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Dopaminergic neurons: simultaneous measurements of dopamine release and single-unit activity during stimulation of the medial forebrain bundle
122
Brain Research, 418 (1987) 122-128 Elsevier
BRE 12773
Dopaminergic neurons: simultaneous measurements of dopamine release and single-unit activity during stimulation of the medial forebrain bundle Werner G. Kuhr l, R. Mark Wightman 1, and George V. Rebec 2 Departments of I Chemistry and 2psychology, lndiana University, Bloomington, IN47405 (U.S.A.) (Accepted 6 January 1987) Key words: Antidromic activation; Dopaminergic neuron; Medial forebrain bundle; Neostriatum; In vivo voltammetry; Unit activity
Simultaneous electrical and chemical recordings have been made of dopamine neuronal activity in the rat brain during electrical stimulation of the medial forebrain bundle. Tungsten recording electrodes were placed at the level of the substantia nigra and carbonfiber, Nation-coated, voltammetric electrodes were placed in the neostriatum. Dopamine units, verified by histology to be in the zona compacta of the substantia nigra, were identified by previously established electrophysiological criteria. Dopamine release was detected by fast-scan cyclic voltammetry, a technique which allows dopamine to be determined in vivo on a sub-second time scale. The majority of dopamine cells examined (7 out of 10) were antidromically activated by 60 Hz stimulation of the medial forebrain bundle. The same stimulus also elicits dopamine overflow in the caudate nucleus. Following stimulation, dopamine concentrations in the extracellular fluid of the neostriatum rapidly declined to prestimulus levels. In addition, impulse flow in dopaminergic neurons was inhibited for 20 s following stimulation. These measurements represent the first direct observation from a neuronal tract of simultaneous unit activity and chemical release of a neurotransmitter in real time. INTRODUCTION Electrical stimulation of the medial forebrain bundle (MFB) appears to activate the nigroneostriatal dopaminergic pathway. Unilateral stimulation at 60 Hz produces maximal contraversive circling behavior which is inhibited by dopamine receptor blockade or dopamine synthesis inhibition 36. Postmortem biochemical studies have shown that dopamine metabolism 22,24 and biosynthesis 3° are accelerated by MFB stimulation. More direct evidence for dopamine release in the neostriatum during MFB stimulation has come from the use of in vivo sampling techniques such as ventricular perfusion 3s, brain dialysis is, and in vivo voltammetry 4. Single-unit recordings in the pars compacta of the substantia nigra (SNC) have demonstrated that antidromic activation of dopamine cell bodies can occur as a result of medial forebrain bundle stimulation 35. Recently, improvements in the temporal resolu-
tion 29 and sensitivity 26 of in vivo voltammetry have made it possible to measure the synaptic overflow of dopamine in the neostriatum in real time during MFB stimulation. In fact, with fast-scan cyclic voltammetry, chemical information can be obtained in vivo on a subsecond time scale. Thus, concentrations of dopamine in the extracellular fluid now can be measured on a time scale similar to that at which electrophysiological events occur. In this report we describe the combined measurement of single unit activity in the SNC with simultaneous voltammetric recording of dopamine release in the neostriatum during MFB stimulation. Although the coincidence of these two events is an expected result, this is the first direct, temporally resolved measurement of these two events in vivo. Because the release of neurotransmitter is the primary consequence of neuronal spike activity, correlation of neurotransmitter release with unit activity is necessary for a complete characterization of neuronal events.
Correspondence: R.M. Wightman, Department of Chemistry, Indiana University, Bloomington, IN 47405, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
123 MATERIALS AND METHODS
Surgery Male Sprague-Dawley rats were anesthetized with urethane (1500 mg/kg -1, i.p.) and placed in a stereotaxic frame housed inside a faraday cage. Body temperature was maintained at 37 °C with a heating pad and temperature control unit ( E K E G Electronics Co., Vancouver, Canada). Holes were drilled at appropriate locations for insertion of electrodes 19'25. After careful removal of the dura, electrodes were inserted to the desired depth. A carbonfiber microvoltammetric electrode was placed in the striatum (1.0 mm anterior and 2.5 mm lateral to bregma) and a bipolar stimulating electrode in the MFB (2.0 mm posterior and 2.0 mm lateral to bregma). A tungsten electrode was placed in the SNC to record single-unit activity (2.5 mm posterior and 1.8 mm lateral to bregma).
Voltarnmetry Microvoltammetric electrodes were prepared from carbon fibers (radius = 5 am), Thornell P-55, Union Carbide, New York, NY) inserted into a glass capillary2°. The electrode tips were bevelled with 1.0 pm diamond paste, and were coated with a thin film of Nation, a cation exchange polymer6. Electrodes were implanted into the striatum at an angle of 10° from normal to a vertical depth o f - 4 . 0 mm as measured from dura. Subsequently the position of the voltammetric electrode was adjusted for maximal release. Potentials are measured vs a saturated calomel electrode (SCE) that was placed in contact with fluid on the skull. Fast-scan cyclic voltammetry (300 V.s -1, -0.4 to 0.8 V vs SCE) was performed under computer control with a locally constructed, 3-electrode potentiostat 17. Scans were repeated at a programmed interval of 50 ms. Voltammograms of released dopamine were obtained as described previously29. The concentration of dopamine at each time interval was obtained by integrating the current at the oxidative wave of dopamine and converting to concentration based on a postcalibration of the electrode in solutions containing between 0.5 and 2.0 pM dopamine in phosphate-buffered saline (pH 7.4).
