Irreversible receptor inactivation reveals differences in dopamine receptor reserve between A9 and A10 dopamine systems: an electrophysiological analysis

Brain Research, 534 (1990) 273-282

273

Elsevier BRES 16093

Irreversible receptor inactivation reveals differences in dopamine receptor reserve between A9 and A10 dopamine systems: an electrophysiological analysis Richard E Cox and Barbara L. Waszczak Pharmacology Section, College of Pharmacy and Allied Health Professions, Northeastern University, Boston, MA 02115 (U.S.A.) (Accepted 12 June 1990)

Key words: Dopamine neuron; Single unit recording; N-Ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline; R-(-)-N-n-Propylnorapomorphine; Receptor reserve; Substantia nigra pars compacta; Ventral tegmental area; A9 cell; A10 cell

Partial receptor inactivation was used as a tool to examine whether differences in receptor reserve exist between the dopamine receptor populations which mediate responses of substanfia nigra (A9) and ventral tegmental area (A10) dopamine neurons to dopamine agonist drugs. The irreversible receptor inactivator, N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ), was administered to rats intraperitoneally at a dose of 6 mg/kg (in an ethanol-water vehicle). Approximately 24 h after EEDQ treatments, extracellular, single-unit recording experiments were carried out. In the first series of experiments, dose-response curves were constructed for the inhibition of A9 and A10 dopamine cell firing by intravenous administration of the potent dopamine agonist, R-(-)-N-n-propylnorapomorphine (NPA). For the A9 dopamine cell group, EEDQ pretreatments caused a 3-fold rightward shift in the NPA dose-response curve (EDsos, 0.3 vs 0.8/~g/kg for vehicle- and EEDQ-treated rats, respectively), but there was no change in the maximum attainable response (>95% inhibition of cell firing). For A10 neurons, the same EEDQ treatments produced a greater rightward shift in the dose-response curve to NPA (EDsos, 0.6 vs 5.4/~g/kg for vehicleand EEDQ-treated rats), and also depressed the maximum response by about 25% relative to the control (vehicle) curve. The dose-response curves from each region were subjected to Furchgott analysis to determine relative receptor occupancy-response relationships for NPA. For the A9 system, a steep, hyperbolic occupancy-response plot revealed that a 50% inhibitory response required only 4% receptor occupancy, while complete (>95%) inhibition of cell firing required about 30% occupancy. This suggests about a 70% receptor reserve for this agonist in inhibiting A9 dopamine cell firing. The occupancy-response curve for A10 cells was less steep with 50% and maximal (>95%) responses occurring when 11 and 70% of receptors were occupied by the agonist, indicating only about a 30% reserve for A10 cell responses to NPA. While the level of 'spare' receptors differed substantially between the two areas, calculated pseduo-KA values were similar (7.7/ag/kg for A9 cells and 5.5/ag/kg for A10 cells), suggesting no regional differences in receptor affinity. To explore where the differences in receptor reserve might reside, a second series of studies evaluated the effects of iontophoretically applied dopamine and NPA on both cell groups in vehicleand EEDQ-treated rats. EEDQ (6 mg/kg, i.p.) treatments lessened the ability of both iontophoresed agonists to inhibit firing in both cell groups, but caused equivalent parallel shifts to the right in the current-response curves to both dopamine and NPA for both cell groups. This finding suggests that the apparent difference in receptor reserve between A9 and A10 systems is probably not attributable to differences at the level of the somatic autoreceptors reachable by iontophoresed drugs, but may be due to differences at more distal dendritic autoreceptors or at other sites in the circuitry which may indirectly mediate midbrain neuronal responses to systemically adminstered agonists.

