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Biochemical correlates of motor changes caused by the manipulation of dopamine function in the substantia nigra of the mouse
Neuropharmacoiogy Vol. 24, No. 12, pp. 1155-I 161, 1985 Printed
in Great
Britain.
All rights reserved
Copyright
OOZS-3908/85 $3.00 + 0.00 Q 1985 Pergamon Press Ltd
BIOCHEMICAL CORRELATES OF MOTOR CHANGES CAUSED BY THE MANIPULATION OF DOPAMINE FUNCTION IN THE SUBSTANTIA NIGRA OF THE MOUSE A. J. BRADBURY,’ B. COSTALL,’ M. E. KELLY,’ R. J. NAYLOR’ and J. A. SMITH* ‘Postgraduate School of Studies in Pharmacology and %chool of Pharmaceutical Chemistry, University
of Bradford, (Accepted
Bradford
BD7 IDP, England
16 April 1985)
Summary-2-Di-n-propylamino-5,6-dihydroxytetralin, injected bilaterally into the substantia nigra of the mouse, caused dose-dependent motor inhibition which was associated with decreased levels of DOPAC and increased levels of dopamine in the striatum. (-)Sulpiride, injected into the substantia nigra, antagonised the locomotor depression although the partial antagonism of the elevation in the level of dopamine in the striatum and of the reduction in levels of DOPAC did not achieve significance. The specificity of the action of tetralin on dopamine receptors was shown by the failure of prazosin and yohimbine to antagonise the locomotor depression induced by tetralin and the reduction in levels of DOPAC. The selectivity of the action of tetralin for the dopamine system was shown by its failure to affect levels of noradrenaline, serotonin and 5-hydroxyindoleacetic acid in the striatum. The injection of tetralin into the substantia nigra also caused biochemical changes in limbic areas (nucleus accumbens and tuberculum olfactorium), where the levels of dopamine and DOPAC were elevated, and in the frontal cortex where the levels of DOPAC were reduced. These changes were antagonised by a concomitant injection of (-)sulpiride into the substantia nigra. It is concluded that the action of dopamine agonists in the midbrain can decrease the functional activity in the ascending dopaminergic pathways. Key words: substantia nigra, intracerebral injection, 2-di-n-propylamino-5.6-dihydroxytetralin, neous locomotor activity, dopamine turnover. striatum.
of small doses of dopamine agonists to reduce locomotor activity may reflect an action on dopamine “autoreceptors” to cause a decrease in the synthesis or release of dopamine (Di Chiara, Porceddu, Argiolas and Gessa, 1976; Carlsson, 1981: Bradbury, Costall and Naylor, 1983). That dopamine-containing cells in the midbrain respond to the administration of dopamine agonists with an inhibition of firing (Bunney, 1979), and that locomotor activity is reduced following the injection of dopamine agonists into the midbrain (Bradbury et al., 1983) indicates that the dopamine-containing cell bodies may be an important locus of action for dopamine agonists. The present study investigated whether a dopamine agonist-antagonist action in the midbrain could be detected as changes in the levels of dopamine and its metabolites in the forebrain. The ability
METHODS
The studies used male albino mice, BKW strain, weighing 35-45 g at the time of stereotaxic surgery. Chronically indwelling bilateral guide cannulae were stereotaxically implanted to allow subsequent intracerebral injections into the substantia nigra (ant. 1.1, vert. - 5.4, lat. f 1.6; the anterior reading was determined from the zero of the Kopf instrument, the vertical reading indicated the depth beneath the skull
sponta-
surface and the lateral placements the distance from the midline of the skull). The coordinates were selected with the aid of the atlases of Lehmann (1974) and Slotnick and Leonard (1975), and full details of the technique are reported elsewhere (Bradbury et al., 1983). After a ICday recovery period the mice were manually restrained as the stylets were removed from the guide cannulae and replaced bilaterally by injection units (0.3 mm diameter stainless steel) extending below the guide tips (located 2 mm higher) to the substantia nigra. A volume of 0.25 ~1 of drug or vehicle was delivered from 5~1 Hamilton syringes over a 5 set period with the units remaining in position for a total of 60 set before being withdrawn and replaced by the stylets. After intracerebral injection, the mice were placed in individual screened perspex cages (10 x 14 x 24 cm fitted with two photocell units located 2.5 cm above the floor of the cage and 3.0cm from the sides) for the measurement of their spontaneous locomotor activity. Interruptions of the two light beams were recorded separately and noted every Smin: when a mouse exhibited locomotor activity the numbers of interruptions of the two beams were approximately equal and these were summed. Mice were also observed visually for the presence or absence of any body asymmetry/circling, muscular hypo-
