partial dopamine depletions result in an enhanced sensitivity of residual dopamine neurons to...

SYNAPSE 9:144-155 (1991)

Partial Dopamine Depletions Result in an Enhanced Sensitivity of Residual Dopamine

Neurons to Apomorphine M.L. PUCAK AND A.A. GRACE

Departments of Behavioral Neuroscience and Psychiatry, Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

KEY WORDS Autoreceptor, Substantia nigra, Zona compacta, 6-Hydroxydopamine

ABSTRACT Extracellular recordings from identified dopamine neurons were used to assess the effect of 6-hydroxydopamine-induced partial lesions of the nigrostriatal dopamine system on the sensitivity of the residual dopamine neurons to the dopamine agonist apomorphine. This was done by testing the response of identified nigral dopamine neurons in control and lesioned rats to systemic apomorphine administration at two time points: 1) 6-10 days post-lesion, when the loss of dopamine cells is nearly complete, and 2) 4-43 weeks post-lesion, which should be sufficient time for changes in dopamine receptor density to occur. As reported previously, dopamine neurons in control rats were inhibited by systemic administration of apomorphine, with their sensitivity being inversely related to their initial firing rate. The sensitivity of the residual dopamine neurons to apomorphine was unaltered in rats tested 6-10 days after depletions of at least 60% of striatal dopamine. However, by 4-8 weeks post-lesion, there was a significant increase in the sensitivity to apomorphine; furthermore, sensitivity was no longer related to baseline firing rate, but instead was uniformly high in all dopamine neurons tested at this time. This enhanced sensitivity was not altered by hemisection of the striatonigral projection, suggesting that the increased sensitivity to apomorphine was most likely a result of a time-dependent up-regulation of somatodendritic autoreceptors on the residual dopamine neurons.

INTRODUCTION Dopamine (DA) neurons have been shown to possess

somatodendritic autoreceptors (Morelli et al., 1988; Nagy et al., 1978) that, in response to stimulation by DA agonists, cause an inhibition of DA neuron activity (Aghajanian and Bunney, 1973; Bunney et al., 1973). The inhibition of DA neuron firing which is caused by the systemic administration of the DA agonist apomorphine (APO) probably occurs via a direct stimulation of these somatodendritic autoreceptors, which have been shown to be of the D, subtype (Morelli et al., 1988). This direct action of APO is supported by studies showing that APO-induced inhibition is not abolished by hemisection of the brain between the striatum and the substantia nigra (Aghajanian and Bunney, 1974; Pucak and Grace, 1990) or by kainic acid lesion of the striatum (Baring et al., 1980). Moreover, DA neurons recorded in the in vitro preparation, in which afferent connections are severed, still exhibit hyperpolarization in response to APO or DA (Grace, 1988; Lacey et al., 1987). In addition, although APO-induced inhibition of DA neurons is completely blocked by the administration of a D, antagonist (Carlson et al., 1986; Mereu et al., 19831, this 0 1991 WILEY-LISS. INC.

inhibition is not attenuated by the administration of a D1 antagonist (Carlson et al., 1986; Mereu et al., 1985; Napier et al., 19861, suggesting that D, receptors located in either the striatum or on striatonigral terminals do not contribute to the APO-induced inhibition. Therefore, the striatonigral projection does not appear to contribute to the inhibition of DA neuron activity that occurs in response to the systemic administration of low doses of APO. This is consistent with the observation that DA neurons are more sensitive to systemic APO than are striatal neurons (Skirboll et al., 1979). Thus, the APO-induced inhibition of DA neurons appears to be mediated by a direct action of APO on somatodendritic autoreceptors.

Electrophysiological studies show that the sensitivity of DA neurons to the systemic administration of APO or amphetamine is related to their baseline firing rates, such that neurons with higher spontaneous firing rates are less sensitive to systemic administration of these DA agonists (Staunton et al., 1980; White and Wang, 1984a1. It has been suggested that this relationship

Received March 22,1991; accepted in revised form May 14,1991

DOPAMINE NEURON SENSITMTY AFTER DEPLETIONS 145

occurs because both the sensitivity to APO and the spontaneous firing rate of a given DA neuron are con- trolled by a common factor; i.e., the density of somatodendritic autoreceptors on the neuron (White and Wang, 1984a). Indeed, the high firing rate and the relative insensitivity to APO reported for neurons that project to the prefrontal cortex has been proposed to result from the absence of firing rate-modulating autoreceptors on this population of neurons (Chiodo et al., 1984; White and Wang, 1984a). However, although such observations suggest that stimulation of somatodendritic autoreceptors by endogenously released DA ex- erts potent control over DA neuron activity, the importance of this process in the normal regulation of DA neuron firing has not been directly demonstrated.