INTRODUCTION D o p a m i n e n e u r o n s of the m a m m a l i a n midbrain are k n o w n to play a central role in the expression of a range of vertebrate behaviors. For example, an intact nigrostriatal dopaminergic projection (A9 cell group) is crucial for the execution of normal voluntary movements. Destruction of this pathway underlies the pathology of Parkinson's disease in humans. A d j a c e n t mesolimbic dopamine cells of the ventral tegmental area (VTA, A10 cell group) are involved in motivational, affective and locomotor processes. A functional excess of dopaminergic transmission in the mesolimbic system has long

been suspect in the symptoms of psychotic disorders 26. Recently, the A10 system has been further implicated as playing a role in the reward and reinforcement properties of drugs of abuse such as cocaine 2°. Given the importance of d o p a m i n e n e u r o n s in both extrapyramidal and limbic functions, it is not surprising that interest has centered on exploring whether differences exist between the regulation of the A9 and A10 dopamine cell groups which could be exploited in the development of drugs with selectively for one or the other cell population. Despite a continuous lateralto-medial anatomical distribution of midbrain d o p a m i n e cells 11, nigral and ventral tegmental n e u r o n s are k n o w n

Correspondence: R.F. Cox, Room 312 Mugar Hall, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, U.S.A. 0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

274 to differ from one another by their projection sites 4"27, afferent innervations 23, and roles in normal and pathological states 26. H o w e v e r , one still unresolved physiological issue is the question of possible differences between A9 and A10 d o p a m i n e neurons in terms of their sensitivity to exogenous dopaminergic agonists. It has been p r o p o s e d that, on the whole, A10 cells may be less sensitive than A 9 cells to the rate slowing actions of systemically administered d o p a m i n e agonists. The basis for this regional sensitivity difference has been attibuted to a relative deficit of somatodendritic autoreceptors on particular subgroups of V T A d o p a m i n e cells. Specifically, d o p a m i n e neurons that project from the V T A to the prefrontal and cingulate cortices are thought to lack s o m a t o d e n d r i t i c as well as terminal autoreceptors 3"8. Not every investigation substantiates r e p o r t e d A 9 A10 differences, however. In some studies where the responses of nigral and V T A d o p a m i n e cells to i.v. a p o m o r p h i n e were m o n i t o r e d , regional differences in sensitivity were not detected in either awake 13 or in chloral hydrate-anesthetized rats 16. Even when d o p a m i n e and a p o m o r p h i n e were directly i o n t o p h o r e s e d onto nigral and V T A d o p a m i n e cells, differences in sensitivity based on anatomical location of cells were sometimes not found 2. The present study was designed to explore further whether differences might exist in the d o p a m i n e receptor populations involved in mediating the inhibitory responses of A 9 and A10 d o p a m i n e neurons to the directly acting d o p a m i n e agonist, R - ( - ) - N - n - p r o p y l n o r a p o m o r phine ( R ( - ) - N P A ) . O u r previous studies have shown that both nigral and V T A d o p a m i n e neurons are inhibited by similar doses of i.v. N P A (EDs0s of 0.53 and 0.50/~g/kg, respectively1°). O n superficial analysis, this might seem to suggest that densities of functional receptors between nigral and limbic circuits do not differ. H o w e v e r , the presence of a large r e c e p t o r reserve for N P A could potentially obscure any differences in receptor density which might exist. A large reserve has already been d e m o n s t r a t e d to exist within the nigrostriatal system at the a u t o r e c e p t o r sites thought to mediate the effects of systemic d o p a m i n e agonists on d o p a m i n e cell firing 9, d o p a m i n e synthesis 21, and release 33. Similar studies have not been carried out to characterize the existence or size of a ' s p a r e ' r e c e p t o r population in the limbic d o p a m i n e system. T h e current studies, therefore, m a k e use of the technique of partial r e c e p t o r inactivation in o r d e r to, first, explore w h e t h e r such a receptor reserve exists for N P A in inhibiting the firing of A10 d o p a m i n e neurons, second, to c o m p a r e the relative sizes of the r e c e p t o r pools involved in mediating the actions of i.v. N P A on the two d o p a m i n e cell groups, and third, to examine w h e t h e r any a p p a r e n t differences in receptor densities

between the two d o p a m i n e systems exist at the level of the somatic autoreceptors reachable by iontophoretically applied d o p a m i n e or N P A . MATERIALS AND METHODS

