167

Journal of Physiology (1992), 453, pp. 167-183 With 6 figures Printed in Great Britain

NEURONAL SELECTIVITY OF ATP-SENSITIVE POTASSIUM CHANNELS IN GUINEA-PIG SUBSTANTIA NIGRA REVEALED BY RESPONSES TO ANOXIA BY KERRY P. S. J. MURPHY AND SUSAN A. GREENFIELD From the University Department of Pharmacology, Mansfield Road, Oxford OX1 3QT

(Received 22 July 1991) SUMMARY

1. Two sub-populations of pars compacta substantia nigra neurones were identified with very different electrophysiological properties and rostral-caudal distribution. Both cell types were identified by biocytin intracellular dye injection and found to be located within pars compacta containing tyrosine hydroxylasepositive cells. These sub-populations displayed distinctly different responses to transient anoxia. 2. The first group ('Phasic' neurones) exhibited a low threshold calcium conductance LTS gc. associated with bursts of action potentials, were located at the level of the mammillary bodies and were highly sensitive to anoxia. The second group ('rhythmic' neurones) fired in a rhythmic pattern, were located at the level of the accessory optic tract and were relatively insensitive to anoxia. 3. The anoxic response of phasic cells was characterized by membrane hyperpolarization (mean 12 mV), a decrease in input resistance (mean 36 %) and cessation of action potential firing. The axonic response of these neurones was not blocked by TEA (5-10 mM), haloperidol (100 /SM), the removal of extracellular calcium or depletion of endogenous dopamine. However, this effect was blocked by both the sulphonylurea tolbutamide (50-500 /tM), and also by quinine (100 /tM) and could be mimicked by application of diazoxide (1 mM). 4. Rhythmic cells displayed a variable response to anoxia consisting of either modest depolarization, hyperpolarization or no change in membrane potential, in all cases accompanied by little or no change in input resistance. The polarity of the membrane potential shift during anoxia was reversed by TEA (5-10 mM) or the removal of calcium. These cells were also relatively insensitive to diazoxide (1 mM). 5. It is concluded from the neuronal responses to anoxia and the pharmacological modification of these responses, that the ATP-sensitive potassium channel (KATP channel) is functionally operative in the substantia nigra and is primarily distributed on the phasically discharging cells of the rostral pars compacta. The relevance of this recently discovered ionic channel is discussed with regard to the normal and abnormal functioning of the substantia nigra.

MS 9570

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K. P. S. J. MURPHY AND S. A. GREENFIELD INTRODUCTION

The ATP-sensitive potassium channel (KATP) is an apparently unique ion channel in that it serves as a direct link between cellular metabolism and transmembrane events (Ashcroft, 1988). Recent binding studies (Mourre, Ben Ari, Bernardi, Fosset

& Lazdunski, 1989; Mourre, Widmann & Lazdunski, 1990b; Treherne & Ashford, 1991) using the sulphonylurea [3H]glibenclamide, which is believed to be a good marker for the KATP channel, have shown that within the CNS, the nucleus where the distribution of the binding sites is most dense is that which is primarily lesioned in Parkinson's disease, the substantia nigra. It could be the case therefore that the KATP channel plays a vital part in the aetiology of Parkinson's disease. Indeed, it was reported long ago, though the rationale is not understood, that the sulphonylurea tolbutamide, is an efficacious treatment in Parkinson's disease (Gates & Hyman, 1960; Hansten & Kristensen, 1965). However, the contribution of the KATP channel to nigral function and dysfunction has only been indirectly inferred from antagonist binding studies (Mourre et al. 1989; Mourre et al. 1990b; Mourre, Smith, Siesj & Lazdunski, 1990a), net neurochemical release (Amoroso, Schmidt-Antomarchi, Fosset & Lazdunski, 1990) or highly non-physiological treatments such as complete removal of intracellular ATP or application of cromakalim, pinacedil (Hausser, de Weille & Lazdunski, 1991) or cyanide (Murphy & Greenfield, 1991). In none of these experimental protocols has it been possible to simulate a situation in which the KATP channel might be normally activated, nor has it been feasible to monitor cell metabolism or the state of the extracellular environment, in simultaneous conjunction with transmembrane events. A further difficulty is that the localization of the KATP channel within the substantia nigra has to date been tentative: it has been suggested that it is located presynaptically on GABA afferent axon terminals (Amoroso et al. 1990) and also postsynaptically on dopaminoceptive neurones (Roeper, Hainsworth & Ashcroft, 1990) with uniform electrophysiological characteristics (Hausser et al. 1991; Hainsworth, Roeper, Kapoor & Asheroft, 1991). None the less, previous experiments employing cyanide application have suggested that there may be a neuronal selectivity for the KATP channel, in the substantia nigra, in a particular subdivision of pars compacta cells (Murphy & Greenfield, 1991). The aim of this study was thus to investigate the electrophysiological and pharmacological profile of neurones differentially sensitive to transient anoxia monitored on-line. In addition, attempts were made to see whether the responses to anoxia were specifically mediated by the KATP channel, by observing the effects of agents known to act as selective channel agonists (diazoxide) (Trube, Rorsman & Ohno-Shosaku, 1986), or antagonists (tolbutamide) (Ashcroft & Ashcroft, 1990) or be relatively inefficacious (TEA) (Ashcroft, 1988). The possibility that any responses to anoxia could be linked to the dopamine receptor, was investigated by employing reserpine and c-methyl-p-tyrosine (AMPT) to deplete the neurones of endogenous dopamine or haloperidol to block the dopamine receptor (Kapoor, Webb & Greenfield, 1989). In order to see whether anoxic responses were dependent on the trans-synaptic release of neurochemicals (Amoroso et al. 1990), the effects were observed of blockade of impulse flow by administration of TTX, and abolition of

