0306-4522/79/1201-2021f02.00/0
Neuruxienw Vol. 4. PP, 2021 to 2029 Pcrgamon Press Ltd 1979. Printed in Great Britain
OlBRO
DOPAMINE RELEASE FROM THE RAT SUBSTANTIA NIGRA IN 1/‘fTRO. EFFECT OF RAPHE LESIONS AND VERATR~DINE STIMULATION S. E. 0. TAGEEUD’ and A. C. CUELLO’ MRC Neut‘ochemical Pharmacology Unit, Department of Pharmacology, Medical School, Hills Road, Cambridge, U.K. Abstract-In order to eliminate the 5-hydroxytryptaminergic input to the substantia nigra lesions were placed in the dorsal and medial raphe nuclei in a number of rats. The release of exogenously applied [3H]dopamine from the partially denervated substantia nigra was determined in I&O and found to be very similar to the release observed from slices of control substantia nigra. These results fend further support to the theory that the release of exogenously applied C3H]dopamine at the level of the substantia nigra occurs mainly from dopaminergic dendrites, rather than from terminals of 5hydroxytryptamine-containing neurons. A veratridine-induced release of E3H]dopamine from the pars reticulata of the subst~tia nigra is also described. An almost complete blockade of veratridine (3.0 FM) stimulation was observed with 100 IIM tetrodotoxin. Similar effects of veratridine and tetrodotoxin were also observed on [3H)dopamine release from slices of corpus striatum. These results suggest that dendrites of the dopaminergic neurones in the substantia nigra contain fast, tetrodotoxin-sensitive sodium channels.
THE PRESENCE of dopamine in dendrites of dopaminergic neurones in the subst~tia nigra was first described by BJ~~RKLCWD& LINDVALL (1975). The glyoxylic acid induced catecholamine fluorescence in dendrites was found to disappear after reserpine treatment, but reappeared after in~bation of nigral tissue in the presence of dopamine, thus indicating a dendritic uptake mechanism for dopamine. This has been further supported in an autoradiographic study in which E3H]dopamine microinjected into the substantia nigra of rats was found to accumulate in dendrites with very little uptake into nerve terminals (CUELLO & KELLY, 1977). It is also possible to demonstrate a c~cium-de~ndent, potassium-evoked release of exogenously applied C3H]dopamine from the rat substantia nigra in vitro (GEFFEN, JESSELL,CUELLO & IVERSEN, 1976; CUELLO & IVERSEN, 1978), and a potassiumevoked release of [‘Hldopamine newly synthesized from E3H]tyrosine has been observed from the cat substantia nigra in viuo (NIEOULLON, CHJZRAMY & GLOWINSKI, 1977). In both cases the release is believed to occur from the dendrites of dopaminergic neurones. The dopaminer~c cell bodies and dendrites, however, are not the only aminergic elements present in the substantia nigra. Neuroanatomical and histofluorescence data indicate that axons from the me~n~phalic raphe nuclei course through and terminate in the substantia nigra (AND&N, DAHLSTR~M,
' Present address: Department of Pharmacology, University of Lund, Soivegatan 10, S-22352 Lund, Sweden. * To whom correspondence should be addressed. Present address: Department of Pharmacology, University of Oxford, South Parks Road, Oxford. Abbreviation: EDTA, ethylenedjamine tetra-acetate.
FUXE, OLSON & UNGERS~DT, 1966; CONRAD, LEONARD& PEAE, 1974; FUXE, 1965). Neurochemical studies indicate the presence of relatively high concentrations of 5hydroxytryptamine and its synthesizing enzyme tryptophan hydroxylase in the substantia nigra (PALKOVITS, BRO~S~IN & SAAVEDRA, 1974; BROWNSTEIN,PALKOVIY$ SAAVEDRA& KIZER, 1975). Recent experiments using horseradish peroxidase histochemistry and autoradiographic tracing have provided good evidence for the existence of a direct serotoninergic projection from the dorsal raphe nucleus to the substantia nigra in the rat (F~EIGER & MILLER, 1977). These data together with electrophysiological (DRAY, GONYE, OAKLEY & TANNER, 1976) and immunohistochemical evidence (PXCKEL, JOH & REI$ 1975) suggest a direct innervation of neurones in the substantia nigra by the 5-hydroxytryptamine projection system (see review by DRAY, 1979). Since the neuronal uptake mechanisms for biogenic amines are not completely specific for the amine contained within the neurones (see review by IVERSEN, 1971) it was important to determine whether or not the serotoninergic nerve terminals present in the substantia nigra could account for a major proportion of the release of C3H]dopamine observed from the rat substantia nigra in vitro. In the present study this possibility was investigated by destroying the dorsal and medial raphe nuclei in rats. The potassium-evoked release of exogenously applied C3H]dopamine from slices of substantia nigra was then determined 7-10 days following the lesions and compared to that from n&rat slices of control rats. In order to further explore the mechanisms involved in the release of dopamine, the effects of veratridine, an agent which selectively increases mem-
2021
2022
S. E. 0. TAGEIWD and .4. (‘. C‘W.I.O
brane permeability to sodium ions (see review by ULBRICHT, 1969), and tetrodotoxin on the release of [3W]dopamine from slices of rat substantia nigra were investigated.
then washed three times with 2 3 ml of Ihe temperature. and transferred to ik cmall superfused with Krebs’ bicarbonate solution rate of 500 pil min _ ’ as described previously
buffer al room chamber and at + 37 (’ at :k ((&XII (‘I &r/..
11176).
EXPERIMENTAL
PROCEDURES
Lesion.5
Male Sprague Dawley rats, weighing 250_36Og, kept on a standard diet and tap water ad fihirum, were used. The rats were anaesthetized by intraperitoneai injections of Equithesin’ and placed in a Kopf stereotaxic frame. Radiofrequency lesions were made with the top of the electrode kept at 55-6O’C for 30s and both the dorsal and medial raphe nuclei were destroyed using the coordinates A.P. -6.0, L.O.0, V. -6.3 and A.P. -6.0. L.O.O. V. -8.0 respectively (PELLEGRINO & CUSHMAN, 1967).
Several of the brains from which the substantia nigra had been microdissected were fixed either with 5”;, glutaraldehyde in 0.1 M phosphate buffer or with 4”, formaldehyde. The brains were then stored in a phosphate buffered solution of 5”” sucrose at +4’ C. Regions of the brain containing the raphe nuclei were cut into 18 itrn thick sections on a cryostat at -20 C. sections being collected at 101%200/1m intervals. Nissl staining of the sections was performed at room temperature using a O.l”,, filtered solution of cresyt violet. The sections were examined under low power in a light microscope.
Rats were used 7 IO days following placement of the raphe lesions. Non-operated rats were used as controls. Rats were killed by decapitation, the brain rapidly removed and serial transverse slices containing the substantia nigra were obtained using either a Mcilwain tissue chopper (300-350 pm thick slices) or razor blades (approx. 0.5 mm thick sIices). The remainder of the brains from animals which had a lesion were kept for later determination of the extent of the lesions (see above). Nigral tissue was microdissected from the slices under a stereomicroscope equipped with a cold stage. For some experiments only the pars reticulata of the substantia nigra was microdissected from 300 to 350 pm thick slices obtained in rhe tissue chopper. When razor blades were used to obtain slices. the microdissected nigral tissue was further cut at 200-300 pm intervals in two directions in the Mcllwain tissue chopper. All handling of the tissue prior to incubation was carried out either on ice or in a cold room kept at -t-4’.C. When striatal tissue slices were required these were obtained at the level of the optic chiasma. Half the nigral tissue from one rat (approx 3 mg). or an approximately equal amount of striatal tissue, was preincubated for 5 min in 1.5 ml Krebs bicarbonate or Krebs’ Henseleit at 37’C. C3H]dopamine was then added to give a final concentration of 1 PM E3H]dopamine, and the incubation was continued Tar a further 15 min. The tissue was
’ Equithesin is made up in the following way: For 100 ml: (1) 0.972 g sodium pentobarbital in 11.5 ml 959,) alcohol: (2) 4.25 g of chloral hydrate in 42.8 ml propylene glycol (propane, l.Ldiol); (3) 2.126g of magnesium sulphate in 45.7 mi distilled water. Mix the above three solutions in the order I. 2 and 3.
The tissue was exposed to either raised potassium concentrations or veratridine. 20. 42 or 44 and somc[lmc\ 64 min after beginning the superfusion. ‘T‘hi~was achieved by switching the superfusion to a Krebs’ bicarbonate buffet containing additional KC1 or added veratridinc in conccntratittnc necessary to give en‘ective c~~~ccxltr~lt~on~ t,f I i( 0~ 26 rnht K’ or 3.0 3.8 PM veratridinc in rhc medium bathing the tissue. In the same manner the effect of tetrodotoxin (I 10On~) was studied by exposing the tissue to this huhstance for 2 min preceding and during a I! min exposure to veratridine. Superfusate sample5 were collected 3t _7mm interbals and 250 ~1 aliquots wcrc taken for measurements ol’ radioactivity by liquid scirlt~ll~~ti~~r~ counting, AI the crtd of the experiment the tissue was recovered and the radio;clivlty remaining in the tissue determined.
