Summary. In the present study the functional role of the striato-nigral dynorphin and substance P path- ways in rat brain has been studied using the rota-.
Ex mental BranResearch
Exp Brain Res (1986) 64:193-207
9 Springer-Verlag 1986
Striato-nigral dynorphin and substance P pathways in the rat II. Functional analysis M. Herrera-Marschitz 1, I. Christensson-Nylander 3, T. Sharp 1, W. Staines 2, M. Reid 1, T. H6kfelt 2, L. Terenius 3, and U. Ungerstedt 1 1 Departments of Pharmacologyand ZHistology,Karolinska Institutet, S-10401 Stockholm, Sweden 3 Department of Pharmacology, Universityof Uppsala, S-75124Uppsala, Sweden
Summary. In the present study the functional role of the striato-nigral dynorphin and substance P pathways in rat brain has been studied using the rotational behavioural model and an intracerebral dialysis technique complemented with brain lesions and immunohistochemical analysis. Attempts were made to evaluate whether these striato-nigral neurons have a feed-back modulatory action on the dopaminergic nigro-striatal system, or whether they represent an outflow pathway conveying motor information from the striatum. Unilateral injection of dynorphin A into the substantia nigra reticulata of naive rats induced contralateral rotational behaviour. This effect was dose-dependent and mimicked by the kappa-opioid receptor agonist, U50,488H. Intranigral injection of substance P, as well as substance K, also produced dose-dependent contralateral rotational behaviour. Unilateral injections of ibotenic acid into various sites of the striatum were used to destroy the striato-nigral pathways. The lesions produced a depletion of dynorphin- and substance P-like immunoreactivity in the pars reticulata of the substantia nigra ipsilateral to the lesion and markedly affected the behavioural responses to intranigral peptide injections. Dynorphin A more potently induced contralateral rotation in the lesioned compared to naive non-lesioned rats, suggesting development of supersensitivity for this peptide. Substance P on the other hand, was markedly less potent in inducing rotation in lesioned animals. The rotational responses to both dynorphin A and substance P were potentiated by injection of amphetamine 1 h later, suggesting that both peptides act via nigro-striatal dopamine neurons. However, in rats with unilateral nigro-striatal dopamine denervation, produced with 6-hydroxy-dopamine, dynorphin A retained its potency to induce rotational behaviour; substance P Offprint requests to: M. Herrera-Marschitz (address see above)
was again much less potent. Thus, both the ibotenic acid and 6-hydroxy-dopamine lesions differently affect the action of dynorphin A and substance P in the zona reticulata of the substantia nigra. The data suggests that substance P requires an intact dopamine pathway to produce the rotational response, while dynorphin A does not. Direct evidence that behavioural activation produced by dynorphin A is not dependent upon dopamine stimulation was obtained by intrastriatal dialysis experiments in which changes in striatal dopamine release were measured following intranigral injection of dynorphin A or substance P. Intranigral dynorphin A in fact reduced, while substance P increased the release of dopamine. It is concluded that the dynorphin and substance P striatonigral pathways have different functions. Thus, substance P in the striato-nigral pathway may have a role in a positive feed-back loop regulating the firing of nigro-striatal dopamine neurons, while dynorphin might be important in negative feed-back control. The rotational behaviour produced by DYN A is probably due to direct stimulation of receptors located on nigro-thalamic and nigro-tectal pathways.
Key words: Basal ganglia - - Dopamine - Dynorphin - Substance P - 6-hydroxy-dopamine - Ibotenic acid - Rotational behaviour - Intracerebral dialysis technique - Feedback regulation - Efferent pathways
Introduction A high density of dynorphin (DYN) (Goldstein et al. 1979, 1981; Tachibana et al. 1982) immunoreactive nerve terminals has been observed in the zona reticulata of the substantia nigra (SNR) in the rat (Vincent et al. 1982a, b; Weber et al. 1982), and this
194
area also contains high levels of DYN peptides as measured by radioimmunoassay in rats and other species (Gramsch et al. 1982; Maysinger et al. 1982; Zamir et al. 1983, 1984a-e; Pittius et al. 1984; Christensson-Nylander and Terenius 1985; Dores et al. 1985; Christensson et al. 1986). Thus, these peptide-containing fibers surround the dendrites of the nigro-striatal DA neurons (Dahlstr6m and Fuxe 1964, Anden et al. 1966, Ungerstedt 1971, Lindvall and Bj6rklund 1974). There is evidence that the DYN fibers in the SNR originate in the striatum (Vincent et al. 1982a, b; Zamir et al. 1984a-e, Fallon et al. 1985), constituting yet another component of the striato-nigral pathway (for review see McGeer et al., 1984). These results suggest the presence of receptors in the SNR sensitive to DYN stimulation. Indeed, unilateral injections of DYN-(1-17) (DYN A) into the SNR have been found to cause dose dependent contralateral rotation (HerreraMarschitz et al. 1983, 1984a, b; Morelli and Di Chiara 1985). This effect is mimicked by the shorter fragments DYN-(1-8) and DYN-(1-13), but also by DYN-(6-17), a fragment that lacks opioid activity (Walker et al. 1982a, b). We have now analysed the striato-nigral DYN pathway in more detail by examining the effect of intrastriatal injections of the neurotoxin ibotenic acid (IBA) (Schwarcz et al. 1979) on both nigral levels of DYN and DYN-induced functional responses. In an accompanying paper (Christensson-Nylander et al. 1986), the effects of striatal IBA lesions on the nigral DYN innervation were studied with immunohistochemistry, radioimmunoassay and HPLC, and a comparison with the effects on substance P (SP) and y-aminobutyric acid (GABA) systems was performed. For the anatomical and biochemical data we refer the reader to the accompanying paper (Christensson-Nylander et al. 1986). In the present paper we focus on the effect of IBA lesions of the striatum on the rotational behaviour produced by intranigral injections of DYN A. The behavioural effects of intranigral SP injections were also analysed in view of (1) the existence of a striato-nigral SP pathway (Brownstein et al. 1977; Gale et al. 1977; Hong et al. 1977; HOkfelt et al. 1977; Kanazawa et al. 1977; Kohno et al. 1984); (2) the close relationship between SP nerve endings and dopamine (DA) dendrites in the zona reticulata (Ljungdahl et al. 1978b), and (3) the demonstration that SP can be released in the substantia nigra (Schenker et al. 1976, Jessell 1978, Michelot et al. 1979, Torrens et al. 1981). Furthermore, it has previously been reported that SP injections into the substantia nigra induce rotational behaviour in the rat (Olpe and Koella 1977; James and Starr 1977,
1979; Waldmeier et al. 1978; Kelley and Iversen 1979). It has recently been discovered that the mammalian central nervous system contains, in addition to SP, a number of other tachykinins, such as substance K (= neurokinin A, = neuromedin L) (SK) and neurokinin B (Kimura et al. 1983, Kanagawa et al. 1983, Minamino et al. 1984), whereby SP and SK have a common precursor (Nawa et al. 1984). Therefore, the effect of SK on rotational behaviour was also analysed. In view of the possibility that proenkephalin Bderived peptides (Herrera-Marschitz et al. 1984a, H6kfelt et al. 1984) and protachykinin-derived peptides (Starr et al. 1978, 1983; James and Starr 1979) exert their functional effects by an action on the dendrites of nigro-striatal DA neurons, we have studied whether peptide-induced behavioural responses are altered by unilateral nigro-striatal DA denervation produced with 6-hydroxy-DA (6-OHDA) (Ungerstedt 1971). Finally, the effect of intranigral injections of DYN A and SP on DA release in the striatum has been directly studied using an intracerebral dialysis method (Ungerstedt 1984). A preliminary account of this work was presented at the 14th C.I.N.P. congress, Florence (HerreraMarschitz et al. 1984b).