The sensitivity of DA neurons to inhibition by DA agonists is not an invariant property of individual DA neurons, because it can be altered by pharmacological manipulation. Repeated administration of dopamine agonists, such as amphetamine (Kamata and Rebec, 1983, 1984b1, APO (Rebec and Lee, 1982), or L-DOPA (Jackson et al., 1982), has been reported to decrease the sensitivity of nigral DA neurons to subsequent doses of amphetamine or APO. DA neurons of the ventral tegmental area have been reported to undergo similar changes in sensitivity after chronic DA agonist administration (Kamata and Rebec, 1984a; White and Wang, 1984b). This decreased response of DA neurons to APO is generally interpreted to reflect a decreased sensitivity of DA neuron somatodendritic autoreceptors (Jackson et al., 1982; Kamata and Rebec, l983,1984a,b; Rebec and Lee, 1982; White and Wang, 1984b). Such a decreased sensitivity of autoreceptors has been proposed to underlie the activation of DA neurons that is observed after chronic amphetamine administration (Kamata and Rebec, 1984b; White and Wang, 1984b).

In contrast to the reported changes in autoreceptor sensitivity that occur in response to chronic DA agonist administration, little evidence exists to support an up- regulation of DA neuron somatodendritic autoreceptors in response to decreases in DA levels in the substantia nigra. Because 6-hydroxydopamine (6-OHDA) has been demonstrated to cause the destruction of nigral DA neurons (Hokfelt and Ungerstedt, 1973; Hollerman and Grace, 1990; Onn et al., 1986) as well as a decrease in the level of extracellular nigral DA as measured by microdialysis (J.P. Bennett, Jr., personal communica- tion), we have examined the effects of 6-OHDA on the sensitivity of the residual DA neurons to systemic administration of APO. Of course, although evidence indicates that systemic APO inhibits DA neurons directly via autoreceptor stimulation in control rats, the induction of striatal DA receptor up-regulation by DA depletions (Creese and Snyder, 1979; Creese et al., 1977; Zigmond and Stricker, 1980) may cause APO to exert additional, indirect effects on DA neuron activity via the striatonigral feedback pathway. In order to control for

this possibility, the response of nigral DA neurons to APO in DA-depleted rats also was tested after acute hemisections of the striatonigral projection. Prelimi- nary results of this work have been presented in abstract form (Pucak and Grace, 1988).

MATERIALS AND METHODS Experiments were performed on Sprague-Dawley al-

bino male rats (Zivic-Miller, Allison Park, PA) weighing between 250 and 350 g. Handling of rats and surgical procedures were performed in accordance with the pro- tocols outlined in the NIH Guide to the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Rats were maintained on a 12 : 12 hour photoperiod with food and water available ad libitum.

Depletion of striatal DA Partial lesions of the nigrostriatal DA projection were

performed by infusing the neurotoxin 6-OHDA into the lateral ventricles as previously described (Hollerman and Grace, 1990; Keefe et al., 1989; Zigmond and Stricker, 1973). Briefly, rats were first anesthetized with Equithesin (0.3 mVO.l kg, i.p.). Des-methyl-imi- pramine (DMI; 25 mgkg, i.p.1 was administered to all rats in order to prevent lesions of norepinephrine-containing neurons (Breese and Traylor, 1971). In some rats, more extensive depletions were obtained by co- administering the monoamine oxidase inhibitor pargyline (40 mgkg, i.p.; Breese and Traylor, 1971). DMI and pargyline were administered 25-45 minutes prior to 6-OHDA infusions. The rats were then mounted in a stereotaxic apparatus (Kopf 1. A burr hole was drilled overlying each lateral ventricle (coordinates 1.2 mm posterior to bregma, 1.5 mm lateral to midline; Paxinos and Watson, 1986) and a 30 gauge cannula lowered stereotaxically into each ventricle (3.2 mm ventral to dura). Three minutes after inserting the cannulae, 6- OHDA (150 or 200 p,g in 10% ascorbic acid in a total volume of 20 pl; 75 or 100 p,g per ventricle) was infused slowly into the ventricles over a period of 5 minutes using a syringe pump (Sage Instruments, Model 341) fitted with two 50 pl Hamilton microsyringes. After cessation of the 6-OHDA infusion, cannulae were left in place for a t least 3 minutes before withdrawal. Recovery from anesthesia was assessed by monitoring locomotor activity and the ability of the rats t o maintain body temperature. After the lesion, the rats were fed a mixture of high-protein baby cereal and powdered hot choc- olate via gastric intubation until they began to eat the mixture independently. To maintain hydration, rats were injected with a solution of 5% dextrose in 0.9% sodium chloride (5.0 ml, i.p., twice daily) until they exhibited signs of appropriate hydration (Le., normal color urine).

146 M.L. PUCAK AND A.A. GRACE

Determination of DA neuron sensitivity to APO

The response of DA neurons to APO was assessed either 6-10 days or 4-8 weeks post-lesion using extracellular recording techniques. By 6-10 days post-lesion, the loss of DA cells is nearly complete (Hokfelt and Ungerstedt, 1973; Onn et al., 19861, while any changes in DA receptor density should have occurred by 4-8 weeks post-lesion (Creese and Snyder, 1979). Rats were anesthetized with chloral hydrate (400 mgkg, i.p.1 and mounted in a stereotaxic instrument (Kopf). Body temperature was maintained at 37°C with a heating pad. A burr hole was drilled overlying the substantia nigra (coordinates 2.9 mm anterior to lambda, 2.2 mm lateral to midline; Paxinos and Watson, 1986). All drugs, including supplemental chloral hydrate, were administered via a lateral tail vein.