FUNCTIONAL KATP CHANNELS IN THE CNS 169 transmitter release by substitution in the perfusing medium of magnesium for calcium ions. METHODS

In vitro slice preparation Male albino guinea-pigs (350-500 g) were anaesthetized with halothane (4%), decapitated and the whole brain removed and rinsed in cold oxygenated artificial cerebrospinal fluid containing HEPES (HEPES-ACSF). A block of mesencephalon containing the substantia nigra was then isolated, mounted on the cutting carriage of a vibratome (Lancer, series 1000) and bathed in cold oxygenated HEPES-ACSF. Coronal sections of mesencephalon (400 #sm thick), containing the substantia nigra, were cut and individually placed in separate vials of oxygenated HEPES-ACSF and maintained at 22 TC for at least 2 h prior to transferral to a recording chamber. The recording chamber consisted of a Perspex well with a Sylgard base on which the slices were placed, anchored by silver wire and superfused at 3 ml/min with either oxygenated or anoxic ACSF maintained at 32-34 0C. Solutions The HEPES-ACSF contained (mM): NaCl, 120; KCl, 5; NaHCO3, 20; CaCl2, 2; MgSO4, 2; glucose, 10; HEPES acid, 6-7; HEPES salt, 3-3. The standard ACSF used in the recording chamber contained (mM): NaCl, 123; KCl, 2; NaHCO3, 26; KH2PO4, 1P25; MgSO4, 13; CaCl2, 2-4; glucose, 10. The calcium-free solution was the same as the standard ACSF except for the omission of CaC12 and the replacement of 1-3 mM-MgSO4 with 3-7 mM-MgCl2. Anoxic ACSF was prepared by bubbling the solution with 95 % nitrogen and 5 % carbon dioxide gas (BOC) for 20 min prior to entry into the recording chamber. The oxygen scavenger, sodium hydrosulphite (500 /M, Aldrich), was added to the anoxic solution 30 s prior to use. The level of anoxia was monitored in the recording chamber with an oxygen-sensitive electrode placed next to the brain slice, downstream to the flow of ACSF (Strathkelvin Instruments, Model 781; Clark-type polargraphic electrode (1302); response time of 18 s for 90% change in oxygen level). The output of the oxygen meter was displayed on a chart recorder in vertical register with membrane voltage (see below) and recorded on magnetic tape. Sodium hydrosulphite added to oxygenated ACSF was without effect. All drugs were bath applied via the perfusing ACSF to the effective concentrations stated in the text. Tetraethylammonium chloride (TEA, Sigma), tetrodotoxin (TTX, Sigma), quinine (Sigma), a-methyl-p-tyrosine (AMPT, Sigma) haloperidol (as Serenace, Searle) and diazoxide (Sigma) were freshly prepared for each experiment. Tolbutamide (Sigma) was prepared from a 0-5 M stock solution in dimethyl sulphoxide (DMSO). DMSO when applied alone was without effect. Intracellular recording and analysis Recordings were made using glass microelectrodes filled with either 3 M-potassium acetate or biocytin (Sigma), (series resistances measured in ACSF: 50-120 Mfl). The microelectrodes were mounted in a micromanipulator and placed with the aid of a dissecting microscope in the region of the pars compacta of the substantia nigra. Intracellular potentials were passed to a Neurodat (IR-283) preamplifier with an active bridge circuit to allow simultaneous measurements of membrane potential and intracellular injection of current through the recording electrode. Input current and voltage signals were stored on magnetic tape for off-line analysis. When an intracellular impalement was made, a 05-1 0 nA hyperpolarizing holding current was applied for 5-10 min to encourage a tight seal. On removal of the holding current, only those neurones that had an apparent resting membrane potential equal to or greater than -45 mV and displayed an overshoot action potential were retained for experimentation. Square pulses of hyperpolarizing current (0-1-1-0 nA, 200 ms duration, at 0-2-0-5 Hz) were applied throughout an experiment to monitor apparent input resistance in control, anoxic and drug-containing ACSF. The majority of retained neurones fired action potentials spontaneously, making it difficult to measure resting membrane potential. However, by constructing voltage-current relationships for each neurone it was possible to ascertain a 'measure' of the resting membrane potential from the voltage intercept at zero current (simple regression line fitted to data, in each case correlation coefficient r2 > 0.99). Input resistances were calculated from the slope of the voltage-current relationship. At the beginning of an experiment each neurone was tested for the presence of a low threshold calcium

