SYNAPSE 7:221-234 (1991)

Regulation of Spontaneous Activity and Oscillatory Spike Firing in Rat Midbrain Dopamine Neurons Recorded In Vitro ANTHONY A. GRACE Departments of Behavioral Neuroscience and Psychiatry, Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

KEY WORDS

Electrophysiology, Pacemaker, Anomalous rectification, Afterhyperpolarization

ABSTRACT

Intracellular recordings were obtained from identified dopamine (DA) neurons in rat midbrain slices maintained in vitro. DA neuron membranes exhibited pronounced instantaneous and time-dependent anomalous rectification that showed evidence of maximal activation at average membrane potentials of -63 and -78 mV, respectively. Action potentials were followed by prominent afterhyperpolarizations (AHP) that consisted of two components. The fast component showed evidence of inactivation at -63 mV independent of the initial membrane potential, whereas the longer-duration, later component increased in amplitude at hyperpolarized potentials. Unlike DA neurons recorded in vivo, there was no evidence of spike frequency adaptation or summation of AHPs with prolonged depolarization-induced spike trains. Spontaneous spike discharge occurred via an endogenous pacemaker potential that was dependent on both T?xsensitive and cobalt-sensitive processes. Hyperpolarizing prepulses could activate rebound pacemaker discharge, but this rebound activity was progressively blocked with larger-amplitude hyperpolarizing prepulses. DA neurons recorded in the anesthetized animal, freely moving animal, and in vitro preparations have been shown t o exist in two states of activity: 1) spontaneously discharging action potentials or 2) hyperpolarized, quiescent, and nonfiring. Furthermore, although it is rare to find DA neurons in the untreated animal in transitional states of activity, quiescent neurons can be activated by stimuli that place a demand on the DA system. The evidence presented here is consistent with the hypothesis that the special combination of membrane properties of DA neurons contribute to the segregation of their activity into active or inactive states.

INTRODUCTION Changes in the membrane potential of neurons exert a number of influences on their levels of spontaneous dischar e and their res onsivity to stimuli. This effect of mem rane potentia is believed to occur via the interaction of several voltage-dependent membrane conductances, includin the low-threshold calcium s ike (LTS; Llinas and arom, 1981,b), activation and &activation of potassium conductances such as the A-current (IA;Connor and Stevens, 1971)and the anomalous rectifier (Adrian, 1969; Constanti and Galvan, 1983; Halliwell and Adams, 19821, as well as some

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Spike firin in midbrain dopamine (DA) neurons is driven by suc an endogenous pacemaker-like de olarization (Grace, 1987; Grace and Bunney, 19 3a,b 1984a; Grace and Onn, 1989; Kita et al., 1986), and these cells possess several uni ue membrane properties that regulate their activity ( race, 1987, 1990; Grace and Onn, 1989). Spontaneous s ike firing in DA neurons recorded in vivo consists o irregular single spike firing or burst firing (Bunney et al., 1973; Grace and , 1984a,b), with firing frequencies showing a norma distribution around an average rate of 4.5 2 1.7 Hz (mean i S.D.; Grace and Bunney, 1984a). Furthermore, this distribution trails off ra id1 at both low and high frequencies, with very few DX c e h firin above 8 Hz or below 1 Hz. Nonetheless, the distri ution of actiuit of DA neurons is actually bimodal, since approximate$ one-third to one-half of the DA neurons are hyperpolarized and inactive (Bunney and Grace, 1978), with no evidence of spontaneously occurring depolariz-

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ating spike discharge, such as those neurons which fire in response to endogenous pacemaker depolarizations. 0 1991 WILEY-LISS. INC.