Single unit recordings were performed from identified DA cells using glass microelectrodes pulled from 2.0 mm O.D. Omegadot glass tubing (WPI) using a vertical electrode puller (Narishige) and broken back under microscopic control to a tip diameter of approximately 1 pm. Electrodes were filled with 2 M NaCl containing 2% Pontamine sky blue, with electrode impedances ranging from 5 to 10 megohms as measured at 135 Hz using a Micro Electrode Tester (Winston Electronics Co., model BL-1000). Electrode potentials were amplified by a high input impedance headstage amplifier connected to a preamplifier (Fintronics, Orange, CT) and were moni- tored via a Hitachi Storage Oscilloscope (V-134) and a Grass Instruments AM-8 audio monitor. Spikes were isolated using a time and amplitude window discrimina- tor (Fintronics) and were counted over successive 10 second intervals. Electrodes were lowered into the brain using a hydraulic microdrive (Kopf, Model 640) until spike activity consistent with that reported for identified DA neurons was encountered in the zona compacta region of the substantia nigra, approximately 6.5 to 8.5 mm ventral to the surface of the brain. DA cells were identified on the basis of previously established criteria which included long duration biphasic action potentials (1.8-4.5 msec) usually with a prominent initial segment notch and a large negative component, moderately slow firing rates (1-8 Hz), and an irregular single spiking or burst-firing pattern (Bunney et al., 1973; Grace and Bunney, 1980, 1983). Once a DA cell was encountered, its baseline firing rate was recorded for a t least 5 minutes before administration of -0. APO was administered in logarithmically increasing doses, starting with an initial dose of 0.5 pgkg. Subsequent doses were administered at two-minute intervals until the spontaneous activity of the recorded cell ceased for at least 30 seconds. In the majority of neurons tested, the APO- induced inhibition was reversed by administering the DA antagonist haloperidol in a dose that was equal to the cumulative dose of APO injected. AF'O sensitivity was tested in only one cell in each animal. Only neurons

showing 100% inhibition to APO were included in statistical analyses.

Determination of extent of striatal DA depletion Immediately after each experiment, the rat was killed

by decapitation and both striata were rapidly removed, frozen on dry ice, and stored at -70°C. The concentra- tion of DA in striata from control and lesioned rats was determined by a modification of the methods of Keller et al. (1976) and Mefford et al. (1980). Briefly, superfu- sates and tissue samples were partially purified by alumina extraction. One hundred microliters of an internal standard, 3,4-dihydroxybenzylamine (DHBA), 10 mg of activated alumina (Anton and Sayre, 19621, and 300 pl of 1 M Tris buffer (pH 8.6) containing 0.2 mM ethylenediaminetetraacetic acid (EDTA) was added to each sample. After centrifugation and washing with dilute Tris buffer (200 pl of 1 mhf Tris, pH 8.61, catecholamines were eluted from the alumina with 200 p1 of 0.1 N perchloric acid. A 25 to 50 p1 aliquot of the perchloric acid eluate was injected into a high perfor- mance liquid chromatography system. DA and DHBA were separated on a Novapak C18 column (Waters Associates, Milford, MA), and the DA content of the samples was measured using an ESA Model 5100A Coulochem detector (ESA, Inc., Bedford, MA) equipped with a glassy carbon electrode maintained at +.38 V. The DA in each sample was quantified by comparing DA peak areas with those of a standard DA solution using a Waters 740 Integrator (Waters Associates, Bedford, MA). DA values were corrected for recovery, which was 6545%.

Hemisection of the striatonigral projection In order to assess the contribution of the striatonigral

projection to the response of DA neurons to APO, hemisections of the brain between the striatum and the substantia nigra were performed in a subset of control and DA-depleted rats. A microknife constructed from a glass coverslip (thickness grade 1; cut to dimensions of approximately 0.5 x 3 cm) was inserted into the brain perpendicular to the rostro-caudal axis at approximately 1.0 mm anterior to the rostra1 pole of the substantia nigra and immediately lateral to the hypothalamus. The knife was inserted ventrally until contact was made with the skull underlying the brain, and then moved laterally to extend the hemisection to the external capsule (approximate coordinates of hemisection: 4.3 mm anterior to lambda, extending from 1.0 mm to 5.5 mm lateral from the midline). Recordings were performed one hour after the transection was com- pleted.

Electrophysiological determination of extent of hemisection

In a subset of experiments, the extent of the hemisection was assessed by stimulation of the striatum with

DOPAMINE NEURON SENSITIVITY AFTER DEPLETIONS 147

concomitant recording of field potentials in the ipsilat- era1 substantia nigra. Prior to hemisection, a concentric bipolar stimulating electrode (MS303/1; Plastics One, Roanoke, VA) was lowered into the striatum (coordinates 8.6 mm anterior to lambda, 3.5 mm lateral to the midline, 4.5 mm ventral to the surface of the brain; Paxinos and Watson, 1986) and constant current square wave stimuli consisting of single pulses of 50 psec duration and 100 G - 1 mA amplitude were delivered using a stimulator (model S88; Grass Instrument Co.) coupled to a Photoelectric Stimulus IsolatiodConstant Current Unit (PSIU6; Grass Instrument Co.). Record- ings of nigral field potentials were made using a glass micropipette (impedance = 3-8 Ma). Pulses of stimuli delivered to the striatum produced prominent field potentials within the substantia nigra. The stimulation was repeated after the hemisection, with the absence of stimulation-evoked field potentials in the substantia nigra serving as an initial criterion that the hemisection had completely transected the striatonigral pathway. Sweeps were averaged using the Cambridge Electronics Devices (Cambridge, England) 1401 interface.