170

K. P. S. J. MURPHY AND S. A. GREENFIELD

conductance (LTS gca, Llinas, Greenfield & Jahnsen, 1984) and associated burst firing of action potentials; this procedure was performed by firstly hyperpolarizing the neurone with constant current (0 2-05 nA) to de-inactivate the LTS gca and then secondly applying a step depolarization to activate it. Records were obtained on-line as a DC trace played out on a two-channel chart recorder (Tekman, TE850). The other channel was used to display the output of the oxygen meter (see above). Data was analysed off-line using a D/A interface (CED 1401), personal computer (Research Machines, Nimbus VX/2) connected to a laser printer and employing Sigavg software (CED). Unless otherwise stated, all numerical data are expressed as the mean+ standard error of the mean, and all statistical significance values are calculated using Student's paired t test. Dopamine depletion Some animals were depleted of dopamine prior to the preparation of slices. These animals were injected intraperitoneally with reserpine (5 mg/kg, dissolved in 20 % ascorbic acid) (Burn & Rand, 1960; Boarder & Fillenz, 1979) 24 h before halothane anaesthesia. To prevent de novo synthesis and release of dopamine in the slices, AMPT (100 /SM) was added to HEPES-ACSF and all ACSF solutions (Leviel, Cheramy & Glowinski, 1979). Dopamine concentrations were measured in striatal tissue taken from both control and reserpinated animals. Once the slices had been prepared, the striata were dissected out on ice, homogenized in 0 5 ml of 20 mM-perchloric acid and centrifuged (1000 g, 5 min). The supernatant was removed and analysed by HPLC for dopamine as previously described by Greenfield, Appleyard & Bloomfield (1986), while the pellet was analysed for protein content (Lowry, Rosenbrough, Fair & Randall, 1957). The dopamine content was then calculated as ng/mg protein. Tyro8ine hydroxyla8e immunocytochemi8try Following intracellular recording, the slices were transferred to a solution of sucrose (30%) in phosphate buffered saline (PBS), and resectioned in 50 ,um slices. The tissue was then washed twice for 5 min in PBS and placed in 0-2 % H202 in 100 % methanol for 30 min. Following two further 5 min washes in PBS (pH 7 4), the slices were incubated in 5 % bovine serum albumin (BSA) and 0-2 % Triton in PBS for 30-60 min. After two further 5 min PBS washes, the tissue was incubated in 1 % tyrosine hydroxylase antibody for 40 h at room temperature. The tissue was subsequently washed twice in PBS containing 10 % goat serum for 5 min each time. The tissue was then incubated for 30-60 min in rabbit peroxidase anti-peroxidase (PAP) (1:100 in PBS and 1 % goat serum). After two further washes in PBS, the slices were incubated to visualize reactive neurones by incubation for 6 min in diaminobenzideine (DAB) (6 mg), 10 ml PBS (pH 7-4) 13-3 ,1 H202 (30 % w/v). The sections were again washed in PBS and mounted on gelatinized slides and air dryed. They were then dehydrated and mounted on slides for light microscope examination. Intracellular filling with dye At the end of experiments employing a biocytin-filled microelectrode, the impaled neurones were filled with dye by injection of depolarizing current pulses (0 5 nA, duration 500 ms, at 1 Hz for 5-10 min). The slices were then removed from the experimental chamber and the nigral hemisphere not containing the filled cell removed and processed for tyrosine hydroxylase immunoreactivity. The hemisphere containing the filled cell was washed twice in PBS for 5 min and transferred for 1 h to 0 2 % H202 in 100 % methanol. Following two further PBS washes, sections were incubated for 15 h in avidin-biotin complex (Dako Co.) in 10 ml PBS. After two more 5 min rinses in PBS, biocytin-filled neurones were visualized by incubation for 6-10 min in 6 mg DAB, 10 ml PBS, 250 #1 1 % CoCl2, 200 ,u 1 % ammonium nickel sulphate, 100 jul DMSO and 13-3 #1 H202 (30% w/v). Following a further rinse in PBS, the tissue was dehydrated in a series of solutions of methyl salicylate (50, 70 and 90%, and absolute alcohol twice), then blotted. RESULTS

Location of neurones within the substantia nigra Long-lasting and stable recordings (30 min-5 h) which were subsequently fully analysed, were obtained for a total of 113 cells located in the pars compacta of the substantia nigra at either the level of the mammillary bodies or of the accessory optic

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FUNCTIONAL KATP CHANNELS IN THE CNS 171 tract. Subsequent histochemical examination showed that all cells were located in regions of nigra displaying tyrosine hydroxylase immunoreactivity (Fig. 1). At the level of the light microscope, a clear difference was apparent in tyrosine hydroxylase immunoreactivity between rostral sections and those at the level of, and caudal to,

Fig. 1. Histological characterization of phasic neurones. A, coronal section (50,um) of guinea-pig mesencephalon at the level of the mammillary bodies, showing tyrosine hydroxylase immunoreactivity in the pars compacta region of the substantia nigra. Phasic neurones were recorded at this level. Scale bar: 2 mm. B, typical tyrosine hydroxylase immunoreactive neurones recorded from the rostral pars compacta, as in A. C, a phasic neurone injected with biocytin in rostral pars compacta. Note similarity in shape, size and dendritic arborization to cell in B. Magnification in B and C: x 80.

the accessory optic tract. In rostral sections, the density of tyrosine hydroxylase reactive 'apical' dendrites appeared greater than in the more caudal sections. Tyrosine hydroxylase immunoreactive neurones displayed variety in the shape of the soma and possessed sparsely branching dendrites extending both within the pars compacta and the pars reticulate (Fig. LB). The morphology of cells filled with biocytin, at both the levels of the mammillary bodies (Fig. 1 C) and accessory tract was indistinguishable from that of the neurones positively identified as dopaminergic pars compacta neurones (Fig. 1B).