Electrical stimulation Electrical stimulation of the MFB was used to increase the overflow of dopamine in the ipsilateral neostriatum and to cause antidromic activation of dopaminergic neurons 4,24'35. A concentric bipolar stimulating electrode was implanted in the brain at the level of the MFB, and its position was optimized by adiusting the vertical depth until maximum release was observed in the striatum by voltammetry24. Electrical stimulations consisted of bursts of 300-/~A biphasic pulses (2-ms each phase) at the indicated frequency. Stimulations were computer generated (IBM PC, Scientific Solutions laboratory interface, Solon, OH) and synchronized with the voltammetry to prevent cross-talk between the 3 electrical systems. Additionally, the stimulus system was optically isolated (Neurolog NL-800, Medical Systems Corp, Greenvale, NY) from the electrochemical system. The faraday cage, stereotaxic frame and auxiliary electrode of the electrochemical system were maintained at ground potential 5.
Electrophysiology Tungsten microelectrodes with impedances of ca. 20 Mff2 were lowered to a point just above the SNC (ca. 6.0 mm below dura). After stimulated dopamine overflow was observed in the neostriatum, the tungsten electrodes were lowered further and the search was begun for spontaneously active singleunit discharges. Neuronal activity was amplified (Grass P-15) and integrated over 10-s intervals. Voltage traces of unit activity isolated to a signal-to-noise ratio of 5:1 or more were recorded on a digital oscilloscope (Nicolet Model 3091, Madison, WI). Neurons that showed a constant latency following stimulation (less than 5% variation in the observed time interval between stimulus and spike) were tested for collisional extinction. In this test, a pulse is delivered immediately (within 1 ms) after a spontaneously occurring spike. If collisional extinction occurs, this stimulus pulse will not induce a spike. To illustrate that antidromic conduction is possible, a second pulse was delivered after a sufficient rest interval to produce a spike with a latency consistant with that observed under normal stimuli. The activation, generation and timing of the stimulus pulses were under computer control.
124
Histology At the completion of each experiment, current was passed through the unit-recording and stimulating electrodes to make a small lesion. Brain tissue was fixed by transcardial perfusion with 10% formalin in isotonic saline after an initial rinse with isotonic saline. The brain was removed, frozen, sectioned, and stained with Cresyl violet for microscopic inspection. Frequently, data were obtained from more than one cell per animal. In these cases, verification of electrode placements was reconstructed from the histological data and stereotaxic placements. RESULTS
Characterization of unit activity A total of 35 neurons was examined in 12 animals. These cells were separated into two categories according to their waveform and firing pattern. Type I cells (n = 10), which were found exclusively in the SNC, generated long-duration action potentials
(2.0-3.5 ms) with an initial segment-somatodendritic break on the rising phase of the spike, as shown in Fig. 1. These neurons also displayed an irregular, slow baseline firing rate ( 1 - 7 spikes/s). Type II cells, on the other hand, were characterized by short-duration action potentials (1.0-2.0 ms) and rapid spontaneous activity (10-20 spikes/s). These neurons were found in the SNR and in regions dorsal to the SNC, but not in the SNC itself.
Antidromic activation All neurons were recorded during electrical stimulation of the MFB and tested for antidromic activation. According to previous reports 9'14, antidromically activated neurons display only one spike/stimulus pulse, follow repetitive pulses with a constant latency (+ 10%) between each stimulus pulse and recorded
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Fig. 1. An action potential recorded from a Type I cell. Note the long duration of the spike (>2.0 ms) and the notch separating the initial segment and somatodendritic spike components. These are characteristic features of dopaminergic neurons.
Fig. 2. Collisional extinction as evidence for direct antidromic activation of dopaminergic neurons. A pair of stimulus pulses (2 ms biphasic, 300/zA peak current with a delay of 16 ms) was delivered to the MFB immediately after a spontaneously active dopaminergic neuron was recorded in the SNC (A). The first stimulus pulse would have induced a unit at (B), had the action potential it produced not collided with the action potential initially recorded (A). The second stimulus produces a unit at the required time (C), indicating that the stimulus is successfully driving the neuron. S, stimulus artifact.
125
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spike, and show collisional extinction. Seven of the 10 Type I cells in our sample met these criteria. A demonstration of collisional extinction as it occurred in one of these cells is shown in Fig. 2. Antidromic spikes were recorded 6.2 (+ 1.0) ms after the onset of the stimulus pulse in the MFB. Type II cells also were activated antidromically but less frequently. In fact, only one quarter of these neurons (5 of 20) met the criteria for antidromic activation. In this small group of neurons, the latency for the occurrence of antidromic spikes was 3.9 (+0.8) ms after MFB stimulation. Electrode placements for all antidromic and non-antidromic Type I and Type II cells are summarized in Fig. 3.
Simultaneous recording of chemical activity Electrically stimulated chemical release was measured voltammetrically in the neostriatum when neuronal activity was recorded from antidromically activated Type I cells. MFB stimulation was applied at a frequency of 60 Hz, which is known to cause maxim u m overflow of dopamine in the neostriatum 24. A typical result is shown in Fig. 4. Note that chemical release, identified by individual voltammograms as
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Fig. 3. Electrode placements for all Type I (circles) and Type II (triangles) cells. Filled symbols indicate antidromically activated neurons. Histological sections are drawn after K6nig and Klippe121
Fig. 4. Simultaneous recording of stimulus-evoked release of dopamine in the neostriatum and spike activity of dopaminergic neurons in the SNC. The same stimulus (60 Hz, 2 ms biphasic pulses, 300/xA peak amplitude, 2 s duration) used to elicit dopamine release (monitored with voltammetry at an interval of 100 ms, upper left) also induced electrical activity in dopaminergic ceils (right). S, stimulus artifact; A, unit. The unit activity shown was recorded during the time indicated by the brackets. A single voltammogram (lower right) recorded during the stimulation is identical to that for dopamine.
