n m o s t behavioral experiments, a - M S H elicited a m a r k e d increase i n the fluorescence intensity o f b o t h D A n e u r o n g r o u p s within 30 rain (Table II). The two D A n e u r o n groups did n o t appear to be equally sensitive, however, as
TABLEII.
FLUORESCENCE INTENSITY OF THE ARCUATE AND NIGRAL DA NEURONS IN GROUPS OF RATS INJECTED WITH PEPTIDE HORMONES OR SALINE(;)
Treatment(2)
Saline, 30 min Phenol-containingvehicle(4J, 30 min a-MSH, 100 tLg/kg, 30 rain a-MSH, 33 t~g/kg, 30 min a-MSH, 10 ~g/kg, 30 min a-MSH, 3 ttg/kg, 30 win ACTH 1-24, 175 tzg/kg, 30 min Prolactin, 2 mg/kg, 30 rain Atropine, 10 mg/kg, 15 min before a-MSH, 100/~g/kg, 30 rain Area postrema lesion q- saline, 30 rain Area postrema lesion + a-MSH, 100 ttg/kg, 30 min(~)
Arctmte DA neurons Nigral DA neurons mean 4- S . D . (3) cell count mean -4- S.D. (a) cell count (No. of rats) (No. of rats) 3.666 4- 0.4357 3.655 4- 0.4394 3.882 4- 0.4468 3.944 -4-0.4466 3.988 4- 0.4142 3.972 4- 0.4828 3.625 4- 0.4593 3.855 4- 0.4330
1266(6) 706 (3) 1074(5) 431 (2) 863 (4) 155 (1) 780 (4) 985 (5)
4.0754- 0.3510 4.037 4- 0.4083 4.2454- 0.3290 4.2154- 0.3309 3.995+ 0.3500 4.034 4- 0.3355 4.141 4- 0.3485 4.232 4- 0.3394
3.797 4- 0.4149 3.718 4- 0.4101
981 (4) 1016(4)
3.9114- 0.4739 3.9774- 0.3406
640 (4) 1285(4)
3.659 4- 0.4204
886 (4)
3.900 4- 0.3661
1044(4)
940 (6) 481 (3) 840 (5)(s) 320 (2) 700 (4) 160 (1) 639 (4) 790 (5)
(1) The two DA neuron groups were ~tudied in the same rats. Data from Lichtensteiger and Lienhart (1975b, 1977). For values of individual rats, of. these references. (2) All substances i.p. except atropine (s.c.). (3) Mean values of (normalized) distributions of logarithmically transformed intensity values. In substantia nigra, both sides were studied only in the lesioned rats which explains the higher cell counts in these animah. No side differences were observed after area postrema lesions, however. (4) Used as a vehicle for prolactin. (5) After inclusion of 2 additional rats with no data on the arcuate DA neuron group, the nigral mean would be 4.259 -4- 0.3503 1159 (7). (6) Rats with complete destruction of area postrema. If 2 rats with small remnants of this area are included, mean of arcuate DA neurons = 3.769 -4- 0.4099 1398 (6), mean of nigral DA neurons = 3.888 4- 0.3601 1625 (6). lower doses of the p e p t i d e failed to influence the n i g i a l D A n e u r o n s whereas the arcuate D A n e u r o n s r e s p o n d e d to the whole dose range studied so far. Provided the relationship which we have repeatedly observed between intensity change a n d s t i m u l a t i o n o f D A n e u r o n s existed also u n d e r the present experimental conditions, o u r findings indicate that b o t h D A n e u r o n groups were stimulated by the peptide. Since M S H secretion is generally
240
WALTER LlcrrrENSTEXOER, RUTH LIENHARTand HANS GEORG KOPP
inhibited by catecholamines (Taleisnik, Tomatis & Celis, 1972; Bower, Hadley & Hruby, 1974; see also Tilders, Mulder & Smelik, 1975), the response of the arcuate DA group probably reflects the activation of a negative feedback mechanism. On the other hand, the nigral response may be related to some of the extrahypothalamic effects of a-MSH. In intact male rats injected with 100/zg/kg a-MSH on 3 days, the decrease in DA concentration of midbrain and striatum after inhibition of tyrosine hydroxylase was found unchanged (Kostrzewa, Kastin & Spirtes, 1975). It cannot be decided as yet whether the discrepancy with our results is due to differences in the experimental situation or in the sensitivity of the methods used. We tried to get some further insight into the effect of a-MSH on the DA systems by checking their reaction to two related peptides, ACTH 1-24 and ACTH 4-10. In a dose that is approx equimolar to the highest dose of a-MSH used, ACTH 1-24 remained completely ineffective on the arcuate DA neurons. In half of the animals, fluorescence intensity increased somewhat in the rtigral neurons but the group as a whole did not differ significantly from the controls (Table H, Lichtensteiger & Lienhart, 1977). While this result does not exclude an effect of ACTH 1-24 at higher doses, it indicates that this hormone apparently is considerably less effective than a-MSH. The lack of a demonstrable feedback action of ACTH 1-24 on the arcuate DA neurons is in agreement with the view that the tubero-infundibular DA neurons are of minor importance in the control of ACTH secretion (Scapagnini, Van Loon, Moberg, Preziosi & Ganong, 1972). This view has been questioned, however (Gouget, Duvernoy & Bugnon, 1973). If, under certain conditions, ACTH is indeed preferentially released from the intermediate lobe as suggested by MialheVoloss (1958), this special situation at least might depend upon the DA innovation of the pars intermedia. Quite in contrast to ACTH 1-24, ACTH 4-10 induced a very strong response of both DA neuron groups (Fig. 1). This observation is of special interest because
-4'-+-
4.4
E 4.2 t3 4-~ 4.0
+
eSoline, 30min C] ACTH 1 - 2 4 , 1 7 5 / z g / k g , 3 0 m i n , v ACTH 4 - 1 0 , 50/.Lg/kg, 50rain
.C
zx
60min
8
ac 3',4
Areuofe nucleus
' 3'.6 ' 3'.8 410 ' 412 ' Relo~rive infensi+y ( m e o n , + 9 5 % conf. lira.; nor. log)
FIG. 1. Effect of i.p. injection of ACTH 4-10 (30 or 60 min) and ACTH 1-24 (30 min) on the fluorescence intensity of the arcuate and nigral DA neuron groups in ovadectomized rats pretreated for one day with estrogen and progesterone. For each rat, the mean value of the arcuate DA group is plotted on the abscissa, that of the nigral D A group on the ordinate (means of logarithmically transformed intensity distributions, cf. Table II). ACTH 4-10 elicited a marked response of both DA systems.