1155
1156
A. J.
BRADBURYet
/hypertonia, seizures or stereotyped behaviour which may non-specifically interfere with the measurement of locomotor activity. Animals demonstrating any of these non-specific effects were excluded from the behavioural and biochemical assessments. Two series of experiments were carried out, one to establish the behavioural parameters and one to take brains for biochemical analysis at a time of maximum behavioural change. Those animals taken for biochemical assessment were subject to careful behavioural analysis before killing. If “control” mice (see below) in an experimental session failed to show “normal” levels of spontaneous activity then these mice were not taken for assessment at the biochemical level. Groups of animals were killed for biochemical analyses only when their behaviour was shown to conform with established responses; 20 min after intracerebral injection these animals were taken from the behavioural room and killed by cervical dislocation. The brains were immediately removed and dissected to obtain tissue from the striatum, limbic area (tuberculum olfactorium + nucleus accumbens) and frontal cortex. Samples were placed in dry ice, weighed and immediately homogenised in 0.2 M perchloric acid for storage at < -70°C and subsequent determination of levels of dopamine, 3,4_dihydroxyphenylacetic acid (DOPAC), noradrenaline, serotonin and 5hydroxyindoleacetic acid (5-HIAA), measured using HPLC with electrochemical detection. Indoleamines from each area of brain were determined simultaneously using a reverse-phase system (5 p hypersil ODS with 7.5% methanol in 0.2 M phosphate/O.1 M citrate buffer pH 6.1 at +0.7 V). Catecholamines were extracted (by absorption and desorption on alumina) before simultaneous determination using a reverse-phase system, containing an ion pairing agent (5 p hypersil ODS with 8.5% methanol and 1.8 mM octane sulphonic acid in 0.2 M phosphate/O.1 M citrate buffer pH4.5 at +0.7V). Initial behavioural studies establishing the dose-response relationships for motor depression or stimulation by 2-di-n-propylamino-5,6-dihydroxytetralin (tetralin) and (-)sulpiride, or showing the ineffectiveness of yohimbine and prazosin, used 3 groups of mice receiving (a) no treatment (cannulated mice providing control data), (b) intracerebral injection of vehicle, (c) intracerebral injection of tetralin, (-)sulpiride, yohimbine or prazosin. To determine the potential of (-)sulpiride, yohimbine and prazosin to antagonise a response to tetralin 5 groups of cannulated mice were used on any one occasion and received (a) no treatment, (b) intracerebral injection of vehicle, (c) intracerebral injection of tetralin, (d) intracerebral injection of (-)sulpiride, yohimbine or prazosin, (e) intracerebral injection of tetralin combined with (-)sulpiride, yohimbine or prazosin. This design of 5 groups was used for the selection of mice for biochemical analyses; 5-6 mice were used in each group. All behavioural testing was carried out be-
al.
tween 12.30 and 4.30 p.m. The statistical significance of the effects of drugs, behaviourally, or biochemically, was determined by application of one-way analysis of variance (ANOVA) followed by Dunnett’s test or Student’s r-test. 2 - Di -n - propylamino - 5,6 - dihydroxytetralin HCI (Glaxo) was prepared in nitrogen-bubbled pyrogen free water containing 0.01% sodium metabisulphite; (-)sulpiride.HCI (SESIF), yohimbine.HCI (Sigma) and prazosin . HCI (Pfizer) were prepared in pyrogenfree water. RESULTS
Modification of locomotor activity after bilateral injection of drug into the substantia nigra of the mouse brain
In each experiment the activities of control, nontreated mice and mice receiving injections of vehicle into the substantia nigra were indistinguishable (see Fig. 1). 2-Di-n-propylamino-5,6_dihydroxytetralin (0.0025-o. 1 pg), injected into the substantia nigra, caused a dose-related decrease in spontaneous locomotor activity whilst similar injections of (-)sulpiride (0.025-0.2 fig) caused a dose-related increase which diminished at a dose of 0.4 pg (Fig. 1). A dose of 0.05pg of tetralin was selected as causing a near-maximal inhibition of locomotor activity and was administered in combination with (-)sulpiride (0.2 pg, a dose selected as causing maximal stimulation), yohimbine or prazosin (1 pg). (-)Sulpiride was shown to significantly antagonise the inhibition of locomotion caused by the tetralin compound, whilst yohimbine and prazosin were ineffective (Fig. 1). Groups of animals so treated were killed at a time of maximal behavioural change (20 min after injection) for biochemical assessment of the effects of the drugs in the substantia nigra. The midbrain of these animals was taken for histological confirmation of the injection site (Fig. 2). ModiJication of levels of noradrenaline, dopamine, serotonin and metabolite in forebrain structures after injection of drug into the midbrain
The injection of tetralin into the midbrain caused an increase in the level of dopamine in the striatum and limbic region (27 and 67% respectively) and a reduction in DOPAC in the striatum (34%), but an increase in the level of DOPAC in the limbic region (38%; Table 1). The injection of tetralin failed to modify the levels of noradrenaline, serotonin and 5-HIAA in either striatal or limbic tissue. The injection of (-)sulpiride into the midbrain failed to cause any significant biochemical change in striatal or limbic tissue (the 28% reduction in levels of dopamine in limbic tissue was not significant, the levels of P > 0.05). The changes in dopamine/DOPAC in the limbic region, induced by tetralin were antagonised by concomitant administration of (-)sulpiride (P < 0.01). There were also trends for (-)sulpiride to antagonise the reduction in
1157
Dopamine action in substantia nigra 2-di-n-pmpylamino-5,6dihydroxytetralin (tetrelln)
*win (T)+Su,,,iride(S)
Tetralin (T) +yohimbine
C
V
Y
T
T
Tetrah (T)
lPrazmin
(P)
CVPTT
Fig. 1. Dose-related reductions in spontaneous locomotor activity in the mouse after the bilateral injection of 2-di-n-propylamino-5,6-dihydroxytetralin (tetralin) into the substantia nigra, and dose-related enhancements caused by similar injections of (-)sulpiride. From this data a dose of 0.05 pg tetralin (T) and 0.2 pg (-)sulpiride (S) were selected for combined administration. Since yohimbine (Y) and prazosin (P) alone failed to modify spontaneous locomotion in the mouse, I .Opg of Y and P was selected for the studies assessing potential antagonism of T (see lower set of histograms). C = responses of untreated cannulated mice, V = responses of mice receiving injection of vehicle into the substantia nigra. n = 5-6. SEMs given. Significant reductions or enhancements in spontaneous locomotion (compared with V) are indicated as *p <0.01-P < 0.001 (one-way ANOVA followed by Dunnett’s test), significant reversal of the response to tetralin as tP < 0.001 (Student’s f-test).
