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Neuronal responses to 5-hydroxytryptamine and dorsal raphe stimulation within the globus pallidus of the rat
EXPERIMENTAL
NEUROLOGY
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Neuronal Responses to 5-Hydroxytryptamine and Dorsal Raphe Stimulation within the Globus Pallidus of the Rat M. N. PERKINS AND T. W. STONE’ Department of Physiology, St. George’s Hospital Medical School, Cranmer Terrace, Tooting, London S WI 7 ORE, United Kingdom Received May 3, 1982; revision received August 20, 1982 The projection from the dorsal raphe nucleus (DRN) to the globus pallidus (GP) was investigated electrophysiologically, in the urethane-anesthetized rat together with the responsiveness of cells in the GP to 5-hydroxytryptamine (5-HT) and noradrenaline (NA). The majority of spontaneously active cells in the GP had high regular firing rates. They were unaffected by both DRN stimulation (69/83 cells) and iontophoretically applied 5-HT (38/63 units) or NA (30/42 units) but were inhibited by GABA. A few cells (N = 10) were recorded from, that were spontaneously active but with a much lower and less regular firing rate, which, however, seemed to be much more responsive to 5-HT. In addition, DL-homocysteic acid (DLH) was used to activate silent cells and all seven cells activated in this manner were inhibited by 5-HT. In addition 5/6 cells that had their firings maintained by DLH were inhibited by stimulation of the dorsal raphe. The results show a lack of responsiveness to both 5HT and DRN stimulation of the typically regular spontaneously active pallidal neurons. There seems to be a small population of normally quiescent cells, however, that is sensitive to 5-HT and receives an input from the DRN.
INTRODUCTION Increasing interest is being paid to the ascending Shydroxytryptamine (SHT)-containing pathways in the forebrain. To date most studies have been concerned either with defining the projections anatomically using a variety of techniques (3, 4, 13, 14, 18, 24, 29), or with the behavioral consequences of manipulation of 5-HT metabolism (8, 9, 12). Electrophysiological studies of 5-HT projections have been fewer, dealing with structures Abbreviations: GP-globus pallidus, 5-HT-5-hydroxytryptamine, NA-noradrenaline, GABA--y-aminobutyric acid, RF-regular firing, DLH-DL-homocysteic acid, DRN-dorsal raphe nucleus. ’ This work was supported by the National Fund for Research into Crippling Diseases. 118 0014-4886/83/010118-12$03.00/O Copyright Q 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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such as the hippocampus, neocortex, and some parts of the basal ganglia (9, 17, 20, 25, 26, 31, 32). The globus pallidus (GP) is one of the major nuclei of the basal ganglia intimately connected to the other components of this system with the possibility for integration that this entails (7). It has been heavily implicated in several motor disorders, in particular Huntington’s chorea (15), although the precise nature of the control it exerts over the motor activity is not clear. The GP contains high concentrations of 5-HT (2 1) and immunofluorescent and autoradiographic work has demonstrated the existence of serotonergic terminals within that nucleus (1, 2, 18). A physiologic relevance for these findings is suggested by the reduction of 5-HT content and receptors in the pallidum associated with certain choreatic diseases (6, 16). Furthermore, a variety of techniques including retro- or anterograde labeling and autoradiographic studies have shown a clear projection to the GP from the dorsal raphe nucleus (DRN) (3, 4, 18, 29). An electrophysiological study of this projection, however, does not seem to have been undertaken. Our interest in extrapyramidal motor disorders (23) (in particular hemiballismus) had drawn our attention to this pathway owing to the possible involvement of the raphe nuclei complex and 5-HT in behavior and motor control. We therefore studied electrophysiologically the projection from the DRN to the GP and of the responsiveness of pallidal neurons to exogenous 5-HT. MATERIALS
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Male Wistar rats were anesthetized with urethane (1.3 g kg-‘, i.p.) and placed in a stereotaxic frame adjusted for use with the Pellegrino et al. (22) stereotaxic atlas. The brain was then exposed above the recording and stimulating sites and the dura removed or slit respectively. Body temperature was maintained at 37°C by means of a heating blanket and rectal probe. Stimulation. A single coaxial bipolar electrode (Rhodes Medical Instruments, SNE 100) with an outside diameter of 0.25 mm was stereotaxicahy positioned in the dorsal raphe nuclei (22) (5.8 mm posterior to bregma, 5.6 mm below the surface of the brain, and 0.1 mm lateral to midline). Monophasic, square wave pulses of 0.5 ms duration were delivered from an isolated constant current stimulator controlled by a programmable clock and Grass S44 stimulator. Initially response testing was with single pulses at 1 Hz but if no response emerged this was increased to a burst of 9 pulses at 200 Hz delivered once per second. Stimulating currents were 80 to 700 PA. Recording and Drug Application. Seven-barrel micropipettes were used to deliver the following drugs by conventional microiontophoretic methods: Gamma-aminobutyric acid (GABA, 100 mM); DL-homocysteic acid, (DLH,
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100 m&Q; L-glutamic acid sodium (200 mM); 5-hydroxytryptamine creatinine sulphate (5-HT, 200 mM) noradrenaline bitartrate (NA, 200 mM); and methysergide hydrogen maleinate (15 IYIM). In addition, one barrel always contained 165 mM NaCl for current balancing. Recording of extracellular single-unit activity was accomplished with a single microelectrode attached to the side of the drug pipette (28). Amplified action potentials were displayed on an oscilloscope and passed through a window discriminator, the output pulses of which generated poststimulus time histograms with either a Neurolog or Ortec histogram generator. The histograms were a composite of 32,64, or 128 sweeps with a variable sweep time depending on the time course of the response. The stability of the recordings of action potentials both spatially and temporally, together with the presence of responses to putative neurotransmitters applied iontophoretically, identified them as somal, not axonal recordings. Localization ofRecording and Stimulating Electrodes.At the termination of each experiment, the position of the recording electrode was marked by inserting into its last “track” a single micropipette with its tip broken down to 8 to 10 pm, containing pontamine sky blue 6BX. A 1-mA negative current was passed through the barrel for 5 min, thus ejecting the dye which was subsequently located under light microscopy (see Fig. 1). Checks were made using two dye-filled electrodes to ensure that this indirect method of marking was accurate to within 100 to 200 pm. Only cells located within the boundaries of the globus pallidus were included in the analysis of results. Direct current (100 PA) was passed through the stimulating electrode for 5 s thus depositing a small quantity of iron from the tip. The animal was then perfused through the aorta with 20 ml 10% potassium ferrocyanide in 10% Formalin saline. After fixation, frozen sections were cut at 40 to 50 pm, stained with cresyl violet, and examined to confirm placement of electrodes (see Fig. 1). RESULTS Recordings were made from 105 cells in the GP which were spontaneously active, with a very regular firing pattern ranging from 20 to 40 spikes/s (3 1 f 1.Ol spikes/s, mean + SE). We noted elsewhere that these cells are characteristic of the pallidum (23) and refer to them as regular firing or RF cells. In total, 63 RF cells were tested with 5-HT using currents of 14 to 100 nA applied for as long as 60 s. Only seven (11%) neurons showed a clear inhibitory response, 18 (29%) were weakly inhibited by as much as approximately 50%; in these latter cases increasing the 5-HT ejecting current rarely produced any further decrease in firing rate. With currents in excess
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FIG. 1. Photomicrographs of coronal sections through the rat brain. upper-marking of the recording electrode with pontamine sky blue. Lower-kxalization of the stimulating electrode to the dorsal raphe.