MSH ONNmRALANDHYPOTHAIAMICD o P ~
NEURONS
241
ACTH 4-10 is devoid of peripheral hormonal activity (de Wied, 1969). One may say that in our system it resembled a-MSH rather than ACTH. In addition to these two peptides, prolactin, whose secretion is inhibited by DA (Kamberi, Mical & Porter, 1970; Lu, Koch & MeRes, 1971 ; Macleod, Fontham & Lehmeyer, 1970; Macleod & Lehmeyer, 1974), was studied in order to check the reaction of the arcuate DA neurons which had earlier been reported to respond to this hormone by an increase in DA turnover (Htkfelt & Fuxe, 1972). The result--a rise in cellular fluorescence intensity which we interpret as indicative of an activation fully agrees with the notion of an activation of a negative feedback mechanism. It demonstrates that the change in the arcuate DA neurons is not specific for a-MSH, which is in keeping with the fact that these neurons control various hormone axes. Surprisingly, the nigral DA neurons were also influenced (Table II). We cannot decide as yet whether the reaction observed in this DA system is due to prolactin itself or to some indirect effect of the administration of this hormone. It seems conceivable that prolactin itself might influence extrahypothalmic DA systems. Fuxe has seen an effect on part of the mesolimbic DA system (Fuxe et al., 1977). PARTICIPATION OF BRAINSTEM STRUCTURES Since Bohus and de Wied (1967a) demonstrated that small bilateral lesions of the parafascicular nucleus of the thalamus abolished the effect of a-MSH on extinction of a conditioned avoidance behavior, we were interested to see whether the arcuate and nigral DA neuron groups could be influenced from this area. Unilateral electrical stimulation of the parafascicular nucleus in urethane anesthesia indeed elicited an increase in fluorescence intensity of both DA neuron groups (Table I; Lichtensteiger & Lienhart, 1975b). A similar response was obtained by stimulation in neighboring posteromedial thalamic regions and in the dorsal hippocampns. The bigger posteromedial thalamic region has been implicated in the control of acquisition while the parafascicular nucleus was related more specifically to extinction (Delacour, Albe-Fessard & Libouban, 1966; Bohus & de Wied, 1967b). Although we used relatively low current intensities, it is obviously not possible to precisely identify the structures influenced by the stimulation. The results of the stimulation experiments demonstrated that coordinate responses of both DA neuron groups can be elicited from areas apparently involved in the behavioral peptide effects. They do not prove, however, that these regions are required for the manifestation of the effect of a-MSH on the two DA systems. Although 3H-a-MSH has recently been found to accumulate in various brain regions (Kastin, Nissen, Nikolics, Medzihradszky, Coy, Teplan & Schally, 1976), the site(s) of action of systemically administered peptide remains uncertain. The posteromedial thalamic region may well act as a relay area for projections from other brain sites. Before trying to eliminate this region, we have therefore started a series of lesion experiments aimed at projections from the lower brainstem. Dorsomedial tegmental lesions at the level of the fascial nucleus prevented the response of either the nigral DA system or both DA neuron groups to a-MSH (Fig. 2). This suggested that an even more caudally located structure, the area postrema, might be involved, as its situation outside the blood-brain barrier (Wislocki & Leduc, 1952; Wilson, Murray & Titus, 1962; Lichtensteiger & Langemann, 1966) would be particularly favorable for a peptide effect. Complete lesions of this area which spared
242
WALTERLICHTENSTEIGER,RUTHLIENHARTand HANSGEORGKOPP
"
~
~
r
a
l
SH in DA neurones .x\~ NJgrol and arcuate DA neurones r ~ N o blockade of response
Fro. 2. Lower brainstem lesions affecting the reaction of the arcuate and nigral DA neuron groups to aMSH. Lesions were made 2 weeks after ovariectomy and a-MSH (100/~g/kg) injected i.p. one week later after the one-day estrogen-progesterone-pretreatment. In addition to the ventral part of the medial longitudinal fasciculus, the medial zone of the nuc. reticularis gigantocellularis (rg) was most constistently involved, ra = hue. raphes, rp = nuc. reticularis parvocellularis, ph = n u c . praepositus hypoglossi, vm nuc. vestibularis med., so = hue. tractus solitarii, VII = nuc. nervi facialis, co = nuc. cochlearis dorsalis, XII = hue. nervi hypoglossi, ol = n u c . olivaris, pgl = n u c . paragigantocellularis lateralis. - - Mean intensities (4- S.D.; cell count) of the nigral D A neurons (L = left side, R = right side): rat 459 L ~ 4.029 40.3068 (160), R = 3.975 4- 0.3139 (160); rat 461 L = 3.897 4- 0.5768 (140), R = 4.003 4- 0.6120 (141); rat 466 L = 3.920 4- 0.4137 (140), R = 4.015 4- 0.3476 (136); rat 465 L : 4.142 4- 0.3053 (161), R = 4.146 4- 0.3478 (160). Mean intensities of the arcuate D A neurons: rat 459 L = 4.132 4- 0.4230 (70), R = 3.966 4- 0.4427 (75); rat 461 L = 3.541 4- 0.3918 (54), R ~ 3.578 :E 0.4445 (82); rat 466 L = 3.748 40.4296 (75), R = 3.770 4- 0.4138 (116); rat 465 L = 3.884 4- 0.4766 (53), R = 3.881 4- 0.3310 (56). (cf. Table II). the s u r r o u n d i n g structures (Fig. 3), entirely prevented the rise in intensity otherwise induced by a - M S H in the two D A n e u r o n groups (Table I I ; Lichtensteiger & Lienhart, 1975b, 1977). Yet, the role of area p o s t r e m a in the m a n i f e s t a t i o n of the effect exerted by the peptide o n the D A systems, m a y be more complex t h a n was originally thought. Whereas the intensity of the arcuate D A n e u r o n s of lesioned rats treated with a - M S H r e m a i n e d in the range of saline-injected intact a n d lesioned controls, the intensity of the nigral D A n e u r o n g r o u p actually decreased to below control levels in lesioned rats injected with a - M S H . This m e a n s that the response of the nigral D A n e u r o n s was reversed rather t h a n being simply abolished by the lesion. As far as the nigral D A system is concerned, a lesion of area p o s t r e m a m a y thus have induced a change in the responsiveness to a - M S H rather t h a n preventing any effect of the peptide. Preliminary observations made in u r e t h a n e anesthesia likewise suggest that the direction of response of the nigral D A system to aM S H m a y depend u p o n the f u n c t i o n a l state of the central nervous system: I n urethaneanesthetized female rats, 100/~g/kg a - M S H caused a reduction in firing of z o n a c o m p a c t a units recorded extracellularly which was accompanied by a slight decrease in fluorescence intensity of the nigral D A n e u r o n s (Felix & Lichtensteiger, u n p u b l i s h e d observations). A different effect of a - M S H had also been n o t e d previously in the male mouse, where the peptide did n o t elicit a rise b u t rather, if anything, a slight decrease of fluorescence intensity
243
FIG. 3. Representative lesion o f area postrema. T h e lesion completely destroyed area p o s t r e m a (ap) b u t left the s u r r o u n d i n g structures practically intact, g = nuc. gracilis, so = nuc. tractus solitarii, XII = nuc. nervi hypoglossi.