Electrophysiological properties The neurones reported here displayed two distinctly different electrophysiological profiles with a marked rostral-caudal distribution. Pars compacta neurones (n = 81) found at the level of the mammillary bodies generated a low threshold calcium conductance (LTS 7ca; Llinas et al. 1984; Kostyuk, 1989), activated by membrane depolarization from a hyperpolarized state (Fig. 2). Activation of the LTS gc. evoked an accompanying burst of sodium action potentials (Llinas et al. 1984) (Fig. 2). Due to this ability to switch firing pattern, and the intermittent nature of spontaneous discharge, these neurones are referred to as 'phasic'. A second class of pars compacta neurone was found at the level of the accessory optic tract (n = 32). These neurones did not possess a LTS 9can and therefore showed no tendency to burst fire (Fig. 2). Rather, action potentials were generated at a constant, rhythmic rate; consequently these neurones are referred to as 'rhythmic'.

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These cells were also characterized by a greater amplitude action potential, a pronounced afterhyperpolarization and lower firing rate (Table 1). They also displayed marked 'anomalous' IQ-type and outward 'Ia-type' rectifiers (Fig. 2) (see also Grace & Onn, 1989; Harris, Webb & Greenfield, 1990; Yung, Hausser & Jack,

-

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I 1 nA 500 ms Fig. 2. The two characteristic cell types recorded in this study: A, phasic neurone (apparent resting membrane potential -47 mV, input resistance 68 MQ, firing frequency 18 Hz). Left: superimposition of membrane voltage responses to injections of hyperpolarizing current (0-2, 0 4, 0-6, 0-8 nA). Centre: generation of LTS gca-evoked burst firing, activated by depolarizing phase of a 04 nA hyperpolarizing pulse from a membrane potential hyperpolarized from rest (dashed line). Right: generation of LTS gCa-evoked burst firing, activated by a brief period of depolarization from a hyperpolarized membrane potential. B, rhythmic neurone (apparent membrane potential -56 mV, input resistance 146 M2, firing rate 3 Hz). Left: superimposition of membrane responses to injections of hyperpolarizing current (0-2, 0 4, 0-6, 0-8 nA); note characteristic inward and outward rectification. Centre: membrane response to an injection of 0-6 nA hyperpolarizing current from a hyperpolarized membrane potential; note the absence of LTS 9Ca; note marked increase in membrane rectification, input resistance and magnitude of action potential afterhyperpolarization. Right: membrane response to an injection of depolarizing current (0 4 nA) from a hyperpolarized membrane potential; note absence of LTS gca evoked burst firing.

1991). The electrophysiological properties of both phasic and rhythmic neurones are summarized in Table 1.

Neuronal selectivity of anoxia The basal concentration of dissolved oxygen in control ACSF, monitored in the bath chamber, was in the range of 10-25 ml oxygen/l. During each experiment, the basal oxygen concentration remained constant. However, from experiment to

FUNCTIONAL KATP CHANNELS IN THE CNS

173

experiment there was a variation in the measured oxygen concentration, primarily due to small differences in the placement of the oxygen electrode, variation in the bath ACSF level and the evolution of gas bubbles which occasionally collected in the vicinity of the oxygen probe. On exchange of control ACSF with anoxic ACSF, the oxygen level within the recording chamber approached zero within 2-5 min. On TABLE 1. Comparison of phasic and rhythmic neurones Rhythmic cells Phasic cells (n = 32) Significance (n = 81) Firing rate (Hz) P < 0001 1-98+0-212 15-6+1-12 Input resistance (MC) P > 0-5 106-1 +6-03 107-7 +3-80 Membrane potential (mV) -55-4+1 25 -55-7+0-56 P > 0-5 Threshold (mV) 0-5 > P > 0-1 -42-3+ 1-16 -43-1 +048 Action potential width (measured at threshold) (ms) 2-21 +0-066 P < 0-001 0-97 +0-022 Action potential width (measured half-way between threshold and peak amplitude) (ms) P < 0-001 0-51+0-019 1P23+0-053 Action potential amplitude (measured from threshold) (mV) 0-02 > F> 0-001 50-9+0-86 55-5+1-67 Action potential maximum amplitude (mV) P < 0-001 82-4+1-59 73-2+0-94 NB All values are the mean+standard error of the mean; P values from Student's unpaired t test.

return to oxygenated ACSF, the bath oxygen concentration rapidly returned to normal, within 1-2 min. Within each experiment, the time course and extent of anoxia was highly reproducible, making it possible to examine and compare the effects of anoxia under varying pharmacological conditions. Phasic and rhythmic cells exhibited highly selective membrane responses to periods of anoxia (3-5 min each).

