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The electrophysiology of dopamine (D2) receptors: A study of the actions of dopamine on corticostriatal transmission
03064522/8393.00+ 0.00 Pergamon Press Ltd Q 1983IBRO
Neuroscience Vol. 10, No. 2, pp. 349-355, 1983
Printed in Great Britain
THE ELECTROPHYSIOLO~Y OF DOPAMINE (Dz) RECEPTORS: A STUDY OF THE ACTIONS OF DOPAMINE ON CORTICOSTRIATAL TRANSMISSION J. R. BROWN* and G. W. ARBUTHNOTT MRC Brain Metabolism Unit, 1 George Square, Edinburgh, Scotland EH8 9JZ Abstract-Electrophysioiogical recordings from the cells of the neostriatum in rats anaesthetised with halothane revealed only inhibitory actions of dopamine applied iontophoretically close to the cells. Inhibition of cortical driving seemed to have a slightly higher threshold in most cells but dopamine inhibited spontaneous action potentials, glutamate-induced responses, and cortical driving in the cells studied. Fluphenazine applied iontophoretically blocked the actions of dopamine but was itself without effect on the neuronal responses. Sulpiride, in contrast, was without effect on the spontaneous activity of the cells and was ineffective in blocking the action of applied dopamine. Sulpiride, nevertheless, increased the response to cortical stimulation though it had no action on the response to applied glutamate. These results suggest that the sub-class of dopamine receptors on the terminals of the corticostriatal pathway may be inhibitory on glutamate release and preferentially sensitive to blockade by sulpiride.
While several different binding sites for dopamine have been described in the central nervous system6,” the number of receptors at which dopamine can be shown to exert a physiological effect is considerably smaller in mammals.‘3.27,40In their simpler scheme,
Kebabian and Calne17 suggested that sites at which there is a known biochemical effect of dopamine on adenylate cyclase be called D, receptors while the binding sites for dopamine-at that time not associated with known biochemical changes in the post synaptic elements-be called D, receptors. With this simplified scheme it is possible to suggest from lesion studies that the two “receptors” in the neostriatum may in fact be preferentially located on different neuronal elements. The D, receptors are associated with neurones of the neostriatum and with those which project to the substantia nigra (SN). They appear to be associated with the outer membrane of these neurones and are present on their terminals in the SN as well as on their cell bodies in the striatum.32 Kainic acid lesions which destroy the neurones of the striatum, also reduce the activity of adenylate cyclase in the nucleus and result in a 94% reduction in the stimulation of the enzyme by dopamine? Such lesions of the striatum lead to a reduction of the dopamine ligand binding sites in the striatum, presumably as a result of the destruction of striatal neurones.20 -._ Reprint requests to: G. W. Arbuthnott, MRC Brain Meta~lism Unit, Universitv Denartment of Pharmacology, 1 George Square, Edinburgh, Scotland EH8 9JZ. *Present address: Glaxo Group Research, Department of Neuropharmacology, Ware, Herts, England. Abbreviations: PSTH, post stimulus time histogram; SN, substantia n&a. NSC1012
At least some of the D, receptors on the other hand seem to be located on the terminals of the cortical input to the striatum. Decortication reduced the number of binding sites in striatal homogenates for [3N]apomorphine3’ for [‘H]haloperido129 for [-‘HIspiro~rido133 and for ~3H]sulpiride.3* Some 60% of the binding remains after decortication, and although some of this is sensitive to kainic acid treatment29’* the source of the remainder is uncertain.6*30 Nevertheless, there did seem to be some reason to suppose that there might be dopamine receptors on the corticostriatal afferents. We have attempted a pharmacolo~cal dissection of the responses to stimulation of the different dopamine receptors by examining striatal cells neurophysiologically. Any study of striatal cells in normal anaesthetised animals would need to use some means of activating these normally silent, or at least extremely slowly firing cells.‘~‘5~28~34 There were three means of doing this at our disposal. Stimulation of the cerebral cortex excited the cells by a presumably monosynaptic pathway*’ whose transmitter may be an amino acid34,36possibly glutamate!~“~2’ Iontophoresis of excitant amino acids also induces the cells to fireclearly, in view of the possible physiolo~cal action of glutamate, this is the substance of choice. Finally, since the route of the output pathway in the rat is known in some detail,39 it is possible to stimulate striatonigral cells antidromically and thus to examine the pharmacological properties of these cells in particular. In this study all three methods have been used. Dopamine iontophoresis inhibited all the modes of stimulation employed. In order to differentiate between an action on the D, sites on the cortical terminals land a direct action on striatal neurones the
349 k
350
J. R. Brown
putative D, antagonist by iontophoresis.
(-)
sulpiride
and G. W. Arbuthnott
has been applied
EXPERIMENTAL PROCEDURES
Male albino Wistar rats weighing between 210 and 250 g were used for these experiments. They were anaesthetised with Fluothane (ICI), induction being with a 3% halothane in air mixture which was reduced to 1.5% during surgery and then maintained at 0.8% with a Vapor-Halothan (Drager) vapouriser. The anesthetic was administered in the 200ml/min air flow delivered to a tracheal canula from which it was scavenged by a suction pump and absorbed into liquid paraffin. The animal’s body temperature was maintained at 37°C with a thermostatically controlled blanket. The stereotaxic implantation of the stimulating electrodes and the recording micropipettes were carried out with a Kopf stereotaxic frame in which the animal’s head was clamped in the plane described in the Kiinig and Klippel’9 atlas of the rat brain. In all experiments a concentric stimulating electrode (0.25 mm external diameter) was placed in the frontal cortex (co-ordinates: A 10.3 mm; V + 3.0 mm; L 2.7 mm) and in some, a similar electrode was introduced into the crus cerebri at an angle of 45’ (coordinates of the tip of the electrode were A 3.2mm; V - 2.8 mm; L 2.0 mm). Recording and microiontophoresis was carried out with electrodes fabricated as already described,’ the recording barrel being glued parallel to the iontophoresis pipette with UV setting glue. Tip separation was approximately 2&3Opm. The pipettes were attached to a hydraulic microdrive and recordings made from an area of the head of the striatum bounded by the co-ordinates: A 8.&9.0mm; V + 2.0 to - I.0 mm; L 2.4-2.8 mm. Iontophoresis was controlled by a neurophore unit (Digitimer Ltd.) and current balancing was routinely employed as well as current control injections being made in each animal. Recording electrodes were filled with Pontamine sky blue and the position of the track identified histologically by iontophoresis of dye at the top and bottom of each recording track. The position of the stimulating electrodes was noted from histological preparationsI by finding the lesion made by passing 6 volts DC for IO s through the electrode at the end of the experiment. Antidromic responses were identified by collision with orthodromic activation initiated by stimulation of the cortex. In each experiment the action of iontophoreticallyapplied substances was compared with an immediately preceeding control period. In those cases where the actions
Table
I. Dopamine
Type of neuronal activity Spotaneous firing Glutamate-induced firing Firing induced by cortical stimulation
were on spontaneous activity the 2 min preceding drug application were treated as 100%. In the case of the experiments on glutamate-induced activity, two preceding applications of glutamate were averaged as control. In the case of cortically-evoked activity the results from two immediately preceding post-stimulus histograms (PSTH) were averaged to give a number of spikes in response to the cortical stimulus. Materials The following drugs were dissolved in distilled water and the pH adjusted with 0.1 N HCl and NaOH. SodiumL-glutamate (0.2 M, pH 7.0, Sigma Chemical Company); Acetvlcholine chloride (ACh. 0.5 M. nH 4.5, Sigma): Dopamme (0.5 M, pH 4.0, Sigma) y-aminobutyric acid (GABA 0.5 M. pH 4.0); Fluphenazine (0.1 M, pH 4.0, Squibb); Sulpiride (0.1 M, pH 5.5, ( + ) and (-) racemates from Dr. Lewis, Chemitechna Ltd.)