MSH ONNIGRALANDHYPOTHALAMICDoPxur~ENEURONS
245
in nigral DAneurons (Lichtensteiger & Lienhart, 1975a). Interestingly, in this species, the peptide delayed by about 15 rain the onset of the rise in intensity induced in the same neuron group by 40 mg/kg morphine. This was accompanied by a delay in the typical locomotor activation. INVOLVEMENT OF CHOLINERGIC SYSTEMS We have previously noted that atropine reduced or blocked the intensity response of the arcuate DA neurons to electrical stimulation in various extrahypothalamic regions. In the case of stimulation in the medial preoptic area, the effect of different doses of the drug was studied and a linear log dose-response relation was observed (Lichtensteiger & Keller, 1974). The same drug has now been found also to suppress the reaction of both the arcuate and nigral DA neuron groups to stimulation in the parafascicular nucleus (Lichtensteiger & Lienhart, 1975b). Moreover, atropine completely prevented the rise in intensity induced by a-MSH in the nigral DA neurons, whereas the reaction of the arcuate DA neuron group was partially reduced. Thus, it appears that cholinergic systems play a role in both, the effect of a-MSH on the two DA systems as well as the transmission of the stimulatory signal from various extrahypothalamic areas, notably the posteromedial thalamic region, to the arcuate or both DA neuron groups, respectively. Because of the wide distribution of cholinergic systems, these observations do not enable us to decide whether the same cholinergic projection was involved in the various situations. The manifestation of the effects elicited by stimulation or peptide injection may as well depend upon a modulatory cholinergic influence at various levels. The clear-cut quantitative difference in the drug effect on the response of the two DA neuron groups to a-MSH further suggests that there may exist certain differences between the two DA systems with respect to the neuronal circuitry involved. CONCLUSIONS The present observations clearly indicate that both, the tubero-hypophyseal and the nigrostriatal DA systems respond, to systemic administration of a-MSH and ACTH 4-10 as well as to prolactin. The effectiveness of ACTH 4-10 appears to rule out a participation of peripheral endocrine mechanisms in this response, while it is not yet completely certain whether the peptide effects on the two DA systems were due exclusively to the peptides themselves or brought about by some interaction with pituitary function. It may be mentioned that a subacute effect of higher doses of ACTH 4-10 (ca 500/~g/kg, at 72 and 24 hr before examination) on 3H-tyrosine incorporation into whole brain catecholamines was abolished by hypophysectomy (Versteeg & Wurtman, 1975). To what extent changes in DA systems were included in this observation, remains uncertain, however. In any case, the a-MSH-induced responses do not appear to result from increased prolactin levels--which evidently might have exerted an influence (cf. above), since prolactin levels tended to decrease after s-MS H (Fig. 4). The effect of a-MSH was found to depend upon the integrity of projections ascending from the lower brainstem and originating in area postrema. Whether this ascending system transmits a signal caused by an action of a-MSH at the level of the area postrema or whether it simply modulates an effect of a-MSH at a higher level, remains to be elucidated. Cholinergic systems appear to be involved in the action of a-MSH which is of interest in
246
WALTERLICHTENSTEIGER,RUTI-I LIENHARTand HANSGEORGKOPP LH GH, proloctin ng/mt nq/mt
(4)
LH
(9)
200
GH.
II
Prolacfin
(4)
20
(7)
(4) T
I
(hi
IOC
C =°MSHAtACTHI-24 I0 I00 + Fg/k~l aMSH
C aMSHAtACTHI-24 I0100 + /~g/kg aMSH
C c=MSHACACTHI-24 I0100 i#g/kg aMSH
FIG. 4. Serum levels (mean 4- S.E.M., No. of rats in brackets) of luteinizing hormone (LH), growth hormone (GH) and prolactin 30 min after i.p. injection of saline (C), a-MSH 10 or 100/~g/kg, 100/~g/kg a-MSH 15 min after 10 mg/kg atropine s.c. (At + a-MSH), or ACTH 1-24 (175/Lg/kg). Hormones were determined by radioimmunoassay using NIH kits (2-3 determinations/rat). view of their participation in the transmission of influences from extrahypothalamic areas to both D A systems. The response of the arcuate D A neurons to a-MSH can be viewed primarily as a reflection of a feedback mechanism aimed at the control of M S H secretion. However, the possibility of crossed feedback effects on other hormone axes of the pituitary should also be considered, even if there may exist some functional specialization within the tubero-inftmdibular D A system (cf. Lichtensteiger & Keller, 1974; Fuxe, L6fstr6m, Eneroth, Gustafsson, Skett, H6kfelt, Wiesel & Agnati, 1977). An analysis of the distribution of individual fluorescence intensities (variance, uni- vs bimodality etc.) did not reveal an increased heterogeneity of the arcuate D A neuron group after a-MSH. The hormone levels of our a-MSH-injected rats (Fig. 4) suggest that especially prolactin secretion may have been reduced through a crossed negative feedback mechanism. Because of the high variability of prolactin concentrations, the effect was not significant. However, it may be noted that A C T H 1-24, which did not influence the arcuate D A neurons, remained without effect on these hormone levels, a-MSH appeared to affect prolactin levels despite atropine pretreatment. As mentioned above, the drug did not abolish the response of the arcuate D A neurons to a - M S H but simply reduced it. The peptide effects on the nigral D A neuron group are probably linked with some extrahypothalamic actions. These need not be restricted to the caudate-putamen, as the rostral part of the nigral D A neurons--which we investigated--has recently been reported to project also to limbic cortical areas (Lindvall & Bj6rklund, 1974). In conclusion, it seems conceivable that the neuroendocrine circuit in which the two D A neuron groups are incorporated, may play a role in adaptive and emotional processes. We wish to acknowledge the excellent technical assistance of Miss F. Boller, Miss R. B6swald and Mrs. Z. Hefti. The investigations were supported by SNF grants (3.1870.73 and 3.669-0.75), by the HartmannMfiiler Stiftung and by the Jubilfiumsspende of the University of Zfirich.