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FIG. 2. Ratemeter records of neuronal activity. A-the loss of sensitivity of cells in the globus pallidus (GP) to 5hydroxytryptamine (5-HT, squares) and noradrenaline (NA, circles) during a single electrode penetration. The figures above the individual records are the depth in micrometers from the surface of the cortex. As seen in the upper two traces, both 5-HT (3 1 nA) and NA (53 nA) clearly inhibited cell firing in the cortex. The following two traces, at a depth that places the cells in the GP (subsequently confirmed histologically), there was almost no response to either 5-HT (6 1 nA) or NA (53 nA). These two cells were good examples of “RF cells” (see text). The last record shows the response of a cell after removal and reinsertion of the same electrode in an adjacent region of cortex to both 5-HT (61 nA) and NA (53 nA). Ba ratemeter record of another RF cell in the GP showing that although no response could be obtained with 5-HT (57 nA) or NA (105 nA), the cell was strongly inhibited by GABA (triangles, 23 nA).
of 80 to 100 nA the 5-HT barrel tended to block, thus limiting the maximum amount of 5-HT which could be ejected. On each of 16 of these cells tested, GABA produced a powerful inhibition of spontaneous activity when ejected with currents of 15 to 40 nA for as long as 5 to 6 s (see Fig. 2). Noradrenaline was also included in some of the experiments and was tested on 42 RF cells. Thirty (7 1%) of these were not responsive to NA when applied with currents to 105 nA for 60 to 90 s (Fig. 2). The remaining cells responded with either a clear inhibition of activity ( 15 units, 12%) or a weak reduction in firing rate (seven cells, 17%). With these latter cells, as with SHT, usually it was not possible to produce complete cessation of
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FIG. 3. Ratemeter record of a regular firing cell in the globus pallidus illustrating partial antagonism of responses to S-hydroxytryptamine (5-HT, 45 nA) by methywgide (MEW, 20 nA). Note the reduction in firing rate of the cell during the application of METH.
activity by increasing the ejection current. GABA was included as an agonist on eight cells not responding to NA and in all cases produced a clear, inhibitory response (Fig. 2). The possibility had to be considered that, although numerous electrodes were used, the lack of neuronal responsiveness to 5-HT and NA could be due to a failure of ejection. Therefore on three occasions unitary activity was recorded in the cortex and 5-HT and/or NA tested on these cells, before descending into the pallidum. Finally, responses of cortical cells (in an adjacent region of undamaged cortex) to 5-HT and NA were tested again upon withdrawal of the electrode from the pallidum. One of these electrode tracks is illustrated in Fig. 2. As seen, 5-HT and NA were clearly inhibitory in the cortex but at the depth of the pallidum there were no responses to the agonists. Upon retesting cells in an adjacent area of cortex, inhibitory responses were again obtained. The firing rate of 2 RF cells was increased by ejecting DL-homocysteic acid (DLH) from one barrel of the micropipette. The iontophoresis of 5HT did not inhibit this, increased firing rate or the spontaneous activity of these cells. To determine the specificity of the few 5-HT inhibitions that were obtained, an attempt was made to antagonize these responses by concurrent iontophoretic application of methysergide. Twelve RF cells that responded to 5-HT (either weakly or strongly) were tested with methysergide. It proved to be very difficult to eject methysergide, especially with the currents and duration of application (to 80 nA for 8 min) that were sometimes required to yield any effect. Furthermore, methysergide itselfhad variable but marked effects on spontaneous activity. On six of these 12 cells, methysergide depressed the firing rate and on two of the remaining units excited the cell. Only on 4/12 cells was there any apparent reduction of the response to 5HT. A typical example is shown in Fig. 3 where there seemed to be antagonism of 5-HT but also a general depression of firing rate rendering interpretation difficult. Nonregular Firing Cells. Very few neurons (N = 10) were encountered
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FIG. 4. Peristimulus time histograms of cells in the globus pallidus (GP) after stimulation of the dorsal raphe nucleus (DRN), the point of stimulation being indicated by the arrow head. a and b-Orthodromic excitation and inhibition respectively; b also illustrates a postinhibition excitation, typical of these responses. c-An example of a longer duration inhibitory response (note the change in time scale). All histograms are a composite of 128 sweeps; the ordinate is in spikes per bin.
