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Intracellular studies of dopamine neurons in vitro: pacemakers modulated by dopamine
European Journal of Pharmacology, 149 (1988) 307-315
307
Elsevier UP 50263
Intracellular studies of dopamine neurons in vitro: pacemakers modulated by dopamine N a n c y L. Silva * ' * * a n d B e n j a m i n S. B u n n e y Department of Pharmacology and Psychiatry, Yale University, School of Medicine, New Hat~en, CT 06510, and * * Laboratory of Neurophysiology, NIH-N1NCDS. Building 36, Room 2 C02. Bethesda, MD 20892, U.S.A.
Received 9 February 1988, accepted 23 February 1988
Intracellular recordings from dopamine (DA)-sensitive neurons in rat substantia nigra tissue slices revealed that these neurons exhibit spontaneous pacemaker-like activity. DA-sensitive neurons had higher input resistances, larger time constants and less linear voltage responses to current injection than did non-DA-sensitive neurons in the zona compacta. The administration of DA produced an inhibition of firing rate, a hyperpolarization and a decrease in input resistance. These effects were blocked by (-)sulpiride, a selective D 2 antagonist. A reversal potential of - 8 8 _+ 14 mV was calculated for the DA-induced hyperpolarization suggesting the involvement of potassium ions in the mechanism of DA action. Dopamine; Substantia nigra; Pacemaker activity; Dopamine neurons; (Intracellular recording, Brain slice)
1. Introduction Midbrain dopamine (DA) neurons have been extensively studied electrophysiologically and pharmacologically. In whole animal studies, it has been demonstrated that the electrical activity of these neurons possess a characteristic waveform and firing pattern (Aghajanian and Bunney, 1973; Bunney et al., 1973; Grace and Bunney, 1983). These cells are inhibited by their own neurotransmitter in vivo (Aghajanian and Bunney, 1973; Bunney et al., 1973) and in vitro (Pinnock, 1983a; Sanghera et al., 1984). This autoinhibition appears to occur through a mechanism involving somatic or dendritic autoreceptors as the response to DA is maintained in the midbrain tissue slice preparation under conditions in which synaptic activity has been blocked (Pinnock, 1983b).
* To whom all correspondence should be addressed.
In vivo, DA neurons, identified following recording with fluorescence histochemical techniques, exhibit long duration ( > 2 ms) action potentials and fire at slow rates (1-5 Hz) (Grace and Bunney, 1984a,b). D A neurons have been observed in vivo to fire in two types of patterns: (1) a slow irregular spiking mode; (2) a burst firing mode. Investigators have described DA neurons as having a spontaneous slow depolarization which results in an action potential followed by an afterhyperpolarization. It has been suggested that D A neurons possess an intrinsic pacemaker potential which is modulated by afferents to produce the irregular and burst firing modes which are characteristic of their firing pattern in vivo. Investigators utilizing extracellular electrophysiological recordings in substantia nigra tissue slices have reported that DA sensitive neurons exhibit waveform and firing rate characteristics which are quite similar to those which have been described in vivo, except that they have a very regular firing pattern
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
308 (Sanghera et al., 1984; Silva and Bunney, 1986). This firing pattern was observed to become even more regular when synaptic activity was eliminated with high magnesium, low calcium medium (Sanghera et al., 1984) indicating that it is intrinsic to DA neurons. In order to understand the physiology of the D A system, an understanding of the membrane properties which underlie their intrinsic electrical activity and its modulation by DA are essential. To accomplish this, we used intracellular recordings to study the electrophysiology of DA neurons in the midbrain slice, a preparation which allowed us to examine the neurons more directly in a situation where most of their afferent input had been eliminated. The effects of dopamine were also investigated.
2. Materials and methods
Midbrain tissue slices were obtained from 200250 g male Sprague-Dawley rats. Following decapitation, the brains were quickly removed and a block containing the midbrain was cut. A razor blade slicer was used to section 300-400/~m thick frontal slices. Typically two slices were transferred to the recording-incubation chamber (Kelso et al., 1983) and allowed to incubate for 1-2 h prior to recording. The tissue slices rested on a nylon net above the inflow of the perfusion medium. Before entering the chamber, the perfusion medium was oxygenated (95% 02-5% CO 2) and heated to 36 ° C. The perfusion medium flowed hydrostatically through polyethylene tubing at a flow rate of 1.5-2 m l / m i n and three way stopcocks were used to exchange perfusion media. The normal nutrient medium (pH 7.35-7.40) consisted of (in mM): 124 NaCI; 4 KC1; 1.25 NaH2PO4; 1.22 MgSO4; 26 NaHCO3; 2.5 CaC12; 2 ascorbic acid; 11 glucose. Ascorbic acid was used as an antioxidant at a physiological concentration (Schenk et al., 1983) which was previously reported to have no effect on either the spontaneous activity of DA-sensitive neurons or the inhibitory action of DA on these cells (Silva et al., 1985). DA (10-200 ~M) and the D 2 antagonist, ( - ) s u l p i r i d e (0.5-1.5 ~M), were bath applied.
Detailed intracellular recording techniques have previously been described by Grace and Bunney (1983). Briefly, microelectrodes were pulled from 1.2 m m (O.D.) Omega dot glass tubing on a Brown and Flaming electrode puller. Electrodes were filled with 3 M KCI and had resistances of 50-90 M~2. Intracellular recordings were made from DA neurons which were identified by their slow firing rate, regular firing pattern, anatomical location and inhibitory response to DA. Recordings were made by using a conventional bridge amplifier (WPI M707). Data was collected on a Nicolet digital oscilloscope, a Gould Brush chart recorder and a Vetter F M tape recorder for off-line analysis on a PDP 1 1 / 3 4 computer. Only cells which had resting membrane potentials exceeding - 5 0 mV, action potentials greater than 50 mV and stable input resistances were included in the analysis. Resting input resistance (Ri) was determined by injecting small (0.05-0.1 nA) pulses of hyperpolarizing current and measuring steady state voltage (250 ms). For all resistance measurements care was taken to use only small hyperpolarizing current injections to avoid evoking rectification. The membrane time constants were calculated by plotting the log percent change of membrane voltage with respect to time as was observed following a constant current hyperpolarizing pulse (0.05-0.1 nA, 250 ms). In all neurons resting membrane potential was defined as the voltage that was observed between the afterhyperpolarization and the slow depolarization.