MSH ON NIGRALAND HYPOTHALAM~CDOPAMINENEURONS
247
REFERENCES BJ6RKLUND, A., FALCK,B., HROMEK,F., OWMAN,C. & WEST, K. A. (1970) Identification and terminal distribution of the tubero-hypophyseal monoamine fibre systems in the rat by means of stereotaxic and microspectrofluorimetric techniques. Brain Res. 17, 1-23. BJ6RKLUND, A., MOORE, R. Y., Nosn~, A. & STE~vI, U. (1973) The organization of tubero-hypophyseal and reticulo-infundibularcatecholamine neuron systems in the rat brain. Brain Res. 51, 171-191. BOHUS,B. & DE WIED,D. (1967a) Failure of a-MSH to delay extinction of conditioned avoidance behavior in rats with lesions in the parafascicular nuclei of the thalamus. Physiol. Behav. 2, 221-223. BOHUS,B. & DE WI~D, D. (1967b) Avoidance and escape behavior following medial thalamic lesions in rats. J. camp. Physiol. PsychoL 64, 26-29. BOWER,A., HADLEY,M. E. & HRUBY,V. J. (1974) Biogenic amines and control of melanophore stimulating hormone release. Science 184, 70-72. DE WrieD, D. (1969) Effects of peptide hormones on behavior. In Frontiers in Neuroendoerinology, W. F. Ganong and L. Martini (Eds.), pp. 97-140. Oxford University Press, New York. DELACOUR,J., ALBE-FESSARD,D. & LIBOUBAN,S. (1966) Role chez le rat de denx noyaux thalamiques dans le conditionnement instrumental. Neuropsychologia 4, 101-112. FuxE, K., I.,6PSTR6M,A., ENEROTH,P., GUSTAFSSON,J..~., SKETF,P., H6K~LT, T., WIESEL,F.-A. &AONATI, L. (1977) Involvement of central catecholamines in the feedback actions of 17fl-estradiolbenzoate on luteinizing hormone secretion in the ovariectomized female rat. Psyehoneuroendocrinology 2, 203-225. Gouo~r, A., DUVF.RNOY,J. & BU~NON,C. (1973) Etude cin6tique compar~ en fluorescence de la d6pl~tion en monoamines de la zone externe de l'6minence me,diane apr~s bloc.age de leur synth~se par l'a-m~thylpara-tyrosine, chez des rats t6moins et bisurr~nalectomis6s. C.r. S~ane. Sac. Biol. 167, 919-923. H6K~PZLT,T. & FUXE,K. (1972) Effects of prolactin and ergot alkaloids on the tubero-infundibulardopamine (DA) neurons. Neuroendocrinology 9, 100-122. KASTIN, A. J., MILLER,L. H., NOCKTON, R., SANDMAN,C. A., SCHALLY,A. V. & STRATTON,L. O. (1973) Behavioral aspects of melanocyte-stimulatinghormone (MSH). In Drug Effects on Neuroendoerine Regulation, E. Zimmermann, W. H. Gispen, B. H. Marks and D. de Wied (Eds.), pp. 461-470. Elsevier, Amsterdam. KASTIN, A. J., NISSEN,C., NIKOLICS,K., MEDZIHRADSZKY,K., Coy, D. H., TEPLAN, I. & SCHALLY,A. V. 09?6) Distribution of 3H-a-MSH in rat brain. Brain Res. Bull. 1, 19-26. KAMBERI,I. A., MICAL,R. S. & PORTER,J. C. (1970) Prolactin-inhibitingactivity in hypophysial stalk blood and elevation by dopamine. Experientia 26, 1150-1151. KOS'TRZEWA,R. M., KASTn~,A. J. & SPmTEs, M. A. (1975) a-MSH and MIF-I effects on catecholamine levels and synthesis in various rat brain areas. Pharmac. Biochem. Behav. 3, 1017-1023. LICHTENS1~JOER,W. (1969a) The catecholamine content of hypothalamic nerve cells after acute exposure to cold and thyroxine administration.J. Physiol., Lond. 203, 675-687. LXCHTENST~I6ER,W. (1969b) Cyclic variations of catecholamine content in hypothalamic nerve cells during the estrous cycle of the rat, with a concomitant study of the substantia nigra. J. Pharmac. exp. Ther. 165, 204-215. LICHTENSTEIOER,W. (1970) Katecholaminhaltige Neurone in der neuroendokrinen Stenerung. Prinzip und Anwendung der Mikrofluorimetrie. Prog. Histochem. Cytochem. 1, 185-276. LICHTENSTEI6ER,W. (1971) Effect of electrical stimulation on the fluorescence intensity of catecholaminecontaining tuberal nerve cells. J. Physiol., Lond. 218, 63-84. LICHTENST~I6ER,W. & Ir~LLER, P. J. (1974) Tubero-infundibulardopamine neurons and the secretion of luteinizing hormone and prolactin: extrahypothalamic influences, interaction with cholinergic systems and the effect of urethane anesthesia. Brain Res. 74, 279-303. LICHTENSTEIGER,W. & LAN6EMANN,H. (1966) Uptake of exogenous catecholamines by monoaminecontaining neurons of the central nervous system: uptake by arcuato-infundibularneurons. J. Pharmac. exp. Ther. 151, 400-408. LICnTENSTEIGER,W. &Lr~r~IART,R. (1975a) Opiates and hypothalamic mechanisms: an ascending influence on diencephalic and mesencephalic dopamine (DA) systems and its interaction with a-MSH. Symposium on Acute Effects of Narcotic Analgesics; Sites and Mechanisms of Action, Nokkala, Espoo, Abstracts. pp. 26-27. LIcrrrENsTEI6ER,W. & LIENnART,R. (1975b) Central action of a-MSH and prolactin: simultaneous responses of hypothalamic and mesencephalic dopamine (DA) systems. In Cellular and Molecular Bases of Neuroendocrine Processes, E. Endr6czi (Ed.), pp. 211-221. Akad6miai Kiad6, Budapest. LIcrrrENsTEIGER, W. & LI~,tART, R. (1977) Response of mesencephalic and hypothalamic dopamine neurones to ~-MSH : mediated by area postrema? Nature Lond. 266, 635-637.
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LICHTENSTEIGER,W., FELIX,D., LIENHART,R. & HEl~rl, F. (1976) A quantitative correlation between single unit activity and fluorescence intensity of dopamine neurones in zona compacta of substantia nigra, as demonstrated under the influence of nicotine and physostigmine. Brain Res. 117, 85-103. LIENHART, R., LICH~NST~IGER,W. & LANGEMANN,H. (1975) Response of nigral dopamine neurons to acute and prolonged morphine treatment: effect of exposure to cold, physostigmine and nicotine. Naunyn Schmiedebergs Arch. exp. Path. Pharmak. 286, 353-369. LINDVALL,O. & BJ6RKLUND,A. (1974) The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence method. Acta physiol, scand. Suppl. 412, 1-48. Lu, K.-H., KOCH, Y. & M~rrEs, J. (1971) Direct inhibition by ergocornine of pituitary prola~tin release. Emlocrinology 89, 229-233. MACLU~D,R. M. & LEm~VER, J. E. (1974) Studies on the mechanism of the dopamine-mediated inhibition of prolactin secretion. Endocrinology 94, 1077-1085. MACLF.~D,R. M., FOlCrttAM,E. H. & LEHIV~YER,J. E. (1970) Prolactin and growth hormone production as inttuenced by catecholamin~ and agents that affect brain catecholamincs. Neuroendocr#tology 6, 283-294. MXALm~-VoLoss,C. (1958) Posthypophyse et activit6 corticotrop¢. Acta endocr. Suppl. 35, 1-96. ScApA~Nrm, U., VAN LOON, G. R., MOB~O, G. P., Pin,zion, P. & GANONG,W. F. (1972) Evidence for central norepinephrine-mediated inhibitionof ACTH secretion in the rat. Neuroendocrinology 10, 155-160. TALEmNm,S., TO~IIS, M. E. & C-~Lm,M. E. (1972) Role of catecholamines in the control of melanocytestimulating hormone secretion in rats. Neuroendocrinology 10, 235-245. TILOE~, F. J. H., Mut.D~t, A. H. & S~Lm, P. G. (1975) On the presence of a MSH-release inhibiting system in the rat neurointermediatelobe. Neuroendocrinology 18, 125-130. VEaS~G, D. H. G. & WUll1~vtm~,R. J. (1975) Effect of ACTH 4-10 on the rate of synthesis of (3H) catecholamines in the brains of intact, hypophys~tomi~d and a ~ l e c t o m i z e d rats. Brain Res. 93, 552-557. WILSON,C. W. M., MURRAY,A. W. & Trrus, E. (1962) The effect of r ~ r p i n e on uptake of epinephrine in brain and certain areas outside the blood-brain barrier, at. Pharmac. exp. Ther. 135, 11-16. WtSLOC~I,G. B. & LEDUC,E. H. (1952) Vital staining of the hematt~ncephalic barrier by silver nitrate and trypan blue, and cytological comparisons of the neurohypophysis, pineal body, area postreana, intercolumnar tubercle and supraoptic crest. J. comp. Neurol. 96, 371-413.