in the pallidum that were not RF cells and their spikes were typically very small, making discrimination above baseline difficult. In a few experiments, however, an attempt was made to ascertain whether or not such cells were responsive to 5-HT. In two of three spontaneous active cells (firing rates 1 to 15 Hz) which were recorded for long enough to test 5-HT, clear inhibitory responses were observed using currents of 21 to 33 nA to 10 s. On a few occasions DLH was included in the electrode and was used to fire quiescent cells using currents of 1 to 10 nA. Seven cells were activated in this manner, and 5-HT with currents similar to those described above inhibited all of them, although two were only weakly responsive. Stimulation of the Dorsal Raphe. Dorsal raphe stimulation was tested on 83 spontaneously active pallidal RF cells, the position of the stimulating electrodes being subsequently confirmed to be in the dorsal raphe. The stimulation ranged from single 0.5-ms square wave pulses at 150 PA to a train of nine pulses of 700 @A at 200 Hz. Typically a train of three pulses of 300 PA at 150 Hz was used. Despite these relatively high stimulation currents, only 14 cells responded to the stimulus; 15 cells were orthodromically excited (Fig. 4) with an increase in firing lasting 24 to 70 ms and with variable latencies of 22 to 72 ms, (35.9 + 2.2). A response was deemed to be orthodromically excited if there was variation in the latency of the driven spike, failure to follow three stimuli at 200 Hz and, if spontaneously active, an inability to produce collision between a spontaneous and an evoked action potential. On two of these five cells the excitatory response took the form of an interesting oscillation of firing rate with peaks of activity every 35 to 40 ms and lasting
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FIG. 5. Peristimulus time histograms of the globus pallidus cell (referred to in the text), that responded to a stimulus to the dorsal raphe nucleus consisting of a train of five pulses at 200 Hz (hence the initial arteftiual inhibition) with an oscillatory response (a). b-&r methysergide had been iontophoresed with 100 nA for 4 min. As seen, the oscillatory nature of the response was largely lost. c-recovery of the original response 23 min after termination of the methysergide ejection. All histograms are a composite of 128 sweeps and the ordinate is in spikes per bin.
in total for 230 ms (see Fig. 5). Nine cells were inhibited by the stimulus (Fig. 4) and again both the latency and duration of inhibition were variable, the latency ranging from undetectable (due to stimulus artefact) to 50 ms, and the duration from 20 to 122 ms (52.7 +- 15.0 ms). Typically, the inhibition was followed by an increase in activity lasting as long as 100 to 150 ms. On four cells responding to stimulation of the dorsal raphe, methysergide was applied with currents of 30 to 100 nA for as long as 16 min during successive retesting of the cell. The same problems were encountered as described above with difficulty in ejecting the antagonist, and changes in basal firing rate complicated interpretation. Two of these four cells responded with inhibition to dorsal raphe stimulation and one of these showed a slight reduction in response with methysexgide, the other being unaffected. The remaining two were excited by dorsal raphe stimulation and one of these was, interestingly, an example of the oscillatory type of response just described. This was clearly affected by methysergide with a reduction in the frequency of the peaks of activity (see Fig. 5). Upon terminating methysergide, complete recovery of the original response was obtained. The remaining responsive cell was unaffected by methysergide. Six cells whose firing was maintained by DLH application were tested with dorsal raphe stimulation (3 pulses, 150 Hz, 300 FA); five of these responded with inhibition of firing for 79 to 238 ms with latencies from 26 to 46 ms (36.2 + 3.3 ms). In addition the other cell showed a massive increase in firing rate beginning 105 ms after the stimulus and lasting 456 ms.
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DISCUSSION The results show that there is not very much effect of both dorsal raphe stimulation and of 5-HT and NA application on neuronal activity in the rat globus pallidus. These findings are surprising in view of autoradiographic and fluorescence studies which show that a projection arises from the dorsal raphe nucleus and ascends in the ventral forebrain to terminate in the pallidum (3, 4, 18, 29). We are satisfied that the negative nature of the findings are not a result of the use of the iontophoretic method in that 5-HT responses could be obtained in cortex but not in pallidum using the same electrode. In addition, crossing of electrode tips can be excluded as GABA was still highly effective in depressing cell firing. 5-Hydroxytryptamine has been shown to have a depressant action on neuronal firing rates in most regions of the forebrain studied including the basal ganglia (30). Thus neurons in the neocortex, hippocampus, and caudate-putamen are all responsive to this drug, inhibition being the predominant response (25,30). The possibility that the RF cells recorded here were in fact, axons is most unlikely in view of the spatial and temporal stability of the recordings together with the ability to respond to both GABA and DLH. Nevertheless, the lack of effect of 5-HT is difficult to reconcile with the presence of 5-HT terminals and receptors within the pallidum ( 1, 2, 5, 18). It is possible that in the case of the RF cells the 5-HT receptors are mainly presynaptic, therefore reducing the likelihood of observing an effect to 5HT applied iontophoretically. Such presynaptic actions of 5-HT have been observed in the peripheral nervous system ( 10, 19). Another possibility is that the 5-HT input to the pallidum terminates on cells other than RF cells. This is supported by the apparent responsiveness to 5-HT of the smaller, typically nonspontaneously active cells that were induced to fire with DLH. The failure of 5-HT to inhibit the DLH-induced increment of firing on RF cells supports the conclusions that these cells are not responsive to 5-HT and at the same time indicates that the 5-HT depression of artificially excited non-RF cells is not the result of a pharmacological antagonism of DLH by 5-HT. It might be argued that, as a total of 40% of RF cells responded to 5-HT, albeit mostly very weakly, this constitutes a reasonably positive result. When one considers that 5-HT was strongly inhibitory in the majority of cases in the neocortex and on non-RF cells, however, then the relative paucity of such responses in RF cells is a surprisingly negative finding. The responses after stimulation of the dorsal raphe were consistent with the two sets of iontophoretic data. Thus the RF cells did not appear to receive an input from the dorsal raphe and were not sensitive to 5-HT.