126 V- . . . . . . . . 102±12
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Fig. 5. Effect of MFB stimulation on the spontaneous activity of a dopaminergic neuron. The spontaneous firing rate of a dopaminergic neuron was evaluated before and after a 2-s, 60-Hz stimulation of the MFB (2-ms biphasic pulses with 300-/~Apeak current). A short, but dramatic, depression in firing rate is observed following the stimulation. Error bars indicate S.E.M. for 4 repetitions. * Significantly different from prestimulus, P < 0.01, Student's t-test.).
dopamine, occurs simultaneously with antidromic activation of the Type I cell. Note also that unit activity follows the stimulus artifact with a constant latency. Non-antidromic Type I cells were unable to follow the 60 Hz stimulation.
Chemical and unit activity following MFB stimulation Dopamine overflow in the neostriatum peaked within 200 (+50) ms after the stimulation and rapidly returned to prestimulation levels. Unit activity also declined. In fact, as shown in Fig. 5, impulse flow remained suppressed for several seconds after MFB stimulation. The gradual return to prestimulation firing rate occurred on the same time scale as the disappearance of extracellular dopamine in the neostriaturn. DISCUSSION Several laboratories have used voltammetry to detect the release of dopamine in the neostriatum of the rat following electrical stimulation of the MFB 4'7' 24-27,29,34. By itself, however, voltammetric detection of dopamine is not sufficient evidence that dopaminergic neurons are activated by MFB stimulation. Indeed, in view of the large number of fibers that comprise the MFB, such stimulation could induce dopamine release indirectly by activating non-dopaminer-
gic neostriatal afferents. Moreover, it is not even clear that dopaminergic neurons can follow the 60Hz stimulation that produces maximal dopamine overflow in the neostriatum. The only definitive way to demonstrate that dopamine release is a direct result of activation of dopaminergic neurons is to combine in vivo voltammetry with single-unit recording. Although previous reports have demonstrated the compatibility of these techniques 5,16,2s,33, this is the first time that they have been used to obtain data simultaneously at both ends of a neuronal network. To accomplish this goal, it was necessary to use a method of electrochemical detection that operates on the same ms time scale as electrophysiological events. For this purpose, we used the method of fastscan voltammetry with microvoltammetric electrodes 26'27. This technique shortens the minimum sampling interval from 2 s for classical in vivo procedures to 50 ms and yet provides complete voltammetric information. Each scan, taken at 300 V.s -l, occurs in less than 10 ms, and the recorded voltammogram contains the current that occurs during both the oxidation of dopamine and the reduction of the generated ortho-quinone. This information gives a unique 'fingerprint' for the indentification of dopamine. Because the waveform causes regeneration of most of the original species, this type of measurement introduces minimal perturbation in the chemical composition of the fluid surrounding the electrode. While dopamine release was being measured in the neostriatum, single-unit electrodes in the SNC recorded the activity of Type I cells, which met the previously established electrophysiologicai characteristics of dopaminergic neurons s'm-lz. Many of these cells were activated antidromically, indicating that indeed dopaminergic neurons are activated by MFB stimulation. Moreover, the relatively long latency that we observed between the onset of MFB stimulation and spike activity in the SNC is consistent with the slow conduction time of these neurons 3. In fact, nearly identical latencies have been reported in previous electrophysiological investigations of dopaminergic neurons during MFB stimulation 35. Thus, we conclude that the release of dopamine in the neostriatum is a direct result of stimulation of dopaminergic neurons, rather than the result of some form of indirect activation.
127 Not all dopaminergic neurons were activated antidromically, indicating that some of these cells were unaffected by MFB stimulation. An examination of electrode tip placements revealed that these non-antidromic neurons were located in the extreme lateral or dorsal extent of the SNC. Thus, these cells may not have been activated by stimulation aimed at the center of the MFB. Alternatively, there may be a subpopulation of dopaminergic neurons that is unable to follow relatively rapid stimulation frequencies. It also is interesting to note that several non-dopaminergic neurons were activated antidromically. The existence of a non-dopaminergic nigral projection to the neostriatum has been postulated for some time15'37; and these cells may be part of this pathway, although they also could project to other forebrain regions. The combined use of voltammetric and single-unit recording techniques to study dopaminergic neurons also may shed some light on the mechanisms that regulate the firing rate of these cells. We found, for example, that when MFB stimulation is terminated, impulse flow in dopaminergic neurons declines and returns to a normal rate with the same time course as the disappearance of extracellular dopamine in the neostriatum. This compensatory reduction in firing rate may be the result of activation of a neostriatonigral negative-feedback pathway, which may modulate the activity of dopaminergic neurons in the SNC, or the release of dopamine from dendritic terminals in the SNC onto inhibitory autoreceptors 1,2. 12,13,23,31,32 In fact, the release of dopamine from both axon and dendritic terminals may contribute to the inhibition of firing rate. Presumably, extracellular dopamine in the SNC disappears after MFB stimulation just as rapidly as extracellular dopamine in the neostriatum. Dopaminergic neurons typically fire at rates below 10 spikes/s 8,9,19, yet with MFB stimulation at this fre-
REFERENCES 1 Bunney, B.S. and Aghajanian, G.K., D-Amphetamine-induced depression of central dopamine neurons: evidence for mediation by both autoreceptors and a striato-nigral feedback pathway, Naunyn-Schmiedeberg's Arch. Pharmacol., 304 (1978) 255-261. 2 Cheramy, A., Leviel, A. and Glowinski, J., Dendritic release of dopamine in the substantia nigra, Nature (Lon-
quency D A overflow in the neostriatum is almost undetectable except after administration of dopamine uptake inhibitors 7'24'26. Consistent with these observations, the steady-state concentration of dopamine in the extracellular fluid of the neostriatum is extremely low 39. Thus, with dopaminergic neurons firing at relatively low rates, dopamine uptake in the neostriatum appears to be rapid and efficient. As we have shown, however, stimulation of dopaminergic neurons at higher frequencies causes substantial overflow of dopamine into the extracellular fluid. Because dopaminergic neurons themselves are capable of firing in short bursts at high frequencies 9,19, dopamine overflow may occur spontaneously under these conditions. Such burst firing would allow dopamine to overwhelm normal uptake mechanisms and thus interact with receptors at some distance removed from the immediate site of synaptic release. Nevertheless, the effect of such extrasynaptic dopamine would be extremely short-lived because, as our voltammetric data indicate, dopamine overflow exists only for brief intervals. Our results demonstrate that the combined use of voltammetric and single-unit recording techniques provides an effective means of studying dopaminergic neurons. The continued application of these techniques in real time promises to increase our understanding of the mechanisms that control not only these cells but also the cells with which they make synaptic contact.