RESULTS
Dopamine was applied iontophoretically to 78 neurones in the rat neostriatum. Spotaneous activity was recorded from 3 1 neurones and 47 were normally silent, but could be excited by cortical stimulation. The effects of dopamine on spontaneous, glutamateinduced, and cortically-evoked activity are now described. Dopamine
and spontaneous
activity
Dopamine (25-80 nA) was tested on 31 neurones which exhibited spontaneous activity. Depression of firing ranging from 50 to IOO’A was observed on 30 cells (97%) (Table 1) (Fig. lb). The onset of depression usually occurred 2-3 s after ejection commenced. The depression persisted for several seconds after the ejecting current was terminated and normal firing resumed 15-30 s later. However, in some cells, the depression persisted for over 60 s after terminating the current. The depression obtained on individual neurones was dependent on the strength of the ejecting current (Fig. la), but the current required to produce a given level of depression varied between neurones. Complete inhibition was obtained by dopamine currents between 40 and 70nA.
inhibition
of striatal
neurone
activity
Number of neurones inhibited
Range of ejection currents (nA)
Mean % depression firing + SEM
30/3 1 43147 29151
25570 25-90 2&80
76.6 + 4.5 74.6 k 3.7 59.5 k 4.2
Cells responding to dopamine/total number of cells tested. None of the cells was excited by dopamine. Neurones can be in more than one class for example; of the 29 ceils in which the cortically-induced firing was inhibited, 9 were spontaneous, I3 glutamate-stimulated and 7 tested only on the response to cortical stimulation. Dopamine application with currents which markedly depressed spontaneous activity (9 neurones) or glutamate-induced firing (IO neurones) were ineffective on cortical driving. The inhibition observed was current-dependent on individual cells (Fig. I) over the range shown, Currents < 2&25 nA were ineffective while those at the upper end of the range caused complete abolition of the response.
351
D, Receptor electrophysiology
Potentiation of the glutamate excitation was never observed. The glutamate excitation was unaffected by dopamine on four cells. Dopamine and cortical stimulation Cortical stimulation evoked an excitation in 51 neurones tested with dopamine. Stimulation currents were adjusted to produce approximately 1 spike per stimulus and post stimulus time histograms (PSTHs) were compiled from 50 consecutive stimuli at l/s. Care was taken to achieve a stable response before testing with dopamine and two or three control histograms were compiled depending on the stability of the response. This procedure often resulted in less than 50 responses in the control histograms (Fig. 2).
_
‘I”
UnA
r
lmin
Fig. 1. The effects of iontophoresis of dopamine on the activity of single units in the striatum. The ratemeter record in B shows the response to the iontophoresis of dopamine close to a spontaneously active striatal unit. The lower current (25 nA) slows the cell obviously while the effect of 35 nA is even more marked. In A is summarised the current/response data from all the cells tested in this way. The error bars on top of each column show the standard error of the mean percentage reduction which the height of the column represents. In C a silent cell is induced to fire with short pulses of glutamate (Glu 44nA). Dopamine (40nA) applied during the bar above the record caused a clear reduction of the response to the standard glutamate stimulation.
Facilitation of spontaneous firing was never seen with the dopamine ejection currents used in this study, and only one neurone was insensitive to dopamine.
DA 40 nA I’3
I
RECOVERY
Dopamine and amino acid-induced excitation
Forty-seven neurones which exhibited no spontaneous activity became active in response to iontophoretically-applied glutamate (15-60 nA). Glutamate was applied in short pulses (1-4 s) at regular intervals (between 10 and 30s apart). Dopamine inhibited the glutamate induced excitation on 43 neurones (Fig. lc, Table 1). The relation between ejection current and response was similar to that described for spontaneously active neurones. Complete depression of the glutamate response was obtained by currents varying from 30 to 90 nA.
IS
Fig. 2. The effect of iontophoretic dopamine on the response of a striatal neurone to cortical stimulation. Responses of the striatal neurone are shown to 50 stimuli applied to the ipsilateral frontal cortex before (A), during (B), and after (C) the application of dopamine iontophoretically (50 nA). The traces on the left of the Figure show the original oscilloscope traces built up during the construction of the post stimulus histograms which are illustrated alongside them. Cortical stimulation was applied once every second.
352
J. R. Brown and G. W. Arbuthnott
Dopamine was applied both before and during the test PSTH. If a change occurred during the test histogram, further tests were not performed until the control response had returned in two consecutive histograms. Dopamine inhibited the number of corticallyevoked responses in 29 out of 51 cells (57%) tested (Fig. 2; Table 1). Recovery of the response was usually complete after 24min. Seven of the 29 neurones on which dopamine was inhibitory were tested with cortical driving alone. On the remaining 22 cells, dopamine ejection currents which depressed cortical driving, also had a marked depressant effect on spontaneous (9 neurones) and glutamate-induced (13 neurones) firing. On 9 spontaneously active cells and 10 neurones activated by glutamate, a dopamine ejection sufficient to markedly depress activity left the cortical excitation unaffected. On 5 silent neurones which were not tested with glutamate, the cortical excitation was unaffected by dopamine. The relationship between ejection current and response to cortical stimulation was current-dependent on individual cells, but varied between cells. Facilitation of cortical driving was never observed on any neurones tested with dopamine. Dopamine
and output cells
Unit responding with constant latency (mean 9.4 ms range 4-16) to electrical stimulation of the crus cerebri were normally silent. The driving was confirmed as antidromic only in those cells where the response to stimulation in the crus could be collided with the response to stimulation through the electrode in the cerebral cortex. Such a fortunate positioning of all three electrodes was not common but dopamine was tested on 7 such antidromically identified striatal output cells and it inhibited the production of an antidromic spike in all 7 cells. Glutamate excitation was inhibited on all 4 of these cells which could be tested and cortical driving was inhibited by dopamine on the 3 cells on which it could be examined. Dopamine
and acetylcholine
To investigate whether the effects of dopamine were specific to glutamate, acetylcholine was tested along with glutamate on 12 cells. Excitation (6 cells) and inhibition (4 cells) was observed with acetylcholine, 2 cells were insensitive to acetylchohne. The acetylchohne excitations were slow in onset and offset in comparison to the glutamate excitations. Dopamine inhibited the effect of both acetylchohne and gluamate on all 6 neurones tested on which acetylcholine was excitatory. Conventional
neuroleptics
The dopamine antagonists, fluphenazine and haloperidol, were tested for their ability to antagonize the effects of dopamine in this preparation.