levels of DOPAC in the striatum and elevation in levels of dopamine induced by tetrahn but these did not achieve significance. Yohimbine, injected into the midbrain, failed to modify the levels of dopamine and DOPAC, causing only a small but significant reduction in the level of noradrenaline in the striatum. The injection of prazosin into the midbrain also reduced the level of noradrenaline in the striatum and limbic tissue but elevated levels of DOPAC in the limbic tissue. Although the injection of yohimbine failed to antago-
nise the ability of the tetralin compound to reduce levels of DOPAC in the striatum, indeed the levels,
of striatal DOPAC were further reduced by the additional treatment with yohimbine, the other actions of tetralin to modify levels of dopamine in the striatum and limbic region and levels of DOPAC were antagonised by yohimbine. Prazosin had a similar spectrum of action to modify the changes in levels of dopamine/DOPAC in striatal and limbic tissue induced by tetralin. (-)Sulpiride, yohimbine or prazosin. administered
1158
A. J.
BRADBURYet al.
Fig. 2. Diagrammatic representation of the sites of injection within the area of the substantia nigra of the brain of the mouse. (0) Indicates the injection sites. Coordinates are according to Slotnick and Leonard (1975). The diagram was constructed using histological data obtained from 20 representative brains. The coordinates were established by histological analyses of 2-3 brains at the commencement of each session of surgery. The locations of the injection sites were then established for all brains taken for biochemical assessment; no location was found to be outside the area of the substantia nigra. One in 5 brains of animals used only for the behavioural studies was subject to histological analysis: of 30 brains examined, only 3 were found to have locations outside the substantia nigra (in the cerebellar peduncles or zona incerta).
alone or in combination with tetralin, failed to modify levels of serotonin or S-HIAA in the striatum and limbic region (control values being 546 + 51, 744 f 43, 324 f 30 and 201 f 10 pg/mg of tissue). Injection of tetralin into the midbrain reduced levels of DOPAC in the frontal cortex (63%; Table 2); there were no other changes in transmitter/ metabolite levels. The injection of (-)sulpiride reduced levels of dopamine in the frontal cortex (56%) but failed to significantly modify levels of DOPAC. (-)Sulpiride significantly antagonised the reduction in levels of DOPAC induced by tetralin. Yohimbine significantly reduced the levels of dopamine (52%) and DOPAC (39%) in the frontal cortex and similar reductions in levels of DOPAC were also observed when yohimbine was combined with tetralin, although the reduction in levels of dopamine induced by yohimbine was less marked. Prazosin also significantly reduced the levels of dopamine (49%) and DOPAC (39%) in the frontal cortex. These reductions were also recorded after the combined treatment with prazosin and tetralin. The levels of noradrenaline, serotonin and 5-HIAA in the frontal cortex (388 + 22, 356 + 16 and 76 +_8 pg/mg tissue) were not consistently modified by any of the treatments with drugs or interactions of the drugs.
DISCUSSION
The present study used the potent dopamine agonist 2-di-n-propylamino-5,6_dihydroxytetralin (tetralin) to determine if the actions of dopamine agonists in the substantia nigra, to reduce locomotor activity in the mouse, could be correlated with biochemical changes in the activity of the dopaminecontaining neurones projecting to the forebrain. It could be proposed that stimulation of dopamine autoreceptors located within the nigra (see introduction and Argiolas, Mellis, Fadda and Gessa, 1982; Glowinski and Cheramy, 1981) would lead to a reduction in the rate of firing of the dopaminergic neurones. A parameter indicative of a reduced rate of firing would be a modified turnover of transmitter, measured in the present study as the concentration of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). Whilst all techniques for measuring turnover have limitations, the attempt to determine a biochemical correlate at the time of maximal behavioural change, precluded the use of other methodologies involving, for example, inhibition of synthesis which would seriously interfere with the behavioural/biochemical measurements. The injection of tetralin was directed at the pars reticulata, that part of the substantia nigra influenced
Dopamine action in substantia nigra
1159
by dendritic release of dopamine and comprising a total volume of approx. 0.5 mm3. Diffusion from such a small area is inevitable. However, in a previous study it was shown that injections of tetralin above, posterior or anterior to the substantia nigra were much less effective in reducing locomotor activity (Bradbury et al., 1983). It was proposed that the tetralin compound may have an important locus of action within the substantia nigra to reduce locomotor activity, and in the present study it was shown that at a time of marked locomotor depression the levels of DOPAC were decreased and levels of dopamine increased in the striatum. A similar spectrum of change was observed following the peripheral administration of the tetralin compound where it was proposed that an inhibition of the firing of cells in the nigra may occur before the stimulation of presynaptic autoreceptors in the striatum (see Westerink, Feenestra, Wirix and Horn, 1984). Also, in an anaesthetised rat preparation and the rabbit, the injection of apomorphine into the nigra is reported to reduce levels of homovanillic acid in the striatum (Maggi, Bruno, Cattabani, Groppetti, Parenti and Racagni, 1978; Wolfarth, Dulska, Golembiowskanikitin and Vetulani, 1978). If it is accepted that decreased levels of DOPAC reflect a reduced dopaminergic neurotransmission, then the depression of locomotor activity observed after the injection of the tetralin compound into the substantia nigra may be due to a decreased dopamine function in the nigrostriatum. The mechanism through which the tetralin compound can cause the behavioural and biochemical changes probably involves an action on dopamine receptors since (-)sulpiride, injected into the substantia nigra, antagonised the behavioural depression although the trend for (-)sulpiride to antagonise the tetralin-induced reduction in the levels of DOPAC in the striatum did not achieve significance. It is also relevant that the antagonism of the reduction in locomotor activity induced by tetralin was not completely reversed by (-)sulpiride which could indicate either (a) the use of too small a dose of (-)sulpiride, or (b) the presence of additional tetralin-sensitive mechanisms within the nigra to moderate behavioural and biochemical changes. Nevertheless, that the injection of (-)sulpiride alone into the nigra could increase locomotor activity and, at least in smaller doses than those used in the present study, increase levels of DOPAC in the striatum (data not shown) suggests the presence of a tonic inhibitory dopaminergic action within the nigra. If additional mechanisms exist within the nigra to mediate the behavioural and biochemical changes induced by tetrahn they are unlikely to be CL,or qadrenoceptor types since prazosin and yohimbine, injected into the substantia nigra, failed to antagonise the depression of locomotor activity and changes in levels of DOPAC induced by tetralin, indeed yohimbine, through non-specified mechanisms actu-
A. J.
1160
hADWRY
et
al.