Received April 12,1990; accepted in revised form August 27,1990

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oxygenated physiological saline at a flow rate of 3 ml per minute via a peristaltic pump (Haake-Biichler model MCP2500). Concentrated drug solutions were added to the chamber using a second peristaltic pum running at 1/100of the speed of the superfusion pump. his allowed accurate control over the final drug concentration in the bath while minimizing the exposure of the drug t o the oxygenated, buffered solution. Calcium conductances were blocked by lowering the calcium concentration in the superfusion solution to 1 mM and adding 2 mM cobalt to physiological saline buffered with tris to diminish precipitation of the cobalt (Llinas and Sugimori, 1980). Electrodes were pulled from 1 mm O.D. Omegadot borosilicate glass tubing (WPI, New Haven, Connecticut) using a Flamin -Brown P80PC electrode puller. The pipettes were fi led with potassium acetate (3 M) and had resistances ranging from 40-65 megohms measured in situ. Electrode potentials were am lified by an adjacent head stage connected to a Neuro ata preamplifier (model IR-283),and current was injected into the cell via an integral bridge circuit balanced by convententials &race, 1978, this report, the effects of altering DA neuron membrane tional means. Potentials were di 'tized continuously at potential on voltage-dependent membrane properties 44 kHz by a NeuroCorder (mo el DR-484) and were and oscillatory firing of DA neurons was examined stored on videotapes using a four-head VHS videocasusing an in vitro brain slice preparation. The data sette recorder. Data were anal zed off-line usin a presented here suggest that the unique membrane Cambridge Electronics Design (ZED, Cambridge, 6K) 1401 Intelligent Laboratory Interface connected to a properties of the DA neuron may be a factor in enablin Compaq microcomputer, and hard copies were obtained spontaneous1 active DA neurons to remain active an attenuating tTle response to depolarizing events in hy- via a Hewlett-Packard 7475A digital plotter for records erpolarized, uiescent DA neurons. These data have less than 3 seconds in duration. Longer-duration traceen presente in part in symposia (Grace, 1987,1988; ings were output in analog form to a Gould RS3400 three-channel chart recorder at 1160th of the capture Grace and Onn, 1988). rate to allow accurate representation of spike ampliMATERIALS AND METHODS tudes. Dopamine-containing cells were identified according Male Sprague-Dawleyrats weighing between 220 and 350 grams were used in all experiments. The animals to their characteristic waveform and pacemaker de owere handled according to the Guide or the Care and larization (Grace and Bunney, 1983a)and by the higI? ly Use o Animals published by the SPHS, and the regular pacemaker firing pattern observed in the in roce ures were approved by the Institutional Animal vitro preparation (Grace and Onn, 1989). In all cases, [are and Use Committee of the University of Pitts- statistics are stated in terms of mean t standard devibur h. Procedures were performed as described previ- ation. ous y (Grace, 1990; Grace and Onn, 1989; Llinas and RESULTS Su 'mori, 1980). Brief1 rats were anesthetized with The data presented here represent the results from soc? ium pentobarbital ( embutal; 50 mgkg i.p.1 and decapitated, and the brain was rapidly removed and more than 70 identified DA neurons recorded intracelplaced in ice-cold h siological saline (composition: lularl in vitro. Both quiescent and spontaneously ac124 mM NaC1, 5 m& kC1, 1.2 mM KH2P04, 2.4 mM tive A neurons were recorded intracellularly in the CaCl,, 1.3 mM MgSO 26 mM NaHC03, and 10 mM midbrain slice. Over half of the neurons impaled exhibglucose)that had beentubbled with 95%:5%C02:02for ited spontaneous activity with chamber temperatures at least 20 minutes. A 5 mm-thick block was sectioned maintained at 36.5" to 37.5"C. The level of activity of DA from the midbrain region between the pons and the neurons has been shown to de end on the temperature infundibulum using a Rat Brain Matrix (RBV-~OOOC, of the chamber (Grace and J n n , 1989; Shepard and Activational Systems, Warren, Michi an) to ensure that Bunney, 1988).The effects of membrane polarization by the section was recisely perpendicu ar to the anterior- constant current injection on DA cell input resistance posterior axis. ections 400 pm thick were cut in ice- and spike activity were tested. cold, oxygenated physiological saline using a Vibratome Interaction between membrane potential and (Pelco model 1000).A brain region ventral to the medial input resistance geniculate and medial to the cortex was isolated using Hyper olarizing or depolarizing the membrane poiris scissors and placed into chambers containing continuously oxygenated hysiological saline at room tem- tential o DA neurons caused reproducible changes in perature for at least 1 our rior to recording. After this cell input resistance and spike activity. In ut resistance incubation period, single syices were transferred to a was determined by measuring the mem rane voltage submerged-slice type recording chamber maintained at change roduced in response to hyperpolarizing current 37°C i 0.5"C and were continuously superfused with pulses. goth the peak membrane voltage deflection and ing events (Grace and Bunney, 1986).Com arable findings have also been reported in recordings rom DA cells in freely moving rats (Freeman et al., 1985; Freeman and Bunney, 1987). Furthermore, quiescent DA neurons can be shifted into a spontaneous firing state by stimuli that place a demand on the DA system, such as the administration of DA-blocking dru s (Bunney and Grace, 1978; Grace and Bunney, 19865, ionto horetic administration of excitatory neurotransmitters YBunne and Grace, 1978; Grace and Bunney, 1986; SkirboJ et al., 1981), artial lesions of DA terminals (Hollerman et al., 1986; Ylollerman and Grace, 19881, or re eated administration of amphetamine (Kamata and tebec, 1984; White and Wang, 1984). In a number of cases, repeated activation by excitatory stimuli will initiate s ontaneous s ike firin in these quiescent DA neurons race, 1987; race an Bunney, 1986). Both nonfirin and s ontaneously active DA neurons have been recor ed in t e in vitro midbrain slice preparation, with

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the stead -state membrane potential produced by the pulse (puze duration = 350 msec) were plotted in order to determine the contribution of the instantaneous and time-dependent anomalous rectification to the overall membrane conductance measured at the soma. As reported previously, DA neurons recorded in vitro typically exhibited input resistances of 80 megohms or eater (average = 168 megohms) at Grace and Onn, 1989). Currentholtage generated at constant depolarized or membrane potentials to examine the brane potential change on the input resistance and rectification. Plotting the am litude of the hyper olarizing current pulses in'ecte against the mem lane voltage responses revea ed that the peak and plateau input resistance curves exhibited different nonlinear

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regions and inflection points that were dependent on the steady-state membrane otential. With increasing levels of h erpolarization, t ere was an increase in the difference etween the peak and plateau input resistances (N = 19; Fig. 1).Furthermore, although both curves exhibited multi le components when elicited from resting potential, [oth the peak and the plateau currentholtage plots were linear at very hyperpolarized membrane potentials. At each membrane potential tested, the peak input resistance exhibited a progressive decrease in slope with increasing levels of hyperpolarization, with the slope reaching a minimum and the regression line becoming linear at membrane potentials averaging -63 r 4 mV (N = 9; Fig. 2A). This linearity of the slope at its minimum value (which would correspond to maximum membrane conductance) has been