Receptor inactivation methods Dopamine receptors were inactivated by N-ethoxycarbonyl2-ethoxy-l,2-dihydroquinoline (EEDQ) according to previously published protocols9'2~. In brief, on the day before each experiment, male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing between 200 and 400 g (mean weight 303 + 5 g), were pretreated with 6 mg/kg EEDQ, i.p. (2 mg/mi in a 1:1 vehicle of ethanol and water) or an equivalent volume of the vehicle alone. On the next day, approximately 24 h later, animals were prepared for single-unit recording experiments. ExtraceUular, single-unit recording techniques Rats were anesthetized with chloral hydrate (400 mg/kg i.p.) and secured in a stereotaxic device. Extracellular single-unit recordings of dopamine neurons of the substantia nigra pars compacta (A9 cells) and the VTA (A10 cells) were carried out using standard electrophysiologicaltechniques, as previously described1°'31. Dopamine neurons were tentatively identified during the recording period by well-documented electrophysiological criteria2a°'2a. For A9 cell recordings, electrodes were inserted into the brain through a 3.0-mm burr hole drilled in the skull 2.0 mm lateral to lambda and 3.0 mm anterior to the lambdoid suture. Nigral dopamine neurons were located within the following stereotaxic boundaries according to the atlas of Paxinos and Watson24: L, 2.0 to 2.4 ram; A, 3.0 to 3.4 mm; V, -6.62 to -7.85 mm (from the surface of the brain). The VTA was approached from a hole drilled 0.5 mm lateral to lambda and 3.0 mm anterior to the lambdoid suture. VTA dopamine cells were located within an area defined by the boundaries: L, 0.5 to 0.9 mm; A, 3.0 to 3.4 ram; V, -6.31 to -8.55 mm. Dopamine cells in both the substantia nigra and the VTA exhibited firing rates between 1 and 9 spikes/s and sometimes fired in a bursting pattern. These neurons exhibit a characteristic triphasic action potential shape with a notched positively deflected initial segment followed by a broad negative deflection and a later positive segment. The durations of the action potentials of the cells recorded exceeded 2 ms. Correct positioning of the tip of the recording electrode in either substantia nigra or the VTA was confirmed by iontophoresis of Pontamine blue dye from the electrode at the end of each recording, followed by later histological examination of tissue sections for a blue spot. Extracellular action potentials of spontaneously active single cells were amplified, filtered, and displayed on an oscilloscope screen. Impulse rates, summed over 10-s intervals, were simultaneously printed by a digital printer and graphed as a rate histogram by a Gould physiological recorder. In addition, the electrical signal was amplified through an audio monitor to aid in signal identification and monitoring. Intravenous drug studies After locating a dopamine cell and recording a stable baseline firing rate for 3-5 min, doses of R(-)-NPA were injected into a lateral tail vein at 1-min intervals such that each successive dose doubled the previous cumulative dose. Additional doses were administered until dopamine cell firing was completely inhibited or a total cumulative dose of 160/~g/kg was reached. Haloperidol, a dopaminergic antagonist, was routinely administered following R(-)-NPA-induced inhibitions of firing to reverse agonist effects and further establish the dopaminergic nature of the response. Only one cell was recorded from each animal in i.v. studies to avoid residual agonist effects. Dose-response relationships for i.v. R(-)-NPA-induced inhibitions of dopamine cell firing were determined for both EEDQ- and vehicle-treated animals. The mean firing rate for the 60-s period

275 after each dose was compared with the mean rate during the initial baseline period for that cell. Response, expressed as percent inhibition relative to baseline rates, was plotted as a function of the log dose. Resulting curves were analyzed using the simultaneous, multiple curve fitting computer program ALLFIT~2as modified for the Apple II computer by M. Teicher (MED-65, ALLFIT, Biomedical Computing Technology Information Center, Nashville, TN).