Table 2. Levels of 3.4.-dihydroxyphenylacetic acid (DOPAC), dopamine (DA) and noradrenaline (NA) in the frontal cortex following bilateral injection of tetralin and potential antagonists into the substantia nigra of the mouse Frontal cortex Injection into substantia nigra Vehicle Tetralin (0.05 pg) (-)Sulpiride (0.2 pg) (-)Sulpiride + tetralin Yohimbine (I p(g) Yohimbine + tetralin Prazosin (I pg) Prazosin + tetralin
DOPAC (A) (pgimg)
DA (W (pgimg)
Ratio A/B
NA (pglmg)
54 f 9 20 f 3’ 33 * 8 43 + 3$ 33 + 2’ 29 +_5’ 33f4’ 29 f 2*
Ilk9 65 + 8 34 f 3t 80+4 37*4t 6Ok6 39+4* 42 + 2’
0.70 0.30 0.97 0.54 0x9 0.48 0.85 0.70
388 It 22 388 + 30 334 + 16 399 f 20 398 +_I3 420 + 26 398 f I8 396 ?r.7
n = 5-6. SEMs given. Significant decreases in levels compared to vehicle controls are indicated as *P < 0.05, tP < 0.01; antagonism of the effect of tetralin significant to $P < 0.01 (Student’s f-test).
ally enhanced the reduction in levels of DOPAC induced by tetralin. The selectivity of the action of tetralin for the dopamine system is shown by its failure to affect levels of noradrenaline, serotonin and S-hydroxyindoleacetic acid in the striatum. While the major dopaminergic projection from the substantia nigra is to the striatum, it is also well established that the nigral dopamine system contributes a more modest innervation to limbic and cortical areas. Conversely, whilst the dopaminergic projection, arising from the midbrain A8 and Al0 cell groups, provides the major innervation to the limbic and cortical areas, a minor component innervates the striatum (Lindvall, 1979). Therefore, it is of interest that the injection of tetralin into the substantia nigra significantly modified levels of DOPAC and dopamine in the limbic region, although both were raised. These effects were antagonised by concomitant injection of (-)sulpiride into the substantia nigra. The increased levels of DOPAC clearly contrast with the reduced levels found in the striatum, and may reflect an actual increase in the function of dopamine in the limbic region in an attempt to compensate and restore the motor behaviour in the presence of a striatal deficit. It remains of interest that yohimbine and prazosin, when injected into the substantia nigra, antagonised the actions of tetralin to enhance the levels of DOPAC and dopamine in the limbic region. The injection of tetralin into the substantia nigra also caused biochemical changes in the frontal cortex where the levels of DOPAC were markedly reduced, a reduction that was antagonised by a concomitant injection of (-)sulpiride into the substantia nigra. The only other significant biochemical change occurring in the frontal cortex was a reduction in levels of DOPAC after the intranigral injection of yohimbine and prazosin. Again, it is difficult to determine whether these changes are a consequence of an action in the substantia nigra, diffusion of drug from the nigra to influence A8 and A10 cell groups located medial to the nigral cells, or reflect compensation between striatal, limbic and cortical systems to maintain motor activity.
In summary, the most important finding of the present study was that the injection of 2-di-n-propylamino-5,6_dihydroxytetralin into the substantia nigra of the brain of the mouse reduced locomotor activity, a behavioural change which correlates with a reduction in levels of DOPAC in the striatum and frontal cortex effected partly through sites sensitive to neuroleptic drugs [( -)sulpiride]. It is concluded that the actions of dopamine agonists in the substantia nigra and/or closely located dopaminecontaining cell body groups can decrease the functional activity in the ascending dopaminergic pathways. Acknowledgements-This work was supported by the Wellcome Trust and the Parkinson’s Disease Society. Gifts of drugs were received from Glaxo, Pfizer and SESIF (France).
REFERENCES
Argiolas A., Mellis M. R., Fadda F. and Gessa G. L. (1982) Evidence for dopamine autoreceptors controlling dopamine synthesis in the substantia nigra. Bruin Res. 234: 177-181. Bradbury A. J., Costall B. and Naylor R. J. (1983) Reduction in motor responding of the mouse by actions of dopamine agonists in the midbrain. Neuropharmacology 22: 1171-1176. Bunney B. S. (1979) The electrophysiological pharmacology of midbrain dopaminergic systems. In: The Neurobiology of Dopamine (Horn A., Korf J. and Westerink B. H. C., Ed.@, pp. 319-342. Academic Press, London. Carlsson A. (1981) Chemical neurotransmission and mental function: pharmacological aspects. In: Chemical Neuretransmission 75 Years (Stjarne L., Hedqrist P. Langercrantz H. and Wennmalm A., Eds), pp. . _ 527-540. Academic Press, London. Di Chiara G., Porceddu M. L., Argiolas A. and Gessa G. L. (1976) Evidence for dopamine receptors mediating sedation in the mouse brain. Nature 264: 564-567. _ Glowinski J. and Cheramv A. (1981) Dendritic release of dopamine: its role in the substantia nigra. In: Chemical Neurotransmission
75 Years (Stjame L., Hedqrist P., Langercrantz H. and Wennmalm A., Eds), pp. 285-299. Academic Press, London. Lehmann A. (1974) Atlas Stereofaxique du Ceroeau de la Souris. Editions du Centre National
de la Recherche Scientifique, Paris. Lindvall 0. (1979) Dopamine pathways in the rat brain. In: The Neurobiology of Dopamine (Horn A., Korf J. and
Dopamine action in substantia nigra Westerink B. H. C., Eds), pp. 319-342. Academic Press, London, Maggi A., Bruno F., Cattabani F., Groppetti A., Parenti M. and Racagni G. (1978) Apomorphine induced inhibition of striatal dopamine release: role of dopaminergic receptors in substantia nigra. Brain Res. 145: 180-184. Slotnick B. M. and Leonard C. A. (1975) A Skreoraxic Allas of le Albino Mouse Forebrain. U.S. Government Printing Office, Washington.