Krebs’ Henseleit containing ascorbic acid (I 30 ~JM)and ethy~enedjanline tetra-acetate (EDTA) (27 ii~) had the foollowing composition (mhl): NaC1 11X. KCI 5.7, CaCl, 0.X. KH,PO, 2.0. MgSO, 2.0. NaHCC), 15. glucose IO. and was gassed with 95”,, 02 S”,, CO,. Krebs’ bicarbonate containing ascorbic acid (170PM) and EDTA (27 /IM)had the following composition (mti): NaCl 134. KC1 5.0, CaCt, 2.0. KHIPOI i.2. MgSOs 1.0. NaHCO, 16. glucose 10.2; II was gassed with 9.Y’, O2 5”,, COz The sodium phosphate buffer, pH 7.4 was 0.1 M. [JH]dopamine, specific activity 5 Ci, mmol and 10 Ci, mmol was obtained from the Radiochemical Centre. Amersham, England. Other chemicals used were obtained from the following sources: Cresyl fast violet acetate (George T. Gurr Ltd.. London); Tetrodotoxin (Sigma Chemical C‘ompany. U.S.A.): Veratridine (Aldrich Chemical Cornpan! inc.. tJ.S.A.); Chloral hydrate (BDH Chemicals. Poole. Dorset): Propylene glycol (propane 1.2-diol) (BDH (‘hemi~1s. Poole. Dorset I; Pentobarbitat (Sigma).
RESULTS
Several brains from animals with lesions were sectioned in order to determine the extent of the raphe lesions. In the majority of cases the lesions were found to extend from the lower portion of the occular motor nucleus (rostra1 end) to the first third of the pontine levels (caudal end). In the ventrolateral plane the lesions reached up to 1 mm on either side of the midline. In all cases examined the dorsal raphe nucleus was completely destroyed, and in only two of the cases examined had the more caudal portions of the media1 raphe nucleus been spared. Figure l(A) shows the extent of the lesions in one representative animal at a level roughly 0.5 mm posterior from the interauricular line according to the atlas of PALKOVITS & JACOBOWITZ (1974). Figure l(B) shows a diagrammatic representation of the localization and maximal extent of the lesions at the same level.
FIG. l(A) A Nissl stained transverse section through the brain roughly 0.5 mm posterior to the interauricular line according to the atlas of PALKOVITS& JACOBOWTZ(1974) showing the extent of the raphe lesions in one representative animal. (B) A diagrammatic representation of the localization and maximal extent of the lesion at approximately the same level as in (A), indicated by the area outlined with a dotted line. Abbreviations: CG, central grey; CT, corticospinal tract; dp, dorsal par&dial nucleus; dr, dorsal raphe nucleus; ic, inferior colliculus; ml, medial lemniscus; mr, medial raphe nucleus; pn, pontine nuclei; prf, pontine reticular formation; scp, superior cerebellar pendunculus.
2023
2025
Dopamine release from substantia nigra
Release of [3H]dopamine Efict of raphe lesions. The release of previously taken up C3H]dopamine was studied using two different depolarizing potassium concentrations, 18 mM and 26 mM. The spontaneous release was initially rapid but became stable after 15 to 20min superfusion. Exposure to the raised potassium concentrations for 2 min evoked large and reproducible increases in C3H]dopamine efflux from both partially denervated substantia nigra tissue slices and control tissue slices (Figs 2 and 3) though the relative peak size decreased with subsequent potassium pulses for both partially denervated and control tissue slices. The calcium dependence of the potassium-evoked release has been shown previously for the same in vitro system (GEFFEN et al., 1976). Although qualitatively the partially denervated tissue slices appeared to behave in a very similar way to the control tissue slices, there were some quantitative differences between the potassiumevoked release from partially denervated and control
substantia nigra tissue slices. This becomes evident when considering the total increase in the amount of [3H]dopamine released over the basal level for each potassium pulse (dashed areas in Figs 2 and 3). The values for this increase in [3H]dopamine release are given in Table 1 for each peak obtained with the two different depolarizing potassium concentrations. The response of the partially denervated tissue to the first potassium pulse was similar to the response of the control tissue. Subsequent potassium pulses, however, were found to evoke somewhat smaller increases in C3H]dopamine release from partially denervated substantia nigra tissue slices (Table 1). The difference becomes evident if the ratio of the total increase in release over basal level for one potassium pulse to that of the preceding pulse is considered (Table 2). As seen in Table 2 there was a significant difference in the ratio S2/S1 between partially denervated and control tissue slices, whereas the ratio S,/S,, obtained only at the higher concentration, was very similar for partially denervated and control tissue. Effects of veratridine and tetrodotoxin. Preliminary experiments showed that exposure of microdissected slices of the entire substantia nigra to 3.8 PM veratridine evoked a large and reproducible increase in C3H]dopamine release. In an attempt to minimize the possibility that some of this evoked release occurred as a result of an antidromic stimulation of the dendrites the following experiments were carried out on microdissected pars reticulata only, an area of the substantia nigra containing mainly the longer branched dendrites of dopaminergic cell bodies located in the pars compacta.
Tim* lmlnl
2. Release of C3H]dopamine from superfused slices of (a) control and (b) partially denervated rat substantia nigra. Tissue slices were superfused at a rate of 500 nl min- ’ with Krebs’ bicarbonate buffer at +37”C, containing EDTA (27 PM) and ascorbic acid (170PM) to prevent spontaneous breakdown of the added dopamine. Superfusate samples were collected at 2 min intervals and radioactivity determined by liquid scintillation counting. At the end of the experiment the tissue slices were recovered and the content of radioactivity determined. C3H]dopamine efflux is expressed as y0 of total tissue stores released per min (d.p.m. released per min in superfusate/d.p.m. in tissue stores at time of collection). Each value represents mean f S.E.M. of four experiments. Horizontal bars indicate 2 min exposures of the tissue to 26 mM KCI. Shaded areas (also labelled S,-SJ) indicate the increase in release of radioactivity induced by the raised potassium concentration. FIG.
Timolminl FIG. 3. As for Fig. 2 but with 18 mM KC1 instead of 26 mM
KCI.
Each
value
represents mean + experiments.
S.E.M.
of
three
2026
S. E. 0. TAGERUDand A. C. CUELLO TABLE1. POTASSIUM IONEVOKED RELEASE OF [‘H]DOPAMINEFROMPARTIALLY DENERVATED AND SUBSTANTIA NIGRATISSUESLICES 26 mu KCI
CONTROL
18 mM KC1
Conditions
S,
S2
S3
S,
S2
Control Partially denervated
29.30 f 1.02
23.00 + 0.46
17.88 + 1.64
5.90 i 0.78
4.34 _t 0.82
32.60 + 5.20
14.86 rf: 4.04
I 1.86 Lt:4.50
7.14 + 1.28
2.8X + 0.32
N.S.
N.S.
N.S.
N.S.
N.S
S,-S, for the two potassium concentrations refer to total increase in release for potassium pulses I--3. Values are expressed as the mean + S.E.M. (n = 4 for 26 mM K+; n = 3 for 18 mM K’) of the total increase in the percentage of C3H]dopamine release over basal level for each pulse. Significance was tested for using the Student’s r-test (unpaired observations). N.S., not significant at the 0.05 confidence limit.
Figure 4(b) shows the effect of two 2 min pulses of 2.0~~ veratridine on the release of C3H]dopamine from microdissected pars reticulata. This response. was qualitatively very similar to the response of striatal tissue slices to the same veratridine concentration (Fig. 4a). Figure 5 shows a comparison of the effect of three tetrodotoxin concentrations on the evoked release of C3H]dopamine from slices of the pars reticulata of the substantia nigra and from striatal tissue slices, using 2 min pulses of 3.0~~ veratridine. A second pulse of veratridine in the absence of tetrodotoxin is also included to show that in all cases the tissue was responsive. As shown in Fig. 5, 1 nM tetro-
dotoxin had little or no effect on the veratridineevoked release either in the striatum or in the substantia nigra. Ten nM tetrodotoxin blocked C3H]dopamine release partially in both cases and
I
a
2
-i--l-
a
b
bk Timalminl
Tim*lminl Effect of veratridine on the release of [‘Hldopamine from superfused slices of the rat corpus striatum (a) and microdissected pars reticulata of the substantia nigra from normal rat (b). Experimental conditions as for Fig. 2. Horizontal bars indicated 2 min exposures to 2.0 PM veratridine. Each value represents the mean of two experiments.