Material and methods Male Sprague-Dawley (Alab, Stockholm, Sweden) rats with free access to food and water were used in all experiments. They were maintained in a temperature controlled environment on a 12 h light/dark cycle when not in experimental sessions.
IBA striatal lesions
Rats weighing 200-250 g were anaesthetized with a mixture of air and halothane and placed in a Kopf stereotaxic frame, with the skull oriented according to the K6nig and Klippel atlas (1963). Four IBA injections (10.0 ~g/~tl) were made into the left striatum. The four injections of IBA were given in the following order: 1.0 I~1into the most caudal part of the tail (coordinates: A 4.9, L -4.4, V -0.6. K6nig and Klippel 1963), 1.0 gl into the tail (coordinates: A 6.2, L -2.6, V 1.4), 1.0 ~1 into the corpus (coordinates: A 7.5, L -2.0, V 0.4), and then 1.0 ~d into the head of the striatum (coordinates: A 8.5, L -2.3, V -0.4). This lesion corresponds to the group VI lesion described in Christenss0nNylander et al. (1986).
6-OHDA nigro striatal lesions
Rats weighing 150-170 g were anaesthetized and placed in the stereotaxic frame. Eight ~g of 6-OHDA in 4.0 ~1 of saline was injected into the left area ventralis tegmenti which contains the bundle of axons leaving the mesencephaiic DA cell bodies (coordinates: A3.2, L-1.2, V-3.1). This lesion corresponds to the group VIII lesion described in Christensson-Nylander et al. (1986).
195
Acute intracerebral injection experiments For the behavioural experiments peptides were administered into the SNR using an acute intracerebral injection technique (Herrera-Marschitz et al. 1984a; 1985a, b). The animals were anaesthetized as indicated above and placed in a stereotaxic frame. An injection cannula, conically shaped with a penetration tip diameter of approximately 0.15 mm, was lowered into the SNR (coordinates: A 1.8, L -2.0, V -2.6). The substances were injected in a total volume of 0.2 pl over a period of 1 min. The halothane anaesthesia was switched off as soon as the injection was complete. The injection cannula was maintained in position for a further min, and then carefuUy retracted. The skin was sutured and the rats were placed in a rotometer (Ungerstedt and Arbuthnott 1970, Herrera-Marschitz and Ungerstedt 1984a, b) in order to continuously record their behaviour. The time from the onset of anaesthesia to when the awake rats were placed in the rotometer was less than 15 min. The same procedure was repeated in control experiments in which rats were injected with vehicle. Rats were used only once in these intranigral injection experiments.
Behavioural evaluations - rotational behaviour Rotational behaviour was measured both in naive rats following a c u t e peptide injections and, in order to verify the lesion, in IBA and 6-OHDA treated rats, following apomorphine. The amount of motor asymmetry was monitored using a modified version of the original rotometer (Ungerstedt and Arbuthnott 1970), which continuously recorded the number of turns to the left or right with a detector utilising infrared photocell beams (Herrera-Marschitz and Ungerstedt 1984a). The rotational behaviour of each individual animal was expressed as the number of 360~ turns/min for the entire duration of the behaviour (total rotation -- Area Under the Curve, AUC), or as turns/10 rain (rotation intensity). Rotometers are sensitive to 180~ right or left movements, therefore counts are recorded whether the rat completes 360~ turns or moves 180~in the left or right direction. The later behaviour is regarded as an exploratory-like behaviour indicative of generalised behavioural activation. Direct observation was used to differentiate between true rotation and activation, since the latter also resulted in an increase of the total counts. The term rotation was ascribed to any asymmetric activation to the left or right. When both right and left counts increased simultaneously, the observed behaviour was named symmetric activation (see Herrera-Marschitz et al. 1984a). The IBA-treated animals were treated with apomorphine, 0.5 mg/kg s.c., 7-10 days after the lesion. Depending upon the extent of the lesion, the rats showed a progressive tendency to rotate ipsilaterally (Schwarcz et al. 1979). Rats were selected according to the magnitude of ipsilateral rotation induced by apomorphine (0.5 mg/kg s.c.). Only rats showing 350 or more total turns after this apomorphine dose were used for intranigral injections (Christensson-Nylander et al. 1986). The 6-OHDAtreated animals were given apomorphine 0.05 mg/kg s.c. two weeks after the lesion. The intensity and duration of apomorphine induced-contralateral rotation was dependent upon the extent of the lesion (Ungerstedt 1971, Ungerstedt and Herrera-Marschitz 1981, Christensson-Nylander et al. 1986). As criterion for a successful denervation we tested for the ability of apomorphine to induce a two-peak rotation which only occurs in animals with more than 95% D A depletion in ~ a t u m (Ungerstedt a~d HerreraMarschitz I981, Herrera-lYIarschitz and Ungerstedt 1984a).