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Fig. 1. Effects of altering the steady-state membrane potential of a DA neuron on its input resistance and rectification properties. A-C: Intracellular injection of negative current pulses (lowertraces) elicited erpolarizations of the membrane (upper traces) of this DA neuron E f d a t stead state depolarized(A) or hyperpolarized (B,C) membrane potentials reyitive to rest. Increasinf the amplitude of the hyperpolarizing current pulse injected produce an initial peak membrane hyperpolarization which then decayed t o a steady-state value. Furthermore,

the delayed re olarization following the current pulse in depolarized DA neurons (Affails to activate when the neuron is hyperpolarized (B). D: A plot ofthe current-voltage relationships of the tracin s illustrated in A-C. The am litude of the hyperpolarizing current pujse is lotted against the e a l (solid lines, o en symbols) and steady state dashed lines, solic? symbols) mernirane hyperpolarizations produced (squares = (A); triangles = (B); circles = (C); baseline current injection: A = +0.1 I& B = -0.1 nA, C = -0.3 nA).

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larizing pulses and became linear at membrane oten= 7). tials more hyperpolarized than -78 ? 3 mV This is reported to reflect the membrane potential at which the time-de endent rectifier is maximally activated (Yarom a n 8 Llinas, 1987). If the steady-state membrane potential is lotted against the total current injected (i.e., pulse p us constant polarization), the

attributed to a maximal activation of the instantaneous rectifier at this membrane potential (Yarom and Llinas, 1987).Analysis of the time-dependent rectification was performed by plotting the plateau membrane potential a ainst the am litude of the current pulse injected (Fig. 2b). The steaiy-state currentholtage plot showed a progressive decrease in slope with increasing hyperpo-

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tude of the hyperpolarizing pulse is plotted against the steady-state membrane potential occurringduring the pulse. In a similar manner as above, the regression line shows a progressive decrease in slope with hyperpolarization, becomin linear at membrane potentials greater than -82 mV in this exampfe. C: In plotting the steady-state current/ voltage curves, the resultant regression lines are found to overlap if each current contributing to the resultant steady-state membrane otential (i.e., both the continuous hyperpolarizing current and the Iyperpolarizing current pulse) is taken into account (symbols are the same as those in Fig. 1).

Fig. 2. Re-plottin of data from Figure 1 enables comparison of instantaneous and ielayed anomalous rectifiers at different mem-

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curves obtained at each membrane potential overlap (Fig. 2C). Thus, unlike the eak input resistance plots, the time-dependent anoma ous rectification is a function of the total membrane hyperpolarization elicited. i.e., 1)the hyperpolarization activated by the pulse and 2) the membrane polarization produced by constant current injection. Interaction between membrane potential, spike firing, and AHPs Depolarization of the membrane of a spontaneously firing DA neuron causes an increase in its firing rate. The relationship between membrane potential and s iking rate is difficult to assess in spontaneously firing IfA neurons due to the difficulty in accurately determining the resting membrane potential durin continuous pacemaker membrane depolarizations. owever, the relationship between current injection and the impulse rate produced was found to be linear (N = 14; Fig. 3). The decrease in the interspike interval was accompanied by an increase in the rate-of-rise of the slow depolarization recedin s ikes and an increase in s ike threshold &race, 18907. Depolarization also cause an ap arent decrease in the amplitude and duration of the! spi e afterhyperpolarization (AHP) measured at the soma (Fi . 4). Two components to the AHP could be identifie : 1)a fast component that exhibits an inflection and inactivation late in the falling phase of the

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rnSec Fi 4 Effects of membrane depolarization on DA cell action potential kP.A DA neuron that was not spontaneously firing a t resting otential initiated pacemaker firin a t three levels of depolarization !+O.l nA; + O X nA; +0.21 nA) ikes captured a t each level of depolarization are superimposed to igustrate the change in the AHP. The fast AHP that repolarizes the action potential under oes inactivation as the membrane potential increases beyond -60 m$, apparently independent of the initial level of cell depolarization. In contrast, the delayed AHP (occurring in this figure between 80 msec and 250 msec) shows a progressive decrease in amplitude as the DA cell is depolarized.

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mSec Fig. 5. At a given membrane potential, the AHPs following spontaneous DA cell action potentials are highly re lar in time course and amplitude. However, ifspike firing is i n i t i a t e g a DAneuron that had previously been inactive, the first s ike of the train (A) consistently exhibits an enhanced AHP (markezby +) when compared to subse-

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action otential, and 2) a delayed component with onset define operationally as the inactivation point of the fast AHP and continuing until the membrane repolarizes to baseline potentials. Overlaying spontaneously occurring action potentials from single DA neurons recorded at different membrane otentials revealed that membrane depolarization in uced a decrease in the delayed component of the AHP. In contrast, inactivation of the fast AHP occurred at approximately the same membrane potential in each neuron tested (average = -64 k 3 mV; N = 7), and this was not altered by hyperpolarization or depolarization of the DA cell membrane (Fig. 4). During rebound activation of spiking, the AHP associated with the first s ike followingthe hy erpolarizing pulse exhibited dif erences in am litu e and time course when compared to the AH s of preceding or subsequent spikes. Thus, the first spike initiated after a rebound train of spikes elicited in either nonfiring DA neurons or after hyper olarization and suppression of activity in spontaneous y firing DA neurons exhibited a