FIG. 5. Effect of three different tetrodotoxin concentrations on the veratridine-induced release of C3H]dopamine from superfused slices of the rat corpus striatum (a) and microdissected pars reticulata of the substantia nigra (b). Experimental conditions as for Fig. 2. Spontaneous outflow of radioactivity before, between and after veratridine pulses is given as the mean + S.E.M. for all experiments combined. Unbroken lines represent the effect of 1 nM tetrodotoxin (n = 1 for both corpus striatum and pars reticulata), broken lines 10 nM tetrodotoxin (n = 1 for corpus striaturn; n = 2 for pars reticulata) and dotted lines 10On~ tetrodotoxin (n = 1 for corpus striatum; n = 2 for pars reticulata). (m) indicates exposure of the tissue slices to tetrodotoxin, (B) indicates exposure to 3.0 PM veratridine. (*) indicates the rather large second peak observed with 3.0 PM veratridine in the pars reticulata after the first peak had been partially blocked by 10 nM tetrodotoxin (see text).
Dopamine release from substantia nigra TABLE 2. RAnO OF ~ElN~~A~ IN TRACTIONAL RELEASE RA~SOF[~H]~PAMINEFORREPEA~DPOTA~IUMPUL~S
Conditions Control Partially denervated
26mM KC S3iS2 SZISI
18mM K+ WSI
0.79 + 0.03
0.78 I: 0.07
0.73 + 0.09
0.47 + 0.10
0.75 f 0.09
0.42 f 0.04
2027
of the f&e output from the medial raphe nucleus was also eliminated. No measurements of the 5-hydroxytryptamine contents of the substantia nigra from controls or animals with lesions were made in the present study. However, judging from the post-mortem histological determination of the extent of the lesions there is little doubt
that the lesions were successful, and in accordance with other lesion studies this would be expected to Sr-S3 corresponds to the values given in Table 1. Values result in a very substantial reduction in the 5-hydroxyare mean f S.E.M.(n = 4 for 26 mM K’ ; n = 3 for 18 mM tryptamine content of the substantia nigra (PALK~K+). VITS, SAAVEDRA,JACOBOWITZ,KIZER, Z~BORSZKY& Significance was tested for using the Student’s r-test (unBRO~S~IN, 1977; DRAY, DAVIES, OAKLEY, TANpaired observation). GROACH& VELLUCCI,1978). Despite this no qualitaN.S., not significant at the 0.05 confidence limit. tive differences were observed between the release of C3H]dopamine from substantia nigra tissue slices of controls and animals with lesions. This is consistent 10On~ tetrodotoxin blocked it almost completely both in the striatum and in the substantia nigra. In all with the theory that the release of previously taken up [‘HJdopamine from the substantia nigra in vitro cases a second exposure to veratridine in the absence of tetrodotoxin evoked a large increase in L3H]dopa- occurs mainly, if not exclusively, from dopaminergic mine release. A particularly large peak was observed dendrites, with little or no contribution from serofrom the pars reticulata of the substantia nigra follow- toninergic elements. Tables 1 and 2 show that, ing partial blockade of the first peak with 10 nM tetro- although qualitatively similar, some quantitative difdotoxin. In these experiments, as seen in Fig. 5, the ferences are observed between partially denervated and control substantia nigra tissue slices in the total baseline release of C3H]dopamine from the substantia nigra was considerably higher than that from the increase of ~3~]dopamine release over basal level for striatal slices, as found previously GEFFENet af. (1976). repeated potassium pulses. The reason for the quantitative differences is not clear but could include a number of factors, not necessarily involving the reDISCUSSION moval of 5-hydroxytryptamine-containing nerve terE@ct of lesions of the raphe nuclei minals per se. An estimation of the total amount of radioactivity present in the tissue slices at the beginThere is good evidence for a serotoninergic projecning of the superfusion revealed no obvious differtion from the mesencephalic raphe nuclei. Experimental evidence also indicates a GABA projection from ences for the [‘H]dopamine uptake in partially the caudoputamen-globus pallidus region to the sub- denervated and control tissue slices, thus suggesting stantia nigra (KIM, BAK, HASSLER& OKADA, 1975; that the raphe lesion does not cause a significant loss HATTORI, MCGEFX, FIBIGER& MCGEER, 1973; FON- of t3H]dopamine uptake sites. The in uivo observaNUM, GROFOV& RAN~IK, S~OM-MA~I~N & WAL- tions of NIEOULLONet al. (1977) and CHERAMY, BERG,1974) and a substance P-containing pathway is NIEOULLON& GLOWINSKI(1978) that L-3,5-[3H]tyroalso believed to originate from this same region of the sine is converted to C3H]dopamine and released from brain (KANAZAWA,EM~C~N & CUELLO,1977). These the cat substantia nigra would also make any particithree neuronal pathways are believed to account for a pation of serotoninergic nerve terminals unlikely since major part of the innervation of the substantia nigra, these lack the enzyme tyrosine hydroxylase. Possibly, and of the three only 5-hydroxy~yptamine containing the removal of tonic inhibitory influences normally neurones would be expected to be able to take up and exerted by 5-hydroxytryptamine (DRAY et at., 1978; possibly release [3H]dopamine to any significant see also NIEOULLONet al., 1977) may be an important extent. Dopaminergic collaterals arising from the factor related to the observed quantitative differences nigrostriatal pathway and projecting back into the in release between partially denervated and control substantia nigra have not been observed (JURASKA, substantia nigra tissue slices. WILsCiN& GROVES,1977). In conclusion these results strongly suggest that the In the present study the possible con~ibution by main part of the reiease occurs from dopaminergic serotoninergic elements to the reported uptake and dendrites in the substantia nigra. release of C3H]dopamine from dendrites of substantia EfSects of veratridine and tetrodntoxin nigra neurones (see Introduction) was assessed by performing in vitro uptake and release studies following The release of dopamine from dendrites of substanlesions of the raphe nuclei. Lesions were placed in tia nigra neurones was further explored by studying both the dorsal and the medial raphe nuclei. In two the effects of veratridine and tetrodotoxin. Veratridine animals the caudal part of the medial raphe nucleus depolarizes nervous tissue by selectively increasing was spared but since the fibres pass in a caudal to the membrane permeability to sodium ions and this rostra1 direction it is most likely that the major part effect is normally blocked by tetrodotoxin. PrelimiP < 0.02
N.S.
P < 0.05
2028
S. F. 0
‘TAGF.R~Wand .A C. c’Ilr;I.Lo
nary experiments showed that veratridine potently induces the release of exogenously applied r3H]dopamine from microdissected slices of the entire substantia nigra, suggesting that the dendritic membrane, like most excitable membranes. contains fast sodium channels which can be activated by veratridine. The possibility was also considered. however. that the dendrites may become depolarized by an antidromic stimulation arising from the action of veratridine on the small axon segments most probably still attached to the cell bodies after microdissection of the entire substantia nigra. This possibility may seem unlikely in view of work carried out on Purkinje cell dendrites (LIJNAS. NICHOLSOK & PKECHT, 1969) which suggests that dendrites, due to a differential excitability. tend to propagate depolarizations mainly in the somatopetat direction. Consequently. an antidromic activation of the cell would be expected to invade only the lower parts of the main dendritic branches. Nevertheless, in order to minimize the possibility 01”any antidromic invasion of the dendrites the experiments reported here were carried out on microdissected slices of the pars reticulata only. an area of the substantia nigra containing the longer branched dendrites arising from cell bodies located in the pars compacta (DAHLSTK~~M & Fllx~, 1964). As shown in Fig. 4, veratridine evoked the release of C3H]dopamine both from slices of the pars reticulata of the substantia nigra and from slices of the striatum. In both cases the veratridine-induced release was blocked by low concentrations of tetrodotoxin (Fig. 5). The conclusion would seem to be that the dopaminergic dendrites, like other parts of the neurone. contain fast tetrodotoxin-sensitive sodium channels. Other studies are not entirely consistent with this interpretation. LLIN~S & HESS (1976) managed to record tetrodotoxin-resistant dendritic spikes from avian Purkinje cells. and the suggestion was put forward that the dendritic spikes were generated by calcium currents. The in r.iro study in NI~OIII,LOI\:et 111. (1977) in which the spontaneous release of dopamine from the cat substantia nigra was not blocked by tctrodotoxin may seem to carry similar implications. A possible explanation of these apparent discrepancies is provided if the dendritic membrane contains fast sodium channels. normally present in a latent state but susceptible to activation by veratridine and to subsequent block by tetrodotoxin. These channels would not normally participate in electrical conduction. and the addition of tetrodotoxin would therefore not block electrical activity or transmitter release
from the dendrites but may even increase it if inhibitory influences are suppressed (NIEOULLON YI (II.. 1977). On the other hand. exposure to veratridine would activate these latent channels. causing depolarization and transmitter release from the dendrites, and this effect of veratridine would be blocked by tetrodotoxin as reported in the present study. Further experimentation either in rim under electrical stimulation or with a more physiological in vim system could probably yield more information on these questions. More direct evidence for the existence of latent fast sodium channels, which can be activated by veratridine and blocked by tetrodotoxin, has recently been obtained by LOWE, BUSH & RIPLEY (1978) in an elecn-ophysiological study on the large sensory dendrites innervating the thoracic-coxal muscle receptor organ of the crab Cur&us muma.\. A further observation made in the present study was that after partial blockade of the first veratridineinduced release of [jH]dopamine from the microdissected pars reticulata tissue slices with IO nM tetrodotoxin. the second veratridine-induced release, in the absence of tetrodotoxin. appeared to be unexpectedly large (Fig. 5). This was found in two separate experiments on pars reticulata but not on striatum. tissue slices. Further experimentation would again be required in order to determine the significance of this observation. In this series of experiments it was also noticed that the spontaneous release observed from substantia nigra tissue slices was considerably higher than that from the striatum tissue slices. This is in agreement with what was found by GEFFEN c’t trl. ( 1976) and may suggest some differences in the dopamine storage -release mechanisms between nerve terminals and dendrites. In this regard it is worth commenting on the absence of vesicular elements in dendrites of the substantia nigra neurones (SOTELO. 1972: CIXLLO & IVERSEK. 1978) and the fact that the monoamine marker 5hydroxydopamine injected intracerebrally can be identified in the interior of cisternac of smooth endoplasmic reticulum of dendrites of the suhstantia nigra neurones (MERVER. DEL FIA(Y‘O & (‘rF.1.1.0, 1979). .4~,knon’/rrlyc,nlent.\ The authors wish to thank Dr L. I.. IVERS~N for reviewing the manuscript. The skilful technical assistance of DAVID CHAPMAN, BRIAN JOHNSON, GEORW MARSHAI.L and ANGIE NELSONis also gratefully acknowledged. We would also like to thank Prof. J. A~WW for many helpful discussions.