PEPTIDE HORMONES A N D CENTRAL DOPAMINE NEURON SYSTEMS WALTER LICHTENSTEIGER,RUTH LIENHART and HANS GEORG KOPP Department of Pharmacology, University of Ziirich, Ziirich, Switzerland (Received 2 August 1976) SUMMARY (1) In ovariectomized, estrogen-progesterone-pretreated rats, the dopamine (DA) neuron group of the arcuate nucleus of the hypothalamus which also projects to the intermediate lobe of the pituitary, and that of the substantia nigra, reacted markedly within 30 rain to single intraperitoneal injections of a-MSH. The response, an increase in cellular fluorescence intensity, was disclosed by histocbemical microfluorimetry. (2) ACTH 4-10 and prolactin had a similar action whereas ACTH 1-24 was ineffective in the dose used. (3) The response of both DA neuron groups was abolished by lesions of the area postrema and by more rostral brainstem lesions, indicating the participation of ascending systems. (4) Both neuron groups were further influenced by electrical stimulation in posteromediai thalami¢ areas known to be involved in central MSH effects. (5) Pretreatment with atropine blocked the stimulationinduced response of both DA neuron groups and the nigral reaction to a-MSH and reduced that of the arcuate DA neurons to the peptide, which suggests the participation of cholinergic systems. Key Words--a-melanocyte-stimulating hormone (a-MSH); dopamine neurons; substantia nigra; area postrema.
INTRODUCTION THE BEHAVIORALand other central effects of the pars intermedia hormone, a-melanocytestimulating hormone (a-MSH), and of related peptides, count among the most striking actions of these substances in mammals (de Wied, 1969; Kastin, Miller, Nockton, Sandman, Schally & Stratton, 1973). The intermediate lobe of the pituitary receives an abundant projection of dopaminergic (DA) fibers which belong to the tubero-infundibular or tuberohypophyseal D A neuron system (Bj6rkltmd, Falck, Hromek, O w m a n & West, 1970; Bj6rklund, Moore, Nobin & Stenevi, 1973). This D A neuron group readily responds to electrical stimulation in various extrahypothalamic areas thought to be involved in the integration of endocrine and behavioxal processes (Table I; Lichtensteiger, 1971; Lichtensteiger & Keller, 1974; Lichtensteiger & Lienhart, 1975b). Although the stimulatory responses were often accompanied by changes in serum levels of anterior pituitary hormones, it appeared to us that some of the extrahypothalamic influences on the tubero-hypophyseal D A system might also be related to the control of the intermediate lobe. In other words, this D A system might constitute a link between extrahypothalamic structures involved in the action of behaviorally active peptides and the intermediate lobe, a site of production o f such peptides. We approached this question first by investigating whether the tuberohypophyseal D A neurons reacted to systemically administered a - M S H and related peptides. 237
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WALTERLICHTENSTF~OER,RUTHLENHARTand HANSGEORGKOPP
TABLE I. EXTRAHYPOTHALAMIC STIMULATION SITES ELICITING AN ACUTE RISE IN FLUORESCENCE INTENSITY OF DA NERVE CELL GROUPS IN URETHANE-ANESTHETIZED RATS
Electrode position Medial preoptic area Diagonal tract nucleus Medial arnygdaloid nucleus Bed nucleus of stria terminalis Ventral tegmental area of midbrain (Tsai) Parafascicular thalamic nuc. Posteromedian and medial thalamic nuclei Med. habenular nucleus Dorsal hippoeampus
Parameters of stimulation (1)
DA neuron groups studied (reference)
100/sec, 100 #A, 5-30 min 100/sec, 100 #A, 10min 100[sec, 100 #A, 10-15 min 100[sec, 100 #A, 10min
Arcuate, nigral (2,3,4) Arcuate (3) Arcuate (3 and unpubl.) Arcuate (3)
100/sec, 100 #A, 10 min 30[sec, I00 t~A, 10min
Arcuate (3) Arcuate, nigral (4)
30/sec, 100/~A, 10 min 30/see, 100/~A, 10rain 30/sec, 100/~A, 10 min
Arcuate, nigral (4) Arcuate, nigral (4) Arcuate, nigral (4)
(1) Rats ovariectomized for 3 weeks, estrogen-progesterone-pretreated. Bipolar concentric electrode, 0.5 msec monophasic positive pulses, 15 see on/off. (2) Lichtensteiger, 1971 ; (3) Lichtensteiger & Keller, 1974; (4) Lichtensteiger & Lienhart, 1975b. In the same animals, an extrahypothalamic D A system, the nigrostriatal system, was also studied. Observations on similar reactions of the arcuate and nigral D A systems to certain environmental and other functional changes (e.g. acute exposure to cold, Lichtensteiger, 1969a; Lienhart, Lichtensteiger & Langemann, 1975) suggested that there might exist coordinate responses of the two systems despite differences in other situations such as the estrous cycle (Lichtensteiger, 1969b). The peptide effects were investigated in female Sprague-Dawley-Ivanovas rats ovariectomized for 3 weeks and pretreated for 1 day with 5 /~g estradiol-dipropionate and 2 mg progesterone (s.c.), since the extrahypothalamic influences had previously been studied on the same animal model. All experiments were performed in the afternoon, the rats being kept at a regular light-dark cycle (14 hr light period; Lichtensteiger & Lienhart, 1975b, 1977). NOTE ON THE MICROFLUORIMETRIC ANALYSIS OF DA NEURON GROUPS The functional state of the two D A neuron groups was assessed by a microfluorimetric technique based on the histochemical fluorescence method of Falck and Hillarp (Lichtensteiger, 1969b, 1970). Thereby, the intensity of the fluorescent light emitted by catecholamine-containing nerve cells is expressed as a fraction of a norepinephrine-containing gelatin standard processed together with the tissue. Account is taken of non-specific fluorescence of brain tissue (tissue blank) and fluorescence of the norepinephrine-free gelatin (standard blank). The distributions of relative fluorescence intensities of individual neurons are characteristic for a given functional state and neuron group. In studies on the nigral D A neuron group, we tlave observed a close non-linear correlation between mean fluorescence intensity and mean firing rate (Lichtensteiger, Felix, Lienhart & Hefti, 1976); in agreement with earlier less direct" evidence (Lichtensteiger, 1971), an increase in mean firing rate was accompanied by a rise in mean cellular fluorescence intensity. Because of certain variations! in fluorescence intensity throughout the DA neuron groups, measure-
239
M S H ON NIGRAL AND HYPOTHALAMIC DOPAMINE NEURONS
m e n t s were always t a k e n at regular intervals between defined antero-posterior levels. I n the arcuate D A n e u r o n group, n e u r o n s of the whole area were studied, while in the s u b s t a n t i a nigra m e a s u r e m e n t s were restricted to the lateral half to two-thirds o f the z o n a c o m p a c t a (for details, see Lichtensteiger & Keller, 1974; L i e n h a r t et aL, 1975). RESPONSE O F T H E A R C U A T E A N D N I G R A L D A N E U R O N G R O U P S TO a-MSH, A C T H 1-24, ACTH 4-10 A N D P R O L A C T I N W h e n a d m i n i s t e r e d i.p. i n doses used i n m o s t behavioral experiments, a - M S H elicited a m a r k e d increase i n the fluorescence intensity o f b o t h D A n e u r o n g r o u p s within 30 rain (Table II). The two D A n e u r o n groups did n o t appear to be equally sensitive, however, as
TABLEII.