Fluphenazine (%X75 nA) iontophoresed for 2-3 min before the ejection of dopamine antagonised the depressant effects of dopamine on both cortical driving and spontaneous activity on 6 neurones, and was ineffective on 2 neurones. During the period of antagonism, fluphenazine did not cause silent cells to fire, nor did it affect the firing rate of spontaneously active cells. The antagonism was reversible within 45 min. Haloperidol was administered peripherally by intraperitoneal injection (i.p. 0.1 mg/kg) in 8 experiments. Only one cell was studied in each experiment after the haloperidol injection. In five experiments several injection of haloperidol were given up to a cumulative dose of 0.5 mg/kg. Haloperidol did not induce spontaneous firing or affect the rate of spontaneous firing in any of the cells. In addition, the depressant effects of iontophoretic dopamine were not antagonized by i.p. haloperidol. Sulpiride Spotaneous and glutamate-induced,firing. The selective Dz antagonist sulpiride’h was used in an attempt pharmacologically to dissect the effects of dopamine on pre- and post-synaptic sites. The effects of iontophoretically-applied (-)sulpiride were investigated on 33 cells. Sulpiride (30-70 nA) did not induce firing from 27 silent cells, neither did it affect the excitatory response to glutamate in all 18 of these cells which were tested. Spontaneous activity was recorded from six cells, four were unaffected by sulpiride but in two there was a decrease in spike height and an increase in firing rate, suggestive of depolarization block. (-)Sulpiride failed to antagonize the depressant effects of iontophoretically-applied dopamine on all 15 neurones tested. Sulpiride and cortical stimulation. The response of striatal neurones to cortical stimulation was tested during the iontophoresis of (-)sulpiride. At stimulation currents which were just threshold for ex(-)sulpiride (30-70 nA) increased the citation, number of cortical stimuli which produced a spike (Fig. 3). This effect was observed on 18 cells with I cell being insensitive. Sulpiride applied for between 30 s and 2 min before the test PSTH reduced the number of stimuli which failed to produce a spike to 22”~ (SEM 6.1) of the control value. The mean number of spikes produced by 50 cortical stimuli was increased from 8.4 (2.5 SEM) to 29.9 (2.8 SEM) (Fig. 3). The increase in response was larger at higher currents but the response rate never exceeded I spike/stimulus. The (+)isomer of sulpiride was ineffective in 3 of the 5 neurones on which it was possible to test it. The facilitation of cortical stimulation by sulpiride was blocked on 6 neurones to which dopamine was applied by iontophoresis simultaneously with the sulpiride. However. this antagonism was only accomplished using ejection currents of dopamine which
D,
Receptor electrophysiology
Fig. 3. The action of sulpiride on the efficacy of cortical stimulation. In A the responses of one cell is illustrated to cortical stimuli of an intensity adjusted to give less than 10 responses in 50 trials. All the responses to 50 stimuli are shown. Calibration bars are 0.2 mV and 2.0 ms. The upper trace shown control responses, the lower the result of sulpiride application (30nA) during the stimulation. In B the effect of sulpiride on all 18 cells is summarised by plotting the total number of action potentials in response to 50 cortical stimuli. The columns are the mean values f standard error of the mean. Although the iontophoresis of dopamine was able to reduce this effect on the 6 cells on which it was tried it never did so at doses which did not themselves inhibit the responsiveness of the ceils.
353
inhibitions, also reported a small proportion of excitations in response to dopamine application. The methods used in this study are almost identical to those of Bevan, Bradshaw and Szabadi3 who reported many (50%) pure excitations with dopamine. The only difference between this study and that of Bevan et al.’ is that different electrodes were used for electrical recording and for iontophoresis; Bevan et aL3 used the same multibarrel electrode for both purposes. The results of the present study are also at variance with those of an earlier study on encephale isole cats which reported a predominance of facilitatory effects on cortical driving by iontophoretically-applied dopamine. 24 Norcross and Spehlman24 also reported that dopamine facilitated excitations evoked from stimulation of the substantia nigra, thalamus and from within the caudate nucleus itself. A minority of cells showed depression of the caudate (20%) and cortical driving (11 ‘A). Facilitation of glutamateinduced and spontaneous firing was also reported. On the basis that the dopamine ejection currents required for 50% inhibition (50nA) were five times greater than those required for a 50% facilitation (9 nA), these authors proposed the existence of distinct excitatory and inhibitory dopamine receptors on the striatal neurones with differing affinities for dopamine. In the present study, no excitatory effects of dopamine were observed over the whole range of ejection currents tested (10-100 nA). There are, however, several methodological differences which may explain the disparity between the results of these two studies. Norcross and Spehlman did not use current balancing during iontophoretic ejections. In addition, Norcross describes that electrodes were filled with drug solutions the day before use and it may be questionable whether the dopamine had remained unoxidized over this period. Dopamine contained in an iontophoretic electrode under similar conditions to those used in the present study has been shown to retain its activity in an adenylate cyclase assay over the timecourse of a typical experiment.” Dopamine and striatal physiology
also depressed cell firing in a similar manner to that described previously. DISCUSSION
The action of iontophoretically-applied dopamine
The results support the hypothesis that dopamine is an inhibitory transmitter in the striatum. Iontophoretic dopamine had a depressant action on spontaneous and cortically driven firing (Table 1), and on glutamate and acetylcholine-induced excitations, as previously described.4,‘4 In agreement with two other studies,7,‘3 no excitatory responses to iontophoretic dopamine were observed. This contrasts with many of the previous reports which, although having a predominance of