3. Results
Intracellular recordings were obtained from 93 zona compacta neurons in midbrain tissue slices. Putative D A neurons were identified by their slow firing rate (1-5 Hz), long duration action potential ( > 2 ms) and regular firing pattern. Cells which responded to DA perfusion with a decrease in firing rate were considered to be DA neurons. Other zona compacta neurons differed from presumed DA neurons as they were observed to have faster firing rates (1-13 Hz), shorter duration action potentials (0.5-1.5 ms) and were not affected by perfusion with DA. Following numerous stable
' 13° --
mV
Io:
309 TABLE 1 Membrane and firing characteristics of zona compacta neurons in vitro. Ri, M~2
Resting
z, ms
Firing rate, H2
Firing pattern
26.4+_2 (n = 46)
0.1-5.5
Regular
16.4+_1.3 (n = 25)
0.1-13
Irregular
membrane potential, mV Neurons inhibited by DA
56.1 +0.9 (n = 63)
172.5 + 4 (n = 60)
Remaining neurons 6 0 ms
57.2+_0.2 (n = 30)
78.4+_3.4 (n = 30)
B cell w h i c h w a s n o t i n h i b i t e d b y D A . I n b o t h c a s e s s p o n t a n e o u s l y f i r i n g cells w e r e h y p e r p o l a r i z e d t o just below firing level ( - 60 mV). Cells which were
A
60
ms
Fig. 1. Membrane properties of zona compacta neurons in vitro. (A) Voltage responses to current injection from a cell which was inhibited by DA. This cell had a resting membrane potential of - 5 6 mV and a R i of 180 M~2. The nonlinearity, indicative of inward rectification, was present in all cells which were inhibited by DA. The small spikes and afterdepolarization which followed the termination of the hyperpolarizing pulse were observed consistently following hyperpolafizing current injections of > 0.2 nA. (B) Voltage responses of a cell which did not respond to DA. Typically this response is linear. The resting membrane potential of this cell was - 5 8 mV and R i was 83 M~2. Overall, cells which were not inhibited by DA exhibited lesser Ri, greater linearity and smaller r.
15S
B
FIRING
RATE
VS MEMBRANE
POTENTIAL 6
4
i m p a l e m e n t s , it w a s e v i d e n t t h a t z o n a c o m p a c t a neurons could be distinguished by their membrane properties. Two types of zona compacta neurons were discerned according to differences in the following parameters: resting membrane potential, resting input resistance (Ri) and time constant
(~). Representative voltage (V) responses to current (I) i n j e c t i o n o f b o t h t y p e s o f n e u r o n s a r e s h o w n i n fig. 1. F i g u r e 1 A is a n e x a m p l e o f a cell t h a t w a s i n h i b i t e d b y D A w h i l e fig. 1 B is a n e x a m p l e o f a
2 ~ 1
-70
-65
~0
55
-50
-45
-40
MEMBRANE POTENTIAL (rnV)
Fig. 2. The firing rate of spontaneously firing DA-sensitive neurons was found to be inversely related to membrane potential. (A) Chart recording depicting firing rate of a cell manipulated by intracellular current injection. (Action potentials are truncated.) (B) The linear relationship between firing and membrane potential represented graphically. These neurons maintain firing rates of 0.1-5.5 Hz.
310
inhibited by DA (fig. 1A) maintained input resistances of 172 + 4 M~2 (S.D. used throughout) (n = 60), r of 26 _+ 2 ms (n = 46) and consistently displayed non-linear V-I relationships at hyperpolarized potentials indicative of inward rectification. Following the termination of hyperpolarizing pulses ( > 0.2 nA) a small spike (30-40 mV) and afterdepolarization were always elicited. In contrast, cells which were not inhibited by DA (fig. 1B) were observed to have input resistances of 78_+3 Mr2 ( n = 3 0 ) , ~'of 1 6 ± 1 m s ( n = 2 5 ) and exhibited linear V-I relationships within these same current injection parameters. In general, as summarized in table 1, all neurons displayed similar resting membrane potentials, however presumed DA zona compacta neurons exhibited greater R~, larger ~- and less linear V-I relationships than did non-OA zona compacta neurons. Membrane potential change over time following a hyper-
polarizing current pulse was fit by a single exponential in both cell groups. Spontaneously active putative DA neurons fired with a slow and regular pattern. Each action potential was preceded by a slow depolarization (8-12 mV, 150-800 ms) and was followed by an afterhyperpolarization. The beginning of the slow depolarization was defined as the point at which the afterhyperpolarization ended and a gradual depolarization could be seen and the spike threshold marked the end of the slow depolarization. (These parameters are most obvious in fig. 4B.) The firing rate of the cells could be increased or decreased by current injection (fig. 2A). The relationship between firing rate and membrane potential (fig. 2B) was found to be linear (correlation coefficient = 0.98, n = 55). Figure 3A-C illustrates the variation in the slow depolarization and the spike threshold which was
A I
,I
500
B
SLOW
| -70
C
/
DEPOLARIZATION
• -65
, -60
RATE
OF RISE
ms
THRESHOLD
• 007 '006
~1
• 005
O'n
.004 -003 -oo2 • 55
, -50
M E M B R A N E P O T E N T I A L (mV)
, 45
0.01
~
70
VS
MEMBRANE
MEMBRANE
POTENTIAL
65
60
J
55
-50
POTENTIAL (mV) -45
35 4o
m =
u)
.5o -55
~<:
60
Fig. 3. Parameters regulating DA cell firing rates were altered proportionally during the manipulation of membrane potential. (A) Two superimposed oscilloscope sweeps of action potentials (truncated) recorded at different membrane potentials. Membrane potential depolarization caused an increase in the rate of rise of the slow depolarization while the threshold for spiking became more depolarized. (B) Changes in the rate of rise of the slow depolarization with membrane potential. As the membrane potential was hyperpolarized from rest, the rate of rise of the slow depolarization was decreased, lengthening the interspike interval and slowing the firing rate, The inverse occurred during depolarizing current injection. (C) Changes in threshold were also a function of m e m b r a n e potential. As the m e m b r a n e potential was hyperpolarized from rest, the threshold for spiking became more hyperpolarized.