FLUORESCENCE INTENSITY OF THE ARCUATE AND NIGRAL DA NEURONS IN GROUPS OF RATS INJECTED WITH PEPTIDE HORMONES OR SALINE(;)
Treatment(2)
Saline, 30 min Phenol-containingvehicle(4J, 30 min a-MSH, 100 tLg/kg, 30 rain a-MSH, 33 t~g/kg, 30 min a-MSH, 10 ~g/kg, 30 min a-MSH, 3 ttg/kg, 30 win ACTH 1-24, 175 tzg/kg, 30 min Prolactin, 2 mg/kg, 30 rain Atropine, 10 mg/kg, 15 min before a-MSH, 100/~g/kg, 30 rain Area postrema lesion q- saline, 30 rain Area postrema lesion + a-MSH, 100 ttg/kg, 30 min(~)
Arctmte DA neurons Nigral DA neurons mean 4- S . D . (3) cell count mean -4- S.D. (a) cell count (No. of rats) (No. of rats) 3.666 4- 0.4357 3.655 4- 0.4394 3.882 4- 0.4468 3.944 -4-0.4466 3.988 4- 0.4142 3.972 4- 0.4828 3.625 4- 0.4593 3.855 4- 0.4330
1266(6) 706 (3) 1074(5) 431 (2) 863 (4) 155 (1) 780 (4) 985 (5)
4.0754- 0.3510 4.037 4- 0.4083 4.2454- 0.3290 4.2154- 0.3309 3.995+ 0.3500 4.034 4- 0.3355 4.141 4- 0.3485 4.232 4- 0.3394
3.797 4- 0.4149 3.718 4- 0.4101
981 (4) 1016(4)
3.9114- 0.4739 3.9774- 0.3406
640 (4) 1285(4)
3.659 4- 0.4204
886 (4)
3.900 4- 0.3661
1044(4)
940 (6) 481 (3) 840 (5)(s) 320 (2) 700 (4) 160 (1) 639 (4) 790 (5)
(1) The two DA neuron groups were ~tudied in the same rats. Data from Lichtensteiger and Lienhart (1975b, 1977). For values of individual rats, of. these references. (2) All substances i.p. except atropine (s.c.). (3) Mean values of (normalized) distributions of logarithmically transformed intensity values. In substantia nigra, both sides were studied only in the lesioned rats which explains the higher cell counts in these animah. No side differences were observed after area postrema lesions, however. (4) Used as a vehicle for prolactin. (5) After inclusion of 2 additional rats with no data on the arcuate DA neuron group, the nigral mean would be 4.259 -4- 0.3503 1159 (7). (6) Rats with complete destruction of area postrema. If 2 rats with small remnants of this area are included, mean of arcuate DA neurons = 3.769 -4- 0.4099 1398 (6), mean of nigral DA neurons = 3.888 4- 0.3601 1625 (6). lower doses of the p e p t i d e failed to influence the n i g i a l D A n e u r o n s whereas the arcuate D A n e u r o n s r e s p o n d e d to the whole dose range studied so far. Provided the relationship which we have repeatedly observed between intensity change a n d s t i m u l a t i o n o f D A n e u r o n s existed also u n d e r the present experimental conditions, o u r findings indicate that b o t h D A n e u r o n groups were stimulated by the peptide. Since M S H secretion is generally
240
WALTER LlcrrrENSTEXOER, RUTH LIENHARTand HANS GEORG KOPP
inhibited by catecholamines (Taleisnik, Tomatis & Celis, 1972; Bower, Hadley & Hruby, 1974; see also Tilders, Mulder & Smelik, 1975), the response of the arcuate DA group probably reflects the activation of a negative feedback mechanism. On the other hand, the nigral response may be related to some of the extrahypothalamic effects of a-MSH. In intact male rats injected with 100/zg/kg a-MSH on 3 days, the decrease in DA concentration of midbrain and striatum after inhibition of tyrosine hydroxylase was found unchanged (Kostrzewa, Kastin & Spirtes, 1975). It cannot be decided as yet whether the discrepancy with our results is due to differences in the experimental situation or in the sensitivity of the methods used. We tried to get some further insight into the effect of a-MSH on the DA systems by checking their reaction to two related peptides, ACTH 1-24 and ACTH 4-10. In a dose that is approx equimolar to the highest dose of a-MSH used, ACTH 1-24 remained completely ineffective on the arcuate DA neurons. In half of the animals, fluorescence intensity increased somewhat in the rtigral neurons but the group as a whole did not differ significantly from the controls (Table H, Lichtensteiger & Lienhart, 1977). While this result does not exclude an effect of ACTH 1-24 at higher doses, it indicates that this hormone apparently is considerably less effective than a-MSH. The lack of a demonstrable feedback action of ACTH 1-24 on the arcuate DA neurons is in agreement with the view that the tubero-infundibular DA neurons are of minor importance in the control of ACTH secretion (Scapagnini, Van Loon, Moberg, Preziosi & Ganong, 1972). This view has been questioned, however (Gouget, Duvernoy & Bugnon, 1973). If, under certain conditions, ACTH is indeed preferentially released from the intermediate lobe as suggested by MialheVoloss (1958), this special situation at least might depend upon the DA innovation of the pars intermedia. Quite in contrast to ACTH 1-24, ACTH 4-10 induced a very strong response of both DA neuron groups (Fig. 1). This observation is of special interest because
-4'-+-
4.4
E 4.2 t3 4-~ 4.0
+
eSoline, 30min C] ACTH 1 - 2 4 , 1 7 5 / z g / k g , 3 0 m i n , v ACTH 4 - 1 0 , 50/.Lg/kg, 50rain
.C
zx
60min
8
ac 3',4
Areuofe nucleus
' 3'.6 ' 3'.8 410 ' 412 ' Relo~rive infensi+y ( m e o n , + 9 5 % conf. lira.; nor. log)
FIG. 1. Effect of i.p. injection of ACTH 4-10 (30 or 60 min) and ACTH 1-24 (30 min) on the fluorescence intensity of the arcuate and nigral DA neuron groups in ovadectomized rats pretreated for one day with estrogen and progesterone. For each rat, the mean value of the arcuate DA group is plotted on the abscissa, that of the nigral D A group on the ordinate (means of logarithmically transformed intensity distributions, cf. Table II). ACTH 4-10 elicited a marked response of both DA systems.