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Conversely, the much less common smaller, typically nonspontaneous cells were both sensitive to 5-HT and inhibited by stimulation of the raphe. Although it is tempting to suggest from this correlation that the raphe input is serotonergic, it must be remembered that non-5-HT containing cells are situated in the raphe (27) and may be the source of these alTerent fibers. The results with the antagonist, methysergide, are far from clear. Difficulty with the iontophoretic use of 5-HT antagonists has been noted before (11, 26) and may reflect a limitation of this method of ejection. Although there was not a clear reversal of response to either exogenous 5-HT or raphe stimulation this may only be reflecting the lack of sufficient ejection of the antagonist to block the 5-HT receptors coupled with the difficulties arising from the marked effects of methysergide itself. Conversely, it may suggest that 5-HT is not the transmitter involved in the stimulus-evoked responses. This input may therefore arise from the nonserotonergic neurons of the raphe. It should be emphasized, however, that in these experiments it was only RF cells that were tested with methysergide and only a small proportion of these received any input from the dorsal raphe. Further experiments are planned to investigate the much rarer nonspontaneously active cells. The insensitivity of the RF cells to NA is also interesting, implying an absence of receptors for NA as seems to be the case with 5-HT. Unfortunately, the smaller cell type was not tested with NA. The lack of responses to 5-HT of RF cells in the pallidum may be related to the scarcity of classical synaptic profiles (2, 5). In the cerebral cortex the scarcity of classical synaptic arrangements has been proposed as an explanation for the slow and prolonged nature of neuronal responses to exogenous noradrenaline, and for the need for rather intense stimulation of the locus ceruleus to evoke responses (5). The diffise release of transmitter presumed to occur from the varicosities makes it very difficult to elicit events clearly related to a single or double stimulus. As few classical aminergic synaptic profiles have been detected in the striatum (2, 5), a similar explanation may account for the absence of stimulus-related responses following raphe stimulation in the pallidum despite a well defined anatomic projection. The control exerted by the raphe may be of a more modulatory nature; chronic activation of the 5-HT ascending pathways releasing 5-HT into the extracellular milieu in a manner similar to that proposed for NA in the cerebral cortex (5). Acute activation of such a pathway may well not be sufficient to produce a detectable effect at the single-unit level. An alternative explanation for the lack of effect of 5-HT and NA applied iontophoretically is that the receptors for these amines are dendritically situated, whereas the recordings here were mainly somal. This cannot be excluded but is unlikely in our opinion as there did not appear to be any
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change in sensitivity as the electrode was maneuvered closer to the cell and it would not explain the lack of stimulus-induced responses after raphe stimulation. In summary, these results show a surprising lack of responsiveness to 5HT of the predominant cell type recordable electrophysiologically in the pallidum as well as the absence of an input from the dorsal raphe nuclei. There does, however, seem to be a much smaller population of cells, normally silent in our preparations but excitable by DLH which is both sensitive to 5-HT and NA and receives an innervation from the raphe. REFERENCES 1. AGHAJANIAN, G. K., M. S. KUHAR, AND R. H. ROTH. 1973. Serotonin-containing neuronal perikarya and terminals. Differential effects of p-chlorophenylalanine. Brain Res. 54: 85-101. 2. ARKISON, M., AND I. S. DE LA MANCHE. 1980. High-resolution radiographic study of the serotonin innervation of the rat corpus striatum after intraventricular administration of (‘H) 5-hydroxytryptamine. Neuroscience 5: 229-240. 3. A~MITIA, E. C. 1978. The serotonin producing neurons ofthe midbrain median and dorsal raphe nuclei. Pages 233-3 14 in L. L. IVERSEN, S. D. IVERSEN,AND S. H. SNYDER, Bds., Handbook of Psychopharmacology. Plenum, New York. 4. AZMITIA, E. C., AND M. SEGAL. 1978. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. Camp. Neural. 179: 641-667.
5. BEAUDET, A., AND L. DESCARRIES. 1978. The monoamine innervation of rat cerebral cortex: synaptic and non-synaptic axon terminals. Neuroscience 3: 851-860. 6. BERNHEIMER, H., AND 0. HORNYKIEWICZ. 1973. Brain amines in Huntington’s chorea. Adv.
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7. CARPENTER, M. B. 1976. Anatomy of the basal ganglia and related nuclei: a review. Adv. Neural.
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8. DRAY, A. 1980. Serotonin in the basal ganglia: functions and interactions with other neuronal pathways. J. Physiol. (Paris) 77: 393-403. 9. DRAY, A., J. DAVIES, N. R. OAKLEY, P. TONGROACH, AND S. VELLUCI. 1978. The dorsal and medial raphe projections to the substantia nigra in the rat. Electrophysiological biochemical and behavioural observations. Brain Res. 151: 431-442. 10. DUN, N. J., AND A. G. KARCZMAR. 1981. Evidence for a presynaptic inhibitory action of 5-hydroxytryptamine in a mammalian sympathetic ganglion. J. Pharmacol. Exp. Ther. 217: 714-718. 11. PULLER, R. W. 1980. Pharmacology of central serotonin neurons. Ann. Rev. Pharmacol. Toxicol. 20: 11 l-127. 12. JAMES, T. A., AND M. S. STARR. 1980. Rotational behavioural elicited by 5-HT in the rat: evidence for an inhibitory role of 5-HT in the substantia nigra and corpus striatum. J. Pharm. Pharmacol. 32: 196-200. 13. VAN DER KOOY, O., AND T. HATTORI. 1980. Dorsal raphe cells with collateral projection to the caudate-putamen and substantia nigra: a fluorescent retrograde double labelling study in the rat. Brain Res. 186: l-7. 14. VAN DER KOOY, O., AND T. HATTORI. 1980. Bilaterally situated dorsal raphi: cell bodies have only forebrain projections in rat. Brain Res. 192: 550-554.