Despite the wide range used, a dopamine ejection current was never found which could affect the cortical driving without also affecting the cell postsynaptically. Therefore any presynaptic component of the depression of cortical driving was masked. The glutamate-induced and spontaneous firing of several cells (34%) was depressed by dopamine without any effect on the response to cortical driving. A similar difference in the efficacy of iontophoretic dopamine on “synaptic” and “spontaneous” activity has been observed in conscious monkeys.2s The significance of these observations is not yet clear. Thorpe (personal communication) has suggested that dopamine may act to alter the signal-to-noise ratio of striatal cells. Alternatively, the results may indicate that there is a particularly high safety margin in corticostriatal
354
J. R. Brown
and G. W. Arbuthnott
driving or that the position of the iontophoretic pipette was closer to the striatal cell bodies than to the corticostriatal synapses on which dopamine was active. Conventional
neuroleptics
The successful antagonism of the effects iontophoretic dopamine by the iontophoretically-applied antagonist fluphenazine is in contrast to the lack of effect of peripherally-applied haloperidol. Other authors2.3s.4’ have reported a similar failure of peripherally-applied antagonist on iontophoretic dopamine. The dose of haloperidol used initially (0. I mg/kg) is enough to cause a large increase in the firing of dopamine-containing cells in the substantia nigra” yet doses considerably in excess of this were still ineffective. Sulpiride
It is tempting to speculate that the facilitation of cortical driving by sulpiride may reflect the blockade of an action of dopamine on cortical terminals. That dopamine receptors on cortical terminals may exist has already been suggested by a variety of experiments on specific binding of dopamine agonists and antagonists,6.3’.38 although it seems that they may be differentiable from the receptors on the dopamine terminals and from those on the striatal cells. The nomenclature is confusing but these binding sites may be responsible for the effects of dopamine and dopaminergic agonists on glutamate release from slices of striatum “in vitro”.22.26 Recent work suggests that both the binding of sulpiride3* and the effects of dopamine on glutamate release’2.3’ are not easily fitted into simple D,, D, classification of receptors. At the doses they used, Stoof er al.” find sulpiride inactive on glutamate release, a result we have recently confirmed although we were able to show that
sulpiride antagonised the effect of apomorphine on [3H]glutamate release (P. R. Mitchell and J. R. Brown, unpublished). Clearly, because of the uncertainties of applied dose of drug in iontophoretic experiments, our results cannot provide the kind of detailed pharmacological information required to prove that the receptors on cortical terminals are of the D2 type as defined in binding studies. The facilitation of cortical driving shown in the present study supports the notion of an increased amount of transmitter release from cortical terminal during the blockade of dopamine receptors on them. Sulpiride was ineffective in altering the action of dopamine on striatal cells, suggesting that the dopamine receptor on the cell bodies is indeed not of the same type. The lack of effect of sulpiride on the spontaneous activity of striatal cells, or on their response to glutamate, strongly suggests a presynaptic site of action on the cortical terminals although the extracellular recording used in the present experiments would not have detected any change on the membrane potential of the cell. Intracellular studies need to be performed on striatal cells during sulpiride application confirm a presynaptic role for sulpiride. Conclusions
Our results suggest that there is a subclass of dopamine receptors on corticostriatal terminals which may be tonically active in anaesthetised animals. Inhibition of these receptors with sulpiride may enhance the influence of cortical activity on striatal cells. Whether this action is responsible for the therapeutic activity of the benzamides or for their motor side effects remains to be discovered. Acknowledgements-This
postgraduate McQueen
studentship;
work was supported
by an MRC
we are grateful to Dr D. S.
for his encouragement
during
this project.
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22. Mitchell P. R. & Doggett N. S. (1980) Modulation of striatal [3H]glutamic acid release by dopaminergic drugs. Life Sci. 26, 2073-208
1.
23. Norcross K. (1978) Ph.D. Thesis. Northwestern University Medical School, Chicago, U.S.A. 24. Norcross K. & Spehlman R. (1978) A quantitative analysis of the excitatory and depressant effects of dopamine on the firing of caudatal neurones: electrophysiological support for the existence of two distinct dopamine-sensitive receptors. Brain Res. 156, 168-174. 25. Rolls E. T., Thorpe S. J., Perret D. I., Boytim M., Wilson F. A. W. & Szabo I. (1981) Responses of striatal neurons in the behaving monkey: influence of dopamine. Sot. Neurosci. Abs 7, p. 778. 26. Rowlands G. J. & Roberts P. J. (1980) Activation of dopamine receptors inhibits calcium-dependent glutamate release from cortico-striatal terminals ‘in vitro’. Eur. J. Pharmac. 62, 241-242. 27. Scatton B. (1982) Effect of dopamine agonists and neuroleptic agents on striatal acetylcholine transmission in the rat: evidence against dopamine receptor multiplicity. f. Phurmuc. exp. Ther. 220, 197-202. 28. Schultz W. & Ungerstedt U. (1978) A method to detect and record from striatal cells of low spontaneous activity by stimulating the corticostriatal pathway. Brain Res. 142, 357-362. 29. Schwartz R., Creese I., Coyle J. T. & Snyder S. H. (1978) Dopamine receptors localized on cerebra1 cortical afferents to rat corpus striatum. Nature, Land. 271, 766768. 30. Seeman P. (1981) Brain dopamine receptors. Pharmac. Rev. 32, 23&313. 31. Sokoloff P., Martres M. P. & Schwartz J. C. (1980) Three classes of dopamine receptor (D-2. D-3, D-4) identified by binding studies with [3H]apomorphine and [3H]domperidone. Naunyn-Schmiedebergs Arch. Pharmuh. 315, 89-102. 32. Spano P. F., Di Chiara G., Tonan G. C. & Trabucchi M. (1976) A dopamine stimulated adenylate cyclase in rat substantia nigra. J. Neurochem. 27, 1565-l 568. 33. Spano P. F., Govoni S. & Trabucchi M. (1978) Studies on the pharmacological properties of dopamine receptors in various areas of the central nervous system. In Dopamine. Ado. In Biochemical Psychopharmacology (eds Roberts P. J., Woodruff G. N. & Iversen L. L.) Vol. 19, pp. 155-165. Raven Press, New York. 34. Spencer H. J. (1976) Antagonism of cortical excitation of striatal neurones by glutamic acid diethyl ester: evidence for glutamic acid as an excitatory transmitter in the rat striatum. Brain Res. 102, 91-101. 