311
observed during the manipulation of membrane potential. The interspike slow depolarization and the spike threshold displayed a linear relationship with membrane potential, though these two parameters opposed one another. That is, as membrane potential was depolarized, the rate of rise of the slow depolarization was increased, thus decreasing the interspike interval and increasing the firing rate. However, as the membrane potential was depolarized the threshold for spiking became more depolarized. An irregular firing pattern could be demonstrated when the regularly firing neuron was investigated beyond the boundaries which we have already described. A sequence of changes in firing pattern induced by varying membrane potential are shown in fig. 4A-E. If the membrane potential was hyperpolarized (A) slightly from the resting level of - 5 9 mV, a decrease in the firing rate occurred as well as a break in the rhythmic firing pattern which was previously observed at rest (B). It should be noted that even though action potentials were occasionally absent the slow depolarizing potential was still present (arrows). The addition of TTX (1-2 /~M) to the bath eliminated all action potentials while the slow depolarization remained (not shown). When neurons were depolarized to - 5 0 and - 4 4 mV (C and D respectively) they began to fire action potentials irregularly, sometimes becoming quiescent. When neurons were held at - 4 4 mV depolarizing pulse injection elicited small spikes (E). These spikes might be either partially inactivated sodium spikes or they may be high threshold calcium spikes, as previously described in unidentified zona compacta cells in vitro (Kita et al., 1986; Llinas et al., 1984). DA (10-100 /zM) was administered to 93 neurons. In 63 spontaneous firing was inhibited (fig. 5A), in the remaining 30 neurons firing was unchanged. To investigate the changes in membrane resistance, cells were hyperpolarized to below firing threshold prior to DA application and hyperpolarizing current pulses (0.05-0.1 nA, 250 ms) were injected. DA-evoked inhibition was associated with a dose-dependent hyperpolarization and decrease in input resistance which was maintained during the manual 'clamping' of the mem-
J
L
100
ms
- 44mV
50mV
59mV
A
~
~
6
4
m
V
300ms
Fig. 4. Less regular firing patterns were seen at the upper and lower limits of firing rate. (A) W h e n the neurons were hyperpolarized a slow depolarizing potential could still be seen though occasionally threshold for spiking was not reached (arrows). (B) The resting m e m r b a n e potential of this cell was - 5 9 mV and its firing rate was 2.5 Hz. At this potential a rhythmic firing pattern was observed. (C) When this neuron was depolarized to - 50 mV, action potentials were sometimes absent. (D) Further depolarization of this cell to - 4 4 mV induced total cessation of action potentials. (E) When the cell was held at - 44 mV injection of a depolarizing pulse (200 ms, 0.2 nA) could still evoke small spikes.
brane potential to baseline levels (fig. 5B). These effects could be eliminated by (-)sulpiride, a selective D2 antagonist (fig. 5C). Voltage responses to current injection were examined prior to and during DA perfusion in order to calculate the reversal potential for the inhibitory response. From 10 cells tested, a mean reversal potential of - 8 8 + 14 mV was calculated. Hyperpolarizing voltage responses to larger hyperpolarizing current injection (0.4-0.5 nA) recorded in control and during DA perfusion both showed evidence of inward rectification. This parameter was not affected by DA. DA did not alter the relationship between membrane potential and either firing rate, spike threshold or the rate of rise of the slow depolariza-
312 A
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Fig. 5. The effects of DA perfusion. (A) An 85% inhibition of spontaneous firing and a 2-3 mV hyperpolarization of RMP were observed during 20 ~M DA perfusion. (B) This cell was hyperpolarized to below firing threshold and hyperpolarizing pulses (0.1 nA, 250 ms) were applied to examine the effects of DA o n R i. Perfusion of 100/~M DA produced a 6 mV hyperpolarization and 30 m~2 decrease in R i. The decrease in R i w a s maintained when the membrane potential was manually clamped to baseline levels. (C) Simultaneous perfusion of the slice with 100/~M DA and 1.5/~M (-)sulpiride eliminated both the hyperpolarization and changes in R i produced by DA.
tion other than w h a t w o u l d be e x p e c t e d to occur c o n c u r r e n t with a c h a n g e in m e m b r a n e p o t e n t i a l .
4. Discussion I n t r a c e l l u l a r r e c o r d i n g from n e u r o n s in the z o n a c o m p a c t a of rat s u b s t a n t i a nigra tissue slices has revealed that electrical m e m b r a n e p r o p e r t i e s can be used to distinguish D A - s e n s i t i v e n e u r o n s f r o m n o n - D A sensitive n e u r o n s in vitro. I n the m i d b r a i n slice, z o n a c o m p a c t a n e u r o n s exhibit electrophysiological a n d p h a r m a c o l o g i c a l characteristics which are quite similar to those which have been d e s c r i b e d in vivo. M o r e specifically, in the slice these cells have long d u r a t i o n b i p h a s i c extracellular action p o t e n t i a l s a n d they are i n h i b i t e d b y their own t r a n s m i t t e r as seen in whole a n i m a l
studies ( A g h a j a n i a n a n d Bunney, 1973; B u n n e y et al., 1973). U t i l i z i n g these c h a r a c t e r i s t i c p r o p e r t i e s to p u t a t i v e l y i d e n t i f y D A neurons, their memb r a n e p r o p e r t i e s were e x a m i n e d . The R~ of zona c o m p a c t a n e u r o n s in vitro are c o n s i d e r a b l y higher t h a n in vivo: it is a s s u m e d that this is due to the i m p r o v e m e n t of i m p a l e m e n t which is p o s s i b l e in this p r e p a r a t i o n . In this s t u d y we have r e p o r t e d differences b e t w e e n the m e m b r a n e characteristics of n e u r o n s which are i n h i b i t e d b y D A a n d neurons which are n o t affected by D A within the z o n a c o m p a c t a . In s u m m a r y , p u t a t i v e D A neurons possessed greater R~, larger ~- a n d less linear voltage responses to c u r r e n t injection than did n o n - D A neurons. T h e reasons for these differences remain u n d e t e r m i n e d , a l t h o u g h there are at least two possible e x p l a n a t i o n s for the differences in R i a n d T. O n e p o s s i b i l i t y is that the e l e c t r o p h y s i o l o g i c a l dif-