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15. LANGE, H., G. THORNER, A. HOPF, AND K. F. SCHRODER. 1978. Morphometric studies of the neuropatholo&al changes in choreatic diseases. J. Neural. Sci. 28: 401-425. 16. LLOYD, K. G., AND 0. HORN~KIE~ICZ. 1974. Dopamine and other monoamines in the basal ganglia: relation to brain dysfunction. Pages 26-35 in P. SEERMAN AND G. M. BROWN, Eds., Frontiers in neurology and neuroscience research. Toronto University, Toronto. 17. MILLER, J. J., T. L. RICHARDSON,H. C. FIBIGER, AND H. MCLENNAN. 1975. Anatomical and electrophysiolo@cal identification of a projection from the mesencephalic raphe to the caudate-putamen in the rat. Brain Res. 97: 133-138. 18. MOORE, R. Y., A. E. HALARIS, AND B. E. Lo~cs. 1978. Serotonin neurones of the midbmin raphe: ascending projections. J. Comp. Neurol. 180: 417-438. 19. NORTH, R. A., G. HENDERSON, Y. KATAYAMA, AND S. M. JOHNSON. 1980. Electrophysiolo&al evidence for presynaptic inhibition of acetylcholine release by 5hydroxytryptamine in the enteric nervous system. Neuroscience 5: 58 l-586. 20. OLPE, H., AND W. P. KOELLA. 1977. The response of striatal cells upon stimulation of the dorsal and median raphe nuclei. Brain Res. 122: 357-360. 21. PALKOVITS, M., M. BRO~NSTRIN, AND J. M. SAAVEDRA. 1974. Serotonin contents of the brain stem nuclei in the rat. Brain Res. 80: 237-289. 22. ~ELLEGRINO, L. J., A. S. PELLEGRINO, AND A. J. CUSHMAN. 1979. A stereotaxic atlas of the rat brain. Plenum, New York. 23. PERKINS, M. N., AND T. W. STONE. 198 1. Iontophomtic studies on pallidal neurones and the projection from the subthalamic nucleus. Q. J. Exp. Physiol. 66: 225-236. 24. SAKAI, K., 0. GALVERT, M. TOURET, AND M. JOVET. 1977. AtTerent connections of the nucleus raphe dorsalis in the cat as visual&d by the horseradish peroxidase technique. Brain Res. 137: 1 l-35. 25. SASTRY, B. S. R., AND J. W. ~HILLIS. 1977. Inhibition of cerebral cortical neurones by a 5-hydroxytryptamine pathway from median raphe nucleus. Can. J. Physiol. Pharmacol. 55: 737-743. 26. SEGAL, M. 1975. Physiological and pharmacolot@l evidence for a serotonergic projection to the hippocampus. Brain Res. 94: 115-131. 27. STEINBRUSCH,H. W. M., 0. VAN DER Koou, A. A. J. VERHOI%TAD, AND A. PELLEGRwo. 1980. Serotonergic and non-serotonergic projection from the nucleus raphe do&is to the camlate.-putamen complex in the rat, studied by a combined immunofluomscence and fluorescent retrograde axonal labelling technique. Neurosci. L&t. 19: 137-142. 28. STONE, T. W. 1973. Cortical pyramidal tract intemeurones and their sensitivity to L-glutamic acid. J. Physiol. (London) 233: 21 l-225. 29. SWBO, J. 1980. Distribution of stiiatal atferents fromthe mesencephalon in the cat. Brain Res. 188: 3-21. 30. TEBECIS, A. K. 1974. Transmitters and identified neurons in the mammalian central nervous system. Scientechnica, Bristol. 3 1. WINSON, J. 1980. InIIuence of raphe nuclei on neuronal transmission from perforant pathway &rough dentate gyrus. J. Neurophysiol. 44: 937-950. 32. YAMAMOTO, M., AND S. MURAYAMA. 1980. Effect of the serotonergic system on the caudate nucleus. Pharmacology u): 3 IO-3 15.
NEUROLOGY
79, 118- 129 ( 1983)
Neuronal Responses to 5-Hydroxytryptamine and Dorsal Raphe Stimulation within the Globus Pallidus of the Rat M. N. PERKINS AND T. W. STONE’ Department of Physiology, St. George’s Hospital Medical School, Cranmer Terrace, Tooting, London S WI 7 ORE, United Kingdom Received May 3, 1982; revision received August 20, 1982 The projection from the dorsal raphe nucleus (DRN) to the globus pallidus (GP) was investigated electrophysiologically, in the urethane-anesthetized rat together with the responsiveness of cells in the GP to 5-hydroxytryptamine (5-HT) and noradrenaline (NA). The majority of spontaneously active cells in the GP had high regular firing rates. They were unaffected by both DRN stimulation (69/83 cells) and iontophoretically applied 5-HT (38/63 units) or NA (30/42 units) but were inhibited by GABA. A few cells (N = 10) were recorded from, that were spontaneously active but with a much lower and less regular firing rate, which, however, seemed to be much more responsive to 5-HT. In addition, DL-homocysteic acid (DLH) was used to activate silent cells and all seven cells activated in this manner were inhibited by 5-HT. In addition 5/6 cells that had their firings maintained by DLH were inhibited by stimulation of the dorsal raphe. The results show a lack of responsiveness to both 5HT and DRN stimulation of the typically regular spontaneously active pallidal neurons. There seems to be a small population of normally quiescent cells, however, that is sensitive to 5-HT and receives an input from the DRN.