REFERENCES ANDCN N.-E.. DAHLSTR~M A., FL~x~: K.. OLSON L. & UNGERSTEDT U. (1966) Ascending monoamine neurones to the telencephalon and diencephalon. Acrtr physiol .scund. 67, 313 326. BIBRKLUNI) A. & LINDVALL 0. (1975) Dopamine in dendrites of substantia nigra neurones: suggestions for a role in dendritic terminals. Bruin Rcs. 83, 531 537. BROWNSTEIN M. J., PALKOVITS M.. SAAVEIIRA J. M. & KIZ~R J. S. (1975) Tryptophan hydroxylase in the rat brain. Brain Rev. 97, 163~ 166.
Dopamine release from substantia nigra
2029
C~RAMY A., NIEOULLO~A, & GLOWINSKII. (1978) In vivo changes in dopamine release in cat caudate Ruckus and substantia nigra induced by nigral application of various drugs including GABAergic agonists and antagonists. In Interactians between Putative Neurotransmitters in the Brain (eds GARATTINIS., PUIOLJ. F. & SAMANIN R.), pp. 175-190. Raven Press, New York. CONRADL. C. A., LEONARD C. M. & PFAFD. W. (1974) Connections of the median and dorsal raphe nuclei in the rat: an autoradiographic and degeneration study. J. camp. Neural. 156, 179-206. Ct.~n~r_oA. C. & IVERsENL. L. (1978) Interactions of dopamine with other neurotransmitters in the rat substantia nigra: a possible functional role of dendritic dopamine. In interacf~~s ~efween Pufafive Neurotra~s~irters in the Brain feds GARATTINIS., P~SOL.I. F. & SAMANINR.), pp. 124-147. Raven Press, New York. CUELLO A. C. & KELLY J. S. (1977) Electron microscopic autoradiographic localization of ‘H-dopamine in the dendrites of the dopaminergic neurones of the rat substantia nigra in uiuo. Br. J. Pharmac. 59, 527-528P. DAHLSTR~M A. & FUXE K. (1964) Evidence for the existence of monoamine neurones in the central nervous system--I. Demonstration of monoamines in the cell bodies of brain stem neurones. Acta physiol. stand. 64, Suppl. 232, l-55. DRAYA. (1979) The striatum and substantia nigra: a commentary on their relationships. Neuroscience 4, 1407-1439. DRAYA., DAVIESJ., OAKLEYN. R., TONGROACH P. & VELLUCCIS. (1978) The dorsal and medial raphe projections to the substantia nigra in the rat: electrophysiological, biochemical and behavioural observations. Brain Res. 151, 431-442. DRAY A., GONYET. J., OAKLEYN. R. & TANNERT. (1976) Evidence for the existence of a raphe projection to the substantia nigra in rat. Brain Res. 113, 45-57. FIBIGERH. C. & MILLERJ. J. (1977) An anatomical and electrophysiological investigation of the serotonergic projection from the dorsal raphe nucleus to the substantia nigra in the rat. Neuroscience 2,97s988. FONNUMF., GROFOV~,I., RANVIKE., STORM-MATHI~NJ. & WAL~~ZRG F. (1974) Origin and dis~ibution of glutamate decarboxylase in the substantia nigra of the cat. Brain Res. 71, 77-92. FUXE K. (1965) Evidence for the existence of monoamine neurones in the central nervous system-IV. Distribution of monoamine nerve terminals in the central nervous system. Acta physiol. stand. 64, Suppl. 247, 37-85. GEFFENL. B., JESSELLT. M., CUELLOA. C. & IVERSENL. L. (1976) Release of dopamine from dendrites in rat substantia nigra. Nature, Lond. 260, 258-260. HATTORIT., MCGEERP. L., FIBIGERH. C. & MCGEERE. G. (1973) On the source of GABA-containing terminals in the substantia nigra: electron microscopic autoradio~aph~c and biochemical studies. Brain Res. S4, 103-114. IVERSENL. L. (1971) The uptake of biogenic amines. In Biogenic Amines and Pkysio~ogiea~ ~e~ranes in Drug Therapy (eds BIEL J. H. & ABAADL. G.), pp, 2.59-327. Dekker, New York. JURASKAJ. M., WILSONC. J. & GROVESP. M. (1977) The substantia nigra of the rat: A Golgi study. J. camp. Neural. 172, 585600. KANAZAWAI., EM~ONP. C. & CUELLOA. C. (1977) Evidence for the existence of substance P-containing fibres in striato-nigral and pallido-nigral pathways in rat brain. Brain Res. 119,447-453. KIM J. S., BAK I. J., HASSLERR. & OKADAY. (1975) Role of gamm~minobutyric acid (GABA) in the extrapyramidal motor systems: some evidence for the existence of a type of GABA-rich strio-nigral neurones. Expi Brain Res. 14, 95-104.
LLINASR. & HESSR. (1976) Tetrodotoxin-resistant
dendritic spikes in avian Purkinje cells. Proc. natn. Acad. Sci. U.S.A.
73,2520-2523. LLIN~SR., NICHOLWNC. & PRECHTW. (1969) Preferred centripetal conduction cells. Science, N.Y. 163, 184187.
of dendritic spikes in alligator Purkinje
LOWED. A., BU~HB. M. H. & RIPLEYS. H. (1978) Pharmacological evidence for ‘fast’ sodium channels in nonspiking neurones. Nature, Load. 274, 289-290. MERCERL., DEL F~ACCOM. & CUELLOA. C. (1979) The smooth endoplasmic reticulum as a possible storage site for dendritic dopamine in substantia nigra neurones. Experientia 35, 101-103. NIEOULLON A., CHERAMY A. & GLOWINSKIJ. (1977) Release of dopamine in viva from cat substantia nigra. Nature, Land. 266, 375-377.
PALKOVITS M., BROWNSTEIN M. & SAAVEDRA J. M. (1974) Serotonin content of the brain stem nuclei in the rat. Brain Res. 80, 237-249.