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large amplitude, longer-duration AHP, with the increase occurrin primarily in the delayed com onent enhancement was more de en ent on (Fig. 5).This A& of the hyperpolarizing repu se than on the amplitude the suppression of spike activity it pro(Puced. Thus, after administration of tetraethylammonium (TEA)to facilitate hyperpolarization of the DA cell soma and its extensive dendritic tree (Llinas et al., 1984),membrane hyperpolarization sufficient to block spike activity did not produce this enhancement of the rebound spike AHP. However, this increase can be produced with larger-amplitude hyperpolarizing re ulses (Fig. 6). This experiment also demonstrates t\ e$ EA-insensitive nature of the delayed AHP, at least at the concentrations of TEA examined (2-5 mM). In the in vitro preparation, long depolarizing pulses elicit trains of spikes that occurred at constant frequency with no evidence of adaptation (Fig. 7). Furthermore, the amplitude of the hyperpolarization following the spike train was not de endent on the number of spikes elicited, but insteal was related to the time

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ever, injection of a hyperpolarizing pulse larger than that required to inhibit spike activity (C) does elicit an enhanced afterhyperpolarization in the rebound s ike (D). This difference in pulse amplitude and rebound is illustratecfby overlaying each of these conditions (E).

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Blockade of spontaneous oscillatory activit was achieved in each case where the amount of T d a d d e d was sufficient to completely block fast spike activity. This was tested by injecting increasing levels of depolardid not exhibit izing current to confirm that fast, cobalt-insensitive s ikes (Grace, 1990) were completely blocked. Often, A X concentrations up to 2 p,M were required to block all fast s ike activity, possibl due to limited enetraOscillatory firing properties of DA neurons tion of T& into the slice. T d also blocked a1 spontaSpontaneously active DA neurons recorded in vitro neous oscillatory activity at resting membrane potenfire in a very regular, pacemaker-like pattern, and tials. Increased levels of membrane depolarization in depolarization of a quiescent DA neuron will cause it to several cases did produce oscillator depolarizations of fire in a similar repetitive manner. Both depolarization- the membrane potential after T d However, in each induced repetitive spiking and most of the subthreshold case the amount of membrane depolarization required pacemaker oscillations are blocked by TTX (Fig. 8). was much larger than that used to elicit spike firing in untreated DA neurons (i.e., depolarizations of the membrane to -39 or -40 mV). Similarly, inhibition of s ontaneous oscillatory spike discharge was produce: by administering cobalt (2 mM)to spontaneously firing DA neurons (Fig. 9).

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Effects of membrane potential on oscillatory spike activity The injection of brief h perpolarizing current pulses into quiescent, nonfiring I3A neurons with resting membrane potentials of approximately -55 to -60 mV elicited several types of rebound activity (Fig. 10): 1)sustained subthreshold oscillations of the membrane potential that dam ened in amplitude with successive oscillations, 2) subt reshold oscillations that increased in amplitude and often led to triggering of an action potential, or 3) a series of repetitively occurring spikes (Fig. 11).However, if the DA neuron was hy erpolarized below approximately -65 mV, in'ections o brief hyperpolarizing current pulses usual y were ineffective in producing rebound activity. Small-amplitude hy erpolarizing current pulses injected into nonfiring A neurons that had membrane potentials more depolarized than -55 mV consistently elicited rebound spikin . However, as the amplitude of the hyperpolarizing pu se was increased, a blockade of rebound spike firing occurred (Fig. 11).This progressive blockade of the rebound de olarization with increasing pulse amplitude war para leled by an increase in the

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DISCUSSION Studies using intracellular recordin DA neurons in vivo have reported an neous epsps in these neurons, with by pacemaker-like slow depolarizations (Grace and Bunne , 1983a, 1984a). This pacemaker activity is a parent y mediated by endogenously generated T and cobalt-sensitive pacemaker de olarizations. Under these conditions, the regulation o spike firing in DA neurons is likely to be more dependent on autoregulatory membrane pro erties than that in neurons that derive their activity y afferent s aptic drive. Changes in the basal, steady-state mem rane potential of DA neurons induce a number of alterations in their state of activity and excitability. The resent study was directed at examining the interaction etween membrane otential and the contribution of passive, active, and ca ciumtriggered membrane conductances to DA cell regulation in vitro. Input resistance and anomalous rectification As shown previously, DA neuron membranes exhibit both instantaneous and time-dependent anomalous rectification (Grace and Onn, 1989; Kita et al., 1986) similar to that reported in other preparations (LopezBarneo and Llinas, 1988; Schwindt et al., 1988a; Yarom and Llinas, 1987).The relative contribution of each type of rectification to the input resistance was de endent on both the initial membrane potential of the A neuron and the amplitude of the hyperpolarizing pulse injected. Activation of the instantaneous rectifier chan ed with respect to the hyperpolarizing pulse amplitu e, showing maximal activation at -63 mV. In contrast, the amplitude of the time-dependent component was correlated with the total current injected into the neuron (i.e., the steady-state olarization plus the pulse am litude). Under these con itions, one may predict that t e influence of the time-dependent component on DA cell excitability would be more dependent on the resting membrane potential, whereas the instantaneous rectifier would play a greater role during brief hyperpolarizing events, such as during a volle of ipsps as are known to occur in DA neurons recorde in vivo (Grace and Bunney, 1985) or during the falling phase of an action potential.