Intracerebral dialysis procedure Rats weighing 230-280 g were anaesthetized and placed in a stereotaxic frame. Halothane anaesthesia was maintained through-
out the experiment, and body temperature was kept at 37~ C using a heating pad controlled via a rectal probe. The skull was exposed and two holes were drilled for the placement of a dialysis probe (coordinates: A 8.9, L -2.4, V -1.4) into the left striatum and for an intracerebral injection of peptide or saline into the left SNR (coordinates: A 1.8, L 2.0, V -2.6). The dialysis probe (Carnegie Medicin AB, Solna, Sweden) consisted of two concentric steel cannulae covered at the tip by a dialysing membrane with an exposed area of 4.0 x 0.4 mm. Physiological Ringer solution, infused through the inner cannula (the inner cannula of 0.1 mm of diameter had two lateral openings at the tip and penetrated to the bottom of the dialysis membrane), flushed the inside of the membrane and left via the outer cannula (see Ungerstedt 1984). The dialysis probe was continuously perfused at 2.0 ~l/min using a microinfusion pump (Carnegie Medicin AB, Solna, Sweden), and the perfusates were collected every 20 min in small Eppendorf tubes inverted over the outlet cannula. Each collection tube contained 10.0 ~1 1 M perchloric acid to prevent oxidation of the monoamines. The perfusates were directly injected onto a reversed phase ion-pair HPLC with 'electrochemical detection system for the measurement of D A and its metabolites, DOPAC and H V A as well as the 5-hydroxy-tryptamine (5-HT) metabolite, 5-HIAA (see Sharp et al. 1986). When basal monoamine levels were stable (approximately 120 rain after the implantation of the dialysis tube), an injection cannula was implanted into the ipsilateral SNR and, following a further 20 min fraction, either peptide or saline vehicle was injected and then the injection cannula was carefully removed. At least 9 more 20 rain fractions were collected. The animals were sacrificed and the brains dissected out for histological verification of the dialysis probe and injection cannula localization. Levels of D A and the metabolites were expressed as the percentage of the 20 min fraction obtained immediately after the implantation of the cannula injection into the SNR.
Immunohistochemistry In the previous paper (Christensson-Nylander et al. 1986) tissue obtained from lesioned brains was analysed using both biochemical and immtmohistochemical techniques. In this paper 20 rats with striatal IBA-lesions and which received intranigral injections of DYN A or SP were sacrificed, and the extent of the peptidedenervation as well as the location of the injection cannula was analysed immunohistochemically using DYN antiserum. For immunohistochemistry, rats were anaesthetized with pentobarbital and perfused with ice-cold 0.9% saline and then the brain was dissected out. About 1.0 mm thick slices containing the substantia nigra were prepared and immersed into a mixture ofpbenzoqninone (0.25%), paraformaldehyde (2%) and 0.05 M phosphate buffer for 90 min before transferring to a buffered sucrose solution (for details see Pearse and Polack 1975, Ltmdberg et al. 1982, Christensson-Nylander et al. 1986). After at least 24 h of rinsing, the brain tissue was frozen and sectioned at a thickness of 14 ~m in a cryostat (Dittes, Heidelberg, FRG). Sections were incubated overnight in a humid atmosphere at 4~ C with antiserum raised against DYN A (antiserum 84). Antiserum was diluted (1:400) in phosphate buffered saline (PBS) containing 0.3% Triton X100. After rinsing in PBS, the primary antiserum was visualized by incubation with swine antirabbit fluorescein isothiocyanate conjugated secondary antibodies (Dakopatts, Copenhagen, Denmark). Sections were coverslipped with glycerol : PBS (3 : 1) containing p-phenylenediamine and examined using a Zeiss fluorescence microscope, equipped with a dark-field oil condensor and proper filter combinations. DYN antiserum preabsorbed with an excess of DYN A (50 ~tg peptide per ml antiserum diluted I : 10) was used as control serum. For informa-
196 Table 1. Contralateral rotation produced by unilateral intranigral injections in naive rats Substance
Dose (~tg)a
N
Total (turns)
Maximum intensity (turns/10 min)
Duration (min) b
DYN A
0.05 0.1 1.0 10.0
4 14 20 6
138 141 339 416
27 39 46 54
7 7 12 7
88+24 95 + 46 243 + 96 > 350
U50,488H
0.1 1.0 10.0
5 5 5
83 + 13 216 _+ 43 342 + 33
21 + 11 38 + 17 37 + 7
74 + 22 238 + 50 316 + 50
DALE
0.1 1.0 10.0
4 5 4
74 _+ 22 212 _+ 50 4O7 _+ 85
32+ 7 40 _+ 11 46 + 9
40+ 9 92 + 14 215 + 45
182 302 393 739 238 328
25 36 44 47 36 61
Substance P
0.00001 0.0001 0.001 0.01 0.1 1.0
4 7 10 7 12 5
_+ 21 _+ 31 + 51 + 20
_+ 41 _+ 62 _+ 86 _+ 69 _+ 44 _+ 63
+ + + +
+ 7 -+ 7 _+ 6 _-+ 7 + 12 + 21
175 + 187 + 254 + > 350 238 + 288 +
40 45 31 44 29
Substance K
0.01 0.1 1.0
5 6 5
130 _+ 11 488 _+ 50 717 + 91
21 + 5 54 _+ 10 35 _-+ 6
172 + 23 208 + 45 > 350
Saline
0.2 gl
4
81 -+ 12
8 _+ 4
68+
6
volume of injections = 0.2 ~tl b total duration was calculated up to the last period showing 5 turns/10 rain
tion on dilutions and characteristics of antiserum and other details of procedure, see Christensson-Nylander et al. (1986). Some rats were also analysed with SP antiserum, but methods and results are mainly reported in the accompanying paper (Christensson-Nylander et al. 1986). For immediate confirmation of the location of the tip of the cannula used for the acute intracerebral injections, the remaining rats were decapitated and the brain rapidly dissected out and frozen on the stage of a Leitz cryomicrotome with carbon dioxide and sectioned.
Chemicals IBA (Sigma, St.Louis, Mo, USA) was dissolved in warm physiological Ringer solution. 6OHDA-HC1 (Sigma, St. Louis, Mo, USA) was dissolved in physiological saline with 0.2% of ascorbic acid. DYN A (DYN 1-17) Peninsula Laboratories, Belmont, California, USA) SP (Bachem, Torrance, California, USA), SK (Bachem, California, USA), D-AlaZ-D-LeuLenkephalin (DALE) (Bachem, California, USA) and U50,488H (Dr. R. Lahti, The Upjohn Co, Kalamazoo, Michigan, USA) were dissolved in physiological saline and injected into the SNR in a total volume of 0.2 ~1. Apomorphine HC1 (Apoteksbolaget, Stockholm, Sweden) was dissolved in warm physiological saline; D-amphetamine sulphate (Sigma, St. Louis, Mo, USA) was dissolved in physiological saline and injected s.c. into the right flank in a volume of 1.0 ml/kg body weight.