INTRODUCTION Increasing interest is being paid to the ascending Shydroxytryptamine (SHT)-containing pathways in the forebrain. To date most studies have been concerned either with defining the projections anatomically using a variety of techniques (3, 4, 13, 14, 18, 24, 29), or with the behavioral consequences of manipulation of 5-HT metabolism (8, 9, 12). Electrophysiological studies of 5-HT projections have been fewer, dealing with structures Abbreviations: GP-globus pallidus, 5-HT-5-hydroxytryptamine, NA-noradrenaline, GABA--y-aminobutyric acid, RF-regular firing, DLH-DL-homocysteic acid, DRN-dorsal raphe nucleus. ’ This work was supported by the National Fund for Research into Crippling Diseases. 118 0014-4886/83/010118-12$03.00/O Copyright Q 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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such as the hippocampus, neocortex, and some parts of the basal ganglia (9, 17, 20, 25, 26, 31, 32). The globus pallidus (GP) is one of the major nuclei of the basal ganglia intimately connected to the other components of this system with the possibility for integration that this entails (7). It has been heavily implicated in several motor disorders, in particular Huntington’s chorea (15), although the precise nature of the control it exerts over the motor activity is not clear. The GP contains high concentrations of 5-HT (2 1) and immunofluorescent and autoradiographic work has demonstrated the existence of serotonergic terminals within that nucleus (1, 2, 18). A physiologic relevance for these findings is suggested by the reduction of 5-HT content and receptors in the pallidum associated with certain choreatic diseases (6, 16). Furthermore, a variety of techniques including retro- or anterograde labeling and autoradiographic studies have shown a clear projection to the GP from the dorsal raphe nucleus (DRN) (3, 4, 18, 29). An electrophysiological study of this projection, however, does not seem to have been undertaken. Our interest in extrapyramidal motor disorders (23) (in particular hemiballismus) had drawn our attention to this pathway owing to the possible involvement of the raphe nuclei complex and 5-HT in behavior and motor control. We therefore studied electrophysiologically the projection from the DRN to the GP and of the responsiveness of pallidal neurons to exogenous 5-HT. MATERIALS
AND
METHODS
Male Wistar rats were anesthetized with urethane (1.3 g kg-‘, i.p.) and placed in a stereotaxic frame adjusted for use with the Pellegrino et al. (22) stereotaxic atlas. The brain was then exposed above the recording and stimulating sites and the dura removed or slit respectively. Body temperature was maintained at 37°C by means of a heating blanket and rectal probe. Stimulation. A single coaxial bipolar electrode (Rhodes Medical Instruments, SNE 100) with an outside diameter of 0.25 mm was stereotaxicahy positioned in the dorsal raphe nuclei (22) (5.8 mm posterior to bregma, 5.6 mm below the surface of the brain, and 0.1 mm lateral to midline). Monophasic, square wave pulses of 0.5 ms duration were delivered from an isolated constant current stimulator controlled by a programmable clock and Grass S44 stimulator. Initially response testing was with single pulses at 1 Hz but if no response emerged this was increased to a burst of 9 pulses at 200 Hz delivered once per second. Stimulating currents were 80 to 700 PA. Recording and Drug Application. Seven-barrel micropipettes were used to deliver the following drugs by conventional microiontophoretic methods: Gamma-aminobutyric acid (GABA, 100 mM); DL-homocysteic acid, (DLH,
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100 m&Q; L-glutamic acid sodium (200 mM); 5-hydroxytryptamine creatinine sulphate (5-HT, 200 mM) noradrenaline bitartrate (NA, 200 mM); and methysergide hydrogen maleinate (15 IYIM). In addition, one barrel always contained 165 mM NaCl for current balancing. Recording of extracellular single-unit activity was accomplished with a single microelectrode attached to the side of the drug pipette (28). Amplified action potentials were displayed on an oscilloscope and passed through a window discriminator, the output pulses of which generated poststimulus time histograms with either a Neurolog or Ortec histogram generator. The histograms were a composite of 32,64, or 128 sweeps with a variable sweep time depending on the time course of the response. The stability of the recordings of action potentials both spatially and temporally, together with the presence of responses to putative neurotransmitters applied iontophoretically, identified them as somal, not axonal recordings. Localization ofRecording and Stimulating Electrodes.At the termination of each experiment, the position of the recording electrode was marked by inserting into its last “track” a single micropipette with its tip broken down to 8 to 10 pm, containing pontamine sky blue 6BX. A 1-mA negative current was passed through the barrel for 5 min, thus ejecting the dye which was subsequently located under light microscopy (see Fig. 1). Checks were made using two dye-filled electrodes to ensure that this indirect method of marking was accurate to within 100 to 200 pm. Only cells located within the boundaries of the globus pallidus were included in the analysis of results. Direct current (100 PA) was passed through the stimulating electrode for 5 s thus depositing a small quantity of iron from the tip. The animal was then perfused through the aorta with 20 ml 10% potassium ferrocyanide in 10% Formalin saline. After fixation, frozen sections were cut at 40 to 50 pm, stained with cresyl violet, and examined to confirm placement of electrodes (see Fig. 1). RESULTS Recordings were made from 105 cells in the GP which were spontaneously active, with a very regular firing pattern ranging from 20 to 40 spikes/s (3 1 f 1.Ol spikes/s, mean + SE). We noted elsewhere that these cells are characteristic of the pallidum (23) and refer to them as regular firing or RF cells. In total, 63 RF cells were tested with 5-HT using currents of 14 to 100 nA applied for as long as 60 s. Only seven (11%) neurons showed a clear inhibitory response, 18 (29%) were weakly inhibited by as much as approximately 50%; in these latter cases increasing the 5-HT ejecting current rarely produced any further decrease in firing rate. With currents in excess
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FIG. 1. Photomicrographs of coronal sections through the rat brain. upper-marking of the recording electrode with pontamine sky blue. Lower-kxalization of the stimulating electrode to the dorsal raphe.
Y
PERKINS
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AND STONE
960pm
2151
1660
ilihk E
i
r
0-
60s
i
FIG. 2. Ratemeter records of neuronal activity. A-the loss of sensitivity of cells in the globus pallidus (GP) to 5hydroxytryptamine (5-HT, squares) and noradrenaline (NA, circles) during a single electrode penetration. The figures above the individual records are the depth in micrometers from the surface of the cortex. As seen in the upper two traces, both 5-HT (3 1 nA) and NA (53 nA) clearly inhibited cell firing in the cortex. The following two traces, at a depth that places the cells in the GP (subsequently confirmed histologically), there was almost no response to either 5-HT (6 1 nA) or NA (53 nA). These two cells were good examples of “RF cells” (see text). The last record shows the response of a cell after removal and reinsertion of the same electrode in an adjacent region of cortex to both 5-HT (61 nA) and NA (53 nA). Ba ratemeter record of another RF cell in the GP showing that although no response could be obtained with 5-HT (57 nA) or NA (105 nA), the cell was strongly inhibited by GABA (triangles, 23 nA).
of 80 to 100 nA the 5-HT barrel tended to block, thus limiting the maximum amount of 5-HT which could be ejected. On each of 16 of these cells tested, GABA produced a powerful inhibition of spontaneous activity when ejected with currents of 15 to 40 nA for as long as 5 to 6 s (see Fig. 2). Noradrenaline was also included in some of the experiments and was tested on 42 RF cells. Thirty (7 1%) of these were not responsive to NA when applied with currents to 105 nA for 60 to 90 s (Fig. 2). The remaining cells responded with either a clear inhibition of activity ( 15 units, 12%) or a weak reduction in firing rate (seven cells, 17%). With these latter cells, as with SHT, usually it was not possible to produce complete cessation of
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3..p tiT45:
_ _ _ . . . . . METH 20
0605
FIG. 3. Ratemeter record of a regular firing cell in the globus pallidus illustrating partial antagonism of responses to S-hydroxytryptamine (5-HT, 45 nA) by methywgide (MEW, 20 nA). Note the reduction in firing rate of the cell during the application of METH.