The effects of anoxia on phasic cells Phasic cells challenged by anoxia (53/58 neurones, 91 %) typically responded by displaying a marked membrane hyperpolarization (apparent membrane potential prior to anoxia, - 56-6 + 0-74 mV; during anoxia, - 68-0 + 0-99 mV (P < 0-001, n = 53)), accompanied by a decrease in membrane input resistance (36 + 1-89 %, n = 53) and the complete cessation of action potential firing. On re-oxygenation, the cells rapidly returned to their control state. However, in some cases, re-oxygenation was accompanied by a further, but transitory hyperpolarization, most readily attributable to re-activation of the ATP-dependent sodium-potassium pump. Often, the LTS gCa and associated burst firing were evident during the initial hyperpolarizing slope of the anoxic response and the subsequent depolarizing slope seen on recovery

(Fig. 3B).

K. P. S. J. MURPHY AND S. A. GREENFIELD Construction of voltage-current relationships for eleven phasic neurones prior to and during the peak effect of anoxia revealed an anoxia reversal potential of -97 + 4-35 mV (Fig. 3 C). This value is very close to the estimated potassium reversal potential of -100 mV for these neurones (at 33 0C, assuming intracellular potassium 174

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5 min Fig. 3. Anoxic responses and voltage-current relationships of a phasic neurone. A, phasic neurone (apparent resting membrane potential -48 mV, input resistance 74 MQ, firing rate 22 Hz). Superimposition of membrane responses to injections of hyperpolarizing current prior to and during anoxia (0-2, 0-4, 016, 0-8 nA). B, chart record of oxygen concentration and membrane response illustrating the time course of action of a 5 min exposure to anoxic ACSF (same cell as in A). Hyperpolarizing current pulses were applied throughout to monitor changes in membrane input resistance (0-2 nA, 200 ms, 0-2 Hz, together with two periods, prior to and during anoxia, when a series of hyperpolarizing currents were applied to allow the construction of voltage-current relationships). Arrowheads denote LTS gc.-evoked burst firing generated during the hyperpolarizing and depolarizing phases of the anoxic response. Action potentials attenuated by frequency response of the chart recorder (this also pertains to all chart record illustrations shown). C, plot of voltage-current relationships prior to (@) and during anoxia (0) (same cell as in A and B). Each data point is an average of measurements made at 175 ms from four successive pulses; standard deviations lost within the symbols. Lines fitted to data are simple regression lines (in both instances, correlation coefficients (r2 > 0-99). Intersect of voltage-current relationships reveal an anoxic reversal potential of -94 mV.

175 FUNCTIONAL KATP CHANNELS IN THE CNS concentration = 140 mV) and strongly suggests that the anoxic response of phasic cells is due to a highly selective increase in membrane potassium conductance.

Characterization of phasic cell anoxic response In nine phasic neurones, TEA (5-10 mm) abolished action potential afterhyperpolarization, dramatically widened the action potential and eventually induced Control

TTX

TTX+10 mM-TEA

155

Oxygen (ml/l)

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3

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Fig. 4. Chart records showing modification of anoxic response in the presence of TTX and TEA. Phasic neurone (apparent membrane potential -48 mV, input resistance 77 MW, firing rate 40 Hz). Left: anoxic response prior to the application of TTX and TEA (hyperpolarizing current pulses were applied: 0 4 nA, 200 ms, at 0-2 Hz). Centre, anoxic response in the presence of 1 /#M-TTX (hyperpolarizing current pulses applied: 0-2 nA, 200 ms, at 0-2 Hz). Right, anoxic response in the presence of TTX and 10 mM-TEA (hyperpolarizing current pulses applied: 0-2 nA, 200 ms, at 0-2 Hz).

depolarizing block (Fig. 4). In five cases the cells then entered a bi-stable state, exhibiting large potential fluctuations from the depolarizing block potential to a hyperpolarized potential, probably due to a cyclic activation of gK(Ca) (Llina's et al. 1984) followed by subsequent activation of the LTS gC.. Figure 4 illustrates the effect of TEA on the anoxic response: in this particular example TTX (10-6 M) was used to prevent TEA-induced bi-stability. Application of TEA did not abolish the anoxic response (Fig. 4) for any of the nine phasic neurones tested. The mean membrane potential prior to anoxia in the absence of TEA was -54-5 + 1-82 mV (n = 9) and during the peak effect of anoxia, -65+2-72 mV (n = 9) (P < 0001, n = 9), accompanied by a 37-11 + 5-13 % (n = 9) decrease in input resistance. In the presence of TEA, the mean membrane potential prior to anoxia was -43 + 3-2 mV (n = 9) and during anoxia, -68+2-88 mV (n = 9) (P < 0001, n = 9), accompanied by a 41-2 + 3-6 % decrease in input resistance. Even though the mean membrane potential was depolarized in the presence of TEA by 11-5 + 3.3 mV (n = 9), there was no significant difference in the final steady-state hyperpolarized potential seen during anoxia, nor in the reduction in input resistance, in control and TEA-containing perfusate, in both cases (0 5 > P > 0 1).