313 ferentiation is due to a variation in the cell sizes between the two populations. Anatomical studies of the basal ganglia have described neurons in the zona compacta as being medium in size ranging from 15-30 t~m (Hanaway et al., 1970). A detailed anatomical analysis concentrating on zona compacta neurons has not been done. An alternative explanation is that the two cell types are under different tonic neurotransmitter, neuromodulator or humoral influences. A few in vitro extracellular experiments have indirectly addressed this question. The addition of GABA antagonists (e.g. bicuculline) but not DA antagonists (e.g. ( - ) s u l p i ride) alter the firing rate of zona compacta neurons in the midbrain slice, suggesting that these cells maybe tonically influenced by G A B A in this preparation (Pinnock, 1983a; 1984) The counterpart experiments on non-DA neurons have not been carried out to date. This is the first intracellular investigation of the electrical activity of spontaneously firing putative DA neurons in the midbrain tissue slice. Our results support the hypothesis, which has been generated from in vivo experiments, that DA neurons have intrinsic pacemaker activity. In vivo, the firing pattern of identified DA neurons has been attributed to an oscillation between a slow depolarization and an afterhyperpolarization which are modulated by extrinsic inputs (Grace and Bunney, 1984a). In slices, a regular firing pattern has been observed in extracellular recording experiments substantiating this hypothesis (Pinnock, 1983a; Sanghera et al., 1984; Silva et al., 1985). The results of the present experiments demonstrate that the regular firing rate of DA-sensitive neurons recorded in vitro is linearly associated with the rate of rise of the slow depolarizing potential, which in conjunction with a variable spike threshold and an afterhyperpolarization determines the spontaneous firing rate of these neurons. Therefore, within a 10-15 mV range of membrane potential these voltage sensitive parameters determine the interspike interval and rate of pacemaker activity of these neurons. Similar oscillatory firing has been described in neurons of the inferior olive and thalamus (Jahnsen and Llinas, 1984; Llinas and Yarom, 1981). The sequence of events promoting firing of these neurons has been
attributed to: a sodium spike which is terminated by an afterhyperpolarization that is generated by one or more potassium currents, the afterhyperpolarization in turn deinactivates a low threshold calcium current providing a rebound depolarizing potential which initiates the next spike. The results of intracellular studies of unidentified quiescent zona compacta neurons in nigral slices have infered the existence of a voltage-sensitive sodium current, three potassium currents and both low threshold and high threshold calcium currents (Kita et al., 1986; Llinas et al., 1984). Thus, the complement of currents necessary for oscillatory firing behavior appears to be present in zona compacta neurons, although whether or not some of these were DA neurons was not determined. In vivo D A neurons do not fire with a regular pattern but in a slow and irregular or bursting mode. We have shown that a slow irregular pattern can be produced in these cells if the membrane potential was hyperpolarized by 3-5 mV or depolarized by 8-10 mV from rest. It is likely that, in vivo, inhibitory and excitatory afferents could modulate an irregular firing pattern. To date, bursting activity has not been observed in vitro and no doubt requires a different and possibly more complex explanation for its occurrence. The effects of DA on zona compacta neurons were also examined. DA perfusion produced inhibition of cell firing rate, a hyperpolarization of the m e m b r a n e potential and a decrease in input resistance. The calculated reversal potential for the response was - 88 mV, suggesting the involvement of potassium ions in the mechanism of DA action. A similar DA effect has been described in growth hormone producing neuroendocrine cells (De Vlieger et al., 1986), prolactin-secreting adenoma cells (Isreal et al., 1985) and in preliminary nigral slice experiments by ourselves and others (Silva and Bunney, 1986; Lacey et al., 1986). These results are in conflict with a previously published report by this laboratory in which Grace and Bunney (1983) described the actions of apomorphine as being hyperpolarizing but associated with an increase in R~. This discrepancy may be because these results were obtained in vivo using i.v. apomorphine administration making it difficult to distinguish direct agonist effects. In
314
our study, the response to DA did not appear to alter the inward rectification which was observed during current injection or to produce changes in the rate of rise of the slow depolarization or spike threshold other than would be expected due to the hyperpolarization of the membrane. The inhibition of the neuronal firing and membrane hyperpolarization could be explained by the enhancement of a calcium-activated potassium current which is associated with the afterhyperpolarization of DA neurons (Grace and Bunney, 1984a). Alternatively, similar to the actions of morphine and clonidine in the locus coeruleus (Aghajanian and Wang, 1986; Andrade and Aghajanian, 1985: North and Williams, 1985) or serotonin and GABA in the hippocampus (Andrade et al., 1986), a neurotransmitter specific potassium channel could be opened by DA to produce a hyperpolarization. In agreement with DA receptor localization studies (Gale et al., 1977; Quik et al., 1979), the effects of DA in our experiments were antagonized by ( - ) s u l p i r i d e , suggesting the involvement of somatic or dendritic autoreceptors of the D 2 type. In conclusion, we have demonstrated that DAsensitive zona compacta neurons are recognizable by their membrane properties in substantia nigra tissue slices. In this preparation DA-sensitive neurons have pacemaker-like firing patterns and possess a functional D 2 receptor. Activation of D 2 receptors apparently stimulates an increase in potassium ion conductance which is inhibitory to action potential generation. Future experiments should be aimed at the identification and characterization of the voltage-sensitive conductance mechanisms that underlie the pacemaker and other properties of DA-sensitive neurons.
Acknowledgements We would like to thank Chen-Lun Pun for laboratory assistance and Neil Harrison and David Lange for their assistance with manuscrupt preparation. This work was supported by United States Public Health Service Grants MH-28849, MH-25642, MH-14276 a research grant from the American Parkinson's Disease Association, James Hudson BrownAlexander B. Coxe Fellowship and the State of Connecticut.
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Pinnock, R.D., 1984, Hyperpolarizating action of baclofen on neurons in the rat substantia nigra slice, Brain Res. 322, 337. Quik, M., P.C. Emson and E. Joyce, 1979, Dissociation between the presynaptic dopamine-sensitive adenylate cyclase and [3H]spiperone binding sites in rat substantia nigra, Brain Res. 167, 355. Sanghera, M.K., M.E. Trulson and D.C. German, 1984, Electrophysiological properties of mouse dopamine neurons: in vivo and in vitro studies, Neuroscience 12, 793. Schenk, J.O., E. Miller, R. Gaddis and R.N. Adams, 1983, Homeostatic control of ascorbate concentration in CNS extracellular fluid, Brain Res. 253, 353. Silva, N . L and B.S. Bunney, 1986, Electrophysiological properties of dopamine-sensitive neurons in substantia nigra tissue slices, Soc. Neurosci. Abstr. 12, 1517. Silva, N.L., J.O. Schenk and B.S. Bunney, 1985, The effect of known concentrations of dopamine on dopamine cell firing rate in midbrain tissue slices, Neurosci. Abstr. 11, 1075.
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Elsevier UP 50263
Intracellular studies of dopamine neurons in vitro: pacemakers modulated by dopamine N a n c y L. Silva * ' * * a n d B e n j a m i n S. B u n n e y Department of Pharmacology and Psychiatry, Yale University, School of Medicine, New Hat~en, CT 06510, and * * Laboratory of Neurophysiology, NIH-N1NCDS. Building 36, Room 2 C02. Bethesda, MD 20892, U.S.A.