3.5. Skirboll L. R. & Bunney B. S. (1979) The effects of acute and chronic haloperidol treatment on spontaneously firing neurones in the caudate nucleus of the rat. Lzfk Sci. 25, 14191434. 36. Stone T. W. (1979) Amino acids as neurotransmitters of cortico-fugal neurones in the rat: a comparison of glutamate and aspartate. Br. J. Phurmcre. 67, 545-551. 37. Stoof J. C., DeBoer T., Sminia P. & Mulder A. H. (1982) Stimulation of D,-dopamine receptors in rat neostriatum inhibits the release of acetylcholine and dopamine but does not affect the release of y-aminobutyric acid, glutamate or serotonin. Eur. J. Pharmac. 84, 21 I-214. 38. Theodorou A., Reavill C., Jenner P. & Marsen C. D. (1981) Kainic acid lesions of striatum and decortication reduce specific [3H]sulpiride binding in rats, so D-2 receptors exist post-synaptically on corticostriate afferents and striatal neurones. J. Pharm. Pharmac. 33, 439-444. 39. Tulloch I. F., Arbuthnott G. W. & Wright A. K. (1978) Topographi~l organization of the striato-nigral pathway revealed by anterograde and retrograde neuroanatomical tracing techniques. J. Anat. 127, 425-441. 40. Woodruff G. N., Freedman S. B. & Poat J. A. (1980) Why does sulpiride not block the effect of dopamine on dopamine-sensitive adenylate cyclase? J. Pharm. Pharmuc. 32, 802-803. 41. Zarzecki P., Blake D. J. & Somjen G. C. (1977) Neurological disturbances, nigrostriate synapses, and iontophoretic dopamine and apormorphine after haloperidol. Expl Neurol. 57, 956970. (Accepted 20 Mzrch 1983)
Neuroscience Vol. 10, No. 2, pp. 349-355, 1983
Printed in Great Britain
THE ELECTROPHYSIOLO~Y OF DOPAMINE (Dz) RECEPTORS: A STUDY OF THE ACTIONS OF DOPAMINE ON CORTICOSTRIATAL TRANSMISSION J. R. BROWN* and G. W. ARBUTHNOTT MRC Brain Metabolism Unit, 1 George Square, Edinburgh, Scotland EH8 9JZ Abstract-Electrophysioiogical recordings from the cells of the neostriatum in rats anaesthetised with halothane revealed only inhibitory actions of dopamine applied iontophoretically close to the cells. Inhibition of cortical driving seemed to have a slightly higher threshold in most cells but dopamine inhibited spontaneous action potentials, glutamate-induced responses, and cortical driving in the cells studied. Fluphenazine applied iontophoretically blocked the actions of dopamine but was itself without effect on the neuronal responses. Sulpiride, in contrast, was without effect on the spontaneous activity of the cells and was ineffective in blocking the action of applied dopamine. Sulpiride, nevertheless, increased the response to cortical stimulation though it had no action on the response to applied glutamate. These results suggest that the sub-class of dopamine receptors on the terminals of the corticostriatal pathway may be inhibitory on glutamate release and preferentially sensitive to blockade by sulpiride.
While several different binding sites for dopamine have been described in the central nervous system6,” the number of receptors at which dopamine can be shown to exert a physiological effect is considerably smaller in mammals.‘3.27,40In their simpler scheme,
Kebabian and Calne17 suggested that sites at which there is a known biochemical effect of dopamine on adenylate cyclase be called D, receptors while the binding sites for dopamine-at that time not associated with known biochemical changes in the post synaptic elements-be called D, receptors. With this simplified scheme it is possible to suggest from lesion studies that the two “receptors” in the neostriatum may in fact be preferentially located on different neuronal elements. The D, receptors are associated with neurones of the neostriatum and with those which project to the substantia nigra (SN). They appear to be associated with the outer membrane of these neurones and are present on their terminals in the SN as well as on their cell bodies in the striatum.32 Kainic acid lesions which destroy the neurones of the striatum, also reduce the activity of adenylate cyclase in the nucleus and result in a 94% reduction in the stimulation of the enzyme by dopamine? Such lesions of the striatum lead to a reduction of the dopamine ligand binding sites in the striatum, presumably as a result of the destruction of striatal neurones.20 -._ Reprint requests to: G. W. Arbuthnott, MRC Brain Meta~lism Unit, Universitv Denartment of Pharmacology, 1 George Square, Edinburgh, Scotland EH8 9JZ. *Present address: Glaxo Group Research, Department of Neuropharmacology, Ware, Herts, England. Abbreviations: PSTH, post stimulus time histogram; SN, substantia n&a. NSC1012
At least some of the D, receptors on the other hand seem to be located on the terminals of the cortical input to the striatum. Decortication reduced the number of binding sites in striatal homogenates for [3N]apomorphine3’ for [‘H]haloperido129 for [-‘HIspiro~rido133 and for ~3H]sulpiride.3* Some 60% of the binding remains after decortication, and although some of this is sensitive to kainic acid treatment29’* the source of the remainder is uncertain.6*30 Nevertheless, there did seem to be some reason to suppose that there might be dopamine receptors on the corticostriatal afferents. We have attempted a pharmacolo~cal dissection of the responses to stimulation of the different dopamine receptors by examining striatal cells neurophysiologically. Any study of striatal cells in normal anaesthetised animals would need to use some means of activating these normally silent, or at least extremely slowly firing cells.‘~‘5~28~34 There were three means of doing this at our disposal. Stimulation of the cerebral cortex excited the cells by a presumably monosynaptic pathway*’ whose transmitter may be an amino acid34,36possibly glutamate!~“~2’ Iontophoresis of excitant amino acids also induces the cells to fireclearly, in view of the possible physiolo~cal action of glutamate, this is the substance of choice. Finally, since the route of the output pathway in the rat is known in some detail,39 it is possible to stimulate striatonigral cells antidromically and thus to examine the pharmacological properties of these cells in particular. In this study all three methods have been used. Dopamine iontophoresis inhibited all the modes of stimulation employed. In order to differentiate between an action on the D, sites on the cortical terminals land a direct action on striatal neurones the
349 k
350
J. R. Brown
putative D, antagonist by iontophoresis.