PALKOVITSM. & JACOEXOWITZ D. M. (1974) Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain-H. Hindbrain (mesencephalon, rhombenceophalon). .I. cotnp. Neural. 157, 2941. PALKOVITS M., SAAVEDRA J. M., JACOBOWITZ D. M., KIZER J. S., ZABORSZKY L. & BROWNSTEIN M. J. (1977) Serotonergic innervation of the forebrain: effect of lesions on serotonin and tryptophan hydroxylase levels, Brain Res. 130, 121-134. PnL~.aGnrNoL. J. & CUSHMANA. J. (1967) A Stereotaxic Atlas of the Rat Brain. Appleton-Century-Crofts, New York, PrcKEL V. M., JOH T. H. & REIS I), J. (1975) Monoamine-synthesizjn~ enzymes in central dopaminergic, noradrenergic and serotonergic neurones: immunocyt~hemical localization by light and electron microscopy. .I. ~istoc~em. Cytathem. 24,792-806. SOTELOC. (1972) The fine structural localization of ‘H-norepinephrine in the substantia nigra and area postrema of the rat: an autoradiographic study. J. Ultmstruct. Res. 36, 824-841. ULBRICHTW. (1969) The effect of veratridine on excitable membranes of nerve and muscle. Rev. Physiol. Biochem. Pharmac. 61, 18-71. (Accepfed 15 June 1979)
Neuruxienw Vol. 4. PP, 2021 to 2029 Pcrgamon Press Ltd 1979. Printed in Great Britain
OlBRO
DOPAMINE RELEASE FROM THE RAT SUBSTANTIA NIGRA IN 1/‘fTRO. EFFECT OF RAPHE LESIONS AND VERATR~DINE STIMULATION S. E. 0. TAGEEUD’ and A. C. CUELLO’ MRC Neut‘ochemical Pharmacology Unit, Department of Pharmacology, Medical School, Hills Road, Cambridge, U.K. Abstract-In order to eliminate the 5-hydroxytryptaminergic input to the substantia nigra lesions were placed in the dorsal and medial raphe nuclei in a number of rats. The release of exogenously applied [3H]dopamine from the partially denervated substantia nigra was determined in I&O and found to be very similar to the release observed from slices of control substantia nigra. These results fend further support to the theory that the release of exogenously applied C3H]dopamine at the level of the substantia nigra occurs mainly from dopaminergic dendrites, rather than from terminals of 5hydroxytryptamine-containing neurons. A veratridine-induced release of E3H]dopamine from the pars reticulata of the subst~tia nigra is also described. An almost complete blockade of veratridine (3.0 FM) stimulation was observed with 100 IIM tetrodotoxin. Similar effects of veratridine and tetrodotoxin were also observed on [3H)dopamine release from slices of corpus striatum. These results suggest that dendrites of the dopaminergic neurones in the substantia nigra contain fast, tetrodotoxin-sensitive sodium channels.
THE PRESENCE of dopamine in dendrites of dopaminergic neurones in the subst~tia nigra was first described by BJ~~RKLCWD& LINDVALL (1975). The glyoxylic acid induced catecholamine fluorescence in dendrites was found to disappear after reserpine treatment, but reappeared after in~bation of nigral tissue in the presence of dopamine, thus indicating a dendritic uptake mechanism for dopamine. This has been further supported in an autoradiographic study in which E3H]dopamine microinjected into the substantia nigra of rats was found to accumulate in dendrites with very little uptake into nerve terminals (CUELLO & KELLY, 1977). It is also possible to demonstrate a c~cium-de~ndent, potassium-evoked release of exogenously applied C3H]dopamine from the rat substantia nigra in vitro (GEFFEN, JESSELL,CUELLO & IVERSEN, 1976; CUELLO & IVERSEN, 1978), and a potassiumevoked release of [‘Hldopamine newly synthesized from E3H]tyrosine has been observed from the cat substantia nigra in viuo (NIEOULLON, CHJZRAMY & GLOWINSKI, 1977). In both cases the release is believed to occur from the dendrites of dopaminergic neurones. The dopaminer~c cell bodies and dendrites, however, are not the only aminergic elements present in the substantia nigra. Neuroanatomical and histofluorescence data indicate that axons from the me~n~phalic raphe nuclei course through and terminate in the substantia nigra (AND&N, DAHLSTR~M,
' Present address: Department of Pharmacology, University of Lund, Soivegatan 10, S-22352 Lund, Sweden. * To whom correspondence should be addressed. Present address: Department of Pharmacology, University of Oxford, South Parks Road, Oxford. Abbreviation: EDTA, ethylenedjamine tetra-acetate.
FUXE, OLSON & UNGERS~DT, 1966; CONRAD, LEONARD& PEAE, 1974; FUXE, 1965). Neurochemical studies indicate the presence of relatively high concentrations of 5hydroxytryptamine and its synthesizing enzyme tryptophan hydroxylase in the substantia nigra (PALKOVITS, BRO~S~IN & SAAVEDRA, 1974; BROWNSTEIN,PALKOVIY$ SAAVEDRA& KIZER, 1975). Recent experiments using horseradish peroxidase histochemistry and autoradiographic tracing have provided good evidence for the existence of a direct serotoninergic projection from the dorsal raphe nucleus to the substantia nigra in the rat (F~EIGER & MILLER, 1977). These data together with electrophysiological (DRAY, GONYE, OAKLEY & TANNER, 1976) and immunohistochemical evidence (PXCKEL, JOH & REI$ 1975) suggest a direct innervation of neurones in the substantia nigra by the 5-hydroxytryptamine projection system (see review by DRAY, 1979). Since the neuronal uptake mechanisms for biogenic amines are not completely specific for the amine contained within the neurones (see review by IVERSEN, 1971) it was important to determine whether or not the serotoninergic nerve terminals present in the substantia nigra could account for a major proportion of the release of C3H]dopamine observed from the rat substantia nigra in vitro. In the present study this possibility was investigated by destroying the dorsal and medial raphe nuclei in rats. The potassium-evoked release of exogenously applied C3H]dopamine from slices of substantia nigra was then determined 7-10 days following the lesions and compared to that from n&rat slices of control rats. In order to further explore the mechanisms involved in the release of dopamine, the effects of veratridine, an agent which selectively increases mem-
2021
2022
S. E. 0. TAGEIWD and .4. (‘. C‘W.I.O
brane permeability to sodium ions (see review by ULBRICHT, 1969), and tetrodotoxin on the release of [3W]dopamine from slices of rat substantia nigra were investigated.
then washed three times with 2 3 ml of Ihe temperature. and transferred to ik cmall superfused with Krebs’ bicarbonate solution rate of 500 pil min _ ’ as described previously
buffer al room chamber and at + 37 (’ at :k ((&XII (‘I &r/..
11176).
EXPERIMENTAL
PROCEDURES
Lesion.5
Male Sprague Dawley rats, weighing 250_36Og, kept on a standard diet and tap water ad fihirum, were used. The rats were anaesthetized by intraperitoneai injections of Equithesin’ and placed in a Kopf stereotaxic frame. Radiofrequency lesions were made with the top of the electrode kept at 55-6O’C for 30s and both the dorsal and medial raphe nuclei were destroyed using the coordinates A.P. -6.0, L.O.0, V. -6.3 and A.P. -6.0. L.O.O. V. -8.0 respectively (PELLEGRINO & CUSHMAN, 1967).
Several of the brains from which the substantia nigra had been microdissected were fixed either with 5”;, glutaraldehyde in 0.1 M phosphate buffer or with 4”, formaldehyde. The brains were then stored in a phosphate buffered solution of 5”” sucrose at +4’ C. Regions of the brain containing the raphe nuclei were cut into 18 itrn thick sections on a cryostat at -20 C. sections being collected at 101%200/1m intervals. Nissl staining of the sections was performed at room temperature using a O.l”,, filtered solution of cresyt violet. The sections were examined under low power in a light microscope.
Rats were used 7 IO days following placement of the raphe lesions. Non-operated rats were used as controls. Rats were killed by decapitation, the brain rapidly removed and serial transverse slices containing the substantia nigra were obtained using either a Mcilwain tissue chopper (300-350 pm thick slices) or razor blades (approx. 0.5 mm thick sIices). The remainder of the brains from animals which had a lesion were kept for later determination of the extent of the lesions (see above). Nigral tissue was microdissected from the slices under a stereomicroscope equipped with a cold stage. For some experiments only the pars reticulata of the substantia nigra was microdissected from 300 to 350 pm thick slices obtained in rhe tissue chopper. When razor blades were used to obtain slices. the microdissected nigral tissue was further cut at 200-300 pm intervals in two directions in the Mcllwain tissue chopper. All handling of the tissue prior to incubation was carried out either on ice or in a cold room kept at -t-4’.C. When striatal tissue slices were required these were obtained at the level of the optic chiasma. Half the nigral tissue from one rat (approx 3 mg). or an approximately equal amount of striatal tissue, was preincubated for 5 min in 1.5 ml Krebs bicarbonate or Krebs’ Henseleit at 37’C. C3H]dopamine was then added to give a final concentration of 1 PM E3H]dopamine, and the incubation was continued Tar a further 15 min. The tissue was
’ Equithesin is made up in the following way: For 100 ml: (1) 0.972 g sodium pentobarbital in 11.5 ml 959,) alcohol: (2) 4.25 g of chloral hydrate in 42.8 ml propylene glycol (propane, l.Ldiol); (3) 2.126g of magnesium sulphate in 45.7 mi distilled water. Mix the above three solutions in the order I. 2 and 3.
The tissue was exposed to either raised potassium concentrations or veratridine. 20. 42 or 44 and somc[lmc\ 64 min after beginning the superfusion. ‘T‘hi~was achieved by switching the superfusion to a Krebs’ bicarbonate buffet containing additional KC1 or added veratridinc in conccntratittnc necessary to give en‘ective c~~~ccxltr~lt~on~ t,f I i( 0~ 26 rnht K’ or 3.0 3.8 PM veratridinc in rhc medium bathing the tissue. In the same manner the effect of tetrodotoxin (I 10On~) was studied by exposing the tissue to this huhstance for 2 min preceding and during a I! min exposure to veratridine. Superfusate sample5 were collected 3t _7mm interbals and 250 ~1 aliquots wcrc taken for measurements ol’ radioactivity by liquid scirlt~ll~~ti~~r~ counting, AI the crtd of the experiment the tissue was recovered and the radio;clivlty remaining in the tissue determined.