Received 9 February 1988, accepted 23 February 1988
Intracellular recordings from dopamine (DA)-sensitive neurons in rat substantia nigra tissue slices revealed that these neurons exhibit spontaneous pacemaker-like activity. DA-sensitive neurons had higher input resistances, larger time constants and less linear voltage responses to current injection than did non-DA-sensitive neurons in the zona compacta. The administration of DA produced an inhibition of firing rate, a hyperpolarization and a decrease in input resistance. These effects were blocked by (-)sulpiride, a selective D 2 antagonist. A reversal potential of - 8 8 _+ 14 mV was calculated for the DA-induced hyperpolarization suggesting the involvement of potassium ions in the mechanism of DA action. Dopamine; Substantia nigra; Pacemaker activity; Dopamine neurons; (Intracellular recording, Brain slice)
1. Introduction Midbrain dopamine (DA) neurons have been extensively studied electrophysiologically and pharmacologically. In whole animal studies, it has been demonstrated that the electrical activity of these neurons possess a characteristic waveform and firing pattern (Aghajanian and Bunney, 1973; Bunney et al., 1973; Grace and Bunney, 1983). These cells are inhibited by their own neurotransmitter in vivo (Aghajanian and Bunney, 1973; Bunney et al., 1973) and in vitro (Pinnock, 1983a; Sanghera et al., 1984). This autoinhibition appears to occur through a mechanism involving somatic or dendritic autoreceptors as the response to DA is maintained in the midbrain tissue slice preparation under conditions in which synaptic activity has been blocked (Pinnock, 1983b).
* To whom all correspondence should be addressed.
In vivo, DA neurons, identified following recording with fluorescence histochemical techniques, exhibit long duration ( > 2 ms) action potentials and fire at slow rates (1-5 Hz) (Grace and Bunney, 1984a,b). D A neurons have been observed in vivo to fire in two types of patterns: (1) a slow irregular spiking mode; (2) a burst firing mode. Investigators have described DA neurons as having a spontaneous slow depolarization which results in an action potential followed by an afterhyperpolarization. It has been suggested that D A neurons possess an intrinsic pacemaker potential which is modulated by afferents to produce the irregular and burst firing modes which are characteristic of their firing pattern in vivo. Investigators utilizing extracellular electrophysiological recordings in substantia nigra tissue slices have reported that DA sensitive neurons exhibit waveform and firing rate characteristics which are quite similar to those which have been described in vivo, except that they have a very regular firing pattern
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
308 (Sanghera et al., 1984; Silva and Bunney, 1986). This firing pattern was observed to become even more regular when synaptic activity was eliminated with high magnesium, low calcium medium (Sanghera et al., 1984) indicating that it is intrinsic to DA neurons. In order to understand the physiology of the D A system, an understanding of the membrane properties which underlie their intrinsic electrical activity and its modulation by DA are essential. To accomplish this, we used intracellular recordings to study the electrophysiology of DA neurons in the midbrain slice, a preparation which allowed us to examine the neurons more directly in a situation where most of their afferent input had been eliminated. The effects of dopamine were also investigated.
2. Materials and methods
Midbrain tissue slices were obtained from 200250 g male Sprague-Dawley rats. Following decapitation, the brains were quickly removed and a block containing the midbrain was cut. A razor blade slicer was used to section 300-400/~m thick frontal slices. Typically two slices were transferred to the recording-incubation chamber (Kelso et al., 1983) and allowed to incubate for 1-2 h prior to recording. The tissue slices rested on a nylon net above the inflow of the perfusion medium. Before entering the chamber, the perfusion medium was oxygenated (95% 02-5% CO 2) and heated to 36 ° C. The perfusion medium flowed hydrostatically through polyethylene tubing at a flow rate of 1.5-2 m l / m i n and three way stopcocks were used to exchange perfusion media. The normal nutrient medium (pH 7.35-7.40) consisted of (in mM): 124 NaCI; 4 KC1; 1.25 NaH2PO4; 1.22 MgSO4; 26 NaHCO3; 2.5 CaC12; 2 ascorbic acid; 11 glucose. Ascorbic acid was used as an antioxidant at a physiological concentration (Schenk et al., 1983) which was previously reported to have no effect on either the spontaneous activity of DA-sensitive neurons or the inhibitory action of DA on these cells (Silva et al., 1985). DA (10-200 ~M) and the D 2 antagonist, ( - ) s u l p i r i d e (0.5-1.5 ~M), were bath applied.
Detailed intracellular recording techniques have previously been described by Grace and Bunney (1983). Briefly, microelectrodes were pulled from 1.2 m m (O.D.) Omega dot glass tubing on a Brown and Flaming electrode puller. Electrodes were filled with 3 M KCI and had resistances of 50-90 M~2. Intracellular recordings were made from DA neurons which were identified by their slow firing rate, regular firing pattern, anatomical location and inhibitory response to DA. Recordings were made by using a conventional bridge amplifier (WPI M707). Data was collected on a Nicolet digital oscilloscope, a Gould Brush chart recorder and a Vetter F M tape recorder for off-line analysis on a PDP 1 1 / 3 4 computer. Only cells which had resting membrane potentials exceeding - 5 0 mV, action potentials greater than 50 mV and stable input resistances were included in the analysis. Resting input resistance (Ri) was determined by injecting small (0.05-0.1 nA) pulses of hyperpolarizing current and measuring steady state voltage (250 ms). For all resistance measurements care was taken to use only small hyperpolarizing current injections to avoid evoking rectification. The membrane time constants were calculated by plotting the log percent change of membrane voltage with respect to time as was observed following a constant current hyperpolarizing pulse (0.05-0.1 nA, 250 ms). In all neurons resting membrane potential was defined as the voltage that was observed between the afterhyperpolarization and the slow depolarization.