Histological verification of recording site and extent of hemisection

At the conclusion of each experiment, a small amount of Pontamine sky blue dye was ejected from the tip of the pipette by applying constant current to the electrode (-30 mA for 20 minutes) to mark the recording site. After removal of the striata for neurochemical analyses, the brain was placed in 10% formalin for at least 48 hours, after which it was transferred to 15% sucrose. The brain was then blocked, cut into 70 pm frontal sections, stained with cresyl violet, and examined mi- croscopically to verify the location of the recording site within the zona compacta of the substantia nigra. After experiments in which hemisections had been made, the brain was sectioned horizontally to allow visualization of the anterior-posterior and medial-lateral extent of the knife cut.

Statistical analyses All data are reported as mean 2 S.E.M. Dose-re-

sponse curves showing the response of DA neurons to APO were constructed for each group, and differences between the groups were analyzed using a 2 x 9 ANOVA with repeated measures for the dose variable. For determining ED50)s, it has been shown that use of the graphical method yields results that are not significantly different from those obtained by other methods such as linear regression or third-order polynomial regression, while log probit analysis was found to underestimate the ED,, (Pitts et al., 1990). Thus, the ED,, was determined using the graphical method. Differ- ences in striatal DA depletions between each of the lesioned groups, as well as differences between ED,, values and baseline firing rates of control and lesioned

groups, were analyzed using a one-way ANOVA which was followed by Fisher’s Protected Least Significant Difference (PLSD) when there was a significant main effect. The effect of hemisection on the ED,, and baseline firing rate of DA neurons in control rats was analyzed using the 2-tailed Student’s t-test. In order to examine the effect of DA depletion on the relationship between baseline firing rate and sensitivity to APO, the ED,, for each DA neuron tested was plotted against its baseline firing rate, and the correlation coefficient between these two variables was calculated for each group. Differences between the correlation coefficients were analyzed by converting the Pearson r scores to z scores.

RESULTS Depletions of striatal dopamine

Intraventricular administration of 6-OHDA caused significant depletions of striatal DA. The level of striatal DA depletion for each group was: 6-10 days post-lesion, 90.4 t 3.7% (range = 81-99%, n = 5); 4-8 weeks post- lesion, 78.4 * 5.4% (range = 60-99%, n = 8); 4-8 weeks post-lesion, hemisected, 83.0 * 2.8% (range = 74-91%, n = 5). None of the groups showed significant differences in the levels of DA depletion (F = 1.7, df = 2,151. The levels of striatal DA depletion could not be determined for two of the rats. However, based on the degree of initial motor impairment and aphagia, and the time course of recovery from these deficits, it was estimated that each of these rats had depletions of approximately 80%, which is well within the range of depletions used in this study (i.e., 60-99%).

Effect of hemisection on DA neuron responses in control rats

Hemisection did not alter the sensitivity of DA neurons to APO in control rats; i.e., there was no significant difference between the dose-response curves (control, n = 11; hemisected, n = 11; F = 0.70, df = 1,20) or the ED,, values (control = 8.1 t 1.8 pgkg; hemisected =

6.4 * 1.9 pg/kg; t = 0.68) for the two groups. The baseline firing rate also was unaffected by the hemisection (control = 3.3 _t 0.5 spikeshec; hemisected = 3.9 2 0.5 spikeshec; t = 0.85). In addition, the correlation coefficients for the relationship between baseline firing rate and sensitivity to APO for these two groups were not significantly different (control, r = 0.83, z = 1.20; hemisected, r = 0.35, z = 0.37; zObs = 0.64). Because hemisection of the brain of non-lesioned rats did not alter the dose-dependent response to APO, the ED,, values for APO, the baseline firing rate, or the relationship between baseline firing rate and sensitivity to APO of the DA neurons tested, these two control groups were com- bined for subsequent comparisons, After grouping, the average ED,, for APO-induced inhibition was 7.2 * 1.3 pgkg, the baseline firing rate was 3.6 f 0.3 spikeslsec, and there was a significant correlation between base-

148 M.L. PUCAK AN

line firing rate and sensitivity to APO (r = 0.53, P < 0.01).

Dose-dependent inhibition of DA neuron firing by systemic APO administration

Systemic administration of APO decreased the spontaneous firing rate of DA neurons in a dose-dependent manner in all groups of rats (control, F = 98.3, df = 8,216, P < 0.01, n = 22; 6-10 days post-lesion, F = 18.2, df = 8,200, P < 0.01, n = 5; 4-8 weeks post- lesion, F = 28.7, df = 8,224, P < 0.01, n = 8; 4-8 weeks post-lesion, hemisected, F = 22.5, df = 8,216, P < 0.01, n = 7). However, the sensitivity of DA neurons to APO and its relationship to baseline firing rate was significantly altered by DA depletions, as outlined below.