Statistics Means and S.E.M. were calculated and differences between means tested with Student's t-test. The changes in striatal DA
release and metabolism in every experimental group were analyzed with one-way F-ANOVA test for replicated data. A level of P < 0.05 for the one-tail test was considered as critical for statistical differences.
Results
Effects of intranigral injections in naive animals A s s h o w n in T a b l e 1, i n t r a n i g r a l i n j e c t i o n o f D Y N A (0.05-10.0 ~g) in n a i v e rats p r o d u c e d d o s e - d e p e n d e n t c o n t r a l a t e r a l r o t a t i o n as i n d i c a t e d by b o t h t h e m a x i m u m i n t en si t y ( m a x i m a l p e a k o f r o t a t i o n ) a n d t h e t o t al r o t a t i o n ( = a r e a u n d e r t h e c u r v e ( A U C ) . T h e effect p r o d u c e d by D Y N A was similar to t h a t i n d u c e d by t h e k a p p a o p i o i d ag o n i st U 5 0 , 4 8 8 H (0.1-10.0 ~g) ( T a b l e 1). T h e stable a n a l o g o f L e u e n k e p h a l i n , D A L E also i n d u c e d d o s e - d e p e n d e n t (0.1-10.0 ~g) c o n t r a l a t e r a l r o t a t i o n w i t h a similar p o t e n c y as D Y N A a n d U 5 0 , 4 8 8 H ( T a b l e 1). SP p r o d u c e d c o n t r a l a t e r a l r o t a t i o n a l b e h a v i o u r in a w i d e dose r a n g e (0.00001-1.0 9g). H o w e v e r , c l e a r dose d e p e n d e n c y was o n l y o b s e r v e d at l o w e r doses o f t h e r a n g e (0.00001-0.01 ~g) ( T a b l e 1), with h i g h e r c o n c e n t r a t i o n s p r o d u c i n g a less p r o n o u n c e d r o t a t i o n . SP a p p e a r e d to b e v e r y p o t e n t , since a d o s e as l ow as
197 Table 2. Contralateral rotation produced by intranigral injections in lesioned rats Treatment
IBA-lesion DYN A
Substance P
6-OHDA-lesion DYN A U50, 488H Substance P
Dose (~g) a
N
Total (turns)
Maximum intensity (turns/10 min)
0.05 0.1 1.0
8 7 17
39 + 18 41+ 5 55 + 12
110 _+ 24 254_+59 249 + 80
0.001 0.01 0.1 1.0
4 4 6 5
172 + 61 272 + 46 532 + 44 39 + 11 53 + 16 123 + 32 198 __+ 40
7+ 16+ 35+ 51__+
2 2 9 9
43+12 5 0 + 13 8 2 + 19 157+77
1.0
8
24+
5
240+20
10.0
4
207 + 42 728 + 103
4 4 4
61 + 15 244 + 54 258 + 52
13+ 1 28+ 7 48 + 14
0.001 0.1 1.0
Duration (min) b
54 + 17
> 350 123+30 265+46 258 + 52
a volume of injections = 0.2 gl u total duration was calculated up to the last period showing 5 turns/10 min a
10.0 pg p r o d u c e d a r o t a t i o n a l r e s p o n s e w h i c h was n o t s e e n f o l l o w i n g i n t r a n i g r a l i n j e c t i o n of saline. I n t e r e s t i n g l y , o n l y 2 o u t o f 10 a n i m a l s i n j e c t e d with 10.0 ~tg of SP s h o w e d c o n t r a l a t e r a l r o t a t i o n , w h i l e t h e o t h e r 8 rats s h o w e d b u r s t s o f i n t e n s i v e sniffing a n d e x p l o r a t o r y - l i k e m o v e m e n t s w i t h p e r i o d s of akinesia. S K also p r o d u c e d c o n t r a l a t e r a l r o t a t i o n w h e n i n j e c t e d i n t o t h e S N R ( T a b l e 1). The. histological data showed that the peptidei n d u c e d c o n t r a l a t e r a l r o t a t i o n a l r e s p o n s e was strictly dependent on the location of the injection. Maximal r o t a t i o n a l b e h a v i o u r was o b s e r v e d w h e n t h e tip o f the i n j e c t i o n c a n n u l a was l o c a t e d in t h e c e n t r a l r e g i o n of t h e S N R , j u s t a b o v e t h e crus c e r e b r i . T h e r e s p o n s e s w e r e less c o n s i s t e n t , w h e n t h e tip of t h e i n j e c t i o n c a n n u l a was l o c a t e d in m e d i a l o r l a t e r a l regions. I n j e c t i o n s into a d j a c e n t a r e a s o f t h e S N R w e r e ineffective. I n j e c t i o n s into t h e p a r s c o m p a c t a p r o d u c e d a n i p s i l a t e r a l bias t h a t was m o r e i n t e n s e t h a n t h a t o b s e r v e d a f t e r saline i n j e c t i o n s .
Dyn A i n t o
Vuros (AOC~ 600
the SNR
)
Hnaive
rats
@-4) IBA-lesioned
rats
.e/
400.
200.
0.01
b T. . . . ~AUC~ 800
0.1
SP
into
1.0
the
10.0
ug
SNR naive ..