activity by increasing the ejection current. GABA was included as an agonist on eight cells not responding to NA and in all cases produced a clear, inhibitory response (Fig. 2). The possibility had to be considered that, although numerous electrodes were used, the lack of neuronal responsiveness to 5-HT and NA could be due to a failure of ejection. Therefore on three occasions unitary activity was recorded in the cortex and 5-HT and/or NA tested on these cells, before descending into the pallidum. Finally, responses of cortical cells (in an adjacent region of undamaged cortex) to 5-HT and NA were tested again upon withdrawal of the electrode from the pallidum. One of these electrode tracks is illustrated in Fig. 2. As seen, 5-HT and NA were clearly inhibitory in the cortex but at the depth of the pallidum there were no responses to the agonists. Upon retesting cells in an adjacent area of cortex, inhibitory responses were again obtained. The firing rate of 2 RF cells was increased by ejecting DL-homocysteic acid (DLH) from one barrel of the micropipette. The iontophoresis of 5HT did not inhibit this, increased firing rate or the spontaneous activity of these cells. To determine the specificity of the few 5-HT inhibitions that were obtained, an attempt was made to antagonize these responses by concurrent iontophoretic application of methysergide. Twelve RF cells that responded to 5-HT (either weakly or strongly) were tested with methysergide. It proved to be very difficult to eject methysergide, especially with the currents and duration of application (to 80 nA for 8 min) that were sometimes required to yield any effect. Furthermore, methysergide itselfhad variable but marked effects on spontaneous activity. On six of these 12 cells, methysergide depressed the firing rate and on two of the remaining units excited the cell. Only on 4/12 cells was there any apparent reduction of the response to 5HT. A typical example is shown in Fig. 3 where there seemed to be antagonism of 5-HT but also a general depression of firing rate rendering interpretation difficult. Nonregular Firing Cells. Very few neurons (N = 10) were encountered
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b
FIG. 4. Peristimulus time histograms of cells in the globus pallidus (GP) after stimulation of the dorsal raphe nucleus (DRN), the point of stimulation being indicated by the arrow head. a and b-Orthodromic excitation and inhibition respectively; b also illustrates a postinhibition excitation, typical of these responses. c-An example of a longer duration inhibitory response (note the change in time scale). All histograms are a composite of 128 sweeps; the ordinate is in spikes per bin.
in the pallidum that were not RF cells and their spikes were typically very small, making discrimination above baseline difficult. In a few experiments, however, an attempt was made to ascertain whether or not such cells were responsive to 5-HT. In two of three spontaneous active cells (firing rates 1 to 15 Hz) which were recorded for long enough to test 5-HT, clear inhibitory responses were observed using currents of 21 to 33 nA to 10 s. On a few occasions DLH was included in the electrode and was used to fire quiescent cells using currents of 1 to 10 nA. Seven cells were activated in this manner, and 5-HT with currents similar to those described above inhibited all of them, although two were only weakly responsive. Stimulation of the Dorsal Raphe. Dorsal raphe stimulation was tested on 83 spontaneously active pallidal RF cells, the position of the stimulating electrodes being subsequently confirmed to be in the dorsal raphe. The stimulation ranged from single 0.5-ms square wave pulses at 150 PA to a train of nine pulses of 700 @A at 200 Hz. Typically a train of three pulses of 300 PA at 150 Hz was used. Despite these relatively high stimulation currents, only 14 cells responded to the stimulus; 15 cells were orthodromically excited (Fig. 4) with an increase in firing lasting 24 to 70 ms and with variable latencies of 22 to 72 ms, (35.9 + 2.2). A response was deemed to be orthodromically excited if there was variation in the latency of the driven spike, failure to follow three stimuli at 200 Hz and, if spontaneously active, an inability to produce collision between a spontaneous and an evoked action potential. On two of these five cells the excitatory response took the form of an interesting oscillation of firing rate with peaks of activity every 35 to 40 ms and lasting
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S-HT IN THE PALLIDUM
0-
100ms
FIG. 5. Peristimulus time histograms of the globus pallidus cell (referred to in the text), that responded to a stimulus to the dorsal raphe nucleus consisting of a train of five pulses at 200 Hz (hence the initial arteftiual inhibition) with an oscillatory response (a). b-&r methysergide had been iontophoresed with 100 nA for 4 min. As seen, the oscillatory nature of the response was largely lost. c-recovery of the original response 23 min after termination of the methysergide ejection. All histograms are a composite of 128 sweeps and the ordinate is in spikes per bin.
in total for 230 ms (see Fig. 5). Nine cells were inhibited by the stimulus (Fig. 4) and again both the latency and duration of inhibition were variable, the latency ranging from undetectable (due to stimulus artefact) to 50 ms, and the duration from 20 to 122 ms (52.7 +- 15.0 ms). Typically, the inhibition was followed by an increase in activity lasting as long as 100 to 150 ms. On four cells responding to stimulation of the dorsal raphe, methysergide was applied with currents of 30 to 100 nA for as long as 16 min during successive retesting of the cell. The same problems were encountered as described above with difficulty in ejecting the antagonist, and changes in basal firing rate complicated interpretation. Two of these four cells responded with inhibition to dorsal raphe stimulation and one of these showed a slight reduction in response with methysexgide, the other being unaffected. The remaining two were excited by dorsal raphe stimulation and one of these was, interestingly, an example of the oscillatory type of response just described. This was clearly affected by methysergide with a reduction in the frequency of the peaks of activity (see Fig. 5). Upon terminating methysergide, complete recovery of the original response was obtained. The remaining responsive cell was unaffected by methysergide. Six cells whose firing was maintained by DLH application were tested with dorsal raphe stimulation (3 pulses, 150 Hz, 300 FA); five of these responded with inhibition of firing for 79 to 238 ms with latencies from 26 to 46 ms (36.2 + 3.3 ms). In addition the other cell showed a massive increase in firing rate beginning 105 ms after the stimulus and lasting 456 ms.