ACKNOWLEDGEMENTS This research was supported by the NSF (BNS8606354) and USPHS Grant D A 02451 (G.V.R.). Brain tissue was prepared for histological analysis by Doris Batson. Leslie May aided in the preparation of figures.
don), 289 (1981) 537-542. 3 Connor, J.D., Electrophysiology of the nigro-caudate dopamine pathway, Pharmacol. Ther. B, 1 (1975) 357-370. 4 Ewing, A.G., Bigelow, J.C. and Wightman, R.M., Direct in vivo monitoring of dopamine released from two striatal compartments in the rat, Science 221 (1983) 169-171, 5 Ewing, A.G., Alloway, K.D., Curtis, S.D., Dayton, M.A., Wightman, R.M. and Rebec, G.V., Simultaneous electrochemical and unit recording measurements: characteriza-
128 tion of the effects of D-amphetamine and ascorbic acid on neostriatal neurons, Brain Research, 261 (1983) 101-108. 6 Gerhardt, G.A., Oke, A.F., Nagy, G., Moghaddam, B. and Adams, R.N., Nation-coated electrodes with high selectivity for CNS electrochemistry, Brain Research, 290 (1984) 390- 395. 7 Gonon, F.G. and Buda, M.J., Regulation of dopamine release by impulse flow and by autoreceptors as studied by in vivo voltammetry in the rat striatum, Neuroscience, 14 (1985) 765-774. 8 Grace, A.A. and Bunney, B.S., Nigral dopamine neurons: intracellular recording and identification with L-dopa injection and histofluorescence, Science, 210 (1980) 654-656. 9 Grace, A. and Bunney, B., Intracellular and extracellular electrophysiology of nigral dopaminergic neurons -1. Identification and characterization, Neuroscience, 10 (1983) 301-315. 10 Grace, A.A. and Bunney, B.S., The control of firing pattern in nigral dopamine neurons: burst firing, J. Neurosci., 4 (1984) 2877-2890. 11 Grace, A.A. and Bunney, B.S., The control of firing pattern in nigral dopamine neurons: single spike firing, J. Neurosci., 4 (1984) 2866-2876. 12 Grace, A.A. and Bunney, B.S., Low doses of apomorphine elicit two opposing influences on dopamine cell electrophysiology, Brain Research, 333 (1985) 285-298. 13 Groves, P.M., Wilson, C.J., Young, S.J. and Rebec, G.V. Self-inhibition by dopaminergic neurons, Science, 190 (1975) 522-529. 14 Guyenet, P.G. and Aghajanian, G., Antidromic identification of dopaminergic and other output neurons of the rat substantia nigra, Brain Research, 150 (1978) 69-84. 15 Guyenet, P.G. and Crane, J.K., Non-dopaminergic nigrostriatal pathway, Brain Research, 213 (1981) 291-305. 16 Hefti, F. and Felix, D., Chronoamperometry in vivo: does it interfere with spontaneous neuronal activity in the brain?, J. Neurosci. Meth., 7 (1983) 151-156. 17 Howell, J.O., Kuhr, W.G., Ensman, R.E. and Wightman, R.M., Background subtraction for rapid scan voltammetry, J. Electroanal. Chem., in press. 18 Imperato, A. and DiChiara, G., Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with electrochemical detection: a new method for the study of the in vivo release of endogenous dopamine and metabolites, J. Neurosci., 4 (1984) 966-977. 19 Kamata, K. and Rebec, G.V., Dopaminergic and neostriatal neurons: dose-dependent changes in sensitivity to amphetamine following long-term treatment, Neuropharmacology, 22 (1983) 1377-1382. 20 Kelly, R.S. and Wightman, R.M., Bevelled carbon-fiber ultra microelectrodes, Anal. Chim. Acta, in press. 21 K6nig, J.F.R. and Klippel, R.A., The Rat Brain: a Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams and Wilkins, Baltimore, 1963. 22 Korf, J., Grasdijk, L. and Westerink, B.H.C., Effects of electrical stimulation of the nigrostriatal pathway of the rat on dopamine metabolism, J. Neurochem., 26 (1976) 579-584. 23 Korf, J., Zieleman, M. and Westerink, B.H.C., Dopamine release in substantia nigra, Nature (London), 260 (1976) 257-258.