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delayed repolarization (Fig. 12). The presence of the delayed repolarization was also dependent on the steady-state membrane otential, showing decreased activation with hyperpo arization of the membrane (Fig. 1; Grace and Onn, 1989). This attenuation of rebound activity is comparatively brief in duration, although the duration of inhibition of spiking could be extended by adding TEA (2-5 mM) to the medium (Fig. 6).

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Fig. 9. Effects of cobalt on s ontaneous oscillatory spike firing in DA neurons. A Spontaneously i r i n g DA neuron at resting membrane otential. B,C: During the addition of cobalt (2 mM) into the trisluffered physiological saline, there was a progressive blockadeof spike activity and membrane oscillatory activity. Further depolarization elicited only a fast, single component TTX-sensitivespike (not shown). D: Overlay of traces A and C to show effects of cobalt on the pacemaker oscillations and membrane potential.

Afterhyperpolarizations In vivo intracellular recordings and intracellular injection of the calcium chelator ethylene glycol-bis (paminoethy1ether)-N,N'-tetraacetic acid (EGTA) have provided evidence that the AHP followingDA cell action otentials is a calcium-dependent hyperpolarization !Grace and Bunney, 1984a), as observed in other reparations (Aghajanian and Vandermaelen, 1982; Zotson and Prince, 1980; Llinas and Sugimori, 1980; Meech,

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in amplitude (toptrace). However, ifthe neuron is first hyperpolarized below -65 mV, no rebound oscillato events are elicited (bottom trace). B: In several cases, the rehounTelicits a pro essive augmentation of the oscillatory events which in this case cuginates in spike discharge.

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Fig. 11. Bi hasic effects of brief hyperpolarizing pulses on the subsequent regound depolarization produced in DA neurons. DA neurons, as in other neurons with significant LTS-like events, exhibit rebound spike activit (bottom trace, first three events) in response to the injection of brief3;lyperpolarizingcurrent pulses (top trace). In-

creasin the amplitude of the h erpolarizing ulse will eventual1 block re%ounds ike firing and sughreshold oscilfato activity (fourti event), althou a rebound cobalt-insensitivefast spxe (Grace, 1990) is often elicite! (fifth and sixth events).

1978). The AHP was composed of at least two components distinguished b their time course: 1) a shortlatency com onent an 2) a delayed component, similar P and the medium-latency com onent, to the fast res ectively, described in other reparations &darns an Galvan, 1986. Lancaster and icoll, 1987;Pennefather et al., 1985; hchwindt et al., 1988a,b). The delayed component exhibited the largest degree of alteration with changes in membrane potential, showing decreasing amplitude with depolarization and increasing amplitude with hyper olarization of the membrane. In contrast, the fast A&P was a parent during the falling phase of the action potentia , and inactivated at the same membrane potential irrespective of the steadystate membrane potential of the DA neuron. Under

these circumstances, the

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control the rate of decay of the AHP (Barrett et al., 1980; Ito and Oshima, 1965; Schwindt et al., 1988b). This correspondence between the AHPs and the anomalous rectifiers is born out by the observations that changes in the activation of the fast AHP and the instantaneous anomalous rectifier occur at similar membrane potentials (i.e., -64 i 3 mV vs. -63 i 4 mV, respectively), and show little dependence on the steady-state membrane potential. In contrast, the activation of both the