3. Results
Intracellular recordings were obtained from 93 zona compacta neurons in midbrain tissue slices. Putative D A neurons were identified by their slow firing rate (1-5 Hz), long duration action potential ( > 2 ms) and regular firing pattern. Cells which responded to DA perfusion with a decrease in firing rate were considered to be DA neurons. Other zona compacta neurons differed from presumed DA neurons as they were observed to have faster firing rates (1-13 Hz), shorter duration action potentials (0.5-1.5 ms) and were not affected by perfusion with DA. Following numerous stable
' 13° --
mV
Io:
309 TABLE 1 Membrane and firing characteristics of zona compacta neurons in vitro. Ri, M~2
Resting
z, ms
Firing rate, H2
Firing pattern
26.4+_2 (n = 46)
0.1-5.5
Regular
16.4+_1.3 (n = 25)
0.1-13
Irregular
membrane potential, mV Neurons inhibited by DA
56.1 +0.9 (n = 63)
172.5 + 4 (n = 60)
Remaining neurons 6 0 ms
57.2+_0.2 (n = 30)
78.4+_3.4 (n = 30)
B cell w h i c h w a s n o t i n h i b i t e d b y D A . I n b o t h c a s e s s p o n t a n e o u s l y f i r i n g cells w e r e h y p e r p o l a r i z e d t o just below firing level ( - 60 mV). Cells which were
A
60
ms
Fig. 1. Membrane properties of zona compacta neurons in vitro. (A) Voltage responses to current injection from a cell which was inhibited by DA. This cell had a resting membrane potential of - 5 6 mV and a R i of 180 M~2. The nonlinearity, indicative of inward rectification, was present in all cells which were inhibited by DA. The small spikes and afterdepolarization which followed the termination of the hyperpolarizing pulse were observed consistently following hyperpolafizing current injections of > 0.2 nA. (B) Voltage responses of a cell which did not respond to DA. Typically this response is linear. The resting membrane potential of this cell was - 5 8 mV and R i was 83 M~2. Overall, cells which were not inhibited by DA exhibited lesser Ri, greater linearity and smaller r.
15S
B
FIRING
RATE
VS MEMBRANE
POTENTIAL 6
4
i m p a l e m e n t s , it w a s e v i d e n t t h a t z o n a c o m p a c t a neurons could be distinguished by their membrane properties. Two types of zona compacta neurons were discerned according to differences in the following parameters: resting membrane potential, resting input resistance (Ri) and time constant
(~). Representative voltage (V) responses to current (I) i n j e c t i o n o f b o t h t y p e s o f n e u r o n s a r e s h o w n i n fig. 1. F i g u r e 1 A is a n e x a m p l e o f a cell t h a t w a s i n h i b i t e d b y D A w h i l e fig. 1 B is a n e x a m p l e o f a
2 ~ 1
-70
-65
~0
55
-50
-45
-40
MEMBRANE POTENTIAL (rnV)
Fig. 2. The firing rate of spontaneously firing DA-sensitive neurons was found to be inversely related to membrane potential. (A) Chart recording depicting firing rate of a cell manipulated by intracellular current injection. (Action potentials are truncated.) (B) The linear relationship between firing and membrane potential represented graphically. These neurons maintain firing rates of 0.1-5.5 Hz.
310
inhibited by DA (fig. 1A) maintained input resistances of 172 + 4 M~2 (S.D. used throughout) (n = 60), r of 26 _+ 2 ms (n = 46) and consistently displayed non-linear V-I relationships at hyperpolarized potentials indicative of inward rectification. Following the termination of hyperpolarizing pulses ( > 0.2 nA) a small spike (30-40 mV) and afterdepolarization were always elicited. In contrast, cells which were not inhibited by DA (fig. 1B) were observed to have input resistances of 78_+3 Mr2 ( n = 3 0 ) , ~'of 1 6 ± 1 m s ( n = 2 5 ) and exhibited linear V-I relationships within these same current injection parameters. In general, as summarized in table 1, all neurons displayed similar resting membrane potentials, however presumed DA zona compacta neurons exhibited greater R~, larger ~- and less linear V-I relationships than did non-OA zona compacta neurons. Membrane potential change over time following a hyper-
polarizing current pulse was fit by a single exponential in both cell groups. Spontaneously active putative DA neurons fired with a slow and regular pattern. Each action potential was preceded by a slow depolarization (8-12 mV, 150-800 ms) and was followed by an afterhyperpolarization. The beginning of the slow depolarization was defined as the point at which the afterhyperpolarization ended and a gradual depolarization could be seen and the spike threshold marked the end of the slow depolarization. (These parameters are most obvious in fig. 4B.) The firing rate of the cells could be increased or decreased by current injection (fig. 2A). The relationship between firing rate and membrane potential (fig. 2B) was found to be linear (correlation coefficient = 0.98, n = 55). Figure 3A-C illustrates the variation in the slow depolarization and the spike threshold which was
A I
,I
500
B
SLOW
| -70
C
/
DEPOLARIZATION
• -65
, -60
RATE
OF RISE
ms
THRESHOLD
• 007 '006
~1
• 005
O'n
.004 -003 -oo2 • 55
, -50
M E M B R A N E P O T E N T I A L (mV)
, 45
0.01
~
70
VS
MEMBRANE
MEMBRANE
POTENTIAL
65
60
J
55
-50
POTENTIAL (mV) -45
35 4o
m =
u)
.5o -55
~<:
60
Fig. 3. Parameters regulating DA cell firing rates were altered proportionally during the manipulation of membrane potential. (A) Two superimposed oscilloscope sweeps of action potentials (truncated) recorded at different membrane potentials. Membrane potential depolarization caused an increase in the rate of rise of the slow depolarization while the threshold for spiking became more depolarized. (B) Changes in the rate of rise of the slow depolarization with membrane potential. As the membrane potential was hyperpolarized from rest, the rate of rise of the slow depolarization was decreased, lengthening the interspike interval and slowing the firing rate, The inverse occurred during depolarizing current injection. (C) Changes in threshold were also a function of m e m b r a n e potential. As the m e m b r a n e potential was hyperpolarized from rest, the threshold for spiking became more hyperpolarized.