24 Kuhr, W.G., Ewing, A.G., Caudill, W.L. and Wightman, R.M., Monitoring the stimulated release of dopamine with in vivo voltammetry. I: Characterization of the response observed in the caudate nucleus of the rat, J. Neurochem., 43 (1984) 560-569. 25 Kuhr, W.G., Ewing, A.G., Near, J.A. and Wightman, R.M., Amphetamine attenuates the stimulated release of dopamine in vivo, J. Pharmacol. Exp. Ther., 232 (1985) 388-394. 26 Kuhr, W.G. and Wightman, R.M., Real-time measurement of dopamine release in rat brain, Brain Research, 381 (1986) 168-171. 27 Kuhr, W., Bigelow, J. and Wightman, R.M., In vivo comparison of the regulation of releasable dopamine in the caudate nucleus and nucleus accumbens of the rat brain, J. Neurosci., 6 (1986) 974-982. 28 Millar, J., Armstrong-James, M. and Kruk, Z.L., Polarographic assay of iontophoretically applied dopamine and low-noise unit recording using a multibarrel carbon fiber microelectrode, Brain Research, 205 (1981) 419-424. 29 Millar, J., Stamford, J.A., Kruk, Z.L. and Wightman, R.M., Electrochemical, pharmacological and electrophysiological evidence of rapid dopamine release and removal in the rat caudate nucleus following electrical stimulation of the medial forebrain bundle, Eur. J. Pharm., 109 (1985) 341-348. 30 Murrin, L.C. and Roth, R.H., Dopaminergic neurons: effects of electrical stimulation on dopamine biosynthesis, Mol. Pharm., 12 (1976) 463-475. 31 Rebec, G.V., Auto- and postsynaptic dopamine receptors in the central nervous system. In J. Marwaha and W. Anderson (Eds.), Neuroreceptors in Health and Disease, Karger, Basel, 1984, pp. 207-223. 32 Rebec, G.V., Electrophysiological pharmacology of amphetamine. In Marwah, J. (Ed.), Neurobiology of Drug Abuse, Karger, Basel, in press. 33 Schenk, J.O. and Adams, R.N., Simultaneous electrochemical and neurophysiological measurements in anesthetized rats, Neurosci. Lett., Supl. 7 (1981) $327. 34 Stamford, J.A., Kruk, Z.L., Millar, J. and Wightman, R.M., Striatal dopamine uptake in the rat: in vivo analysis by fast cyclic voltammetry, Neurosci. Lett., 51 (1984) 133-138. 35 Takeuchi, H., Young, S.J. and Groves, P.M., Dopaminergic terminal excitability following arrival of the nerve impulse: the influence of amphetamine and haloperidol, Brain Research, 245 (1982) 47-56. 36 Van der Heyden, J.A.M., Circling behaviour induced by electrical stimulation of the medial forebrain bundle, importance of stimulus parameters and dopaminergic processes, Pharm. Biochem. Behav., 21 (1984) 567-574. 37 Van der Kooy, D., Coscina, D.V. and Hattori, T., Is there a non-dopaminergic nigrostriatal pathway?, Neuroscience, 6 (1981) 345-357. 38 Von Voightlander, P.F. and Moore, K.E., Nigro-striatal pathway: stimulus-evoked release of [3HI dopamine from caudate nucleus, Brain Research, 35 (1971) 580-583. 39 Zetterstr6m, T., Sharp, T., Marsden, C.A. and Ungerstedt, U., In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after D-amphetamine, J. Neurochem., 41 (1983) 1769-1773.
Brain Research, 418 (1987) 122-128 Elsevier
BRE 12773
Dopaminergic neurons: simultaneous measurements of dopamine release and single-unit activity during stimulation of the medial forebrain bundle Werner G. Kuhr l, R. Mark Wightman 1, and George V. Rebec 2 Departments of I Chemistry and 2psychology, lndiana University, Bloomington, IN47405 (U.S.A.) (Accepted 6 January 1987) Key words: Antidromic activation; Dopaminergic neuron; Medial forebrain bundle; Neostriatum; In vivo voltammetry; Unit activity
Simultaneous electrical and chemical recordings have been made of dopamine neuronal activity in the rat brain during electrical stimulation of the medial forebrain bundle. Tungsten recording electrodes were placed at the level of the substantia nigra and carbonfiber, Nation-coated, voltammetric electrodes were placed in the neostriatum. Dopamine units, verified by histology to be in the zona compacta of the substantia nigra, were identified by previously established electrophysiological criteria. Dopamine release was detected by fast-scan cyclic voltammetry, a technique which allows dopamine to be determined in vivo on a sub-second time scale. The majority of dopamine cells examined (7 out of 10) were antidromically activated by 60 Hz stimulation of the medial forebrain bundle. The same stimulus also elicits dopamine overflow in the caudate nucleus. Following stimulation, dopamine concentrations in the extracellular fluid of the neostriatum rapidly declined to prestimulus levels. In addition, impulse flow in dopaminergic neurons was inhibited for 20 s following stimulation. These measurements represent the first direct observation from a neuronal tract of simultaneous unit activity and chemical release of a neurotransmitter in real time. INTRODUCTION Electrical stimulation of the medial forebrain bundle (MFB) appears to activate the nigroneostriatal dopaminergic pathway. Unilateral stimulation at 60 Hz produces maximal contraversive circling behavior which is inhibited by dopamine receptor blockade or dopamine synthesis inhibition 36. Postmortem biochemical studies have shown that dopamine metabolism 22,24 and biosynthesis 3° are accelerated by MFB stimulation. More direct evidence for dopamine release in the neostriatum during MFB stimulation has come from the use of in vivo sampling techniques such as ventricular perfusion 3s, brain dialysis is, and in vivo voltammetry 4. Single-unit recordings in the pars compacta of the substantia nigra (SNC) have demonstrated that antidromic activation of dopamine cell bodies can occur as a result of medial forebrain bundle stimulation 35. Recently, improvements in the temporal resolu-
tion 29 and sensitivity 26 of in vivo voltammetry have made it possible to measure the synaptic overflow of dopamine in the neostriatum in real time during MFB stimulation. In fact, with fast-scan cyclic voltammetry, chemical information can be obtained in vivo on a subsecond time scale. Thus, concentrations of dopamine in the extracellular fluid now can be measured on a time scale similar to that at which electrophysiological events occur. In this report we describe the combined measurement of single unit activity in the SNC with simultaneous voltammetric recording of dopamine release in the neostriatum during MFB stimulation. Although the coincidence of these two events is an expected result, this is the first direct, temporally resolved measurement of these two events in vivo. Because the release of neurotransmitter is the primary consequence of neuronal spike activity, correlation of neurotransmitter release with unit activity is necessary for a complete characterization of neuronal events.