MSH ONNmRALANDHYPOTHAIAMICD o P ~
NEURONS
241
ACTH 4-10 is devoid of peripheral hormonal activity (de Wied, 1969). One may say that in our system it resembled a-MSH rather than ACTH. In addition to these two peptides, prolactin, whose secretion is inhibited by DA (Kamberi, Mical & Porter, 1970; Lu, Koch & MeRes, 1971 ; Macleod, Fontham & Lehmeyer, 1970; Macleod & Lehmeyer, 1974), was studied in order to check the reaction of the arcuate DA neurons which had earlier been reported to respond to this hormone by an increase in DA turnover (Htkfelt & Fuxe, 1972). The result--a rise in cellular fluorescence intensity which we interpret as indicative of an activation fully agrees with the notion of an activation of a negative feedback mechanism. It demonstrates that the change in the arcuate DA neurons is not specific for a-MSH, which is in keeping with the fact that these neurons control various hormone axes. Surprisingly, the nigral DA neurons were also influenced (Table II). We cannot decide as yet whether the reaction observed in this DA system is due to prolactin itself or to some indirect effect of the administration of this hormone. It seems conceivable that prolactin itself might influence extrahypothalmic DA systems. Fuxe has seen an effect on part of the mesolimbic DA system (Fuxe et al., 1977). PARTICIPATION OF BRAINSTEM STRUCTURES Since Bohus and de Wied (1967a) demonstrated that small bilateral lesions of the parafascicular nucleus of the thalamus abolished the effect of a-MSH on extinction of a conditioned avoidance behavior, we were interested to see whether the arcuate and nigral DA neuron groups could be influenced from this area. Unilateral electrical stimulation of the parafascicular nucleus in urethane anesthesia indeed elicited an increase in fluorescence intensity of both DA neuron groups (Table I; Lichtensteiger & Lienhart, 1975b). A similar response was obtained by stimulation in neighboring posteromedial thalamic regions and in the dorsal hippocampns. The bigger posteromedial thalamic region has been implicated in the control of acquisition while the parafascicular nucleus was related more specifically to extinction (Delacour, Albe-Fessard & Libouban, 1966; Bohus & de Wied, 1967b). Although we used relatively low current intensities, it is obviously not possible to precisely identify the structures influenced by the stimulation. The results of the stimulation experiments demonstrated that coordinate responses of both DA neuron groups can be elicited from areas apparently involved in the behavioral peptide effects. They do not prove, however, that these regions are required for the manifestation of the effect of a-MSH on the two DA systems. Although 3H-a-MSH has recently been found to accumulate in various brain regions (Kastin, Nissen, Nikolics, Medzihradszky, Coy, Teplan & Schally, 1976), the site(s) of action of systemically administered peptide remains uncertain. The posteromedial thalamic region may well act as a relay area for projections from other brain sites. Before trying to eliminate this region, we have therefore started a series of lesion experiments aimed at projections from the lower brainstem. Dorsomedial tegmental lesions at the level of the fascial nucleus prevented the response of either the nigral DA system or both DA neuron groups to a-MSH (Fig. 2). This suggested that an even more caudally located structure, the area postrema, might be involved, as its situation outside the blood-brain barrier (Wislocki & Leduc, 1952; Wilson, Murray & Titus, 1962; Lichtensteiger & Langemann, 1966) would be particularly favorable for a peptide effect. Complete lesions of this area which spared
242
WALTERLICHTENSTEIGER,RUTHLIENHARTand HANSGEORGKOPP
"
~
~
r
a
l
SH in DA neurones .x\~ NJgrol and arcuate DA neurones r ~ N o blockade of response
Fro. 2. Lower brainstem lesions affecting the reaction of the arcuate and nigral DA neuron groups to aMSH. Lesions were made 2 weeks after ovariectomy and a-MSH (100/~g/kg) injected i.p. one week later after the one-day estrogen-progesterone-pretreatment. In addition to the ventral part of the medial longitudinal fasciculus, the medial zone of the nuc. reticularis gigantocellularis (rg) was most constistently involved, ra = hue. raphes, rp = nuc. reticularis parvocellularis, ph = n u c . praepositus hypoglossi, vm nuc. vestibularis med., so = hue. tractus solitarii, VII = nuc. nervi facialis, co = nuc. cochlearis dorsalis, XII = hue. nervi hypoglossi, ol = n u c . olivaris, pgl = n u c . paragigantocellularis lateralis. - - Mean intensities (4- S.D.; cell count) of the nigral D A neurons (L = left side, R = right side): rat 459 L ~ 4.029 40.3068 (160), R = 3.975 4- 0.3139 (160); rat 461 L = 3.897 4- 0.5768 (140), R = 4.003 4- 0.6120 (141); rat 466 L = 3.920 4- 0.4137 (140), R = 4.015 4- 0.3476 (136); rat 465 L : 4.142 4- 0.3053 (161), R = 4.146 4- 0.3478 (160). Mean intensities of the arcuate D A neurons: rat 459 L = 4.132 4- 0.4230 (70), R = 3.966 4- 0.4427 (75); rat 461 L = 3.541 4- 0.3918 (54), R ~ 3.578 :E 0.4445 (82); rat 466 L = 3.748 40.4296 (75), R = 3.770 4- 0.4138 (116); rat 465 L = 3.884 4- 0.4766 (53), R = 3.881 4- 0.3310 (56). (cf. Table II). the s u r r o u n d i n g structures (Fig. 3), entirely prevented the rise in intensity otherwise induced by a - M S H in the two D A n e u r o n groups (Table I I ; Lichtensteiger & Lienhart, 1975b, 1977). Yet, the role of area p o s t r e m a in the m a n i f e s t a t i o n of the effect exerted by the peptide o n the D A systems, m a y be more complex t h a n was originally thought. Whereas the intensity of the arcuate D A n e u r o n s of lesioned rats treated with a - M S H r e m a i n e d in the range of saline-injected intact a n d lesioned controls, the intensity of the nigral D A n e u r o n g r o u p actually decreased to below control levels in lesioned rats injected with a - M S H . This m e a n s that the response of the nigral D A n e u r o n s was reversed rather t h a n being simply abolished by the lesion. As far as the nigral D A system is concerned, a lesion of area p o s t r e m a m a y thus have induced a change in the responsiveness to a - M S H rather t h a n preventing any effect of the peptide. Preliminary observations made in u r e t h a n e anesthesia likewise suggest that the direction of response of the nigral D A system to aM S H m a y depend u p o n the f u n c t i o n a l state of the central nervous system: I n urethaneanesthetized female rats, 100/~g/kg a - M S H caused a reduction in firing of z o n a c o m p a c t a units recorded extracellularly which was accompanied by a slight decrease in fluorescence intensity of the nigral D A n e u r o n s (Felix & Lichtensteiger, u n p u b l i s h e d observations). A different effect of a - M S H had also been n o t e d previously in the male mouse, where the peptide did n o t elicit a rise b u t rather, if anything, a slight decrease of fluorescence intensity
243
FIG. 3. Representative lesion o f area postrema. T h e lesion completely destroyed area p o s t r e m a (ap) b u t left the s u r r o u n d i n g structures practically intact, g = nuc. gracilis, so = nuc. tractus solitarii, XII = nuc. nervi hypoglossi.