[~ ~ ~Oxygen(ml/)

K. P. S. J. MURPHY AND S. A. GREENFIELD

176

Four phasic cells were recorded in slices depleted of dopamine (control animals, 19-6+1-19 ng dopamine/mg protein, n = 8; reserpinated animals, 0-72 + 042 ng dopamine/mg protein, n = 4). Depletion of dopamine did not abolish or modify the anoxic response (mean apparent membrane potential prior to anoxia A

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B

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Fig. 5. The anoxic response of phasic cells is not dependent on the presence of extracellular calcium ions. A, phasic neurone (apparent membrane potential -57 mV, input resistance 121 MC, firing rate 22 Hz). Left: generation of LTS g,.-evoked burst firing from a hyperpolarized membrane potential; note voltage dependency of the response. Centre: generation of LTS gca in the presence of 1 ,/M-TTX. Right: abolition of LTS gca in the absence of extracellular calcium (indicating a near zero concentration for extracellular calcium ions). B, chart records of oxygen concentration and membrane potential, illustrating the anoxic response of the above cell in the control state (left), presence of TTX (centre) and removal of extracellular calcium (right). Hyperpolarizing current pulses were applied: 0-2 nA, 200 ms, at 0-2 Hz.

was -54-2+3317 mV, n = 4; during anoxia -68+3-5 mV, n = 4 (P < 0001), accompanied by a 428 + 48 % (n = 4) decrease in input resistance). In a further three neurones, application of haloperidol (100 /,M), did not affect the anoxic response (data not shown). TTX was applied to nine phasic neurones; in all cases sodium action potentials were abolished, leaving the LTS 9ca unaffected (see Fig. 5A). In seven of these cells, TTX induced a persistent depolarization of 10-9+ 1-96 mV (n = 7). TTX did not

FUNCTIONAL KATP CHANNELS IN THE CNS Control

5

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Recovery

Quinine

Recovery

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Fig. 6. Pharmacology of anoxic response recorded in phasic neurones (chart records of oxygen concentration and membrane potential). A, phasic neurone (resting membrane potential -47 mV; input resistance 68 MO, firing rate 18 Hz). Left: control response to anoxia. Centre: 500 ,SM-tolbutamide blocks the normal anoxic response. Right: recovery of anoxic response 45 min after the removal of tolbutamide. In all, hyperpolarizing current pulses applied: 0 4 nA, 200 ms, at 0-2 Hz. B, phasic neurone (apparent membrane potential -53 mV, input resistance 83 MCI, firing rate 30 Hz). Left: normal anoxic response. Centre: anoxic response in the presence of 100 /iM-quinine. Right: recovery of the normal anoxic response, 28 min after the removal of quinine. In all, hyperpolarizing current pulses applied: 0-2 nA, 200 ms, at 0-2 Hz. C, phasic neurone (apparent membrane potential -57 mV, input resistance 67 MKI, firing rate 10 Hz). Application of 1 mmdiazoxide followed by subsequent co-application of 500 /vM-tolbutamide (hyperpolarizing current pulses applied: 0-2 nA, 200 ms, at 0-2 Hz).

block the anoxic response (Figs 4 and 5B). The apparent resting membrane potential of these cells prior to TTX was -56-2 + 1-78 mV (n = 9), during anoxia -68-7 + 3 0 mV (n = 9) (P < 0001), accompanied by a 33-3 + 5-79 % (n = 9) decrease

K. P. S. J. MURPHY AND S. A. GREENFIELD 178 in input resistance. In the presence of TTX the membrane potential prior to anoxia was -47-7 + 3-29 mV (n = 9), during anoxia -67-6 + 4-16 mV (n = 9) (P < 0-001), accompanied by a 25-9 + 6-5 % (n = 9) decrease in input resistance. There was no significant difference between the final steady-state hyperpolarized potential and decrease in input resistance achieved during anoxia in the presence and absence of TTX; 0 5 > P > 041 (n = 9) and 0 5 > P > 0.1 (n = 9) respectively. The effect of the removal of extracellular calcium on the anoxic response was examined in eight phasic neurones. Disappearance of the LTS 9ca was judged to indicate the near complete removal of extracellular calcium ions (Fig. 5A). Often, the removal of calcium (by substitution with magnesium) caused instability in the recording; however, stability was improved by the addition of TTX (10-6 M). Regardless of whether TTX was present or not, the anoxic response was not abolished by the removal of extracellular calcium ions (Fig. 5B): membrane potential prior to anoxia was -54+1 mV (n = 3), during anoxia -68+1 mV (n = 3), accompanied by a decrease in input resistance of 48-7 + 1-89 % (n = 3); in the absence of extracellular calcium, membrane potential prior to anoxia was -46 + 2-33 mV (n = 3), during anoxia - 64-7 + 3-33 mV (n = 3), accompanied by a decrease in input resistance of 53 +1 /% (n = 3). The action of tolbutamide was examined on the anoxic response of phasic neurones (Fig. 6A). Tolbutamide alone had no effect on passive membrane properties. However, it did drastically modify the anoxic response of these cells. Tolbutamide not only blocked the characteristic hyperpolarizing response seen during anoxia, but also prevented the reduction in input resistance and the cessation of action potential firing (Fig. 6A). In the absence of tolbutamide the normal apparent resting membrane potential of the cells was - 57-2 + 1'51 mV (n = 9), during anoxia -68-1 + 1-95 mV (n = 9), accompanied by a 40-8+ 5-71 % (n = 9) decrease in input resistance. In the presence of tolbutamide, the apparent resting membrane potential was -57*4 + 1-5 mV (n = 9); however, instead of hyperpolarizing during anoxia, the cells now displayed a depolarizing response, - 55-8 +1P78 mV (n = 9), which in turn was accompanied by an increase in input resistance of 8-4 + 10-93 % (n = 9). In five cases, following a full hour of washing after the removal of tolbutamide, it was possible to show complete or partial recovery of the hyperpolarizing anoxic response (Fig. 6A). A second KATP channel antagonist, quinine (100-300 jrm; Ashcroft & Ashcroft, 1990), was used to investigate the anoxic response in an additional four phasic cells. Overall, quinine reversed the polarity of the membrane response to anoxia: - 158+ 1-79 mV (n = 4) and +4-25+ 6-2 mV (n = 4) prior to and during exposure to quinine respectively, and reduced the magnitude of the reduction in input resistance: 37-8 + 2-2 % (n = 4) and 1P5 ± 241 % (n = 4) prior to and during quinine application respectively. Figure 6B illustrates the action of quinine on the anoxic response of a phasic cell. This example was chosen because it shows complete recovery of the anoxic response on the wash-out of quinine. However, it should be noted that it was the only cell of the four tested which did not show a clear depolarization, in the presence of quinine, in response to anoxia. Diazoxide was applied to three phasic neurones which had previously exhibited hyperpolarizing responses to anoxia (apparent membrane potential prior to anoxia