(-)
sulpiride
and G. W. Arbuthnott
has been applied
EXPERIMENTAL PROCEDURES
Male albino Wistar rats weighing between 210 and 250 g were used for these experiments. They were anaesthetised with Fluothane (ICI), induction being with a 3% halothane in air mixture which was reduced to 1.5% during surgery and then maintained at 0.8% with a Vapor-Halothan (Drager) vapouriser. The anesthetic was administered in the 200ml/min air flow delivered to a tracheal canula from which it was scavenged by a suction pump and absorbed into liquid paraffin. The animal’s body temperature was maintained at 37°C with a thermostatically controlled blanket. The stereotaxic implantation of the stimulating electrodes and the recording micropipettes were carried out with a Kopf stereotaxic frame in which the animal’s head was clamped in the plane described in the Kiinig and Klippel’9 atlas of the rat brain. In all experiments a concentric stimulating electrode (0.25 mm external diameter) was placed in the frontal cortex (co-ordinates: A 10.3 mm; V + 3.0 mm; L 2.7 mm) and in some, a similar electrode was introduced into the crus cerebri at an angle of 45’ (coordinates of the tip of the electrode were A 3.2mm; V - 2.8 mm; L 2.0 mm). Recording and microiontophoresis was carried out with electrodes fabricated as already described,’ the recording barrel being glued parallel to the iontophoresis pipette with UV setting glue. Tip separation was approximately 2&3Opm. The pipettes were attached to a hydraulic microdrive and recordings made from an area of the head of the striatum bounded by the co-ordinates: A 8.&9.0mm; V + 2.0 to - I.0 mm; L 2.4-2.8 mm. Iontophoresis was controlled by a neurophore unit (Digitimer Ltd.) and current balancing was routinely employed as well as current control injections being made in each animal. Recording electrodes were filled with Pontamine sky blue and the position of the track identified histologically by iontophoresis of dye at the top and bottom of each recording track. The position of the stimulating electrodes was noted from histological preparationsI by finding the lesion made by passing 6 volts DC for IO s through the electrode at the end of the experiment. Antidromic responses were identified by collision with orthodromic activation initiated by stimulation of the cortex. In each experiment the action of iontophoreticallyapplied substances was compared with an immediately preceeding control period. In those cases where the actions
Table
I. Dopamine
Type of neuronal activity Spotaneous firing Glutamate-induced firing Firing induced by cortical stimulation
were on spontaneous activity the 2 min preceding drug application were treated as 100%. In the case of the experiments on glutamate-induced activity, two preceding applications of glutamate were averaged as control. In the case of cortically-evoked activity the results from two immediately preceding post-stimulus histograms (PSTH) were averaged to give a number of spikes in response to the cortical stimulus. Materials The following drugs were dissolved in distilled water and the pH adjusted with 0.1 N HCl and NaOH. SodiumL-glutamate (0.2 M, pH 7.0, Sigma Chemical Company); Acetvlcholine chloride (ACh. 0.5 M. nH 4.5, Sigma): Dopamme (0.5 M, pH 4.0, Sigma) y-aminobutyric acid (GABA 0.5 M. pH 4.0); Fluphenazine (0.1 M, pH 4.0, Squibb); Sulpiride (0.1 M, pH 5.5, ( + ) and (-) racemates from Dr. Lewis, Chemitechna Ltd.)
RESULTS
Dopamine was applied iontophoretically to 78 neurones in the rat neostriatum. Spotaneous activity was recorded from 3 1 neurones and 47 were normally silent, but could be excited by cortical stimulation. The effects of dopamine on spontaneous, glutamateinduced, and cortically-evoked activity are now described. Dopamine
and spontaneous
activity
Dopamine (25-80 nA) was tested on 31 neurones which exhibited spontaneous activity. Depression of firing ranging from 50 to IOO’A was observed on 30 cells (97%) (Table 1) (Fig. lb). The onset of depression usually occurred 2-3 s after ejection commenced. The depression persisted for several seconds after the ejecting current was terminated and normal firing resumed 15-30 s later. However, in some cells, the depression persisted for over 60 s after terminating the current. The depression obtained on individual neurones was dependent on the strength of the ejecting current (Fig. la), but the current required to produce a given level of depression varied between neurones. Complete inhibition was obtained by dopamine currents between 40 and 70nA.
inhibition
of striatal
neurone
activity
Number of neurones inhibited
Range of ejection currents (nA)
Mean % depression firing + SEM
30/3 1 43147 29151
25570 25-90 2&80
76.6 + 4.5 74.6 k 3.7 59.5 k 4.2
Cells responding to dopamine/total number of cells tested. None of the cells was excited by dopamine. Neurones can be in more than one class for example; of the 29 ceils in which the cortically-induced firing was inhibited, 9 were spontaneous, I3 glutamate-stimulated and 7 tested only on the response to cortical stimulation. Dopamine application with currents which markedly depressed spontaneous activity (9 neurones) or glutamate-induced firing (IO neurones) were ineffective on cortical driving. The inhibition observed was current-dependent on individual cells (Fig. I) over the range shown, Currents < 2&25 nA were ineffective while those at the upper end of the range caused complete abolition of the response.
351
D, Receptor electrophysiology
Potentiation of the glutamate excitation was never observed. The glutamate excitation was unaffected by dopamine on four cells. Dopamine and cortical stimulation Cortical stimulation evoked an excitation in 51 neurones tested with dopamine. Stimulation currents were adjusted to produce approximately 1 spike per stimulus and post stimulus time histograms (PSTHs) were compiled from 50 consecutive stimuli at l/s. Care was taken to achieve a stable response before testing with dopamine and two or three control histograms were compiled depending on the stability of the response. This procedure often resulted in less than 50 responses in the control histograms (Fig. 2).
_
‘I”
UnA
r
lmin
Fig. 1. The effects of iontophoresis of dopamine on the activity of single units in the striatum. The ratemeter record in B shows the response to the iontophoresis of dopamine close to a spontaneously active striatal unit. The lower current (25 nA) slows the cell obviously while the effect of 35 nA is even more marked. In A is summarised the current/response data from all the cells tested in this way. The error bars on top of each column show the standard error of the mean percentage reduction which the height of the column represents. In C a silent cell is induced to fire with short pulses of glutamate (Glu 44nA). Dopamine (40nA) applied during the bar above the record caused a clear reduction of the response to the standard glutamate stimulation.
Facilitation of spontaneous firing was never seen with the dopamine ejection currents used in this study, and only one neurone was insensitive to dopamine.
DA 40 nA I’3
I
RECOVERY
Dopamine and amino acid-induced excitation
Forty-seven neurones which exhibited no spontaneous activity became active in response to iontophoretically-applied glutamate (15-60 nA). Glutamate was applied in short pulses (1-4 s) at regular intervals (between 10 and 30s apart). Dopamine inhibited the glutamate induced excitation on 43 neurones (Fig. lc, Table 1). The relation between ejection current and response was similar to that described for spontaneously active neurones. Complete depression of the glutamate response was obtained by currents varying from 30 to 90 nA.
IS
Fig. 2. The effect of iontophoretic dopamine on the response of a striatal neurone to cortical stimulation. Responses of the striatal neurone are shown to 50 stimuli applied to the ipsilateral frontal cortex before (A), during (B), and after (C) the application of dopamine iontophoretically (50 nA). The traces on the left of the Figure show the original oscilloscope traces built up during the construction of the post stimulus histograms which are illustrated alongside them. Cortical stimulation was applied once every second.