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cation) at the site where the AHP is generated. Thus, if repolarization of the dendrites followinga hy erpolarizing pulse la s behind the repolarization o the soma (e.g., due eit er to its electrotonic distance or to the mV absence of a constant spike-induced depolarization), the first rebound spike may trigger the AHP in dendrites that are comparative1 more hyperpolarized with re0.5 spect to the soma. In eed, an anomalous rectification 0.0 nA activated by hyperpolarization has been proposed to E -0 5 decrease the coupling between the soma and the dendrites of neurons (Llinas et al., 1984). In cortical neurons, a similar phenomenon involving the anomalous 0 rectifier was roposed to cause an increase in the medium-latency A hP if spike activity is temporarily blocked by membrane hyperpolarization (Schwindt et al., 1988c, Spain et al., 1987). It is unclear what effect these phenomena would produce on the overall level of excitability of DA neurons. One possibility is that a larger-am litude AHP would activate a proportionately larger re ound event. This would be particularly effective in quiescent DA neurons with membrane otentials near spontaneous P could facilitate the susfiring levels, where the tained oscillatory firing that occurs following brief -20 O 1 membrane hy erpolarizations (e . Fi . 11).A second possibility is t at this increased p aP7fo7lowing the first spike in a rebound event could attenuate LTS-triggered -60 rebound bursts of spikes in DA cells in vitro by limiting the rate of membrane repolarization following the first -80 rebound s ike. The A€& plays a rominent role in the regulation of spike activity in D neurons observed in vivo (Grace and Bunnev, 1984a.b). However. the AHP following DA cell spiking recorded in vitro differs from that rec&ded in vivo in at least two respects: 1)it does not contribute to a pro essive slowing of spike discharge during prolonged %polarizations, and 2) its am litude following depolarization-induced spiking is not ependent on the number of spikes elicited. Thus, durin in vivo intracel0 00 050 I00 1.50 lular recordings, depolarization elicite a train of spikes Seconds that exhibited a progressive decrease in the frequency of Fig. 12. Decrease in rebound depolarization in DA neurons second- spike firing (i.e., increasing interspike intervals) with ary to increasing the amplitude of the h erpolarizin repulse. A long duration pulses. This phenomenon has been Brief h erpolarizations of the membrane ?a nonfiring 61neuron are termed spike frequency adaptation in other preparafollowe?by rebound depolarizations and the triggering of two spikes. tions (Lancaster and Adams, 1986; Lancaster et al., B-D: Increasin the amplitude of the pulses causes a progressive attenuation of t f e rebound response, culminating in blockade of spike 1986; Madison and Nicoll, 1982, 1984. Partridge and firing. The decrease in rebound activation was accompanied by a Stevens, 1976). Trains of s ikes elicited in DA neurons concomitant delayed repolarization of the membrane following the in vivo also were followed y a long calcium-dependent hyperpolarization. hyperpolarization, with the amplitude and duration of the hyperpolarization being pro ortional to the number of spikes triggered during the epolarization. Furtherdelayed AHP and the time-dependent anomalous recti- more, each of these events could be blocked by intracelfication are altered by changes in the steady-state mem- M a r injection of EGTA (Grace and Bunney, 1984a). brane potential of the DA neuron. However, in DA neurons recorded in vitro, the hyperpoFollowing brief hyperpolarizations of the membrane larization followin a train of spikes was found to and inhibition of spontaneous firing, there is an in- depend only on the atency between the last spike in the crease in the AHP of the first spike elicited upon re- train and the end of the pulse. In this regard, the sumption of spike discharge. This occurs both in quies- post- ulse h erpolarization does not appear to differ cent and in spontaneously firing DA neurons, although signi icantly rom what would be expected for the conDA cells with high levels of spontaneous s ike discharge tinuation of the AHP of the last spike in the train, often did not show this phenomenon un ess TEA was particularly when allowing for the increase in AHP added. It is unclear precisely what events contribute to amplitude that occurs at hyper olarized membrane this phenomenon. One possibility is that the enhanced potentials. In addition, identified E A neurons observed AHP may reflect a difference in the membrane potential in vitro reveal little evidence of adaptation during ex(and thus degree of activation of the anomalous rectifi- tended depolarization-elicited spike trains. Although

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the reason for these differences in spike train characteristics is unclear, the lack of summation of AHPs could contribute to the absence of burst firing and maintenance of a highly regular pacemaker firing pattern in vitro (Grace, 1987; Grace and Onn, 1989). Indeed, intracellular injection of EGTA in DA neurons in vivo, in addition to eliciting pacemaker activity, also blocks accommodation during depolarization (Grace and Bunney, 1984a). Oscillatory spike firing The ability to fire spikes in an oscillatory manner has been proposed to be a major factor in controllin the functional activity of central neurons and may p ay a significant role in synchronizin neuronal activity in the organization of behavior (Gri lner, 1975; Llinas and Yarom, 1986). Spontaneously firing DA neurons recorded in vitro fire in a very regular pacemaker-like attern (Grace, 1987; 1988; Grace and Onn, 1989 . his is contrasted to in vivo recordings of these neurons, in which spontaneously active DA cells fire in either an irregular single spiking attern or a burstin attern of activity (Bunney et a ., 1973; Grace an Eunney, 1984a,b). Although pacemaker activity is rarely observed in substantia nigra DA neurons in vivo under basal conditions, it can be elicited b three manipulations: 1)intracellular injection of EG A (Grace and Bunney, 1984a),2) transection of the athway from the subthalamus to the substantia nigra ffmith and Grace, 1990), or 3) administration of clonidine (Grenhoff and Svensson, 1988).Under these conditions, a hi hly re lar oscillatory pacemaker firing pattern sirni ar to t a t observed in vitro is observed, and is maintained even at relatively fast frequencies of spike firing (e.g., > 7 Hz). Pacemaker firing also has been reported in ventral tegmental DA neurons followinginactivation of afferent projections from the frontal cortex (Svensson and Tung, 1989). Although a small number of studies have reported bursting in substantia nigra neurons in vitro (Harris et al., 1989a; E t a et al., 1986), these investigations were limited by the absence of identification of the neurons as dopaminerDc and poor stability of penetrations. Indeed, the time course of the putative burst activity are marked1 different from those of identified DA neurons recorde in vivo (Grace and Bunney, 1983a, 1984b). Oscillatory membrane activity of DA neurons recorded in vitro can be observed in the form of subthreshold oscillator depolarizations or repetitive pacemaker spike firing. $his pacemaker firing appears to de end on two pharmacologically distinct processes: 1)a TXsensitive depolarization preceding spike firing (Grace and Onn, 1989) and 2) a cobalt-sensitive de olarization and/or subsequent s ike AHP. Thus, botl! spontaneously occurring or iepolarization-elicited oscillatory s ike firing can be blocked with either TTX or cobalt. &hers have reported the presence of s ontaneous de olarizing potentials in DA neurons fo owing TTX ( ujimura and Matsuda, 1989; Harris et al., 198913; E t a et al., 1986). However, as reported by those authors and as observed here, initiation of spontaneous oscillations after TTX can only occur by de olarizing the DA cell membrane far beyond what had een required to elicit spike activity rior to TTX administration; i.e., to -30 or to -40 m$In fact, similar TTX-insensitive membrane potential oscillations can be produced in neocorti-