Krebs’ Henseleit containing ascorbic acid (I 30 ~JM)and ethy~enedjanline tetra-acetate (EDTA) (27 ii~) had the foollowing composition (mhl): NaC1 11X. KCI 5.7, CaCl, 0.X. KH,PO, 2.0. MgSO, 2.0. NaHCC), 15. glucose IO. and was gassed with 95”,, 02 S”,, CO,. Krebs’ bicarbonate containing ascorbic acid (170PM) and EDTA (27 /IM)had the following composition (mti): NaCl 134. KC1 5.0, CaCt, 2.0. KHIPOI i.2. MgSOs 1.0. NaHCO, 16. glucose 10.2; II was gassed with 9.Y’, O2 5”,, COz The sodium phosphate buffer, pH 7.4 was 0.1 M. [JH]dopamine, specific activity 5 Ci, mmol and 10 Ci, mmol was obtained from the Radiochemical Centre. Amersham, England. Other chemicals used were obtained from the following sources: Cresyl fast violet acetate (George T. Gurr Ltd.. London); Tetrodotoxin (Sigma Chemical C‘ompany. U.S.A.): Veratridine (Aldrich Chemical Cornpan! inc.. tJ.S.A.); Chloral hydrate (BDH Chemicals. Poole. Dorset): Propylene glycol (propane 1.2-diol) (BDH (‘hemi~1s. Poole. Dorset I; Pentobarbitat (Sigma).
RESULTS
Several brains from animals with lesions were sectioned in order to determine the extent of the raphe lesions. In the majority of cases the lesions were found to extend from the lower portion of the occular motor nucleus (rostra1 end) to the first third of the pontine levels (caudal end). In the ventrolateral plane the lesions reached up to 1 mm on either side of the midline. In all cases examined the dorsal raphe nucleus was completely destroyed, and in only two of the cases examined had the more caudal portions of the media1 raphe nucleus been spared. Figure l(A) shows the extent of the lesions in one representative animal at a level roughly 0.5 mm posterior from the interauricular line according to the atlas of PALKOVITS & JACOBOWITZ (1974). Figure l(B) shows a diagrammatic representation of the localization and maximal extent of the lesions at the same level.
FIG. l(A) A Nissl stained transverse section through the brain roughly 0.5 mm posterior to the interauricular line according to the atlas of PALKOVITS& JACOBOWTZ(1974) showing the extent of the raphe lesions in one representative animal. (B) A diagrammatic representation of the localization and maximal extent of the lesion at approximately the same level as in (A), indicated by the area outlined with a dotted line. Abbreviations: CG, central grey; CT, corticospinal tract; dp, dorsal par&dial nucleus; dr, dorsal raphe nucleus; ic, inferior colliculus; ml, medial lemniscus; mr, medial raphe nucleus; pn, pontine nuclei; prf, pontine reticular formation; scp, superior cerebellar pendunculus.
2023
2025
Dopamine release from substantia nigra
Release of [3H]dopamine Efict of raphe lesions. The release of previously taken up C3H]dopamine was studied using two different depolarizing potassium concentrations, 18 mM and 26 mM. The spontaneous release was initially rapid but became stable after 15 to 20min superfusion. Exposure to the raised potassium concentrations for 2 min evoked large and reproducible increases in C3H]dopamine efflux from both partially denervated substantia nigra tissue slices and control tissue slices (Figs 2 and 3) though the relative peak size decreased with subsequent potassium pulses for both partially denervated and control tissue slices. The calcium dependence of the potassium-evoked release has been shown previously for the same in vitro system (GEFFEN et al., 1976). Although qualitatively the partially denervated tissue slices appeared to behave in a very similar way to the control tissue slices, there were some quantitative differences between the potassiumevoked release from partially denervated and control
substantia nigra tissue slices. This becomes evident when considering the total increase in the amount of [3H]dopamine released over the basal level for each potassium pulse (dashed areas in Figs 2 and 3). The values for this increase in [3H]dopamine release are given in Table 1 for each peak obtained with the two different depolarizing potassium concentrations. The response of the partially denervated tissue to the first potassium pulse was similar to the response of the control tissue. Subsequent potassium pulses, however, were found to evoke somewhat smaller increases in C3H]dopamine release from partially denervated substantia nigra tissue slices (Table 1). The difference becomes evident if the ratio of the total increase in release over basal level for one potassium pulse to that of the preceding pulse is considered (Table 2). As seen in Table 2 there was a significant difference in the ratio S2/S1 between partially denervated and control tissue slices, whereas the ratio S,/S,, obtained only at the higher concentration, was very similar for partially denervated and control tissue. Effects of veratridine and tetrodotoxin. Preliminary experiments showed that exposure of microdissected slices of the entire substantia nigra to 3.8 PM veratridine evoked a large and reproducible increase in C3H]dopamine release. In an attempt to minimize the possibility that some of this evoked release occurred as a result of an antidromic stimulation of the dendrites the following experiments were carried out on microdissected pars reticulata only, an area of the substantia nigra containing mainly the longer branched dendrites of dopaminergic cell bodies located in the pars compacta.
Tim* lmlnl
2. Release of C3H]dopamine from superfused slices of (a) control and (b) partially denervated rat substantia nigra. Tissue slices were superfused at a rate of 500 nl min- ’ with Krebs’ bicarbonate buffer at +37”C, containing EDTA (27 PM) and ascorbic acid (170PM) to prevent spontaneous breakdown of the added dopamine. Superfusate samples were collected at 2 min intervals and radioactivity determined by liquid scintillation counting. At the end of the experiment the tissue slices were recovered and the content of radioactivity determined. C3H]dopamine efflux is expressed as y0 of total tissue stores released per min (d.p.m. released per min in superfusate/d.p.m. in tissue stores at time of collection). Each value represents mean f S.E.M. of four experiments. Horizontal bars indicate 2 min exposures of the tissue to 26 mM KCI. Shaded areas (also labelled S,-SJ) indicate the increase in release of radioactivity induced by the raised potassium concentration. FIG.
Timolminl FIG. 3. As for Fig. 2 but with 18 mM KC1 instead of 26 mM
KCI.
Each
value
represents mean + experiments.
S.E.M.
of
three
2026
S. E. 0. TAGERUDand A. C. CUELLO TABLE1. POTASSIUM IONEVOKED RELEASE OF [‘H]DOPAMINEFROMPARTIALLY DENERVATED AND SUBSTANTIA NIGRATISSUESLICES 26 mu KCI
CONTROL
18 mM KC1
Conditions
S,
S2
S3
S,
S2
Control Partially denervated
29.30 f 1.02
23.00 + 0.46
17.88 + 1.64
5.90 i 0.78
4.34 _t 0.82
32.60 + 5.20
14.86 rf: 4.04
I 1.86 Lt:4.50
7.14 + 1.28
2.8X + 0.32
N.S.
N.S.
N.S.
N.S.
N.S
S,-S, for the two potassium concentrations refer to total increase in release for potassium pulses I--3. Values are expressed as the mean + S.E.M. (n = 4 for 26 mM K+; n = 3 for 18 mM K’) of the total increase in the percentage of C3H]dopamine release over basal level for each pulse. Significance was tested for using the Student’s r-test (unpaired observations). N.S., not significant at the 0.05 confidence limit.
Figure 4(b) shows the effect of two 2 min pulses of 2.0~~ veratridine on the release of C3H]dopamine from microdissected pars reticulata. This response. was qualitatively very similar to the response of striatal tissue slices to the same veratridine concentration (Fig. 4a). Figure 5 shows a comparison of the effect of three tetrodotoxin concentrations on the evoked release of C3H]dopamine from slices of the pars reticulata of the substantia nigra and from striatal tissue slices, using 2 min pulses of 3.0~~ veratridine. A second pulse of veratridine in the absence of tetrodotoxin is also included to show that in all cases the tissue was responsive. As shown in Fig. 5, 1 nM tetro-
dotoxin had little or no effect on the veratridineevoked release either in the striatum or in the substantia nigra. Ten nM tetrodotoxin blocked C3H]dopamine release partially in both cases and
I
a
2
-i--l-
a
b
bk Timalminl
Tim*lminl Effect of veratridine on the release of [‘Hldopamine from superfused slices of the rat corpus striatum (a) and microdissected pars reticulata of the substantia nigra from normal rat (b). Experimental conditions as for Fig. 2. Horizontal bars indicated 2 min exposures to 2.0 PM veratridine. Each value represents the mean of two experiments.