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Westerink B. H. C., Feenestra M. G. P., Wirix E. and Horn A. S. (1984) Certain dopamine agonists increase dopamine synthesis in the striatum of the rat brain. Eur. J. Pharmac. 101: 235-241.
Wolfarth S., Dulska E., Golembiowska-nikitin K. and Ventulani J. (1978) Role of the polysynaptic system of substantia nigra in the cholinergic-dopaminergic equilibrium in the central nervous system. NaunynSchmiedebergs Arch. Pharmac. 302: 123-l 3 I.
in Great
Britain.
All rights reserved
Copyright
OOZS-3908/85 $3.00 + 0.00 Q 1985 Pergamon Press Ltd
BIOCHEMICAL CORRELATES OF MOTOR CHANGES CAUSED BY THE MANIPULATION OF DOPAMINE FUNCTION IN THE SUBSTANTIA NIGRA OF THE MOUSE A. J. BRADBURY,’ B. COSTALL,’ M. E. KELLY,’ R. J. NAYLOR’ and J. A. SMITH* ‘Postgraduate School of Studies in Pharmacology and %chool of Pharmaceutical Chemistry, University
of Bradford, (Accepted
Bradford
BD7 IDP, England
16 April 1985)
Summary-2-Di-n-propylamino-5,6-dihydroxytetralin, injected bilaterally into the substantia nigra of the mouse, caused dose-dependent motor inhibition which was associated with decreased levels of DOPAC and increased levels of dopamine in the striatum. (-)Sulpiride, injected into the substantia nigra, antagonised the locomotor depression although the partial antagonism of the elevation in the level of dopamine in the striatum and of the reduction in levels of DOPAC did not achieve significance. The specificity of the action of tetralin on dopamine receptors was shown by the failure of prazosin and yohimbine to antagonise the locomotor depression induced by tetralin and the reduction in levels of DOPAC. The selectivity of the action of tetralin for the dopamine system was shown by its failure to affect levels of noradrenaline, serotonin and 5-hydroxyindoleacetic acid in the striatum. The injection of tetralin into the substantia nigra also caused biochemical changes in limbic areas (nucleus accumbens and tuberculum olfactorium), where the levels of dopamine and DOPAC were elevated, and in the frontal cortex where the levels of DOPAC were reduced. These changes were antagonised by a concomitant injection of (-)sulpiride into the substantia nigra. It is concluded that the action of dopamine agonists in the midbrain can decrease the functional activity in the ascending dopaminergic pathways. Key words: substantia nigra, intracerebral injection, 2-di-n-propylamino-5.6-dihydroxytetralin, neous locomotor activity, dopamine turnover. striatum.
of small doses of dopamine agonists to reduce locomotor activity may reflect an action on dopamine “autoreceptors” to cause a decrease in the synthesis or release of dopamine (Di Chiara, Porceddu, Argiolas and Gessa, 1976; Carlsson, 1981: Bradbury, Costall and Naylor, 1983). That dopamine-containing cells in the midbrain respond to the administration of dopamine agonists with an inhibition of firing (Bunney, 1979), and that locomotor activity is reduced following the injection of dopamine agonists into the midbrain (Bradbury et al., 1983) indicates that the dopamine-containing cell bodies may be an important locus of action for dopamine agonists. The present study investigated whether a dopamine agonist-antagonist action in the midbrain could be detected as changes in the levels of dopamine and its metabolites in the forebrain. The ability
METHODS
The studies used male albino mice, BKW strain, weighing 35-45 g at the time of stereotaxic surgery. Chronically indwelling bilateral guide cannulae were stereotaxically implanted to allow subsequent intracerebral injections into the substantia nigra (ant. 1.1, vert. - 5.4, lat. f 1.6; the anterior reading was determined from the zero of the Kopf instrument, the vertical reading indicated the depth beneath the skull
sponta-
surface and the lateral placements the distance from the midline of the skull). The coordinates were selected with the aid of the atlases of Lehmann (1974) and Slotnick and Leonard (1975), and full details of the technique are reported elsewhere (Bradbury et al., 1983). After a ICday recovery period the mice were manually restrained as the stylets were removed from the guide cannulae and replaced bilaterally by injection units (0.3 mm diameter stainless steel) extending below the guide tips (located 2 mm higher) to the substantia nigra. A volume of 0.25 ~1 of drug or vehicle was delivered from 5~1 Hamilton syringes over a 5 set period with the units remaining in position for a total of 60 set before being withdrawn and replaced by the stylets. After intracerebral injection, the mice were placed in individual screened perspex cages (10 x 14 x 24 cm fitted with two photocell units located 2.5 cm above the floor of the cage and 3.0cm from the sides) for the measurement of their spontaneous locomotor activity. Interruptions of the two light beams were recorded separately and noted every Smin: when a mouse exhibited locomotor activity the numbers of interruptions of the two beams were approximately equal and these were summed. Mice were also observed visually for the presence or absence of any body asymmetry/circling, muscular hypo-