-
rats '
rats
600
400"
Effects of intranigral injections in 1BA-lesioned animals D Y N A was i n j e c t e d into t h e S N R o f rats with striatal I B A lesions. T h e c o o r d i n a t e s for t h e injections w e r e t h e s a m e as u s e d for n a i v e rats, e x c e p t t h a t t h e d i s t a n c e f r o m b r e g m a was m o d i f i e d since, as r e p o r t e d in t h e a c c o m p a n y i n g p a p e r ( C h r i s t e n s s o n N y l a n d e r et al. 1986), t h e s u b s t a n t i a n i g r a i p s i l a t e r a l to the lesion side was significantly r e d u c e d . T h e a t r o p h y of t h e s u b s t a n t i a n i g r a r e s u l t e d in a l a c k of
200
0.00001
0.0001
0.001
0.01
0.1
1.0 ug
Fig. la, b. Comparison of dose-response curves of rotational behaviour induced by intranigral injections of DYN A (a) and SP (b) in naive (full lines) or IBA-lesioned rats (dotted lines). Ordinate: Contralateral rotational behaviour calculated from the area under the curve (AUC) of the respective experiments. Abscissa: doses (volume of every injection: 0.2 ~tl). Vertical lines show S.E.M. * = P < 0.05. ** = P < 0.01)
198
Fig. 2a, b. Immunofluorescence micrographs of section of the substantia nigra of control (a) and lesion (b) side after incubation with DYN antiserum. The rat received 4 injections of IBA into the left striatum (group VI, see Christensson-Nylander et al. 1986) and 10 days later I ~tg DYN A was injected into the left substantia nigra. Note almost complete disappearance of DYN-LI in the zona reticulata (zr) (b) as compared to control (a) side. The section shown in b is semiadjacent to the cannnla track and only the location of its tip (arrows) can be seen. The fluorescent structures around the tip represent exogenous DYN-LI, as has been described previously (Herrera-Marschitz et al. 1984a). cc = crus cerebri; zc = zona compacta. Bar indicates 50 ~m. Both micrographs have the same magnification
r e s p o n s e to i n t r a c e r e b r a l i n j e c t i o n s in s e v e r a l anim a l s in w h i c h t h e tip o f t h e c a n n u l a m i s s e d t h e S N R . Such a n i m a l s w e r e d i s m i s s e d f r o m t h e study. W h e n e v e r t h e tip o f t h e n e e d l e w a s l o c a t e d in t h e SNR, intensive and long lasting rotational behaviour
was o b s e r v e d ( T a b l e 2). T h e r o t a t i o n a l r e s p o n s e ( A U C ) p r o d u c e d b y D Y N A ( 0 . 1 - 1 0 . 0 ~tg) i n j e c t e d into t h e S N R of n a i v e a n d l e s i o n e d a n i m a l s is s h o w n in Fig. l a . A significant shift to t h e left o f t h e d o s e r e s p o n s e c u r v e p r o d u c e d b y D Y N A was o b s e r v e d in
199 a Turns 10 min
C Turns 10 rain
Dyn A into SNR/ naive rats
N=3 D-Amphetamine 2 mg/kg s.c,
20,
20.
1 pg
t 0
SP into SNR/naive rats N=4 D-Amphetamine 2mg/kg s.c.
0 60 0
60
120
180
240 rain
[
0.001 pg
60 0
60
120
180
240
600
min
O,
O-
20 20
40, 40
b Turns lOmin
Dyn A into SNR/6OHDA N=3 D-Amphetamine 2 mg/kg s.c.
d Turns 10 rain
2O 1 pg
01
8P into SNR/6OHDA N=3 D-Amphetamine 2 mg/kg s.c.
20
60 180 rain
0.0010 j pg 60
*~! ....
20
~o L~
~ 2 ;0
rain
20 40
Fig. 3a--d. Contralateralrotation induced by DYN A and SP injected into the SNR of naive (a, c) and 6-OHDA-lesioned (b, d) rats. Damphetamine given systemically 60in later enhanced the contralateralrotation induced by the peptides in naive rats,but not in 6-OHDAlesioned rats. Abscissa: rain after the intranigral infusion of the peptides or after subcutaneous administration of D-amphetamine as indicated by the respective arrows. Ordinate: turns/10 rain. Vertical lines show S. E. M.
IBA-lesioned rats (Fig. la). However, the ability of SP (0.001-1.0 ~tg) to produce contralateral rotation following intranigral administration was significantly reduced in IBA-lesioned compared to naive rats, which resulted in a shift of the dose response curve to the right (Fig. lb). Immunohistochemical analysis of IBA-lesioned rats injected intranigrally with various doses of D Y N A and SP showed a marked decrease in D Y N like immunoreactivity (LI) in the zona reticulata (c.f. Fig. 2a and b). However, in several cases dense fiber networks remained in the most lateral and medial parts of the SNR. In the zona compacta a sparse to medium dense D Y N - and SP-immunoreactive network was still seen (see Christensson-Nylander et al. 1986). In the rats analysed, the tip of the injection needle was seen to have been present in the maxi-
mally depleted mid-zone of the zona reticulata, sometimes penetrating into the most dorsal part of the crus cerebri (Fig. 2b).
Effects of D-amphetamine and
6 - O H D A treatment
The rotational behaviour produced by 1.0 ~g of D Y N A injected into the SNR of naive rats was enhanced by 2.0 mg/kg of D-amphetamine injected s.c. 60 rain later (Fig. 3a). However, an even stronger potentiation was observed after the injection of 0.001 ~g of substance P (Fig. 3c). D Y N A produced contralateral rotation after being injected into the SNR of animals lesioned unilaterally with 6 - O H D A , as did US0,488H (Table 2). In 6 - O H D A lesioned rats, the rotation produced by 1.0 ~tg of
200 o----..-oSaline a *
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Fig. 4a--d. Effect of a Dyn A (1.0 btg), a SP (0.01 lxg), and a physiological saline solution (Saline) injected into the left SNR (total volume of injections = 0.2 ~tl) on D A (a), D O P A C (b), H V A (e) and 5 - H I A A (d) levels measured in 20 min perfusates collected from the left striatum using an intracerebral dialysis procedure. The first arrow indicates the 20 rain collection period (control period) when the injection cannula was implanted into the SNR. The second arrow indicates the period when the respective substance was injected. A m o u n t of D A and metabolites found in control periods: (a) DADyn a = 0.722 + 0.088 pmol/40 ~1, N = 4; DAsF = 0.738 + 0.174 pmol/40 ~tl, N = 5; DA~line = 0.698 + 0.130 pmol/40 ~tl, N = 5, (b) DOPACnyn A = 77.48 + 8.6 pmol/40 bd, N = 4; DOPACsp = 72.75 + 5.9 pmol/40 ~tl, N = 5; DOPACsa~,~ = 88.44 _+ 4.83 pmol/40 gl, N = 5. (c) HVADy n A = 46.73 +-- 5.46 pmol]40 t*1, N = 4; H V A s r = 45.87 + 8.71 pmoI/ 40 &l, N = 5; HVA~,~ine = 52.06 + 5.26 pmol/40 gl, N = 5. (d) 5-HIAAny~ A = 9.52 + 0.97 pmol/40 btl, N = 4; 5-HIAAsF = 13.44 _+_2.08 pmol/40 ~l, N = 5, 5-HIAA~lln~ = 14.04 + 4.07 pmol/40 ~tl, N = 5. Abscissa: min after or before the implantation of the cannula injection into the SNR. Ordinate: % of values obtained during the control period. Vertical lines show S. E . M . Comparisons (Student's test) were done with the corresponding values obtained after intranigral saline. * = P < 0.05, ** = P < 0.01 for one tail test
DYN A was not enhanced by D-amphetamine treatment; on the contrary, the rotational behaviour was reversed to ipsilateral rotation (Fig. 3b). The ability of SP to produce contralateral rotation following intranigral injections was significantly diminished in 6-OHDA-lesioned rats (compare tables 1 and 2). Furthermore, D-amphetamine reversed the response to ipsilateral rotation (Fig. 3d).