352
J. R. Brown and G. W. Arbuthnott
Dopamine was applied both before and during the test PSTH. If a change occurred during the test histogram, further tests were not performed until the control response had returned in two consecutive histograms. Dopamine inhibited the number of corticallyevoked responses in 29 out of 51 cells (57%) tested (Fig. 2; Table 1). Recovery of the response was usually complete after 24min. Seven of the 29 neurones on which dopamine was inhibitory were tested with cortical driving alone. On the remaining 22 cells, dopamine ejection currents which depressed cortical driving, also had a marked depressant effect on spontaneous (9 neurones) and glutamate-induced (13 neurones) firing. On 9 spontaneously active cells and 10 neurones activated by glutamate, a dopamine ejection sufficient to markedly depress activity left the cortical excitation unaffected. On 5 silent neurones which were not tested with glutamate, the cortical excitation was unaffected by dopamine. The relationship between ejection current and response to cortical stimulation was current-dependent on individual cells, but varied between cells. Facilitation of cortical driving was never observed on any neurones tested with dopamine. Dopamine
and output cells
Unit responding with constant latency (mean 9.4 ms range 4-16) to electrical stimulation of the crus cerebri were normally silent. The driving was confirmed as antidromic only in those cells where the response to stimulation in the crus could be collided with the response to stimulation through the electrode in the cerebral cortex. Such a fortunate positioning of all three electrodes was not common but dopamine was tested on 7 such antidromically identified striatal output cells and it inhibited the production of an antidromic spike in all 7 cells. Glutamate excitation was inhibited on all 4 of these cells which could be tested and cortical driving was inhibited by dopamine on the 3 cells on which it could be examined. Dopamine
and acetylcholine
To investigate whether the effects of dopamine were specific to glutamate, acetylcholine was tested along with glutamate on 12 cells. Excitation (6 cells) and inhibition (4 cells) was observed with acetylcholine, 2 cells were insensitive to acetylchohne. The acetylchohne excitations were slow in onset and offset in comparison to the glutamate excitations. Dopamine inhibited the effect of both acetylchohne and gluamate on all 6 neurones tested on which acetylcholine was excitatory. Conventional
neuroleptics
The dopamine antagonists, fluphenazine and haloperidol, were tested for their ability to antagonize the effects of dopamine in this preparation.
Fluphenazine (%X75 nA) iontophoresed for 2-3 min before the ejection of dopamine antagonised the depressant effects of dopamine on both cortical driving and spontaneous activity on 6 neurones, and was ineffective on 2 neurones. During the period of antagonism, fluphenazine did not cause silent cells to fire, nor did it affect the firing rate of spontaneously active cells. The antagonism was reversible within 45 min. Haloperidol was administered peripherally by intraperitoneal injection (i.p. 0.1 mg/kg) in 8 experiments. Only one cell was studied in each experiment after the haloperidol injection. In five experiments several injection of haloperidol were given up to a cumulative dose of 0.5 mg/kg. Haloperidol did not induce spontaneous firing or affect the rate of spontaneous firing in any of the cells. In addition, the depressant effects of iontophoretic dopamine were not antagonized by i.p. haloperidol. Sulpiride Spotaneous and glutamate-induced,firing. The selective Dz antagonist sulpiride’h was used in an attempt pharmacologically to dissect the effects of dopamine on pre- and post-synaptic sites. The effects of iontophoretically-applied (-)sulpiride were investigated on 33 cells. Sulpiride (30-70 nA) did not induce firing from 27 silent cells, neither did it affect the excitatory response to glutamate in all 18 of these cells which were tested. Spontaneous activity was recorded from six cells, four were unaffected by sulpiride but in two there was a decrease in spike height and an increase in firing rate, suggestive of depolarization block. (-)Sulpiride failed to antagonize the depressant effects of iontophoretically-applied dopamine on all 15 neurones tested. Sulpiride and cortical stimulation. The response of striatal neurones to cortical stimulation was tested during the iontophoresis of (-)sulpiride. At stimulation currents which were just threshold for ex(-)sulpiride (30-70 nA) increased the citation, number of cortical stimuli which produced a spike (Fig. 3). This effect was observed on 18 cells with I cell being insensitive. Sulpiride applied for between 30 s and 2 min before the test PSTH reduced the number of stimuli which failed to produce a spike to 22”~ (SEM 6.1) of the control value. The mean number of spikes produced by 50 cortical stimuli was increased from 8.4 (2.5 SEM) to 29.9 (2.8 SEM) (Fig. 3). The increase in response was larger at higher currents but the response rate never exceeded I spike/stimulus. The (+)isomer of sulpiride was ineffective in 3 of the 5 neurones on which it was possible to test it. The facilitation of cortical stimulation by sulpiride was blocked on 6 neurones to which dopamine was applied by iontophoresis simultaneously with the sulpiride. However. this antagonism was only accomplished using ejection currents of dopamine which
D,
Receptor electrophysiology
Fig. 3. The action of sulpiride on the efficacy of cortical stimulation. In A the responses of one cell is illustrated to cortical stimuli of an intensity adjusted to give less than 10 responses in 50 trials. All the responses to 50 stimuli are shown. Calibration bars are 0.2 mV and 2.0 ms. The upper trace shown control responses, the lower the result of sulpiride application (30nA) during the stimulation. In B the effect of sulpiride on all 18 cells is summarised by plotting the total number of action potentials in response to 50 cortical stimuli. The columns are the mean values f standard error of the mean. Although the iontophoresis of dopamine was able to reduce this effect on the 6 cells on which it was tried it never did so at doses which did not themselves inhibit the responsiveness of the ceils.