1155
1156
A. J.
BRADBURYet
/hypertonia, seizures or stereotyped behaviour which may non-specifically interfere with the measurement of locomotor activity. Animals demonstrating any of these non-specific effects were excluded from the behavioural and biochemical assessments. Two series of experiments were carried out, one to establish the behavioural parameters and one to take brains for biochemical analysis at a time of maximum behavioural change. Those animals taken for biochemical assessment were subject to careful behavioural analysis before killing. If “control” mice (see below) in an experimental session failed to show “normal” levels of spontaneous activity then these mice were not taken for assessment at the biochemical level. Groups of animals were killed for biochemical analyses only when their behaviour was shown to conform with established responses; 20 min after intracerebral injection these animals were taken from the behavioural room and killed by cervical dislocation. The brains were immediately removed and dissected to obtain tissue from the striatum, limbic area (tuberculum olfactorium + nucleus accumbens) and frontal cortex. Samples were placed in dry ice, weighed and immediately homogenised in 0.2 M perchloric acid for storage at < -70°C and subsequent determination of levels of dopamine, 3,4_dihydroxyphenylacetic acid (DOPAC), noradrenaline, serotonin and 5hydroxyindoleacetic acid (5-HIAA), measured using HPLC with electrochemical detection. Indoleamines from each area of brain were determined simultaneously using a reverse-phase system (5 p hypersil ODS with 7.5% methanol in 0.2 M phosphate/O.1 M citrate buffer pH 6.1 at +0.7 V). Catecholamines were extracted (by absorption and desorption on alumina) before simultaneous determination using a reverse-phase system, containing an ion pairing agent (5 p hypersil ODS with 8.5% methanol and 1.8 mM octane sulphonic acid in 0.2 M phosphate/O.1 M citrate buffer pH4.5 at +0.7V). Initial behavioural studies establishing the dose-response relationships for motor depression or stimulation by 2-di-n-propylamino-5,6-dihydroxytetralin (tetralin) and (-)sulpiride, or showing the ineffectiveness of yohimbine and prazosin, used 3 groups of mice receiving (a) no treatment (cannulated mice providing control data), (b) intracerebral injection of vehicle, (c) intracerebral injection of tetralin, (-)sulpiride, yohimbine or prazosin. To determine the potential of (-)sulpiride, yohimbine and prazosin to antagonise a response to tetralin 5 groups of cannulated mice were used on any one occasion and received (a) no treatment, (b) intracerebral injection of vehicle, (c) intracerebral injection of tetralin, (d) intracerebral injection of (-)sulpiride, yohimbine or prazosin, (e) intracerebral injection of tetralin combined with (-)sulpiride, yohimbine or prazosin. This design of 5 groups was used for the selection of mice for biochemical analyses; 5-6 mice were used in each group. All behavioural testing was carried out be-
al.
tween 12.30 and 4.30 p.m. The statistical significance of the effects of drugs, behaviourally, or biochemically, was determined by application of one-way analysis of variance (ANOVA) followed by Dunnett’s test or Student’s r-test. 2 - Di -n - propylamino - 5,6 - dihydroxytetralin HCI (Glaxo) was prepared in nitrogen-bubbled pyrogen free water containing 0.01% sodium metabisulphite; (-)sulpiride.HCI (SESIF), yohimbine.HCI (Sigma) and prazosin . HCI (Pfizer) were prepared in pyrogenfree water. RESULTS
Modification of locomotor activity after bilateral injection of drug into the substantia nigra of the mouse brain
In each experiment the activities of control, nontreated mice and mice receiving injections of vehicle into the substantia nigra were indistinguishable (see Fig. 1). 2-Di-n-propylamino-5,6_dihydroxytetralin (0.0025-o. 1 pg), injected into the substantia nigra, caused a dose-related decrease in spontaneous locomotor activity whilst similar injections of (-)sulpiride (0.025-0.2 fig) caused a dose-related increase which diminished at a dose of 0.4 pg (Fig. 1). A dose of 0.05pg of tetralin was selected as causing a near-maximal inhibition of locomotor activity and was administered in combination with (-)sulpiride (0.2 pg, a dose selected as causing maximal stimulation), yohimbine or prazosin (1 pg). (-)Sulpiride was shown to significantly antagonise the inhibition of locomotion caused by the tetralin compound, whilst yohimbine and prazosin were ineffective (Fig. 1). Groups of animals so treated were killed at a time of maximal behavioural change (20 min after injection) for biochemical assessment of the effects of the drugs in the substantia nigra. The midbrain of these animals was taken for histological confirmation of the injection site (Fig. 2). ModiJication of levels of noradrenaline, dopamine, serotonin and metabolite in forebrain structures after injection of drug into the midbrain
The injection of tetralin into the midbrain caused an increase in the level of dopamine in the striatum and limbic region (27 and 67% respectively) and a reduction in DOPAC in the striatum (34%), but an increase in the level of DOPAC in the limbic region (38%; Table 1). The injection of tetralin failed to modify the levels of noradrenaline, serotonin and 5-HIAA in either striatal or limbic tissue. The injection of (-)sulpiride into the midbrain failed to cause any significant biochemical change in striatal or limbic tissue (the 28% reduction in levels of dopamine in limbic tissue was not significant, the levels of P > 0.05). The changes in dopamine/DOPAC in the limbic region, induced by tetralin were antagonised by concomitant administration of (-)sulpiride (P < 0.01). There were also trends for (-)sulpiride to antagonise the reduction in
1157
Dopamine action in substantia nigra 2-di-n-pmpylamino-5,6dihydroxytetralin (tetrelln)
*win (T)+Su,,,iride(S)
Tetralin (T) +yohimbine
C
V
Y
T
T
Tetrah (T)
lPrazmin
(P)
CVPTT
Fig. 1. Dose-related reductions in spontaneous locomotor activity in the mouse after the bilateral injection of 2-di-n-propylamino-5,6-dihydroxytetralin (tetralin) into the substantia nigra, and dose-related enhancements caused by similar injections of (-)sulpiride. From this data a dose of 0.05 pg tetralin (T) and 0.2 pg (-)sulpiride (S) were selected for combined administration. Since yohimbine (Y) and prazosin (P) alone failed to modify spontaneous locomotion in the mouse, I .Opg of Y and P was selected for the studies assessing potential antagonism of T (see lower set of histograms). C = responses of untreated cannulated mice, V = responses of mice receiving injection of vehicle into the substantia nigra. n = 5-6. SEMs given. Significant reductions or enhancements in spontaneous locomotion (compared with V) are indicated as *p <0.01-P < 0.001 (one-way ANOVA followed by Dunnett’s test), significant reversal of the response to tetralin as tP < 0.001 (Student’s f-test).