Correspondence: R.M. Wightman, Department of Chemistry, Indiana University, Bloomington, IN 47405, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
123 MATERIALS AND METHODS
Surgery Male Sprague-Dawley rats were anesthetized with urethane (1500 mg/kg -1, i.p.) and placed in a stereotaxic frame housed inside a faraday cage. Body temperature was maintained at 37 °C with a heating pad and temperature control unit ( E K E G Electronics Co., Vancouver, Canada). Holes were drilled at appropriate locations for insertion of electrodes 19'25. After careful removal of the dura, electrodes were inserted to the desired depth. A carbonfiber microvoltammetric electrode was placed in the striatum (1.0 mm anterior and 2.5 mm lateral to bregma) and a bipolar stimulating electrode in the MFB (2.0 mm posterior and 2.0 mm lateral to bregma). A tungsten electrode was placed in the SNC to record single-unit activity (2.5 mm posterior and 1.8 mm lateral to bregma).
Voltarnmetry Microvoltammetric electrodes were prepared from carbon fibers (radius = 5 am), Thornell P-55, Union Carbide, New York, NY) inserted into a glass capillary2°. The electrode tips were bevelled with 1.0 pm diamond paste, and were coated with a thin film of Nation, a cation exchange polymer6. Electrodes were implanted into the striatum at an angle of 10° from normal to a vertical depth o f - 4 . 0 mm as measured from dura. Subsequently the position of the voltammetric electrode was adjusted for maximal release. Potentials are measured vs a saturated calomel electrode (SCE) that was placed in contact with fluid on the skull. Fast-scan cyclic voltammetry (300 V.s -1, -0.4 to 0.8 V vs SCE) was performed under computer control with a locally constructed, 3-electrode potentiostat 17. Scans were repeated at a programmed interval of 50 ms. Voltammograms of released dopamine were obtained as described previously29. The concentration of dopamine at each time interval was obtained by integrating the current at the oxidative wave of dopamine and converting to concentration based on a postcalibration of the electrode in solutions containing between 0.5 and 2.0 pM dopamine in phosphate-buffered saline (pH 7.4).
Electrical stimulation Electrical stimulation of the MFB was used to increase the overflow of dopamine in the ipsilateral neostriatum and to cause antidromic activation of dopaminergic neurons 4,24'35. A concentric bipolar stimulating electrode was implanted in the brain at the level of the MFB, and its position was optimized by adiusting the vertical depth until maximum release was observed in the striatum by voltammetry24. Electrical stimulations consisted of bursts of 300-/~A biphasic pulses (2-ms each phase) at the indicated frequency. Stimulations were computer generated (IBM PC, Scientific Solutions laboratory interface, Solon, OH) and synchronized with the voltammetry to prevent cross-talk between the 3 electrical systems. Additionally, the stimulus system was optically isolated (Neurolog NL-800, Medical Systems Corp, Greenvale, NY) from the electrochemical system. The faraday cage, stereotaxic frame and auxiliary electrode of the electrochemical system were maintained at ground potential 5.
Electrophysiology Tungsten microelectrodes with impedances of ca. 20 Mff2 were lowered to a point just above the SNC (ca. 6.0 mm below dura). After stimulated dopamine overflow was observed in the neostriatum, the tungsten electrodes were lowered further and the search was begun for spontaneously active singleunit discharges. Neuronal activity was amplified (Grass P-15) and integrated over 10-s intervals. Voltage traces of unit activity isolated to a signal-to-noise ratio of 5:1 or more were recorded on a digital oscilloscope (Nicolet Model 3091, Madison, WI). Neurons that showed a constant latency following stimulation (less than 5% variation in the observed time interval between stimulus and spike) were tested for collisional extinction. In this test, a pulse is delivered immediately (within 1 ms) after a spontaneously occurring spike. If collisional extinction occurs, this stimulus pulse will not induce a spike. To illustrate that antidromic conduction is possible, a second pulse was delivered after a sufficient rest interval to produce a spike with a latency consistant with that observed under normal stimuli. The activation, generation and timing of the stimulus pulses were under computer control.