179 FUNCTIONAL KATP CHANNELS IN THE CNS -57x7+2x67 mV (n = 3), during anoxia -72-3+3±18 mV, accompanied by a 41 + 9-06 % (n = 3) decrease in input resistance). In these cells diazoxide mimicked the effects of anoxia (apparent membrane potential prior to diazoxide application - 57-6 + 2-55 mV (n = 3), during diazoxide exposure -70 + 4 50 mV (n = 3) together with a 28 + 150 % decrease in input resistance (Fig. 6C). In two of these cells, the effects of diazoxide were completely blocked on the addition of tolbutamide to the bathing medium (Fig. 6C).

The effects of anoxia on rhythmic cells The effect of transient anoxia was examined for twenty rhythmic cells. Unlike phasic cells which responded in a consistent manner to anoxia, rhythmic cells gave rise to a variety of transient membrane responses; eight cells either depolarized or showed little change in membrane potential whilst displaying either increases or decreases in input resistance; twelve cells exhibited a modest hyperpolarization accompanied by either an increase, decrease or no change in input resistance. Overall, the apparent resting membrane potential prior to anoxia was -54-8+ 146 mV (n =20), during anoxia there was a small hyperpolarization to - 588 + 2-04 mV (n = 20), accompanied by a 9-1 + 418 % (n = 20) decrease in input resistance. The effect of TEA application on rhythmic cell responses to anoxia was examined in five neurones. The main effect of TEA was to reverse the polarity of the membrane responses to anoxia. The mean apparent resting potential for these cells in the absence of TEA was - 55'6 + 3-74 mV (n = 5), during anoxia - 57-8 + 476 mV (n = 5), accompanied by a 2-8 + 7-24 % increase in input resistance; in the presence of TEA, the mean membrane potential prior to anoxia was -55-7 + 3-5 mV (n = 5), during anoxia -53 + 4-21 mV (n = 5), accompanied by a 0 4 + 0 39 % (n = 5) decrease in input resistance. The TEA-induced change in the polarity of the anoxic response was significant, P < 0 05 (n = 5). Removal of extracellular calcium also reversed the polarity of the anoxic response (n = 3 neurones, data not shown). The effect of tolbutamide was examined on the membrane anoxic response (n = 4). In three cells, tolbutamide was without effect, but in one it did reverse the polarity of the anoxic response from a 9 mV hyperpolarization to a 4 mV depolarization. Application of diazoxide to rhythmic neurones induced a marginal hyperpolarization (2-17 +0-88 mV, n = 6); however, there was no accompanying change in input resistance. DISCUSSION

Previous investigations of the KATP channel in the substantia nigra have used treatments that block cellular respiration (Murphy & Greenfield, 1991) or which act directly on the channel (Hausser et al. 1991). The procedure used here to activate the KATP channel, oxygen deprivation, has the advantage of enabling the sensitive recording in real time of the changes in the extracellular milieu: hence the removal of oxygen can be kept as 'physiological' as possible, and the risk of neuronal damage minimized, by reversing the anoxia within a few minutes of its taking full effect. This treatment of transient anoxia does not appear to involve any non-specific effects on the neuronal membrane, as the principle finding of this study is that there