Response of residual DA neurons to APO 6-10 days after DA depletion

The dose-response curve of the response of residual DA neurons to APO in rats tested 6-10 days post-lesion was not significantly different from that of controls (F = 0.71, df = 1,25; Figs. 1,2A). Furthermore, neither the mean ED,, for APO inhibition (10.5 * 3.2 p,g/kg; F = 4.1, df = 3,41, P < 0.025; Fisher's PLSD = 5.2) nor the mean baseline firing rate (3.8 t 0.6 spikeshec; F = 0.8, df = 3,41; Table I) was significantly different from that of controls. However, unlike DA neurons recorded in control rats, DA neurons recorded in rats 6-10 days post-lesion did not exhibit a significant correlation between baseline firing rate and sensitivity to APO (r = -0.54; Fig. 3A).

Response of residual DA neurons to APO 4-8 weeks after DA depletion

The sensitivity of residual DA neurons recorded in rats 4-8 weeks post-lesion was significantly higher than that of controls, as evidenced by the shift to the left of the APO dose-response curve in lesioned rats (F = 5.0, df = 1,28, P < 0.05; Figs. 1, 2B). DA neurons recorded in this group also exhibited a significantly lower ED50 (2.2 t 0.3 pgkg; Fisher's PLSD = 4.3, P < 0.05; Table I) than was found for DA cells in control rats. The firing rate of DA neurons in lesioned rats was not significantly different from that of controls (4.5 t 0.6 spikeshec; Table I). However, the correlation between baseline firing rate and sensitivity to APO was absent in this group; i.e., all DA cells tested exhibited low ED,, values which were independent of baseline firing rates (r = 0.07; Fig. 3B).

Effects of acute hemisections on the enhanced sensitivity of residual DA neurons to AF'O

In rats tested 4-8 weeks post-lesion, the increased sensitivity of residual DA neurons to APO was not altered by the hemisection; the shift to the left in the dose-response curve for this lesioned group with respect to controls was maintained (F = 6.2, df = 1,27, P <

'D A.A. GRACE

B

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Fig. 1. Ratemeter recordings illustrating the response of single DA neurons to APO in control and DA-depleted rats. In this diagram, the vertical lines represent 10-second intervals of spike activity, with the height of each line being proportional to the firing rate of the DA neuron. A. The response of DA neurons in control rats to APO. In this example, administration of increasing doses of APO (solid arrows) caused a dose-dependent decrease in the firing rate of this DA cell, which culminated in a total cessation of spike activity. Subsequent administration of haloperidol (open arrow) reinstated firing. Doses of APO: 0.5, 0.5, 1,2,4,8,16, 32, and 64 pgkg; dose of haloperidol: 128 pgkg. B: The response of DA neurons to APO in rats tested 6-10 days after DA depletion. Administration of APO (solid arrows) at doses similar to those required in control rats caused a dose-dependent inhibition offiring in this DA cell recorded 6 days after the administration of 6-OHDA (striatal DA depletion = 91%). Subsequent administration of haloperidol (open arrow) reinstated firing. Doses ofAPO: 0.5, 0.5,1,2,4,8,16,32,64, and 128 pgkg; dose of haloperidol: 256 pgkg. C: The response of DA neurons to APO in rats tested 4-8 weeks after DA depletion. In a rat recorded 7 1 weeks after administration of 6-OHDA (striatal DA depletion = 69%), lower doses of APO (solid arrows) were required to inhibit the firing of this residual DA neuron. Subsequent administration of haloperidol (open arrow) reinstated firing. Doses ofAF'O: 0.5,0.5,1,2,4, and 8 p e g ; dose of haloperidol: 16 pgkg. Overall, DA neurons recorded in rats 4 - 8 weeks after DA depletions were inhibited by lower doses ofAPO than were DA neurons in control rats or in rats tested within &lo days of the lesion.

0.05; Fig. 2C). The mean ED,, for neurons in this group was significantly lower than that of controls (2.6 2 0.8 Fgkg; Fisher's PLSD = 4.5, P < 0.05; Table I), although the baseline firing rate was not significantly


*- Control ---*--- 6-10 Days Post-lesion

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-- Control 4-8 Weeks Post-lesion, Hemisected

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Fig. 2. Dose-response curves illustrating the effect of DA depletion on the inhibition of the residual DA neurons produced by the systemic administration of APO. DA neurons in all groups showed a dose- dependent inhibition to APO. A The response of DA neurons to APO in rats tested 6-10 days after 6-OHDA administration was not significantly different from that of control rats. B: In rats tested 4-8 weeks after 6-OHDA administration, DA cells were inhibited by significantly

altered (4.5 & 0.7 Hz; Table I). All DA neurons recorded in this group exhibited high sensitivity to APO independent of their baseline firing rates, such that there was no significant relationship between baseline firing rate and sensitivity tom0 (r = 0.27; Fig. 3C). Furthermore, the response of residual DA neurons to APO in rats recorded 4-8 weeks post-lesion was not altered by the hemisection (Figs. 2D, 3D).