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cal neurons with similar levels of depolarization even though these neurons do not fire in a spontaneous pacemaker-like manner in control conditions (Flatman et al., 1986).Therefore, the TTX-insensitive oscillations occurring at very depolarized membrane potentials more likely represent activation of calcium-mediated events other than those involved in pacemaker firing. The relevance of these TTX-sensitive and cobaltsensitive events to spontaneous acemaker activity is unclear. Although speculative at t is stage, one possible interpretation of this co-dependence of oscillato activity on TTX- and cobalt-sensitive processes may e that the TTX-sensitive slow depolarization is necessary to depolarize the soma, proximal dendrites, and initial segment to threshold whereas the calcium-dependent spike AHP is re uiredi to repolarize the membrane and tri e r a reboun! LTS (Grace and Onn, 1989;Kita et al. 1988; Nakanishi et al. 1987) or to produce rebound activation of the slow depolarization that leads to the next spike in the train. Althou h small membrane h perpolarizations or spike A& s may trigger reboun pacemaker spiking, larger membrane hyperpolarizations tend to suppress rebound spiking. This is in marked contrast to reports of rebound spiking in other cell types, where lar er hyperolarizing pulses will elicit more ronounce rebound gepolarizations. In DA neurons, t e rebound depolarization may be masked by a concomitant delayed repolarization of the membrane that is triggered following lar e hyperpolarizin pulses (Fig. 1;Grace, 1987; Grace an Onn, 1989). Alt ough a number of other tests are required to establish its precise identity, this dela ed repolarization has properties similar to those descri ed for the A current (IA)in other preparations (Connor and Stevens, 1971; Haas and Reiner, 1988). Thus, as has been reported in cortical neurons (Bossu et al., 1985), the extent of rebound activation appears to depend on the relative level of activation of the putative IA vs. the LTS at the membrane potential examined. If the steadystate membrane potential is hy erpolarized, however, even small-amplitude hyper o arizing ulses fail to elicit rebound spiking, possib because t e repolarization occurring at the end o f t e hyper olarizing pulse does not de olarize the membrane su iciently to activate the L S. Of course, this failure to activate either the putative IA or the LTS during repolarizations to hyperpolarized membrane potentials should not be confused with the progressive blockade of the rebound LTS that is roduced in comparatively depolarized DA neurons wit increasing hyper olarizing pulse amplitudes (Grace, 1987; Grace and nn, 19891,as these are clearly different phenomena (Harris et al., 1989b).

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Interpretation and synthesis One of the classic tenets of earl research into neuronal physiolo is that hyperpo arized neurons are under tonic in ibitory control, and therefore are less likely to discharge in res onse to depolarizing stimuli. However, recent studies y Llinas and colleagues have shown that some classes of neurons in the mammalian central nervous system exhibit enhanced excitability to stimuli when in a hyperpolarized state. This was attributed to a hyperpolarization-induced de-inactivation of the LTS (Llinas and Yarom, 1981a,b).Thus, for neurons in the inferior olive (Llinas and Yarom, 1981a,b),thala-

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mus (Jahnsen and Llinas, 1984a,b), cortex (Grace and Llinas, 1984), deep cerebellar nuclei (Llinas and Muhlethaler, 1988), substantia nigra (Nedergaard et al., 1988a,b), and habenula (Wilcox et al., 19881, small depolarizing stimuli are more effective in eliciting spike activity when the neurons are hyperpolarized. Indeed, in some neuronal types the rebound activation following brief hyperpolarizing pulses consists of prolonged bursts of rapid spike discharge which can last for periods ranging from tens of milliseconds up to seconds, and is a more owerful excitatory response than can be induced eit er by activation of known excitatory afferents or brief intracellular depolarizations of these neurons (Llinas and Muhlethaler, 1988; Wilcox et al., 1988). Although DA neurons also have been reported to exhibit a rebound de olarization with characteristics similar to the LTS ( d a c e and Onn, 1989; Kita et al., 1986; Nakanishi et al., 19871, hyperpolarizations of the membrane of identified DA neurons have not been observed to produce rebound bursts of spikes. Instead, brief, low-amplitude hy erpolarizations of the DA neuron membrane will at est activate a small rebound spike or initiate slow oscillatory spike activity. Furthermore, larger amplitudes of hyperpolarization are reported here to block rebound depolarizations and spiking in DA neurons. Thus, in contrast to other neuron types with prominent LTSs reported to date, DA cells appear to have membrane properties that tend to support their ongoin state of activity. The more depolarized, spontaneous y firing DA neurons tend to remain in a spontaneously active state, since depolarization would tend to increase their excitability by: 1)increasing the rate-of-rise of the slow depolarization trig ering spike firing (Grace and Bunney, 1984a; Grace anc fOnn, 1989), 2) decreasing the shunting actions of the anomalous rectifier on pacemaker currents, 3) decreasing the amplitude of the AHP following spikes, and 4) facilitating rebound activation of spiking by attenuating the effects ofthe putative IA(Grace, 1987;Grace and Onn, 1989).In contrast, hyperpolarized and inactive DA neurons exhibit membrane properties that would tend t o lock them into this inactive state: 1)slowed rate-of-rise of the slow depolarization, 2) increased membrane conductance secondar to activation of the anomalous rectifiers, 3) increasec? AHP amplitude with hyper olarization and also following the first rebound spi e elicited after hyper olarizing stimuli, and 4) the facilitated activation o the putative IA, which will attenuate rebound depolarizations (Grace and Onn, 1989) and slow membrane re olarization after an AHP (e. ,Schwindt et al., 1988~). &though the increase in spite threshold with depolarization may at first appear to be inconsistent with this model, one must consider that the spike threshold measured at the soma is a reflection of initial segment spike firin in an anatomically distal spikegeneratin region o the neuron (Grace and Bunney, 1983a,b; race and Onn, 1989; Juraska et al., 1977; Preston et al., 1981; Tepper et al., 1987).Therefore, it is likely that the initial segment spike threshold may be constant across membrane potentials, and that the variability in threshold measured in the soma is a reflection of changing voltage-dependent conductances located between the soma and this spike-generating re 'on (Grace, 1990).Thus, the increase in s ike thresh01 with depolarization may be more in icative of a