FIG. 5. Effect of three different tetrodotoxin concentrations on the veratridine-induced release of C3H]dopamine from superfused slices of the rat corpus striatum (a) and microdissected pars reticulata of the substantia nigra (b). Experimental conditions as for Fig. 2. Spontaneous outflow of radioactivity before, between and after veratridine pulses is given as the mean + S.E.M. for all experiments combined. Unbroken lines represent the effect of 1 nM tetrodotoxin (n = 1 for both corpus striatum and pars reticulata), broken lines 10 nM tetrodotoxin (n = 1 for corpus striaturn; n = 2 for pars reticulata) and dotted lines 10On~ tetrodotoxin (n = 1 for corpus striatum; n = 2 for pars reticulata). (m) indicates exposure of the tissue slices to tetrodotoxin, (B) indicates exposure to 3.0 PM veratridine. (*) indicates the rather large second peak observed with 3.0 PM veratridine in the pars reticulata after the first peak had been partially blocked by 10 nM tetrodotoxin (see text).
Dopamine release from substantia nigra TABLE 2. RAnO OF ~ElN~~A~ IN TRACTIONAL RELEASE RA~SOF[~H]~PAMINEFORREPEA~DPOTA~IUMPUL~S
Conditions Control Partially denervated
26mM KC S3iS2 SZISI
18mM K+ WSI
0.79 + 0.03
0.78 I: 0.07
0.73 + 0.09
0.47 + 0.10
0.75 f 0.09
0.42 f 0.04
2027
of the f&e output from the medial raphe nucleus was also eliminated. No measurements of the 5-hydroxytryptamine contents of the substantia nigra from controls or animals with lesions were made in the present study. However, judging from the post-mortem histological determination of the extent of the lesions there is little doubt
that the lesions were successful, and in accordance with other lesion studies this would be expected to Sr-S3 corresponds to the values given in Table 1. Values result in a very substantial reduction in the 5-hydroxyare mean f S.E.M.(n = 4 for 26 mM K’ ; n = 3 for 18 mM tryptamine content of the substantia nigra (PALK~K+). VITS, SAAVEDRA,JACOBOWITZ,KIZER, Z~BORSZKY& Significance was tested for using the Student’s r-test (unBRO~S~IN, 1977; DRAY, DAVIES, OAKLEY, TANpaired observation). GROACH& VELLUCCI,1978). Despite this no qualitaN.S., not significant at the 0.05 confidence limit. tive differences were observed between the release of C3H]dopamine from substantia nigra tissue slices of controls and animals with lesions. This is consistent 10On~ tetrodotoxin blocked it almost completely both in the striatum and in the substantia nigra. In all with the theory that the release of previously taken up [‘HJdopamine from the substantia nigra in vitro cases a second exposure to veratridine in the absence of tetrodotoxin evoked a large increase in L3H]dopa- occurs mainly, if not exclusively, from dopaminergic mine release. A particularly large peak was observed dendrites, with little or no contribution from serofrom the pars reticulata of the substantia nigra follow- toninergic elements. Tables 1 and 2 show that, ing partial blockade of the first peak with 10 nM tetro- although qualitatively similar, some quantitative difdotoxin. In these experiments, as seen in Fig. 5, the ferences are observed between partially denervated and control substantia nigra tissue slices in the total baseline release of C3H]dopamine from the substantia nigra was considerably higher than that from the increase of ~3~]dopamine release over basal level for striatal slices, as found previously GEFFENet af. (1976). repeated potassium pulses. The reason for the quantitative differences is not clear but could include a number of factors, not necessarily involving the reDISCUSSION moval of 5-hydroxytryptamine-containing nerve terE@ct of lesions of the raphe nuclei minals per se. An estimation of the total amount of radioactivity present in the tissue slices at the beginThere is good evidence for a serotoninergic projecning of the superfusion revealed no obvious differtion from the mesencephalic raphe nuclei. Experimental evidence also indicates a GABA projection from ences for the [‘H]dopamine uptake in partially the caudoputamen-globus pallidus region to the sub- denervated and control tissue slices, thus suggesting stantia nigra (KIM, BAK, HASSLER& OKADA, 1975; that the raphe lesion does not cause a significant loss HATTORI, MCGEFX, FIBIGER& MCGEER, 1973; FON- of t3H]dopamine uptake sites. The in uivo observaNUM, GROFOV& RAN~IK, S~OM-MA~I~N & WAL- tions of NIEOULLONet al. (1977) and CHERAMY, BERG,1974) and a substance P-containing pathway is NIEOULLON& GLOWINSKI(1978) that L-3,5-[3H]tyroalso believed to originate from this same region of the sine is converted to C3H]dopamine and released from brain (KANAZAWA,EM~C~N & CUELLO,1977). These the cat substantia nigra would also make any particithree neuronal pathways are believed to account for a pation of serotoninergic nerve terminals unlikely since major part of the innervation of the substantia nigra, these lack the enzyme tyrosine hydroxylase. Possibly, and of the three only 5-hydroxy~yptamine containing the removal of tonic inhibitory influences normally neurones would be expected to be able to take up and exerted by 5-hydroxytryptamine (DRAY et at., 1978; possibly release [3H]dopamine to any significant see also NIEOULLONet al., 1977) may be an important extent. Dopaminergic collaterals arising from the factor related to the observed quantitative differences nigrostriatal pathway and projecting back into the in release between partially denervated and control substantia nigra have not been observed (JURASKA, substantia nigra tissue slices. WILsCiN& GROVES,1977). In conclusion these results strongly suggest that the In the present study the possible con~ibution by main part of the reiease occurs from dopaminergic serotoninergic elements to the reported uptake and dendrites in the substantia nigra. release of C3H]dopamine from dendrites of substantia EfSects of veratridine and tetrodntoxin nigra neurones (see Introduction) was assessed by performing in vitro uptake and release studies following The release of dopamine from dendrites of substanlesions of the raphe nuclei. Lesions were placed in tia nigra neurones was further explored by studying both the dorsal and the medial raphe nuclei. In two the effects of veratridine and tetrodotoxin. Veratridine animals the caudal part of the medial raphe nucleus depolarizes nervous tissue by selectively increasing was spared but since the fibres pass in a caudal to the membrane permeability to sodium ions and this rostra1 direction it is most likely that the major part effect is normally blocked by tetrodotoxin. PrelimiP < 0.02
N.S.
P < 0.05
2028
S. F. 0
‘TAGF.R~Wand .A C. c’Ilr;I.Lo
nary experiments showed that veratridine potently induces the release of exogenously applied r3H]dopamine from microdissected slices of the entire substantia nigra, suggesting that the dendritic membrane, like most excitable membranes. contains fast sodium channels which can be activated by veratridine. The possibility was also considered. however. that the dendrites may become depolarized by an antidromic stimulation arising from the action of veratridine on the small axon segments most probably still attached to the cell bodies after microdissection of the entire substantia nigra. This possibility may seem unlikely in view of work carried out on Purkinje cell dendrites (LIJNAS. NICHOLSOK & PKECHT, 1969) which suggests that dendrites, due to a differential excitability. tend to propagate depolarizations mainly in the somatopetat direction. Consequently. an antidromic activation of the cell would be expected to invade only the lower parts of the main dendritic branches. Nevertheless, in order to minimize the possibility 01”any antidromic invasion of the dendrites the experiments reported here were carried out on microdissected slices of the pars reticulata only. an area of the substantia nigra containing the longer branched dendrites arising from cell bodies located in the pars compacta (DAHLSTK~~M & Fllx~, 1964). As shown in Fig. 4, veratridine evoked the release of C3H]dopamine both from slices of the pars reticulata of the substantia nigra and from slices of the striatum. In both cases the veratridine-induced release was blocked by low concentrations of tetrodotoxin (Fig. 5). The conclusion would seem to be that the dopaminergic dendrites, like other parts of the neurone. contain fast tetrodotoxin-sensitive sodium channels. Other studies are not entirely consistent with this interpretation. LLIN~S & HESS (1976) managed to record tetrodotoxin-resistant dendritic spikes from avian Purkinje cells. and the suggestion was put forward that the dendritic spikes were generated by calcium currents. The in r.iro study in NI~OIII,LOI\:et 111. (1977) in which the spontaneous release of dopamine from the cat substantia nigra was not blocked by tctrodotoxin may seem to carry similar implications. A possible explanation of these apparent discrepancies is provided if the dendritic membrane contains fast sodium channels. normally present in a latent state but susceptible to activation by veratridine and to subsequent block by tetrodotoxin. These channels would not normally participate in electrical conduction. and the addition of tetrodotoxin would therefore not block electrical activity or transmitter release
from the dendrites but may even increase it if inhibitory influences are suppressed (NIEOULLON YI (II.. 1977). On the other hand. exposure to veratridine would activate these latent channels. causing depolarization and transmitter release from the dendrites, and this effect of veratridine would be blocked by tetrodotoxin as reported in the present study. Further experimentation either in rim under electrical stimulation or with a more physiological in vim system could probably yield more information on these questions. More direct evidence for the existence of latent fast sodium channels, which can be activated by veratridine and blocked by tetrodotoxin, has recently been obtained by LOWE, BUSH & RIPLEY (1978) in an elecn-ophysiological study on the large sensory dendrites innervating the thoracic-coxal muscle receptor organ of the crab Cur&us muma.\. A further observation made in the present study was that after partial blockade of the first veratridineinduced release of [jH]dopamine from the microdissected pars reticulata tissue slices with IO nM tetrodotoxin. the second veratridine-induced release, in the absence of tetrodotoxin. appeared to be unexpectedly large (Fig. 5). This was found in two separate experiments on pars reticulata but not on striatum. tissue slices. Further experimentation would again be required in order to determine the significance of this observation. In this series of experiments it was also noticed that the spontaneous release observed from substantia nigra tissue slices was considerably higher than that from the striatum tissue slices. This is in agreement with what was found by GEFFEN c’t trl. ( 1976) and may suggest some differences in the dopamine storage -release mechanisms between nerve terminals and dendrites. In this regard it is worth commenting on the absence of vesicular elements in dendrites of the substantia nigra neurones (SOTELO. 1972: CIXLLO & IVERSEK. 1978) and the fact that the monoamine marker 5hydroxydopamine injected intracerebrally can be identified in the interior of cisternac of smooth endoplasmic reticulum of dendrites of the suhstantia nigra neurones (MERVER. DEL FIA(Y‘O & (‘rF.1.1.0, 1979). .4~,knon’/rrlyc,nlent.\ The authors wish to thank Dr L. I.. IVERS~N for reviewing the manuscript. The skilful technical assistance of DAVID CHAPMAN, BRIAN JOHNSON, GEORW MARSHAI.L and ANGIE NELSONis also gratefully acknowledged. We would also like to thank Prof. J. A~WW for many helpful discussions.