Effect of hemisection on striatally evoked field potentials recorded in the substantia nigra

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lower doses of APO, as evidenced by the parallel shift to the left of the dose-response curve. C: After a hemisection which completely inter- rupted connections between the striatum and the substantia nigra, the sensitivity of DA cells to APO in rats tested 4-8 weeks post-lesion was still significantly greater than that of controls. D: Hemisections in rats tested 4-8 weeks post-lesion did not alter their sensitivity to APO

TABLE I. Baseline firing rate and sensitivity to APO of recorded in control and DA-dedeted rats

Average DA Firing rate N depletion (%) (spikedsec)

Control (22) - 3.6 f 0.3 6-10 days (5) 90.4 f 3.7 3.8 f 0.6

4-8 weeks (9) 78.4 f 5.4 4.5 f 0.6 post-lesion

. . post-lesion

4-8 weeks (7) 83.0 f 2.8 4.5 f 0.7 post-lesion,

DA neurons

EDSO APO ( d k g ) 7.2 f 1.3

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Stimulation of the striatum evoked positive-going field potentials in the substantia nigra that had peak

hemisected *Indicates a significant difference from control, P < 0.05, one-way ANOVA.

M.L. PUCAK AND A.A. GRACE 150

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Fig. 3. Sensitivity of DA neurons to APO as a function of their baseline firing rates. In these scatterplots, the ED,, of each neuron is plotted against its baseline firing rate. A The regression line illus- trates the relationship between the response to APO and the initial firing rates of DAneurons recorded in control rats, with fast-firing cells requiring larger doses of APO to reach 50% inhibition. However, in DA neurons recorded 6-10 days after DA depletions, this relationship was not evident. B: In contrast to control rats, the sensitivity of DAneurons to APO was not significantly correlated with their baseline firing rates.

amplitudes of approximately 0.45 mV occurring at an average latency of 12.4 * 1.1 msec (range = 6.7-17.5 msec; Fig. 4B). These field potentials were occasionally followed by negative-going potentials, and were similar in time course and amplitude to the striatally-evoked field potentials reported previously in the substantia nigra of the rat (Dray et al., 1976) and of the cat (Yoshida and Precht, 1971; Precht and Yoshida, 1971). In the present experiments, stimulation of the striatum evoked field potentials that were observed only when the recording electrode was located within the substantia nigra (6.0 to 8.5 mm ventral to the surface of the

B 3( 0 - Control A - - 4-8 Weeks Post-lesion

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Instead, all DA neurons tested showed a uniformly high sensitivity to APO-induced inhibition. C: This uniformly high sensitivity of DA neurons to APO in rats tested 4-8 weeks post-lesion was still present after hemisection of the striatonigral projection. D: In rats tested 4-8 weeks post-lesion, neither the enhanced sensitivity of DA neurons to APO nor the lack of correlation between this sensitivity and baseline firing rate was affected by hemisection of the brain between the substantia nigra and the striatum.

brain; the peak amplitude was recorded at a depth of approximately 7.0 mm). Nigral field potentials were not observed when the stimulating electrode was moved dorsal to the striatum (8.6 mm anterior to lambda; 3.5 mm lateral to the midline; 1.5 mm ventral to the surface of the brain). There were no apparent differences between the evoked field potentials in control versus DA-depleted rats. After hemisection of the striatonigral projection, the field potential was no longer evident (Fig. 4B). This procedure was not performed on all hemisected rats, but rather was used in the initial stages of the experiments in order to guide the place-


A

Fig. 4. Verification of the extent of the hemisection. A Example of a cresyl violet-stained 70 pm thick horizontal section of the rat brain located approximately 6.7 mm ventral to the surface of the skull. This section shows the placement of the hemisection at approximately 4.3 mm anterior to lambda on the right side of the brain. The hemisection begins approximately 1.5 mm lateral to the midline and extends to the lateral edge of the brain, resulting in complete ipsilateral transection of the internal capsule. B: Effects of hemisection on field potentials

B2

ment and extent of the knife cuts. The actual transection produced was verified histologically in all cases.

Histological confirmation of recording site and extent of hemisection

Microscopic examination of frontal sections cut through the midbrain revealed deposition of Pontamine sky blue within the zona compacta region of the substantia nigra in each rat recorded. Horizontal sections from hemisected rats revealed that the knife cuts were located approximately 4.0-4.5 mm anterior to lambda and extended in the medial-lateral plane from the lateral edge of the hypothalamus to the lateral edge of the external capsule. This extent of the hemisection en- sured that the cut had completely transected the internal capsule (see Fig. 4A). In five control and four lesioned rats, the hemisections were located more than

I

10 msec

recorded in the substantia nigra produced by electrical stimulation of the stnatum. B1: Stimulation ofthe striatum (100 4 - 1 mA, 50 psec) evoked a positive-going field potential followed by a negative-going potential only when the recording electrode was located within the substantia nigra (positive = up). B2: After hemisection, the same amplitude of striatal stimulation failed to elicit a field potential within the substantia nigra. Traces represent stimulus-triggered computer averages of 25 sweeps.