levels of DOPAC in the striatum and elevation in levels of dopamine induced by tetrahn but these did not achieve significance. Yohimbine, injected into the midbrain, failed to modify the levels of dopamine and DOPAC, causing only a small but significant reduction in the level of noradrenaline in the striatum. The injection of prazosin into the midbrain also reduced the level of noradrenaline in the striatum and limbic tissue but elevated levels of DOPAC in the limbic tissue. Although the injection of yohimbine failed to antago-
nise the ability of the tetralin compound to reduce levels of DOPAC in the striatum, indeed the levels,
of striatal DOPAC were further reduced by the additional treatment with yohimbine, the other actions of tetralin to modify levels of dopamine in the striatum and limbic region and levels of DOPAC were antagonised by yohimbine. Prazosin had a similar spectrum of action to modify the changes in levels of dopamine/DOPAC in striatal and limbic tissue induced by tetralin. (-)Sulpiride, yohimbine or prazosin. administered
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BRADBURYet al.
Fig. 2. Diagrammatic representation of the sites of injection within the area of the substantia nigra of the brain of the mouse. (0) Indicates the injection sites. Coordinates are according to Slotnick and Leonard (1975). The diagram was constructed using histological data obtained from 20 representative brains. The coordinates were established by histological analyses of 2-3 brains at the commencement of each session of surgery. The locations of the injection sites were then established for all brains taken for biochemical assessment; no location was found to be outside the area of the substantia nigra. One in 5 brains of animals used only for the behavioural studies was subject to histological analysis: of 30 brains examined, only 3 were found to have locations outside the substantia nigra (in the cerebellar peduncles or zona incerta).
alone or in combination with tetralin, failed to modify levels of serotonin or S-HIAA in the striatum and limbic region (control values being 546 + 51, 744 f 43, 324 f 30 and 201 f 10 pg/mg of tissue). Injection of tetralin into the midbrain reduced levels of DOPAC in the frontal cortex (63%; Table 2); there were no other changes in transmitter/ metabolite levels. The injection of (-)sulpiride reduced levels of dopamine in the frontal cortex (56%) but failed to significantly modify levels of DOPAC. (-)Sulpiride significantly antagonised the reduction in levels of DOPAC induced by tetralin. Yohimbine significantly reduced the levels of dopamine (52%) and DOPAC (39%) in the frontal cortex and similar reductions in levels of DOPAC were also observed when yohimbine was combined with tetralin, although the reduction in levels of dopamine induced by yohimbine was less marked. Prazosin also significantly reduced the levels of dopamine (49%) and DOPAC (39%) in the frontal cortex. These reductions were also recorded after the combined treatment with prazosin and tetralin. The levels of noradrenaline, serotonin and 5-HIAA in the frontal cortex (388 + 22, 356 + 16 and 76 +_8 pg/mg tissue) were not consistently modified by any of the treatments with drugs or interactions of the drugs.