REFERENCES ANDCN N.-E.. DAHLSTR~M A., FL~x~: K.. OLSON L. & UNGERSTEDT U. (1966) Ascending monoamine neurones to the telencephalon and diencephalon. Acrtr physiol .scund. 67, 313 326. BIBRKLUNI) A. & LINDVALL 0. (1975) Dopamine in dendrites of substantia nigra neurones: suggestions for a role in dendritic terminals. Bruin Rcs. 83, 531 537. BROWNSTEIN M. J., PALKOVITS M.. SAAVEIIRA J. M. & KIZ~R J. S. (1975) Tryptophan hydroxylase in the rat brain. Brain Rev. 97, 163~ 166.
Dopamine release from substantia nigra
2029
C~RAMY A., NIEOULLO~A, & GLOWINSKII. (1978) In vivo changes in dopamine release in cat caudate Ruckus and substantia nigra induced by nigral application of various drugs including GABAergic agonists and antagonists. In Interactians between Putative Neurotransmitters in the Brain (eds GARATTINIS., PUIOLJ. F. & SAMANIN R.), pp. 175-190. Raven Press, New York. CONRADL. C. A., LEONARD C. M. & PFAFD. W. (1974) Connections of the median and dorsal raphe nuclei in the rat: an autoradiographic and degeneration study. J. camp. Neural. 156, 179-206. Ct.~n~r_oA. C. & IVERsENL. L. (1978) Interactions of dopamine with other neurotransmitters in the rat substantia nigra: a possible functional role of dendritic dopamine. In interacf~~s ~efween Pufafive Neurotra~s~irters in the Brain feds GARATTINIS., P~SOL.I. F. & SAMANINR.), pp. 124-147. Raven Press, New York. CUELLO A. C. & KELLY J. S. (1977) Electron microscopic autoradiographic localization of ‘H-dopamine in the dendrites of the dopaminergic neurones of the rat substantia nigra in uiuo. Br. J. Pharmac. 59, 527-528P. DAHLSTR~M A. & FUXE K. (1964) Evidence for the existence of monoamine neurones in the central nervous system--I. Demonstration of monoamines in the cell bodies of brain stem neurones. Acta physiol. stand. 64, Suppl. 232, l-55. DRAYA. (1979) The striatum and substantia nigra: a commentary on their relationships. Neuroscience 4, 1407-1439. DRAYA., DAVIESJ., OAKLEYN. R., TONGROACH P. & VELLUCCIS. (1978) The dorsal and medial raphe projections to the substantia nigra in the rat: electrophysiological, biochemical and behavioural observations. Brain Res. 151, 431-442. DRAY A., GONYET. J., OAKLEYN. R. & TANNERT. (1976) Evidence for the existence of a raphe projection to the substantia nigra in rat. Brain Res. 113, 45-57. FIBIGERH. C. & MILLERJ. J. (1977) An anatomical and electrophysiological investigation of the serotonergic projection from the dorsal raphe nucleus to the substantia nigra in the rat. Neuroscience 2,97s988. FONNUMF., GROFOV~,I., RANVIKE., STORM-MATHI~NJ. & WAL~~ZRG F. (1974) Origin and dis~ibution of glutamate decarboxylase in the substantia nigra of the cat. Brain Res. 71, 77-92. FUXE K. (1965) Evidence for the existence of monoamine neurones in the central nervous system-IV. Distribution of monoamine nerve terminals in the central nervous system. Acta physiol. stand. 64, Suppl. 247, 37-85. GEFFENL. B., JESSELLT. M., CUELLOA. C. & IVERSENL. L. (1976) Release of dopamine from dendrites in rat substantia nigra. Nature, Lond. 260, 258-260. HATTORIT., MCGEERP. L., FIBIGERH. C. & MCGEERE. G. (1973) On the source of GABA-containing terminals in the substantia nigra: electron microscopic autoradio~aph~c and biochemical studies. Brain Res. S4, 103-114. IVERSENL. L. (1971) The uptake of biogenic amines. In Biogenic Amines and Pkysio~ogiea~ ~e~ranes in Drug Therapy (eds BIEL J. H. & ABAADL. G.), pp, 2.59-327. Dekker, New York. JURASKAJ. M., WILSONC. J. & GROVESP. M. (1977) The substantia nigra of the rat: A Golgi study. J. camp. Neural. 172, 585600. KANAZAWAI., EM~ONP. C. & CUELLOA. C. (1977) Evidence for the existence of substance P-containing fibres in striato-nigral and pallido-nigral pathways in rat brain. Brain Res. 119,447-453. KIM J. S., BAK I. J., HASSLERR. & OKADAY. (1975) Role of gamm~minobutyric acid (GABA) in the extrapyramidal motor systems: some evidence for the existence of a type of GABA-rich strio-nigral neurones. Expi Brain Res. 14, 95-104.
LLINASR. & HESSR. (1976) Tetrodotoxin-resistant
dendritic spikes in avian Purkinje cells. Proc. natn. Acad. Sci. U.S.A.
73,2520-2523. LLIN~SR., NICHOLWNC. & PRECHTW. (1969) Preferred centripetal conduction cells. Science, N.Y. 163, 184187.
of dendritic spikes in alligator Purkinje
LOWED. A., BU~HB. M. H. & RIPLEYS. H. (1978) Pharmacological evidence for ‘fast’ sodium channels in nonspiking neurones. Nature, Load. 274, 289-290. MERCERL., DEL F~ACCOM. & CUELLOA. C. (1979) The smooth endoplasmic reticulum as a possible storage site for dendritic dopamine in substantia nigra neurones. Experientia 35, 101-103. NIEOULLON A., CHERAMY A. & GLOWINSKIJ. (1977) Release of dopamine in viva from cat substantia nigra. Nature, Land. 266, 375-377.
PALKOVITS M., BROWNSTEIN M. & SAAVEDRA J. M. (1974) Serotonin content of the brain stem nuclei in the rat. Brain Res. 80, 237-249.
PALKOVITSM. & JACOEXOWITZ D. M. (1974) Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain-H. Hindbrain (mesencephalon, rhombenceophalon). .I. cotnp. Neural. 157, 2941. PALKOVITS M., SAAVEDRA J. M., JACOBOWITZ D. M., KIZER J. S., ZABORSZKY L. & BROWNSTEIN M. J. (1977) Serotonergic innervation of the forebrain: effect of lesions on serotonin and tryptophan hydroxylase levels, Brain Res. 130, 121-134. PnL~.aGnrNoL. J. & CUSHMANA. J. (1967) A Stereotaxic Atlas of the Rat Brain. Appleton-Century-Crofts, New York, PrcKEL V. M., JOH T. H. & REIS I), J. (1975) Monoamine-synthesizjn~ enzymes in central dopaminergic, noradrenergic and serotonergic neurones: immunocyt~hemical localization by light and electron microscopy. .I. ~istoc~em. Cytathem. 24,792-806. SOTELOC. (1972) The fine structural localization of ‘H-norepinephrine in the substantia nigra and area postrema of the rat: an autoradiographic study. J. Ultmstruct. Res. 36, 824-841. ULBRICHTW. (1969) The effect of veratridine on excitable membranes of nerve and muscle. Rev. Physiol. Biochem. Pharmac. 61, 18-71. (Accepfed 15 June 1979)