4.5 mm anterior to lambda, leaving intact a significant portion of the striatum posterior to the hemisection, or did not extend sufficiently in the medial plane to completely transect the internal capsule. Although the results from these rats did not differ significantly from those with complete hemisections, the data were nonetheless excluded from the data set.

In several cases, the hemisection was found to extend medially into the hypothalamus, with the result that extensive bleeding and swelling of the brain was observed in the histological analysis. Disturbances of this nature were consistently associated with disruption of the electrophysiological recordings, including increased electrode noise and pulsations, frequent clogging of the electrode, and widespread decreases in cell activity. Data from these animals were therefore excluded from this study.

152 M.L. PUCAK AND A.A. GRACE

DISCUSSION Effect of DA depletion on the response of residual

DA neurons to APO These experiments show that partial destruction of

the nigrostriatal DA system can result in an enhanced sensitivity of the residual DA neurons in the substantia nigra to APO. Although this enhanced sensitivity is evident 4-8 weeks after 6-OHDA-induced DA depletions, it does not appear to be present in rats with similar size depletions when tested 6-10 days after the lesion. This delayed induction of hypersensitivity to APO is similar in time course to that reported for 6-OHDA-induced increases in the density of striatal D, receptors, which also are not observed until approximately one month after 6-OHDA administration (Creese and Snyder, 1979).

DA depletions do not cause a significant change in DA neuron firing rate at either time point, in agreement with our previous work (Hollerman and Grace, 1990). Nonetheless, the DA depletions do alter the relationship between baseline firing rate and sensitivity to APO of the DA neurons tested. Although the small sample size and the variability of the responses recorded in the group of rats tested 6-10 days after DA depletions makes evaluation of that group difficult, it is clear that by 4-8 weeks after 6-OHDA administration the sensitivity to APO exhibited by the residual DA neurons is not related to their baseline firing rates. Instead, these DA neurons demonstrate a uniformly high sensitivity to APO regardless of their initial firing rates. This loss of rate-dependence of DA cell sensitivity to APO after 6-OHDA lesions is probably distinct in nature from the loss in correlation between DA cell firing rate and sensitivity to D, agonists produced by acute D, agonist administration (Kelland et al., 1988); in contrast to the actions of D1 agonists, 6-OHDA lesions produce an overall increase in sensitivity that requires time to develop and is not dependent on an intact striatonigral projection.

Possible contribution of non-autoreceptor sites to the enhanced sensitivity of residual DA neurons

In addition to an action at autoreceptors, systemically administered AF'O could potentially act at two other sites: 1) on the striatal neurons which provide feedback to DA neurons (Bunney and Aghajanian, 1978; Grace and Bunney, 1985b) or 2) on DA receptors located on non-dopaminergic elements within the substantia nigra, such as D, receptors located on the terminals of the striatonigral projection (Aiso et al., 1987; Altar and Hauser, 1987; Altar and Marien, 1987; Spano et al., 1977) or possibly DA receptors located on non-dopaminergic neurons in the substantia nigra (Nagy et al., 1978).

1. The striatonigral projection Because extensive DA depletions have been shown to

increase the sensitivity of striatal neurons to DA

(Creese and Snyder, 1979; Creese et al., 1977; Feltz and De Champlain, 1972), it is possible that low doses of APO administered to lesioned rats may exert direct effects on supersensitive striatal neurons, and might thereby indirectly alter DA neuron activity. However, stimulation of post-synaptic receptors in the striatum by APO does not appear to contribute to the enhanced sensitivity of residual DA neurons after lesions because: 1) hemisection of the striatonigral projection did not alter the magnitude of this enhanced sensitivity, and 2) the enhanced sensitivity of DA neurons was observed even in rats which had only a 60% decrease in striatal DA levels. In contrast, up-regulation of striatal DA receptors is not observed unless at least 80% of striatal DA is depleted (Zigmond and Stricker, 1980). Thus, this evidence suggests that the doses of APO administered in this study exert their effect on DA cells via a local action within the substantia nigra.

2. Non-dopaminergic elements within the substantia nigra

Another indirect influence that possibly could contribute to the alterations in DA neuron sensitivity to APO is the potential effects of APO on DA receptors located on non-dopaminergic elements within the substantia nigra. However, evidence indicates that stimulation of D, receptors located on striatonigral terminals is unlikely to contribute to the enhanced sensitivity of DA neurons t o APO observed here. For example, D, stimulation does not contribute to the response of DA neurons in untreated rats to APO (Carlson et al., 1986; Mereu et al., 1985; Napier et al., 19861, and 6-OHDA does not increase D, binding in the substantia nigra (Altar and Marien, 1987; Morelli et al., 1988). Indeed, evidence suggests that D, responses may be down- regulated in lesioned rats; i.e., lesions are reported to decrease D, binding, decrease DA-stimulated adenylate cyclase (Porceddu et al., 1987), and decrease the produc- tion of messenger RNA for the D, receptor in striatal neurons projecting to the substantia nigra (Gerfen et al., 1990). Moreover, in the present study, the APO-induced inhibition of DA neurons was completely reversed by the administration of moderate doses of haloperidol, a DA antagonist with preferential actions at the Dz receptor (Kebabian and Calne, 1979). It is also unlikely that DA-sensitive non-dopaminergic neurons in the substantia nigra are involved in this response, since previous reports have shown that the administration of 6-OHDA enhances APO-induced inhibition of zona reticulata neurons (Waszczak et al., 1984) which are believed to provide an inhibitory input to DA neurons (Grace and Bunney, 1979, 1985a,b) via their local collaterals (Gro- fova et al., 1982). Thus, the emergence of APO effects on zona reticulata neurons would, if anything, be expected to counteract APO-induced inhibition of DA neuron activity, causing at most an underestimate of the enhanced sensitivity of DA cells to APO. Finally, our