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decrease in the couplin between the soma and the initial segment than a c ange in neuronal excitability per se. This bimodal regulation of DA cell activity into active and inactive states may thus be a consequence of the aforementioned voltage-dependent membrane properties. Therefore, DA neurons appear to have membrane properties that distribute their activity into two states: either maintaining spontaneous spike activity or holding DA cells in a quiescent, nonfirin state. These membrane roperties may account fort e bimodal distribution o DA neuron activity observed in vivo: 1) spontaneously firin DA neurons discharging at an average frequency o 4.5 2 1.7 Hz but rarely below 1.0 Hz, and 2) hyperpolarized, nonfiring DA neurons. This observation may be an electrophysiological parallel to the reported bimodal distribution of tyrosine hydroxylase messenger RNA content in subsets of DA neurons re orted by others (Bayer and Pickel, 1990; Weissd n d e r and Chesselet, 1989). The quiescent pool of DA neurons could thus be activated in times of eater postsyna tic DA demand, as occurs with the al Y ministration o antipsychotic drugs or after partial DA-deleting brain lesions, to substantially augment DA rePease into postsynaptic target areas. ACKNOWLEDGMENTS I thank Jeffrey Hollerman, Eric Nisenbaum, and Michele Pucak for helpful sug estions on the manuscri t. This work was sup ortefby USPHS MH42217, Md 5 1 5 6 , and NS19608. f h e author is a Sloan fellow.

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REFERENCES Adams, P.R., and Galvan, M. (1986) Voltage-dependent currents of vertebrate neurons and their role in membrane excitabilit In Basic Mechanisms of the Epile sies; Advances in Neurolo $01. '44. A. Delgado-Escueta, A.A. &rd, D.M. Woodbury, and iig'porter, eds. Raven Press, New York, pp. 137-170. Adrian, R.H. (1969) Rectification in muscle membrane. Prog. Biophys. Mol. Biol., 19:340-369. Aghajanian, G.K., and Vandermaelen, C.L. (1982)Intracellular recordings from serotoner 'c dorsal raphe neurons: Pacemaker potentials Brain Res., 238:463469. and the effects of Barrett, E.F., Barrett, J.N., and Crill, W.E. (1980) Volta e sensitive outward currents in cat motoneurons. J . Physiol. (Lon6,304:251276. Bayer, V.E., and Pickel, V.M. (1990) Ultrastructural localization of tyrosine hydroxylase in the rat ventral tegmental area: Relationship between immunolabeling density and neuronal associations.J . Neurosci. (in press). BOSSU,J.-L., DuPont, J.-L., and Feltz, A. (1985)I, current com ared to low threshold calcium current in cranial sensory neurons. &urosci. Lett. 62:249-254. Bunney, B.S., and Grace, A.A. (1978) Acute and chronic halo eridol treatment: Comparison of effects on nigral dopaminergic cely activity. Life Sci. 23:1715-1728. Bunney, B.S., Walters, J.R., Roth, R.H., and Aghajanian, G.K. (1973) Dopaminergic neurons: Effect of antipsychotic drugs and amphetamine on single cell activity. J . Pharmacol. Exp. Ther., 185:560-571. Connor, J.A., and Stevens, C.F. (1971) Voltage clamp studies of a transient outward membrane current in gastropod neural somata. J. Phvsiol. (Lond.). 213:21-30. ConGanti A., and Galvan, M.J. (1983) Fast inward-rectifying current accounts for anomalous rectification in olfactory cortex neurons. J . Physiol. (Lond.) 335153-178. Flatman, J.A., Schwindt, P.C., and Crill, W.E. (1986) The induction and modification of voltage-sensitive res onses in cat neocortical neurons by N-methyl-D-aspartate. Brain {es., 363:62-77. Freeman, A.S., and Bunney, B.S. (1987) Activity of A9 and A10 dopaminer 'c neurons In unrestrained rats: Further charactenzation and eFects of apomorphine and cholecystokinin. Brain Res., 405:4&55.

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