MSH ONNIGRALANDHYPOTHALAMICDoPxur~ENEURONS
245
in nigral DAneurons (Lichtensteiger & Lienhart, 1975a). Interestingly, in this species, the peptide delayed by about 15 rain the onset of the rise in intensity induced in the same neuron group by 40 mg/kg morphine. This was accompanied by a delay in the typical locomotor activation. INVOLVEMENT OF CHOLINERGIC SYSTEMS We have previously noted that atropine reduced or blocked the intensity response of the arcuate DA neurons to electrical stimulation in various extrahypothalamic regions. In the case of stimulation in the medial preoptic area, the effect of different doses of the drug was studied and a linear log dose-response relation was observed (Lichtensteiger & Keller, 1974). The same drug has now been found also to suppress the reaction of both the arcuate and nigral DA neuron groups to stimulation in the parafascicular nucleus (Lichtensteiger & Lienhart, 1975b). Moreover, atropine completely prevented the rise in intensity induced by a-MSH in the nigral DA neurons, whereas the reaction of the arcuate DA neuron group was partially reduced. Thus, it appears that cholinergic systems play a role in both, the effect of a-MSH on the two DA systems as well as the transmission of the stimulatory signal from various extrahypothalamic areas, notably the posteromedial thalamic region, to the arcuate or both DA neuron groups, respectively. Because of the wide distribution of cholinergic systems, these observations do not enable us to decide whether the same cholinergic projection was involved in the various situations. The manifestation of the effects elicited by stimulation or peptide injection may as well depend upon a modulatory cholinergic influence at various levels. The clear-cut quantitative difference in the drug effect on the response of the two DA neuron groups to a-MSH further suggests that there may exist certain differences between the two DA systems with respect to the neuronal circuitry involved. CONCLUSIONS The present observations clearly indicate that both, the tubero-hypophyseal and the nigrostriatal DA systems respond, to systemic administration of a-MSH and ACTH 4-10 as well as to prolactin. The effectiveness of ACTH 4-10 appears to rule out a participation of peripheral endocrine mechanisms in this response, while it is not yet completely certain whether the peptide effects on the two DA systems were due exclusively to the peptides themselves or brought about by some interaction with pituitary function. It may be mentioned that a subacute effect of higher doses of ACTH 4-10 (ca 500/~g/kg, at 72 and 24 hr before examination) on 3H-tyrosine incorporation into whole brain catecholamines was abolished by hypophysectomy (Versteeg & Wurtman, 1975). To what extent changes in DA systems were included in this observation, remains uncertain, however. In any case, the a-MSH-induced responses do not appear to result from increased prolactin levels--which evidently might have exerted an influence (cf. above), since prolactin levels tended to decrease after s-MS H (Fig. 4). The effect of a-MSH was found to depend upon the integrity of projections ascending from the lower brainstem and originating in area postrema. Whether this ascending system transmits a signal caused by an action of a-MSH at the level of the area postrema or whether it simply modulates an effect of a-MSH at a higher level, remains to be elucidated. Cholinergic systems appear to be involved in the action of a-MSH which is of interest in
246
WALTERLICHTENSTEIGER,RUTI-I LIENHARTand HANSGEORGKOPP LH GH, proloctin ng/mt nq/mt
(4)
LH
(9)
200
GH.
II
Prolacfin
(4)
20
(7)
(4) T
I
(hi
IOC
C =°MSHAtACTHI-24 I0 I00 + Fg/k~l aMSH
C aMSHAtACTHI-24 I0100 + /~g/kg aMSH
C c=MSHACACTHI-24 I0100 i#g/kg aMSH
FIG. 4. Serum levels (mean 4- S.E.M., No. of rats in brackets) of luteinizing hormone (LH), growth hormone (GH) and prolactin 30 min after i.p. injection of saline (C), a-MSH 10 or 100/~g/kg, 100/~g/kg a-MSH 15 min after 10 mg/kg atropine s.c. (At + a-MSH), or ACTH 1-24 (175/Lg/kg). Hormones were determined by radioimmunoassay using NIH kits (2-3 determinations/rat). view of their participation in the transmission of influences from extrahypothalamic areas to both D A systems. The response of the arcuate D A neurons to a-MSH can be viewed primarily as a reflection of a feedback mechanism aimed at the control of M S H secretion. However, the possibility of crossed feedback effects on other hormone axes of the pituitary should also be considered, even if there may exist some functional specialization within the tubero-inftmdibular D A system (cf. Lichtensteiger & Keller, 1974; Fuxe, L6fstr6m, Eneroth, Gustafsson, Skett, H6kfelt, Wiesel & Agnati, 1977). An analysis of the distribution of individual fluorescence intensities (variance, uni- vs bimodality etc.) did not reveal an increased heterogeneity of the arcuate D A neuron group after a-MSH. The hormone levels of our a-MSH-injected rats (Fig. 4) suggest that especially prolactin secretion may have been reduced through a crossed negative feedback mechanism. Because of the high variability of prolactin concentrations, the effect was not significant. However, it may be noted that A C T H 1-24, which did not influence the arcuate D A neurons, remained without effect on these hormone levels, a-MSH appeared to affect prolactin levels despite atropine pretreatment. As mentioned above, the drug did not abolish the response of the arcuate D A neurons to a - M S H but simply reduced it. The peptide effects on the nigral D A neuron group are probably linked with some extrahypothalamic actions. These need not be restricted to the caudate-putamen, as the rostral part of the nigral D A neurons--which we investigated--has recently been reported to project also to limbic cortical areas (Lindvall & Bj6rklund, 1974). In conclusion, it seems conceivable that the neuroendocrine circuit in which the two D A neuron groups are incorporated, may play a role in adaptive and emotional processes. We wish to acknowledge the excellent technical assistance of Miss F. Boller, Miss R. B6swald and Mrs. Z. Hefti. The investigations were supported by SNF grants (3.1870.73 and 3.669-0.75), by the HartmannMfiiler Stiftung and by the Jubilfiumsspende of the University of Zfirich.
MSH ON NIGRALAND HYPOTHALAM~CDOPAMINENEURONS
247
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