preliminary results suggest that residual DA neurons recorded in rats 4-8 weeks post-lesion exhibit an enhanced response to microiontophoretically applied DA (Pucak and Grace, unpublished results). Therefore, the most likely explanation for the enhanced sensitivity of DA neurons to APO reported here is an up-regulation in the sensitivity of somatodendritic autoreceptors located on the residual DA neurons.

Functional implications of DA-depletion-induced enhancement of the sensitivity of somatodendritic

DA autoreceptors The functional importance of the somatodendritic

autoreceptors in the normal regulation of DA neuron activity has yet to be defined. It has been suggested that dendritically released DA inhibits DA neuron activity via the stimulation of somatodendritic autoreceptors (Cheramy et al., 1981; Groves et al., 1975). This is consistent with our observation that systemic administration of haloperidol increases DA neuron firing rate in rats with hemisections of the striatonigral projection (Pucak and Grace, 1991). The ability of DA depletions to increase somatodendritic autoreceptor sensitivity is consistent with the hypothesis that these autoreceptors are normally stimulated by endogenous DA released from DA neuron dendrites.

Indeed, these results may provide an indication of the site of action of dendritically released DA. DA released from the dendrites of DA neurons could potentially act on autoreceptors at two sites to inhibit DA neuron firing: 1) on autoreceptors located on the neuron releasing the DA (i.e., autoinhibition), and/or 2) on autoreceptors located on DA cells in close proximity to the neuron releasing the DA (i.e., lateral inhibition). If the sole function of dendritic DA is the autoregulation of DA neurons, then the removal of neighboring DA neurons would not be expected to change the level of autoreceptor stimulation occurring on the residual DA cells. In contrast, if autoreceptors are normally stimulated by DA released from a pool of nearby DA cells, loss of these neighboring DA release sites may be expected to induce a compensatory increase in autoreceptor sensitivity. Thus, the observation that DA neuron somatodendritic autoreceptors appear to be up-regulated after partial loss of DA neurons suggests that at least a portion of somatodendritic autoreceptor stimulation in the intact animal is provided by DA released from neighboring DA neurons.

An up-regulation of somatodendritic autoreceptors may account for the finding that the response of DA neurons to compromise of DA transmission depends on the manner in which the compromise is induced. Specif- ically, although both haloperidol administration and DA depletion decrease DA receptor stimulation, only haloperidol causes significant increases in DA neuron firing rate (Bunney et al., 1973; Hollerman and Grace, 1990). One possible explanation for this difference is

that, in the DA-depleted rat, the increased autoreceptor sensitivity may act to offset any striatonigral feedback- mediated excitation of DA neurons produced by the loss of DA. Haloperidol administration, in contrast, would not only block DA receptors in the striatum, but also would block DA neuron autoreceptors in the substantia nigra, thereby preventing any opposing influence of the autoreceptor on feedback excitation of DA neuron firing. Thus, increased autoreceptor sensitivity may constrain DA neuron firing rates to within circumscribed limits, and thereby maintain the long-term stability of the DA system after it has been compromised.

Clinical implications The finding that partial DA depletions may cause

supersensitivity of DA neuron autoreceptors may be relevant to the treatment of the movement disorder Parkinson’s disease, which occurs as a consequence of the degeneration of nigrostriatal DA neurons (Horny- kiewicz, 1971). The motor symptoms associated with this disease are normally attenuated by the administration of L-DOPA. However, L-DOPA has been reported to produce anomalous effects in some Parkinsonian pa- tients. For example, it has been reported that the administration of subthreshold doses of L-DOPA to pa- tients suffering from Parkinson’s disease actually exacerbates the underlying motor impairment when compared to the administration of a placebo (Nutt et al., 1988). One possible explanation for this observation is that the subthreshold doses of L-DOPA, which may not be adequate to stimulate postsynaptic DA receptors in the striatum, may nonetheless be sufficient to inhibit DA neuron activity via stimulation of supersensitive autoreceptors. Thus, preferential stimulation of autoreceptors by low doses of L-DOPA could conceivably lead to an exacerbation of symptoms secondary to a decrease in the release of DA from terminals within the striatum.

ACKNOWLEDGMENTS The authors thank Ms. Beth Vojta for performing

dopamine assays, Ms. Denise Faulx-Adams for histological work, Dr. Robert Pagano for advice on statistical analyses, and McNeil Pharmaceutical for their gener- ous gift of haloperidol. This work was supported by USPHS MH09873 (to M.L.P.) and by NS19608, MH42217, MH45156. A.A.G. is a Sloan Fellow.

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