Effect of intranigral injections on striatal DA release and metabolism studied using intracerebral dialysis Intranigral injection of 0.2 ~1 of a saline solution did not modify the levels of DA, DOPAC, HVA and 5HIAA measured in perfusates collected from the ipsilateral striatum using the intracerebral dialysis procedure (F-ANOVA for intranigral saline experi-
201 ments:
0 . 8 8 , n.s., N,M = 5,13; 1.30, n.s., N,M = 5,13; HVAF-ANOVA = 2.08, n.s., N,M = 5,13; 5HIAAF-ANOVA = 1.28, n.s., N,M = 5,13) (Fig. 4a-d). However, intranigral injection of 1.0 ~tg of DYN A produced a statistically significant decrease of DA whether compared with pre-injection values obtained in the same group of animals ( D A F _ A N O V A = 3.74. P < 0.05, N , M - - 4,13) or with the saline treated group (Fig. 4a). Decrease of D A release was detected during the 0-20 min perfusate collection period (first interval after the DYN A injection), reacheing a maximum (41 + 5%, N = 4) in the 60-80 min period, but did not reverse over the 180 min post injection period (Fig. 4a). In contrast to DA, its major metabolite D O P A C increased after the intranigral injection of 1.0 ~tg of D Y N A (DOPACF-ANOVA = 10.68, P < 0.01, N,M = 4,13). This effect had an onset during the 20-40 min period reaching a maximum (32 + 9%, N = 4) at the 40-60 min period and declining thereafter (Fig. 4b). In comparison to DOPAC, H V A showed a smaller but statistically significant increase after the intranigral injection of DYN A (HVAF_ANOVA ----- 3.26, P < 0.05, N,M = 4,13), reaching a maximum at the 80-100 min period (37 + 13%, N = 4) (Fig. 4c). The 5-HT metabolite, 5-HIAA did not show any statistically significant change after the intranigral injection of DYN A (5-HIAAF_ANOVA= 0.34, n.s., N,M = 4,13) (Fig. 4d). In contrast to DYN A, intranigral injection of 0.01 [xg of SP produced a statistically significant increase of DA (DAv_ANOVA = 6.66, P < 0.01, N,M = 5,13) (Fig. 4a). This effect was detected during the 0-20 min period, reached a maximum at the 60-80 min period (59 +_ 26%, N = 5) and was still present 180min after the injection time, DOPAC and H V A also increased after 0.01 Ixg of SP (DOPACF_ANOVA = 13.89, P < 0.01, N,M = 5,13; HVAF_ANOVA = 4.70, P < 0.05, N,M = 5,13). The increase of D O P A C and H V A reached a maximum at the 40-60 min period (DOPAC = 19 _+ 4%, N = 5, H V A = 18 _+ 4, N = 5) (Fig. 4b, c). Furthermore, intranigral SP also produced an increase in 5-HIAA (5-HIAAF_ANOVA= 10.85, P < 0.01, N,M = 5,13), reaching a maximum at the 60-80 min period (22 _+ 4%, N = 5). Histological analysis revealed that the dialysis probe was located in the rostral portion of the corpus of the striatum (A 8920 Ix according with K6nig and Klippel 1963) with the dialysis membrane (4 mm) exposed to the dorsal and ventral regions of the striatum. The membrane penetrated into the fundus striati, but never into the accumbens. Moreover, it was clear that the changes in D A release produced in the DAF_ANOVA
DOPACF_ANOV A
=
=
dialysis experiments were dependent upon the location of the injection site within the SNR, since misplaced nigral injections were consistently associated with lack of significant changes in D A release and metabolism.
Discussion
There is evidence that SP- (Brownstein et al. 1977, Gale et al. 1977, Hong et. al. 1977, Kanazawa et al. 1977, Jessell et al. 1978) and DYN- (Vincent et al. 1982b, Zamir et al. 1984a, Fallon et al. 1985, Christensson-Nylander et al. 1986) positive fibers originating in the striatum innervate the ipsilateral substantia nigra constituting striato-nigral pathways. Indeed, a very high density of SP- and DYNimmunoreactive nerve terminals have been observed in the SNR of the rat (SP: H6kfelt et al. 1975, 1977; Cuello and Kanazawa 1978; Ljungdahl et al. 1978a, Somogyi et al. 1978. DYN: Weber et al. 1982, Vincent et al. 1982a, b). In the accompanying paper (Christensson-Nylander et al. 1986), biochemical and histochemical analysis of lesions of the striato-nigral DYN, SP and G A B A pathways revealed a high degree of parallelism of these three systems and a distinct topographical relationship between placement of the lesion and localization of the depleted area, in agreement with earlier neuroanatomical (Nauta and Mehler 1966, Szabo 1970, Tulloch et al. 1978, Grofova 1979, Royce and Laine 1984, Getfen 1985) biochemical and immunohistochemical (Brownstein et al. 1977, Jessell et al. 1978, Staines et al. 1980, Kohno et al. 1984) studies. In the present paper, evidence is given for the presence in the substantia nigra of receptors sensitive to stimulation by proenkephalin B- and protachykinin-derived peptides, which however seem to be differently associated to outflow pathways of the substantia nigra.