DISCUSSION
The present study used the potent dopamine agonist 2-di-n-propylamino-5,6_dihydroxytetralin (tetralin) to determine if the actions of dopamine agonists in the substantia nigra, to reduce locomotor activity in the mouse, could be correlated with biochemical changes in the activity of the dopaminecontaining neurones projecting to the forebrain. It could be proposed that stimulation of dopamine autoreceptors located within the nigra (see introduction and Argiolas, Mellis, Fadda and Gessa, 1982; Glowinski and Cheramy, 1981) would lead to a reduction in the rate of firing of the dopaminergic neurones. A parameter indicative of a reduced rate of firing would be a modified turnover of transmitter, measured in the present study as the concentration of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). Whilst all techniques for measuring turnover have limitations, the attempt to determine a biochemical correlate at the time of maximal behavioural change, precluded the use of other methodologies involving, for example, inhibition of synthesis which would seriously interfere with the behavioural/biochemical measurements. The injection of tetralin was directed at the pars reticulata, that part of the substantia nigra influenced
Dopamine action in substantia nigra
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by dendritic release of dopamine and comprising a total volume of approx. 0.5 mm3. Diffusion from such a small area is inevitable. However, in a previous study it was shown that injections of tetralin above, posterior or anterior to the substantia nigra were much less effective in reducing locomotor activity (Bradbury et al., 1983). It was proposed that the tetralin compound may have an important locus of action within the substantia nigra to reduce locomotor activity, and in the present study it was shown that at a time of marked locomotor depression the levels of DOPAC were decreased and levels of dopamine increased in the striatum. A similar spectrum of change was observed following the peripheral administration of the tetralin compound where it was proposed that an inhibition of the firing of cells in the nigra may occur before the stimulation of presynaptic autoreceptors in the striatum (see Westerink, Feenestra, Wirix and Horn, 1984). Also, in an anaesthetised rat preparation and the rabbit, the injection of apomorphine into the nigra is reported to reduce levels of homovanillic acid in the striatum (Maggi, Bruno, Cattabani, Groppetti, Parenti and Racagni, 1978; Wolfarth, Dulska, Golembiowskanikitin and Vetulani, 1978). If it is accepted that decreased levels of DOPAC reflect a reduced dopaminergic neurotransmission, then the depression of locomotor activity observed after the injection of the tetralin compound into the substantia nigra may be due to a decreased dopamine function in the nigrostriatum. The mechanism through which the tetralin compound can cause the behavioural and biochemical changes probably involves an action on dopamine receptors since (-)sulpiride, injected into the substantia nigra, antagonised the behavioural depression although the trend for (-)sulpiride to antagonise the tetralin-induced reduction in the levels of DOPAC in the striatum did not achieve significance. It is also relevant that the antagonism of the reduction in locomotor activity induced by tetralin was not completely reversed by (-)sulpiride which could indicate either (a) the use of too small a dose of (-)sulpiride, or (b) the presence of additional tetralin-sensitive mechanisms within the nigra to moderate behavioural and biochemical changes. Nevertheless, that the injection of (-)sulpiride alone into the nigra could increase locomotor activity and, at least in smaller doses than those used in the present study, increase levels of DOPAC in the striatum (data not shown) suggests the presence of a tonic inhibitory dopaminergic action within the nigra. If additional mechanisms exist within the nigra to mediate the behavioural and biochemical changes induced by tetrahn they are unlikely to be CL,or qadrenoceptor types since prazosin and yohimbine, injected into the substantia nigra, failed to antagonise the depression of locomotor activity and changes in levels of DOPAC induced by tetralin, indeed yohimbine, through non-specified mechanisms actu-
A. J.
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et
al.
Table 2. Levels of 3.4.-dihydroxyphenylacetic acid (DOPAC), dopamine (DA) and noradrenaline (NA) in the frontal cortex following bilateral injection of tetralin and potential antagonists into the substantia nigra of the mouse Frontal cortex Injection into substantia nigra Vehicle Tetralin (0.05 pg) (-)Sulpiride (0.2 pg) (-)Sulpiride + tetralin Yohimbine (I p(g) Yohimbine + tetralin Prazosin (I pg) Prazosin + tetralin
DOPAC (A) (pgimg)
DA (W (pgimg)
Ratio A/B
NA (pglmg)
54 f 9 20 f 3’ 33 * 8 43 + 3$ 33 + 2’ 29 +_5’ 33f4’ 29 f 2*
Ilk9 65 + 8 34 f 3t 80+4 37*4t 6Ok6 39+4* 42 + 2’
0.70 0.30 0.97 0.54 0x9 0.48 0.85 0.70
388 It 22 388 + 30 334 + 16 399 f 20 398 +_I3 420 + 26 398 f I8 396 ?r.7
n = 5-6. SEMs given. Significant decreases in levels compared to vehicle controls are indicated as *P < 0.05, tP < 0.01; antagonism of the effect of tetralin significant to $P < 0.01 (Student’s f-test).
ally enhanced the reduction in levels of DOPAC induced by tetralin. The selectivity of the action of tetralin for the dopamine system is shown by its failure to affect levels of noradrenaline, serotonin and S-hydroxyindoleacetic acid in the striatum. While the major dopaminergic projection from the substantia nigra is to the striatum, it is also well established that the nigral dopamine system contributes a more modest innervation to limbic and cortical areas. Conversely, whilst the dopaminergic projection, arising from the midbrain A8 and Al0 cell groups, provides the major innervation to the limbic and cortical areas, a minor component innervates the striatum (Lindvall, 1979). Therefore, it is of interest that the injection of tetralin into the substantia nigra significantly modified levels of DOPAC and dopamine in the limbic region, although both were raised. These effects were antagonised by concomitant injection of (-)sulpiride into the substantia nigra. The increased levels of DOPAC clearly contrast with the reduced levels found in the striatum, and may reflect an actual increase in the function of dopamine in the limbic region in an attempt to compensate and restore the motor behaviour in the presence of a striatal deficit. It remains of interest that yohimbine and prazosin, when injected into the substantia nigra, antagonised the actions of tetralin to enhance the levels of DOPAC and dopamine in the limbic region. The injection of tetralin into the substantia nigra also caused biochemical changes in the frontal cortex where the levels of DOPAC were markedly reduced, a reduction that was antagonised by a concomitant injection of (-)sulpiride into the substantia nigra. The only other significant biochemical change occurring in the frontal cortex was a reduction in levels of DOPAC after the intranigral injection of yohimbine and prazosin. Again, it is difficult to determine whether these changes are a consequence of an action in the substantia nigra, diffusion of drug from the nigra to influence A8 and A10 cell groups located medial to the nigral cells, or reflect compensation between striatal, limbic and cortical systems to maintain motor activity.
In summary, the most important finding of the present study was that the injection of 2-di-n-propylamino-5,6_dihydroxytetralin into the substantia nigra of the brain of the mouse reduced locomotor activity, a behavioural change which correlates with a reduction in levels of DOPAC in the striatum and frontal cortex effected partly through sites sensitive to neuroleptic drugs [( -)sulpiride]. It is concluded that the actions of dopamine agonists in the substantia nigra and/or closely located dopaminecontaining cell body groups can decrease the functional activity in the ascending dopaminergic pathways. Acknowledgements-This work was supported by the Wellcome Trust and the Parkinson’s Disease Society. Gifts of drugs were received from Glaxo, Pfizer and SESIF (France).
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