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DISCUSSION The results show that there is not very much effect of both dorsal raphe stimulation and of 5-HT and NA application on neuronal activity in the rat globus pallidus. These findings are surprising in view of autoradiographic and fluorescence studies which show that a projection arises from the dorsal raphe nucleus and ascends in the ventral forebrain to terminate in the pallidum (3, 4, 18, 29). We are satisfied that the negative nature of the findings are not a result of the use of the iontophoretic method in that 5-HT responses could be obtained in cortex but not in pallidum using the same electrode. In addition, crossing of electrode tips can be excluded as GABA was still highly effective in depressing cell firing. 5-Hydroxytryptamine has been shown to have a depressant action on neuronal firing rates in most regions of the forebrain studied including the basal ganglia (30). Thus neurons in the neocortex, hippocampus, and caudate-putamen are all responsive to this drug, inhibition being the predominant response (25,30). The possibility that the RF cells recorded here were in fact, axons is most unlikely in view of the spatial and temporal stability of the recordings together with the ability to respond to both GABA and DLH. Nevertheless, the lack of effect of 5-HT is difficult to reconcile with the presence of 5-HT terminals and receptors within the pallidum ( 1, 2, 5, 18). It is possible that in the case of the RF cells the 5-HT receptors are mainly presynaptic, therefore reducing the likelihood of observing an effect to 5HT applied iontophoretically. Such presynaptic actions of 5-HT have been observed in the peripheral nervous system ( 10, 19). Another possibility is that the 5-HT input to the pallidum terminates on cells other than RF cells. This is supported by the apparent responsiveness to 5-HT of the smaller, typically nonspontaneously active cells that were induced to fire with DLH. The failure of 5-HT to inhibit the DLH-induced increment of firing on RF cells supports the conclusions that these cells are not responsive to 5-HT and at the same time indicates that the 5-HT depression of artificially excited non-RF cells is not the result of a pharmacological antagonism of DLH by 5-HT. It might be argued that, as a total of 40% of RF cells responded to 5-HT, albeit mostly very weakly, this constitutes a reasonably positive result. When one considers that 5-HT was strongly inhibitory in the majority of cases in the neocortex and on non-RF cells, however, then the relative paucity of such responses in RF cells is a surprisingly negative finding. The responses after stimulation of the dorsal raphe were consistent with the two sets of iontophoretic data. Thus the RF cells did not appear to receive an input from the dorsal raphe and were not sensitive to 5-HT.
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Conversely, the much less common smaller, typically nonspontaneous cells were both sensitive to 5-HT and inhibited by stimulation of the raphe. Although it is tempting to suggest from this correlation that the raphe input is serotonergic, it must be remembered that non-5-HT containing cells are situated in the raphe (27) and may be the source of these alTerent fibers. The results with the antagonist, methysergide, are far from clear. Difficulty with the iontophoretic use of 5-HT antagonists has been noted before (11, 26) and may reflect a limitation of this method of ejection. Although there was not a clear reversal of response to either exogenous 5-HT or raphe stimulation this may only be reflecting the lack of sufficient ejection of the antagonist to block the 5-HT receptors coupled with the difficulties arising from the marked effects of methysergide itself. Conversely, it may suggest that 5-HT is not the transmitter involved in the stimulus-evoked responses. This input may therefore arise from the nonserotonergic neurons of the raphe. It should be emphasized, however, that in these experiments it was only RF cells that were tested with methysergide and only a small proportion of these received any input from the dorsal raphe. Further experiments are planned to investigate the much rarer nonspontaneously active cells. The insensitivity of the RF cells to NA is also interesting, implying an absence of receptors for NA as seems to be the case with 5-HT. Unfortunately, the smaller cell type was not tested with NA. The lack of responses to 5-HT of RF cells in the pallidum may be related to the scarcity of classical synaptic profiles (2, 5). In the cerebral cortex the scarcity of classical synaptic arrangements has been proposed as an explanation for the slow and prolonged nature of neuronal responses to exogenous noradrenaline, and for the need for rather intense stimulation of the locus ceruleus to evoke responses (5). The diffise release of transmitter presumed to occur from the varicosities makes it very difficult to elicit events clearly related to a single or double stimulus. As few classical aminergic synaptic profiles have been detected in the striatum (2, 5), a similar explanation may account for the absence of stimulus-related responses following raphe stimulation in the pallidum despite a well defined anatomic projection. The control exerted by the raphe may be of a more modulatory nature; chronic activation of the 5-HT ascending pathways releasing 5-HT into the extracellular milieu in a manner similar to that proposed for NA in the cerebral cortex (5). Acute activation of such a pathway may well not be sufficient to produce a detectable effect at the single-unit level. An alternative explanation for the lack of effect of 5-HT and NA applied iontophoretically is that the receptors for these amines are dendritically situated, whereas the recordings here were mainly somal. This cannot be excluded but is unlikely in our opinion as there did not appear to be any
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change in sensitivity as the electrode was maneuvered closer to the cell and it would not explain the lack of stimulus-induced responses after raphe stimulation. In summary, these results show a surprising lack of responsiveness to 5HT of the predominant cell type recordable electrophysiologically in the pallidum as well as the absence of an input from the dorsal raphe nuclei. There does, however, seem to be a much smaller population of cells, normally silent in our preparations but excitable by DLH which is both sensitive to 5-HT and NA and receives an innervation from the raphe. REFERENCES 1. AGHAJANIAN, G. K., M. S. KUHAR, AND R. H. ROTH. 1973. Serotonin-containing neuronal perikarya and terminals. Differential effects of p-chlorophenylalanine. Brain Res. 54: 85-101. 2. ARKISON, M., AND I. S. DE LA MANCHE. 1980. High-resolution radiographic study of the serotonin innervation of the rat corpus striatum after intraventricular administration of (‘H) 5-hydroxytryptamine. Neuroscience 5: 229-240. 3. A~MITIA, E. C. 1978. The serotonin producing neurons ofthe midbrain median and dorsal raphe nuclei. Pages 233-3 14 in L. L. IVERSEN, S. D. IVERSEN,AND S. H. SNYDER, Bds., Handbook of Psychopharmacology. Plenum, New York. 4. AZMITIA, E. C., AND M. SEGAL. 1978. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. Camp. Neural. 179: 641-667.
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