Effects of intranigral injections of D Y N and SP in naive rats The present study shows that intranigral injection of both DYN A and SP in naive rats produces a contralateral rotational response. The finding that DYN A induces a dose-dependent rotational response agrees with earlier reports (Herrera-Marschitz et al. 1983, 1984a, b; Morelli and Di Chiara 1985). Furthermore, this effect is mimicked by the kappa opioid agonist, U50, 488H (Von Voigtlander et al. 1982), which is consistent with the proposal that kappa-receptors mediate the DYN effect (HerreraMarschitz et al. 1983, 1984a). As discussed in the
202 accompanying paper (Christensson-Nylander et al. 1986), DYN peptides may be metabolically transformed to leucine (Leu)enkephalin in the striatonigral pathway (Zamir et al. 1984b) and this peptide is selective for delta opioid receptors. In agreement, the metabolically stable enkephalin analog, DALE also produced rotational behaviour following intranigral injection, which has also been reported by Morelli and Di Chiara (1985). The possibility that both DYN and enkephalin peptides produce the same behavioural response via different opioid receptors remains open. However, since the receptor selectivity of these peptides is not absolute (cf. Paterson et al. 1983), it is also possible that enkephafins would work through kappa receptors. Intranigral injection of SP also produced contralateral rotation, in agreement with earlier findings (Olpe and Koella 1977, James and Starr 1977; Kelley and Iversen 1979). Furthermore, SP was approximately 1000 times more potent than DYN A in inducing rotational behaviour.
Effects of intranigral injections of D Y N and SP in IBA-lesioned rats A unilateral lesion of the descending striato-nigral projection using IBA produces an increase in sensitivity to the application of DYN A in the ipsilateral SNR. The increased sensitivity to DYN A might well reflect stimulation of receptors rendered supersensitive by the DYN denervation, a phenomenon equivalent to the DA receptor supersensitivity found after denervation of the nigro-striatal DA pathway with 6-OHDA (Ungerstedt 1971). The ability of SP to produce rotational behaviour was, in contrast to DYN A, significantly diminished in animals with striatal IBA lesions. This finding is unlikely to be explained by a differential depletion of these peptides, since nigral SP- and DYN-LI disappear in a parallel fashion after striatal IBA lesions (Christensson-Nylander et al. 1986). Instead, the IBA injections may have impaired the pathway by which SP produces rotational behaviour. Thus, the present experimental model does not allow to test a possible SP receptor supersensitivity induced by intrastriatal IBA lesion. Involvement of DA neurons in peptide-induced rotation We have previously reported (Herrera-Marschitz et al. 1984a, H6kfelt et al. 1984) that the rotational behaviour induced by nigral injection of DYN frag-
ments in naive rats is enhanced by D-amphetamine given 60 min later. In the present study Damphetamine also strongly potentiated the rotational response to nigral injection of SP. These data suggested that the two peptides, when intranigrally injected, increase the activity of nigro-striatal DA neurons via an action on the DA dendrites in the zona reticulata. With regard to SP, this view is in agreement with James and Starr (1979), who reported that the rotational behaviour elicited by intranigral injection of SP was blocked by haloperidol and potentiated by the monoamine oxidase inhibitor, nialamide, and with Kelley and Iversen (1979), who showed that striatal 6-OHDA-lesions blocked behaviours elicited by bilateral injections of SP into the substantia nigra. In the present experiments, peptides were in addition injected into the left SNR of rats with 6-OHDA denervation of the nigro-striatal D A pathway of the left side (Ungerstedt 1971, Ungerstedt and Herrera-Marschitz 1981). DYN A (and U50, 488H) still produced strong contralateral rotational behaviour and similar results have been obtained with the shorter DYN A fragments (unpublished data). Thus, in contrast to our previous view (see above), the rotational response elicited by the proenkephalin B-derived peptides does not seem to be mediated by stimulation of nigro-striatal DA transmission. However, the contralateral rotation produced by intranigral administration of SP was significantly diminished in 6-OHDA-lesioned animals, which supports the hypothesis that this effect of SP is mediated through the nigro-striatal DA system (James and Start 1977, 1979; Cheramy et al. 1978; Waldmeier et al. 1978; Kelley and Iversen 1979; Glowinski et al. 1982; Kelley et al. 1985; Quirion et al. 1985). The exact neuroanatomical relationship between nerve endings of the striato-nigral peptidergic pathways and their targets in the substantia nigra has only been partly analysed (see also Discussion in Christensson-Nylander et al. 1986). Somogyi et al. (1981) have demonstrated that nigral neurons projecting to the caudate nucleus receive monosynaptic inputs from striatal neurons. Furthermore, it has been shown that such monosynaptic inputs can contact dopaminergic dendrites (Wassef et al. 1981), as well as nigro-tectal neurons (William and Faull 1985). SP-immunoreactive nerve endings can form direct contacts with dendrites and cell bodies in the substantia nigra (Somogyi et al. 1982), but the transmitter identity of the postsynaptic contacts has not been clearly established (see Gerfen 1985). Recent electrophysiological studies by Innis et al. (1985) indicate that SK excites both DA and non-dopaminergic
203
neurons in the substantia nigra, and perhaps this is also relevant for effects induced by intranigral SP injections.
Effect of D Y N and SP on striatal DA release and metabolism
A direct way to analyze whether the behavioural response produced by intranigral administration of SP but not DYN is dependent upon an increase in nigro-striatal DA transmission is by using a new dialysis technique which enables measurement of extracellular DA and monoamine metabolites in rat brain in vivo (Zetterstr6m et al. 1983, 1984, Ungerstedt 1984). Indeed, intranigral injection of SP caused an immediate and long-lasting increase of DA release in the ipsilateral striatum, which was accompanied by an increase in DA metabolism. In contrast, the striatal DA release decreased following injection of DYN A into the SNR. However, DOPAC and HVA increased after DYN A. Preliminary data indicate that intranigral injections of the kappa opioid agonist U-50,488H and shorter DYN A fragments produce similar effects on striatal release (Sharp et al. in preparation). These results give further evidence for differences in the mechanisms by which SP and DYN produce rotational behaviour. The decrease in extracellular DA levels by DYN may be explained by an inhibition of the nigrostriatal DA neurons. Such inhibitory effects of DYN have been observed electrophysiologically in various models (Brookes and Bradley 1984, Werz and Mac Donald 1984, Sutor and Zieglgfinsberger 1984, Vidal et al. 1984). These results suggest that the striato-nigral negative feed-back loop for the nigrostriatal DA pathway (Carlsson and Lindqvist 1963, Groves et al. 1975, Bunney and Aghajanian 1976a, b, Nissbrandt et al. 1985) not only involves GABA (Anden and Stock 1973, Anden 1974, James and Starr 1978, Grace and Bunney 1979, Starr et al. 1983; for further refs., see Dray 1979), but also DYN. Moreover, there may exist a functional connection between the GABAergic negative feed-back loop and the parallel, postulated positive SP feed-back system, since it has been shown by several groups that GABA can inhibit nigral SP release (Jessell 1978, Torrens et al. 1981). Such an arrangement would allow the GABA system to exert its negative feed-back action in two ways in the substantia nigra: (1) by direct inhibition of DA neurons and (2) by inhibiting SP release and in this way SP-induced stimulation of DA neurons. The paradoxical increase of extracellular levels of DOPAC and HVA in striatum after intranigral
DYN A adds further evidence to our observations that these metabolites do not provide a direct index of changes in extracellular levels of DA itself (Zetterstr6m et al. 1984, Westerink 1985). The increase in metabolites after the DYN injection probably reflects a greater availability of intraneuronal DA to MAO. In addition, 5-HIAA was increased after intranigral SP, but not after DYN A. This effect may be secondary to the increase in DA (Williams and Davis 1983).