124
Histology At the completion of each experiment, current was passed through the unit-recording and stimulating electrodes to make a small lesion. Brain tissue was fixed by transcardial perfusion with 10% formalin in isotonic saline after an initial rinse with isotonic saline. The brain was removed, frozen, sectioned, and stained with Cresyl violet for microscopic inspection. Frequently, data were obtained from more than one cell per animal. In these cases, verification of electrode placements was reconstructed from the histological data and stereotaxic placements. RESULTS
Characterization of unit activity A total of 35 neurons was examined in 12 animals. These cells were separated into two categories according to their waveform and firing pattern. Type I cells (n = 10), which were found exclusively in the SNC, generated long-duration action potentials
(2.0-3.5 ms) with an initial segment-somatodendritic break on the rising phase of the spike, as shown in Fig. 1. These neurons also displayed an irregular, slow baseline firing rate ( 1 - 7 spikes/s). Type II cells, on the other hand, were characterized by short-duration action potentials (1.0-2.0 ms) and rapid spontaneous activity (10-20 spikes/s). These neurons were found in the SNR and in regions dorsal to the SNC, but not in the SNC itself.
Antidromic activation All neurons were recorded during electrical stimulation of the MFB and tested for antidromic activation. According to previous reports 9'14, antidromically activated neurons display only one spike/stimulus pulse, follow repetitive pulses with a constant latency (+ 10%) between each stimulus pulse and recorded
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Fig. 1. An action potential recorded from a Type I cell. Note the long duration of the spike (>2.0 ms) and the notch separating the initial segment and somatodendritic spike components. These are characteristic features of dopaminergic neurons.
Fig. 2. Collisional extinction as evidence for direct antidromic activation of dopaminergic neurons. A pair of stimulus pulses (2 ms biphasic, 300/zA peak current with a delay of 16 ms) was delivered to the MFB immediately after a spontaneously active dopaminergic neuron was recorded in the SNC (A). The first stimulus pulse would have induced a unit at (B), had the action potential it produced not collided with the action potential initially recorded (A). The second stimulus produces a unit at the required time (C), indicating that the stimulus is successfully driving the neuron. S, stimulus artifact.
125
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spike, and show collisional extinction. Seven of the 10 Type I cells in our sample met these criteria. A demonstration of collisional extinction as it occurred in one of these cells is shown in Fig. 2. Antidromic spikes were recorded 6.2 (+ 1.0) ms after the onset of the stimulus pulse in the MFB. Type II cells also were activated antidromically but less frequently. In fact, only one quarter of these neurons (5 of 20) met the criteria for antidromic activation. In this small group of neurons, the latency for the occurrence of antidromic spikes was 3.9 (+0.8) ms after MFB stimulation. Electrode placements for all antidromic and non-antidromic Type I and Type II cells are summarized in Fig. 3.
Simultaneous recording of chemical activity Electrically stimulated chemical release was measured voltammetrically in the neostriatum when neuronal activity was recorded from antidromically activated Type I cells. MFB stimulation was applied at a frequency of 60 Hz, which is known to cause maxim u m overflow of dopamine in the neostriatum 24. A typical result is shown in Fig. 4. Note that chemical release, identified by individual voltammograms as
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Fig. 3. Electrode placements for all Type I (circles) and Type II (triangles) cells. Filled symbols indicate antidromically activated neurons. Histological sections are drawn after K6nig and Klippe121
Fig. 4. Simultaneous recording of stimulus-evoked release of dopamine in the neostriatum and spike activity of dopaminergic neurons in the SNC. The same stimulus (60 Hz, 2 ms biphasic pulses, 300/xA peak amplitude, 2 s duration) used to elicit dopamine release (monitored with voltammetry at an interval of 100 ms, upper left) also induced electrical activity in dopaminergic ceils (right). S, stimulus artifact; A, unit. The unit activity shown was recorded during the time indicated by the brackets. A single voltammogram (lower right) recorded during the stimulation is identical to that for dopamine.
126 V- . . . . . . . . 102±12
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Fig. 5. Effect of MFB stimulation on the spontaneous activity of a dopaminergic neuron. The spontaneous firing rate of a dopaminergic neuron was evaluated before and after a 2-s, 60-Hz stimulation of the MFB (2-ms biphasic pulses with 300-/~Apeak current). A short, but dramatic, depression in firing rate is observed following the stimulation. Error bars indicate S.E.M. for 4 repetitions. * Significantly different from prestimulus, P < 0.01, Student's t-test.).
dopamine, occurs simultaneously with antidromic activation of the Type I cell. Note also that unit activity follows the stimulus artifact with a constant latency. Non-antidromic Type I cells were unable to follow the 60 Hz stimulation.