311
observed during the manipulation of membrane potential. The interspike slow depolarization and the spike threshold displayed a linear relationship with membrane potential, though these two parameters opposed one another. That is, as membrane potential was depolarized, the rate of rise of the slow depolarization was increased, thus decreasing the interspike interval and increasing the firing rate. However, as the membrane potential was depolarized the threshold for spiking became more depolarized. An irregular firing pattern could be demonstrated when the regularly firing neuron was investigated beyond the boundaries which we have already described. A sequence of changes in firing pattern induced by varying membrane potential are shown in fig. 4A-E. If the membrane potential was hyperpolarized (A) slightly from the resting level of - 5 9 mV, a decrease in the firing rate occurred as well as a break in the rhythmic firing pattern which was previously observed at rest (B). It should be noted that even though action potentials were occasionally absent the slow depolarizing potential was still present (arrows). The addition of TTX (1-2 /~M) to the bath eliminated all action potentials while the slow depolarization remained (not shown). When neurons were depolarized to - 5 0 and - 4 4 mV (C and D respectively) they began to fire action potentials irregularly, sometimes becoming quiescent. When neurons were held at - 4 4 mV depolarizing pulse injection elicited small spikes (E). These spikes might be either partially inactivated sodium spikes or they may be high threshold calcium spikes, as previously described in unidentified zona compacta cells in vitro (Kita et al., 1986; Llinas et al., 1984). DA (10-100 /zM) was administered to 93 neurons. In 63 spontaneous firing was inhibited (fig. 5A), in the remaining 30 neurons firing was unchanged. To investigate the changes in membrane resistance, cells were hyperpolarized to below firing threshold prior to DA application and hyperpolarizing current pulses (0.05-0.1 nA, 250 ms) were injected. DA-evoked inhibition was associated with a dose-dependent hyperpolarization and decrease in input resistance which was maintained during the manual 'clamping' of the mem-
J
L
100
ms
- 44mV
50mV
59mV
A
~
~
6
4
m
V
300ms
Fig. 4. Less regular firing patterns were seen at the upper and lower limits of firing rate. (A) W h e n the neurons were hyperpolarized a slow depolarizing potential could still be seen though occasionally threshold for spiking was not reached (arrows). (B) The resting m e m r b a n e potential of this cell was - 5 9 mV and its firing rate was 2.5 Hz. At this potential a rhythmic firing pattern was observed. (C) When this neuron was depolarized to - 50 mV, action potentials were sometimes absent. (D) Further depolarization of this cell to - 4 4 mV induced total cessation of action potentials. (E) When the cell was held at - 44 mV injection of a depolarizing pulse (200 ms, 0.2 nA) could still evoke small spikes.
brane potential to baseline levels (fig. 5B). These effects could be eliminated by (-)sulpiride, a selective D2 antagonist (fig. 5C). Voltage responses to current injection were examined prior to and during DA perfusion in order to calculate the reversal potential for the inhibitory response. From 10 cells tested, a mean reversal potential of - 8 8 + 14 mV was calculated. Hyperpolarizing voltage responses to larger hyperpolarizing current injection (0.4-0.5 nA) recorded in control and during DA perfusion both showed evidence of inward rectification. This parameter was not affected by DA. DA did not alter the relationship between membrane potential and either firing rate, spike threshold or the rate of rise of the slow depolariza-
312 A
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Fig. 5. The effects of DA perfusion. (A) An 85% inhibition of spontaneous firing and a 2-3 mV hyperpolarization of RMP were observed during 20 ~M DA perfusion. (B) This cell was hyperpolarized to below firing threshold and hyperpolarizing pulses (0.1 nA, 250 ms) were applied to examine the effects of DA o n R i. Perfusion of 100/~M DA produced a 6 mV hyperpolarization and 30 m~2 decrease in R i. The decrease in R i w a s maintained when the membrane potential was manually clamped to baseline levels. (C) Simultaneous perfusion of the slice with 100/~M DA and 1.5/~M (-)sulpiride eliminated both the hyperpolarization and changes in R i produced by DA.
tion other than w h a t w o u l d be e x p e c t e d to occur c o n c u r r e n t with a c h a n g e in m e m b r a n e p o t e n t i a l .
4. Discussion I n t r a c e l l u l a r r e c o r d i n g from n e u r o n s in the z o n a c o m p a c t a of rat s u b s t a n t i a nigra tissue slices has revealed that electrical m e m b r a n e p r o p e r t i e s can be used to distinguish D A - s e n s i t i v e n e u r o n s f r o m n o n - D A sensitive n e u r o n s in vitro. I n the m i d b r a i n slice, z o n a c o m p a c t a n e u r o n s exhibit electrophysiological a n d p h a r m a c o l o g i c a l characteristics which are quite similar to those which have been d e s c r i b e d in vivo. M o r e specifically, in the slice these cells have long d u r a t i o n b i p h a s i c extracellular action p o t e n t i a l s a n d they are i n h i b i t e d b y their own t r a n s m i t t e r as seen in whole a n i m a l
studies ( A g h a j a n i a n a n d Bunney, 1973; B u n n e y et al., 1973). U t i l i z i n g these c h a r a c t e r i s t i c p r o p e r t i e s to p u t a t i v e l y i d e n t i f y D A neurons, their memb r a n e p r o p e r t i e s were e x a m i n e d . The R~ of zona c o m p a c t a n e u r o n s in vitro are c o n s i d e r a b l y higher t h a n in vivo: it is a s s u m e d that this is due to the i m p r o v e m e n t of i m p a l e m e n t which is p o s s i b l e in this p r e p a r a t i o n . In this s t u d y we have r e p o r t e d differences b e t w e e n the m e m b r a n e characteristics of n e u r o n s which are i n h i b i t e d b y D A a n d neurons which are n o t affected by D A within the z o n a c o m p a c t a . In s u m m a r y , p u t a t i v e D A neurons possessed greater R~, larger ~- a n d less linear voltage responses to c u r r e n t injection than did n o n - D A neurons. T h e reasons for these differences remain u n d e t e r m i n e d , a l t h o u g h there are at least two possible e x p l a n a t i o n s for the differences in R i a n d T. O n e p o s s i b i l i t y is that the e l e c t r o p h y s i o l o g i c a l dif-