Substance P versus substance K
The discovery of several tachykinins in the mammalian central nervous system (Kimura et al. 1983, Kangawa et al. 1983, Nawa et al. 1983, Minamino et al. 1984) has raised several important issues of special interest for the present paper. Several biochemical studies have given evidence for the occurrence of both SP and SK in the SNR (Kanazawa et al. 1984, Maggio et al. 1984, Minamino et al. 1984, Brodin et al. 1985, Lindefors et al. 1985, Shults et al. 1985), which are supported by immunohistochemistry (Kurazawa et al. 1984, Brodin et al. 1985) and are in agreement with the view of a common precursor for SP and SK (Nawa et al. 1983). In the present study, strong rotational behaviour was induced by intranigral injections of both SP and SK in naive rats, indicating an activation of tachykinin receptors in this region. The observation that SP produces contralateral rotation is in agreement with earlier findings (Olpe and Koella 1977, James and Starr 1977, 1978, 1979; Kelley and Iversen 1979). However, it has been recently reported that the rat substantia nigra has a high density of SK but few SP binding sites (Mantyh et al. 1984, Quirion and Dam 1985, Quirion and Than-Vinh 1985). Furthermore, electrophysiological studies have shown that SP causes a low proportion of neuronal responses in the substantia nigra (Davies and Dray 1976, Walker et al. 1976, Collinridge and Davies 1982, Pinnoch and Dray 1982), while SK activates many units in this region (approximately 50% of both dopaminergic and non-dopaminergic neurons) (Innis et al. 1985). Also in the ventral tegmental area SK seems to be more potent than SP in increasing motor activity (Kalivas et al. 1985). Against this background it may appear surprising that not only SK but also SP, as shown here, exerts potent effects on rotational behaviour. It is, at present, not clear whether SP and SK produce these behavioural effects via the same mechanism. The fact that the dose-response curve for intranigral injections
204
of SP was biphasic, suggests the participation of several mechanisms of actions. Furthermore, it has been discussed that SP may be metabolized (Jessell et al. 1976) into functionally active fragments (Sakurada et al. 1985); therefore the effects produced by intranigral injections of SP may be due to shorter tachykinin fragments active on SK-preferring receptors (Quirion and Than-Vinh 1985).
Do DYN, SP and GABA coexist?
We have previously raised the question whether DYN, SP and GABA, or a combination of two of these compounds, coexist in striato-nigral neurons (Herrera-Marschitz et al. 1984, Christensson-Nylander et al. 1986). Our recent biochemical and immunohistochemical analysis of DYN and SP after striatal IBA lesions (Christensson-Nylander et al. 1986) did not provide an answer to this question. The present results indicating that DYN and SP have opposite actions on nigro-striatal DA neurons could be taken as argument against colocalization of these peptides, but there are examples that apparently coexisting compounds have opposite effects on effector cells (see Li 1984). In contrast, direct evidence for coexistence of opioid peptides and GABA in striatal neurons has been given (Oertel et al. 1983, Aronin et al. 1984), although their exact relation to the striatonigral DYN system is not known. In the rotational model GABA has been shown to cause similar effects to DYN peptides (Herrera-Marschitz et al. 1984a), and our preliminary data indicate that intranigral GABA injection gives rise to a similar decrease in striatal D A as intranigral DYN does. However, further studies are needed to define the striato-nigral systems containing SP, DYN and/or GABA with regard to possible colocalization as well as to positive and negative cooperativity of substances released from the same or different nerve terminals.
Conclusion
The DYN A and SP neuronal components of the descending striato-nigral pathway are closely linked anatomically (Christensson-Nylander et al. 1986) and involved in the conveying of the flow of information elicited by striatal dopaminergic stimulation. Consequently, both DYN A and SP intranigral stimulation elicit contralateral rotational behaviour, a response associated with enhanced dopaminergic transmission. The present study indicates, however, that these pathways represent different functional units in
the nigro-striato-nigral system. Thus SP in the stfiato-nigral pathway may be involved in a positive feed-back loop regulating the firing of nigro-striatal DA neurons, while the DYN might exert a negative feed-back action. The rotational behaviour produced by DYN A is probably due to direct stimulation of receptors located on non-dopaminergic nigrothalamic and nigro-tectal pathways (Vincent et al. 1978, Chevalier et al. 1981, Kilpatrick et al. 1982, Starr et al. 1983).
Acknowledgements.We gratefully acknowledge the skillful technical assistance of Ms. A. Eliasson, Ms. M. Alexandersson, Ms. W. Hiort, Ms. A. Peters, Ms. S. Nilsson, Ms. M. Karlsson, and Ms. A.K. Collin. The study was supported by grants from the Swedish Medical Research Council (14X-0357, 04X-2887, 04X-3776, 81/ 2136 A1-5/253), Magnus Bergvalls Stiftelse, Knnt och Alice Wallenberg Stiftelse, Fredrik och Ingrid Thurings Stiftelse, Luridbeck & Co. A/S and Karolinska Instituters Fonder. We thank Dr. R. Lahti, The Upjohn Co, Kalamazoo, Michigan, USA, for generous supply of U50,488H. W. Staines is a fellow of the Canadian Medical Research Council. T. Sharp is supported by a post doctoral fellowship from the Science and Engineering Research Council (U. K.).
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Received January 24, 1986 / Accepted May 27, 1986