353
inhibitions, also reported a small proportion of excitations in response to dopamine application. The methods used in this study are almost identical to those of Bevan, Bradshaw and Szabadi3 who reported many (50%) pure excitations with dopamine. The only difference between this study and that of Bevan et al.’ is that different electrodes were used for electrical recording and for iontophoresis; Bevan et aL3 used the same multibarrel electrode for both purposes. The results of the present study are also at variance with those of an earlier study on encephale isole cats which reported a predominance of facilitatory effects on cortical driving by iontophoretically-applied dopamine. 24 Norcross and Spehlman24 also reported that dopamine facilitated excitations evoked from stimulation of the substantia nigra, thalamus and from within the caudate nucleus itself. A minority of cells showed depression of the caudate (20%) and cortical driving (11 ‘A). Facilitation of glutamateinduced and spontaneous firing was also reported. On the basis that the dopamine ejection currents required for 50% inhibition (50nA) were five times greater than those required for a 50% facilitation (9 nA), these authors proposed the existence of distinct excitatory and inhibitory dopamine receptors on the striatal neurones with differing affinities for dopamine. In the present study, no excitatory effects of dopamine were observed over the whole range of ejection currents tested (10-100 nA). There are, however, several methodological differences which may explain the disparity between the results of these two studies. Norcross and Spehlman did not use current balancing during iontophoretic ejections. In addition, Norcross describes that electrodes were filled with drug solutions the day before use and it may be questionable whether the dopamine had remained unoxidized over this period. Dopamine contained in an iontophoretic electrode under similar conditions to those used in the present study has been shown to retain its activity in an adenylate cyclase assay over the timecourse of a typical experiment.” Dopamine and striatal physiology
also depressed cell firing in a similar manner to that described previously. DISCUSSION
The action of iontophoretically-applied dopamine
The results support the hypothesis that dopamine is an inhibitory transmitter in the striatum. Iontophoretic dopamine had a depressant action on spontaneous and cortically driven firing (Table 1), and on glutamate and acetylcholine-induced excitations, as previously described.4,‘4 In agreement with two other studies,7,‘3 no excitatory responses to iontophoretic dopamine were observed. This contrasts with many of the previous reports which, although having a predominance of
Despite the wide range used, a dopamine ejection current was never found which could affect the cortical driving without also affecting the cell postsynaptically. Therefore any presynaptic component of the depression of cortical driving was masked. The glutamate-induced and spontaneous firing of several cells (34%) was depressed by dopamine without any effect on the response to cortical driving. A similar difference in the efficacy of iontophoretic dopamine on “synaptic” and “spontaneous” activity has been observed in conscious monkeys.2s The significance of these observations is not yet clear. Thorpe (personal communication) has suggested that dopamine may act to alter the signal-to-noise ratio of striatal cells. Alternatively, the results may indicate that there is a particularly high safety margin in corticostriatal
354
J. R. Brown
and G. W. Arbuthnott
driving or that the position of the iontophoretic pipette was closer to the striatal cell bodies than to the corticostriatal synapses on which dopamine was active. Conventional
neuroleptics
The successful antagonism of the effects iontophoretic dopamine by the iontophoretically-applied antagonist fluphenazine is in contrast to the lack of effect of peripherally-applied haloperidol. Other authors2.3s.4’ have reported a similar failure of peripherally-applied antagonist on iontophoretic dopamine. The dose of haloperidol used initially (0. I mg/kg) is enough to cause a large increase in the firing of dopamine-containing cells in the substantia nigra” yet doses considerably in excess of this were still ineffective. Sulpiride
It is tempting to speculate that the facilitation of cortical driving by sulpiride may reflect the blockade of an action of dopamine on cortical terminals. That dopamine receptors on cortical terminals may exist has already been suggested by a variety of experiments on specific binding of dopamine agonists and antagonists,6.3’.38 although it seems that they may be differentiable from the receptors on the dopamine terminals and from those on the striatal cells. The nomenclature is confusing but these binding sites may be responsible for the effects of dopamine and dopaminergic agonists on glutamate release from slices of striatum “in vitro”.22.26 Recent work suggests that both the binding of sulpiride3* and the effects of dopamine on glutamate release’2.3’ are not easily fitted into simple D,, D, classification of receptors. At the doses they used, Stoof er al.” find sulpiride inactive on glutamate release, a result we have recently confirmed although we were able to show that
sulpiride antagonised the effect of apomorphine on [3H]glutamate release (P. R. Mitchell and J. R. Brown, unpublished). Clearly, because of the uncertainties of applied dose of drug in iontophoretic experiments, our results cannot provide the kind of detailed pharmacological information required to prove that the receptors on cortical terminals are of the D2 type as defined in binding studies. The facilitation of cortical driving shown in the present study supports the notion of an increased amount of transmitter release from cortical terminal during the blockade of dopamine receptors on them. Sulpiride was ineffective in altering the action of dopamine on striatal cells, suggesting that the dopamine receptor on the cell bodies is indeed not of the same type. The lack of effect of sulpiride on the spontaneous activity of striatal cells, or on their response to glutamate, strongly suggests a presynaptic site of action on the cortical terminals although the extracellular recording used in the present experiments would not have detected any change on the membrane potential of the cell. Intracellular studies need to be performed on striatal cells during sulpiride application confirm a presynaptic role for sulpiride. Conclusions
Our results suggest that there is a subclass of dopamine receptors on corticostriatal terminals which may be tonically active in anaesthetised animals. Inhibition of these receptors with sulpiride may enhance the influence of cortical activity on striatal cells. Whether this action is responsible for the therapeutic activity of the benzamides or for their motor side effects remains to be discovered. Acknowledgements-This
postgraduate McQueen
studentship;
work was supported
by an MRC
we are grateful to Dr D. S.
for his encouragement
during
this project.
REFERENCES I. Arbuthnott G. W. (1974) Spontaneous activity of single units in the striatum after unilateral destruction of the dopamine input. J. Physiol., Lond. 339, I2 I-122P. 2. Ben-Ari Y. and Kelly J. S. (1975) Dopamine-evoked inhibiton of single cells of the feline putamen and basolateral amygdala. J. Physiol., Land. 256, l-22. 3. Bevan P., Bradshaw C. M. & Szabadi E. (1975) Effects of desipramine on neuronal responses to dopamine. noradrenaline, 5-hydroxytryptamine and acetylcholine in the caudate nucleus of the rat. Br. J. Pharmac. 54, 285-293. 4. Bloom F. E., Costa R. & Salmoraghi G. C. (1965) Anaesthesia and the responsiveness of individual neurones of the caudate nucleus of the cat to acetylcholine, norepinephrine and dopamine, administered by microelectrophoresis. J. Pharmac. exp. Ther. 150, 244-252: 5. Brown J. R.. Maver M. L. & Arbuthnott G. W. (1980) The use of ultra-violet setting-- glue for microelectrode fabrication. J. qf Neurosci. ð. 3, 203-204. 6. Creese 1. (1982) Dopamine receptors explained. Trend.y Neurosci. 5, 4G43. 7. Feltz P. & De Champlain J. (I 972) Persistence of caudate unitary responses to nigral stimulation after destruction and functional impairment of the striatal dopaminergic terminals. Brain Res. 43, 595-600. 8. Fields J. Z., Reisine T. D. & Tamamura H. (1978)Loss of striatal dopaminergic receptors after intrastriatal kainic acid injection. L{fe Sci. 23, 569-574. 9. Fonnum F., Storm-Mathisen J. & Divac 1. (1981) Biochemical evidence for glutamate as neurotransmitter in corticostriatal and corticothalamic fibres in rat brain. Neuroscience 6, 863-873. 10. Garcia Munoz M. (1978) The striatonigral pathway: its role in the regulation of dopamine metabolism. Ph.D. Thesis, University of Edinburgh. I I. Hassler R., Haug P., Nitsch C.. Kim J. S. & Paik K. (1982) Effect of motor and premotor cortex ablation on concentrations of amino acids, monoamines and acetylcholine and on the ultrastructure in rat striatum. A confirmation of glutamate as the specific corticostriatal transmitter. J. Neurochem. 38, 1087-1098.
D? Receptor el~trophysioIo~
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