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is a selectivity of response which corresponds to a specific electrophysiological and pharmacological neuronal profile. One group of neurones was relatively insensitive to anoxia: these cells were located in the pars compacta neurones at the level of the accessory optic tract, displayed a wide action potential, inward and outward rectifiers, and a rhythmic firing rate (described in this study as 'rhythmic' cells). It is noteworthy that the modest anoxia-induced responses of these neurones were not uniform in terms of the polarity of shift in the membrane potential: furthermore, neither increases nor decreases in membrane potential were accompanied by any clear changes in input resistance. On the other hand, both types of response were Ca2+-dependent: it is conceivable therefore that the effects of anoxia on these cells were a result of trans-synaptic events from intact neuronal circuits. The effects of anoxia on these neurones were sensitive to TEA and insensitive to tolbutamide. In addition, these neurones were also insensitive to diazoxide application. Since the KATP channel is relatively insensitive to TEA, sensitive to tolbutamide and activated by diazoxide (Findlay, Dunne, Ullrich, Wollheim & Petersen, 1985; Ashcroft, 1988), it is difficult to regard the spasmodic hyperpolarizing responses as unequivocal examples of activated KATP channels. On the other hand, neurones with similar electrophysiological characteristics to the above group of cells have been reported to possess the KATP channel due to their responses to cromakalim, pinacedil or removal of ATP (Hausser et al. 1991) or because tolbutamide reversed the hyperpolarizing action of a dopamine agonist (Roeper et al. 1990). However, the neurones used in both these studies (Roeper et al. 1990; Hausser et al. 1991) were dissociated and thus any direct comparison of membrane properties with those of neurones in intact slices, such as in this study, may not be strictly justified. An additional consideration is that in these earlier reports, the purported activation of the KATP channel was not fully characterized, in that the actions of the relatively ineffective blocker TEA were not investigated nor was the response compared with that of any second population of neurones displaying an even greater sensitivity, as in these experiments. In one study, no second population of neurones was examined (Roeper et al. 1990) whilst in the other (Hausser et al. 1991) no other type of neurone showed any sensitivity at all to any of the KATP channel activating treatments. It is concluded from the data presented here that this subpopulation of rhythmically firing pars compacta neurones is not the prime location of a physiologically active KATP channel within the substantia nigra. By contrast, in rostrally distributed, phasically discharging pars compacta neurones (described in this study as 'phasic' cells), anoxia induced a marked and consistent response, characterized by membrane hyperpolarization, decrease in input resistance and cessation of action potential firing. This response was clearly due to a direct effect on the phasic cell membrane, since it persisted after blockade of impulse flow by TTX and of transmitter release by removal of calcium ions. The persistence of the anoxic response of phasic neurones after the removal of extracellular calcium, and indeed to TEA application, would be consistent with mediation of the effect by a KATP channel (see Ashcroft, 1988). Indeed the observations that the anoxic response could be blocked by tolbutamide and quinine, two chemically diverse KATP channel antagonists (Findlay et al. 1985; Ashcroft, 1988; Ashcroft & Ashcroft, 1990), and mimicked by the KATP channel opener

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diazoxide (Trube et al. 1986), would provide fairly conclusive evidence that the potassium conductance activated by anoxia in these neurones is indeed mediated by the KATP channel. It is noteworthy that this presumed activation of the KATP channel on phasic cells persisted following the depletion of endogenous dopamine by reserpine and amethyl-p- tyrosine application. In addition, the experiments involving removal of calcium ions from the perfusate have demontrated that the activation of the KATP channel on these cells is independent of transmitter release of any kind, including the dendritic release of dopamine from the pars compacta neurones themselves (Neioullon, Cheramy & Glowinski, 1977). In this regard it should be noted that Roeper et al. (1990) have shown that the action of the D2 receptor antagonist, quinpirole can be reversed by tolbutamide, and hence concluded that endogenous dopamine might activate the KATP channel. The results presented here, however, would suggest that endogenous dopamine would act via an alternative potassium channel. It has been shown that the KATP channel-mediated anoxic response here is resistant to TEA, yet the hyperpolarizing actions of dopamine are sensitive to this particular potassium channel blocker (Lacey, Mercuri & North, 1988; Nedergaard, Webb & Greenfield, 1989). Indeed tolbutamide antagonism per se cannot be presumed to be a completely faithful index of functioning KATP channels (Doroshenko, Kostyuk, Martynyuk, Kursky & Vorobetz, 1984). There is a growing body of evidence that there are at least two sub-populations of nigral dopaminergic cells, in terms of their location and morphology (Gerfen, Baimbridge & Thibault, 1987 a; Gerfen, Herkenham & Thibault, 1987 b; Graybiel, Hirsch & Agid, 1987; Ostergaard, Schou & Zimmer, 1990), degree of tyrosine hydroxylase immunoreactivity (Gerfen et al. 1987a, b), electrophysiology (Chiodo, Antelman, Caggiula & Lineberry, 1980; Shepard & German, 1988; Kubota, Kang & Kitai, 1989; Hainsworth et al. 1991) and pathology (Gerfen et al. 1987 a; Gibb & Lees, 1991). The present findings suggest a further criterion could be sensitivity to anoxia. This work was funded by Bristol-Myers Squibb Co. (USA). We would like to thank Chris Webb and Michael Bertoz for their excellent technical assistance, Pat Coudery for her histological services, Claire Wardell for her HPLC expertise, Lesley Annetts for her photographic skill and Elizabeth Clarke for her secretarial aid.

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