Chemical and unit activity following MFB stimulation Dopamine overflow in the neostriatum peaked within 200 (+50) ms after the stimulation and rapidly returned to prestimulation levels. Unit activity also declined. In fact, as shown in Fig. 5, impulse flow remained suppressed for several seconds after MFB stimulation. The gradual return to prestimulation firing rate occurred on the same time scale as the disappearance of extracellular dopamine in the neostriaturn. DISCUSSION Several laboratories have used voltammetry to detect the release of dopamine in the neostriatum of the rat following electrical stimulation of the MFB 4'7' 24-27,29,34. By itself, however, voltammetric detection of dopamine is not sufficient evidence that dopaminergic neurons are activated by MFB stimulation. Indeed, in view of the large number of fibers that comprise the MFB, such stimulation could induce dopamine release indirectly by activating non-dopaminer-
gic neostriatal afferents. Moreover, it is not even clear that dopaminergic neurons can follow the 60Hz stimulation that produces maximal dopamine overflow in the neostriatum. The only definitive way to demonstrate that dopamine release is a direct result of activation of dopaminergic neurons is to combine in vivo voltammetry with single-unit recording. Although previous reports have demonstrated the compatibility of these techniques 5,16,2s,33, this is the first time that they have been used to obtain data simultaneously at both ends of a neuronal network. To accomplish this goal, it was necessary to use a method of electrochemical detection that operates on the same ms time scale as electrophysiological events. For this purpose, we used the method of fastscan voltammetry with microvoltammetric electrodes 26'27. This technique shortens the minimum sampling interval from 2 s for classical in vivo procedures to 50 ms and yet provides complete voltammetric information. Each scan, taken at 300 V.s -l, occurs in less than 10 ms, and the recorded voltammogram contains the current that occurs during both the oxidation of dopamine and the reduction of the generated ortho-quinone. This information gives a unique 'fingerprint' for the indentification of dopamine. Because the waveform causes regeneration of most of the original species, this type of measurement introduces minimal perturbation in the chemical composition of the fluid surrounding the electrode. While dopamine release was being measured in the neostriatum, single-unit electrodes in the SNC recorded the activity of Type I cells, which met the previously established electrophysiologicai characteristics of dopaminergic neurons s'm-lz. Many of these cells were activated antidromically, indicating that indeed dopaminergic neurons are activated by MFB stimulation. Moreover, the relatively long latency that we observed between the onset of MFB stimulation and spike activity in the SNC is consistent with the slow conduction time of these neurons 3. In fact, nearly identical latencies have been reported in previous electrophysiological investigations of dopaminergic neurons during MFB stimulation 35. Thus, we conclude that the release of dopamine in the neostriatum is a direct result of stimulation of dopaminergic neurons, rather than the result of some form of indirect activation.
127 Not all dopaminergic neurons were activated antidromically, indicating that some of these cells were unaffected by MFB stimulation. An examination of electrode tip placements revealed that these non-antidromic neurons were located in the extreme lateral or dorsal extent of the SNC. Thus, these cells may not have been activated by stimulation aimed at the center of the MFB. Alternatively, there may be a subpopulation of dopaminergic neurons that is unable to follow relatively rapid stimulation frequencies. It also is interesting to note that several non-dopaminergic neurons were activated antidromically. The existence of a non-dopaminergic nigral projection to the neostriatum has been postulated for some time15'37; and these cells may be part of this pathway, although they also could project to other forebrain regions. The combined use of voltammetric and single-unit recording techniques to study dopaminergic neurons also may shed some light on the mechanisms that regulate the firing rate of these cells. We found, for example, that when MFB stimulation is terminated, impulse flow in dopaminergic neurons declines and returns to a normal rate with the same time course as the disappearance of extracellular dopamine in the neostriatum. This compensatory reduction in firing rate may be the result of activation of a neostriatonigral negative-feedback pathway, which may modulate the activity of dopaminergic neurons in the SNC, or the release of dopamine from dendritic terminals in the SNC onto inhibitory autoreceptors 1,2. 12,13,23,31,32 In fact, the release of dopamine from both axon and dendritic terminals may contribute to the inhibition of firing rate. Presumably, extracellular dopamine in the SNC disappears after MFB stimulation just as rapidly as extracellular dopamine in the neostriatum. Dopaminergic neurons typically fire at rates below 10 spikes/s 8,9,19, yet with MFB stimulation at this fre-
REFERENCES 1 Bunney, B.S. and Aghajanian, G.K., D-Amphetamine-induced depression of central dopamine neurons: evidence for mediation by both autoreceptors and a striato-nigral feedback pathway, Naunyn-Schmiedeberg's Arch. Pharmacol., 304 (1978) 255-261. 2 Cheramy, A., Leviel, A. and Glowinski, J., Dendritic release of dopamine in the substantia nigra, Nature (Lon-
quency D A overflow in the neostriatum is almost undetectable except after administration of dopamine uptake inhibitors 7'24'26. Consistent with these observations, the steady-state concentration of dopamine in the extracellular fluid of the neostriatum is extremely low 39. Thus, with dopaminergic neurons firing at relatively low rates, dopamine uptake in the neostriatum appears to be rapid and efficient. As we have shown, however, stimulation of dopaminergic neurons at higher frequencies causes substantial overflow of dopamine into the extracellular fluid. Because dopaminergic neurons themselves are capable of firing in short bursts at high frequencies 9,19, dopamine overflow may occur spontaneously under these conditions. Such burst firing would allow dopamine to overwhelm normal uptake mechanisms and thus interact with receptors at some distance removed from the immediate site of synaptic release. Nevertheless, the effect of such extrasynaptic dopamine would be extremely short-lived because, as our voltammetric data indicate, dopamine overflow exists only for brief intervals. Our results demonstrate that the combined use of voltammetric and single-unit recording techniques provides an effective means of studying dopaminergic neurons. The continued application of these techniques in real time promises to increase our understanding of the mechanisms that control not only these cells but also the cells with which they make synaptic contact.
ACKNOWLEDGEMENTS This research was supported by the NSF (BNS8606354) and USPHS Grant D A 02451 (G.V.R.). Brain tissue was prepared for histological analysis by Doris Batson. Leslie May aided in the preparation of figures.
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