313 ferentiation is due to a variation in the cell sizes between the two populations. Anatomical studies of the basal ganglia have described neurons in the zona compacta as being medium in size ranging from 15-30 t~m (Hanaway et al., 1970). A detailed anatomical analysis concentrating on zona compacta neurons has not been done. An alternative explanation is that the two cell types are under different tonic neurotransmitter, neuromodulator or humoral influences. A few in vitro extracellular experiments have indirectly addressed this question. The addition of GABA antagonists (e.g. bicuculline) but not DA antagonists (e.g. ( - ) s u l p i ride) alter the firing rate of zona compacta neurons in the midbrain slice, suggesting that these cells maybe tonically influenced by G A B A in this preparation (Pinnock, 1983a; 1984) The counterpart experiments on non-DA neurons have not been carried out to date. This is the first intracellular investigation of the electrical activity of spontaneously firing putative DA neurons in the midbrain tissue slice. Our results support the hypothesis, which has been generated from in vivo experiments, that DA neurons have intrinsic pacemaker activity. In vivo, the firing pattern of identified DA neurons has been attributed to an oscillation between a slow depolarization and an afterhyperpolarization which are modulated by extrinsic inputs (Grace and Bunney, 1984a). In slices, a regular firing pattern has been observed in extracellular recording experiments substantiating this hypothesis (Pinnock, 1983a; Sanghera et al., 1984; Silva et al., 1985). The results of the present experiments demonstrate that the regular firing rate of DA-sensitive neurons recorded in vitro is linearly associated with the rate of rise of the slow depolarizing potential, which in conjunction with a variable spike threshold and an afterhyperpolarization determines the spontaneous firing rate of these neurons. Therefore, within a 10-15 mV range of membrane potential these voltage sensitive parameters determine the interspike interval and rate of pacemaker activity of these neurons. Similar oscillatory firing has been described in neurons of the inferior olive and thalamus (Jahnsen and Llinas, 1984; Llinas and Yarom, 1981). The sequence of events promoting firing of these neurons has been
attributed to: a sodium spike which is terminated by an afterhyperpolarization that is generated by one or more potassium currents, the afterhyperpolarization in turn deinactivates a low threshold calcium current providing a rebound depolarizing potential which initiates the next spike. The results of intracellular studies of unidentified quiescent zona compacta neurons in nigral slices have infered the existence of a voltage-sensitive sodium current, three potassium currents and both low threshold and high threshold calcium currents (Kita et al., 1986; Llinas et al., 1984). Thus, the complement of currents necessary for oscillatory firing behavior appears to be present in zona compacta neurons, although whether or not some of these were DA neurons was not determined. In vivo D A neurons do not fire with a regular pattern but in a slow and irregular or bursting mode. We have shown that a slow irregular pattern can be produced in these cells if the membrane potential was hyperpolarized by 3-5 mV or depolarized by 8-10 mV from rest. It is likely that, in vivo, inhibitory and excitatory afferents could modulate an irregular firing pattern. To date, bursting activity has not been observed in vitro and no doubt requires a different and possibly more complex explanation for its occurrence. The effects of DA on zona compacta neurons were also examined. DA perfusion produced inhibition of cell firing rate, a hyperpolarization of the m e m b r a n e potential and a decrease in input resistance. The calculated reversal potential for the response was - 88 mV, suggesting the involvement of potassium ions in the mechanism of DA action. A similar DA effect has been described in growth hormone producing neuroendocrine cells (De Vlieger et al., 1986), prolactin-secreting adenoma cells (Isreal et al., 1985) and in preliminary nigral slice experiments by ourselves and others (Silva and Bunney, 1986; Lacey et al., 1986). These results are in conflict with a previously published report by this laboratory in which Grace and Bunney (1983) described the actions of apomorphine as being hyperpolarizing but associated with an increase in R~. This discrepancy may be because these results were obtained in vivo using i.v. apomorphine administration making it difficult to distinguish direct agonist effects. In
314
our study, the response to DA did not appear to alter the inward rectification which was observed during current injection or to produce changes in the rate of rise of the slow depolarization or spike threshold other than would be expected due to the hyperpolarization of the membrane. The inhibition of the neuronal firing and membrane hyperpolarization could be explained by the enhancement of a calcium-activated potassium current which is associated with the afterhyperpolarization of DA neurons (Grace and Bunney, 1984a). Alternatively, similar to the actions of morphine and clonidine in the locus coeruleus (Aghajanian and Wang, 1986; Andrade and Aghajanian, 1985: North and Williams, 1985) or serotonin and GABA in the hippocampus (Andrade et al., 1986), a neurotransmitter specific potassium channel could be opened by DA to produce a hyperpolarization. In agreement with DA receptor localization studies (Gale et al., 1977; Quik et al., 1979), the effects of DA in our experiments were antagonized by ( - ) s u l p i r i d e , suggesting the involvement of somatic or dendritic autoreceptors of the D 2 type. In conclusion, we have demonstrated that DAsensitive zona compacta neurons are recognizable by their membrane properties in substantia nigra tissue slices. In this preparation DA-sensitive neurons have pacemaker-like firing patterns and possess a functional D 2 receptor. Activation of D 2 receptors apparently stimulates an increase in potassium ion conductance which is inhibitory to action potential generation. Future experiments should be aimed at the identification and characterization of the voltage-sensitive conductance mechanisms that underlie the pacemaker and other properties of DA-sensitive neurons.
Acknowledgements We would like to thank Chen-Lun Pun for laboratory assistance and Neil Harrison and David Lange for their assistance with manuscrupt preparation. This work was supported by United States Public Health Service Grants MH-28849, MH-25642, MH-14276 a research grant from the American Parkinson's Disease Association, James Hudson BrownAlexander B. Coxe Fellowship and the State of Connecticut.
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315 hyperpolarizes neurones in the rat substantia nigra zona compacta in vivo by increasing a potassium conductance, J. Physiol. 381, 51P. Llinas, R., S.A. Greenfield and H. Jahnsen, 1984, Electrophysiology of pars compacta cells in the in vitro substantia nigra a possible mechanism for dendritic release, Brain Res. 294, 127. Llinas~ R. and Y. Yarom, 1981, Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurons in vitro, J. Physiol. 315, 569. North, R.A. and J.T. Williams, 1985, On the potassium conductance increased by opioids in rats locus coeruleus neurones. J. Physiol. 364, 265. Pinnock, R.D., 1983a, Sensitivity of compacta neurons in the rat substantia nigra slice to dopamine agonists, European J. Pharmacol. 96. 269. Pinnock, R.D., 1983b, Inhibition of pars compacta neurons by dopamine in slices of rat substantia nigra maintained in vitro, J. Physiol. 334, 88P. -
Pinnock, R.D., 1984, Hyperpolarizating action of baclofen on neurons in the rat substantia nigra slice, Brain Res. 322, 337. Quik, M., P.C. Emson and E. Joyce, 1979, Dissociation between the presynaptic dopamine-sensitive adenylate cyclase and [3H]spiperone binding sites in rat substantia nigra, Brain Res. 167, 355. Sanghera, M.K., M.E. Trulson and D.C. German, 1984, Electrophysiological properties of mouse dopamine neurons: in vivo and in vitro studies, Neuroscience 12, 793. Schenk, J.O., E. Miller, R. Gaddis and R.N. Adams, 1983, Homeostatic control of ascorbate concentration in CNS extracellular fluid, Brain Res. 253, 353. Silva, N . L and B.S. Bunney, 1986, Electrophysiological properties of dopamine-sensitive neurons in substantia nigra tissue slices, Soc. Neurosci. Abstr. 12, 1517. Silva, N.L., J.O. Schenk and B.S. Bunney, 1985, The effect of known concentrations of dopamine on dopamine cell firing rate in midbrain tissue slices, Neurosci. Abstr. 11, 1075.