1ontophoresis studies Five barrelled glass micropipettes were prepared in a standard way2'3L32. The central recording barrel was filled with 2 M sodium chloride containing 1% Pontamine sky blue dye. One of the 4 outer barrels contained 4 M sodium chloride solution and was used for automatic current balancing. At least one outer barrel contained R(-)-NPA (0.01 M in 0.02 M NaCI, pH 4.0-4.5), and at least one contained dopamine (0.2 M, pH 4.0). After filling the micropipettes, electrode tips were broken back under light microscopy so that the in vitro impedences of recording barrels were between 3 and 12 MS'2, measured at 135 Hz, corresponding to approximate tip diameters of 3-5 /~m. Resistances of the balance channel were between 20 and 30 MI2, while impedances of drug-containing barrels ranged between 30 and 110 Mr2. Ejection currents were applied to the drug barrels for 60-s intervals in a discontinuous, stepwise manner (5, 10, 20, 40, 80, 100 nA) and neuronal firing rates were allowed to return to baseline levels for at least 60 s before advancing to the next higher current. Between periods of iontophoresis, a retaining current of -10 nA was applied through drug-containing barrels. Response was measured by comparing the mean firing rate during the 60-s drug application with the mean firing rate during the 60-s period immediately preceding each ejection. Responses were plotted as the percent inhibition of dopamine cell firing versus ejection current for further comparisons.

Materials R(-)-NPA (6aR-N-n-propylnorapomorphine, Research Biochemicals Inc., Natick, MA), chloral hydrate (Sigma Chemical Co., St. Louis, MO) and haloperidol (premixed injection, McNeil Pharmaceutical Co., Spring House, PA) were dissolved and diluted in distilled water. EEDQ (N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline,Research Biochemicals, Inc., Natick, MA and Aldrich Chemical CO., Milwaukee, WI) was dissolved in 100% ethanol and diluted with distilled water to a final ethanol concentration of 50% (v/v). RESULTS

Responses of substantia nigra (A9) dopamine neurons to i.v. R(-)-NPA Representative rate histograms and cumulative d o s e response curves for inhibition of substantia nigra dopamine cell firing by R ( - ) - N P A are shown in Figs. 1 and 2 for both vehicle- and E E D Q - t r e a t e d rats. The i.v. adminstration of R ( - ) - N P A resulted in inhibition of A9 n e u r o n a l firing with an A L L F I T - d e r i v e d EDso value of 0.27 _+ 0.02 pg/kg. Nigral d o p a m i n e cells from rats in the vehicle-treated group were maximally inhibited (99.3 + 0.5%) by doses of R ( - ) - N P A less than or equal to 10 pg/kg. After partial receptor inactivation by systemic E E D Q (6 mg/kg), the d o s e - r e s p o n s e curve to R ( - ) - N P A was shifted 3-fold to the right (ED5o, 0.79 _+ 0.04/~g/kg) with no significant decline in maximal response (96.0 +

Receptor occupancy-response calculations

4.0% inhibition of firing at the 10 /~g/kg cumulative

Receptor occupancy-response relationships were determined for the effects of i.v. R(-)-NPA on firing of A9 and A10 dopamine neurons using the methods of Furchgon and Burstyn 14 as modified for in vivo comparisons9"z1'22. The doses of agonist required to elicit specific levels of response (i.e. 30, 40, 50, 60, 70% inhibition) were determined for control R(-)-NPA dose-response curves and for curves generated from EEDQ-pretreated rats. These equieffective doses, A and A', were graphed as a double-reciprocal plot, related by the formula:

dose).

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where [A] is the dose of agonist needed to produce a given response, [A'] is that dose which produces the same degree of response after partial receptor inactivation, q is the fraction of receptors not inactivated by EEDQ treatment, and KA is the apparent agonist dissociation constant. Although this KA is not a true constant as are KA values derived from in vitro equilibrium ligand-receptor binding studies, this value can be used to estimate relative occupancyresponse relationships for in vivo experiments (see refs. 9,21). Fractional receptor occupancy (f) at various agonist doses was determined by substituting doses (A) from control dose-response curves and KA derived above into the following equation: f = iRA] =

[RT]

[A]

Response of VTA (AIO) dopamine neurons to i.v.

R(-)-NPA D o p a m i n e cells from the ventral tegmental area of vehicle-treated rats were moderately less sensitive to i.v. R ( - ) - N P A than were cells from the A 9 region (EDso for A10 cells, 0.63 + 0.07/~g/kg; P < 0.05). However, partial receptor inactivation by E E D Q uncovered a substantial regional difference in response to this agonist. After p r e t r e a t m e n t with the same 6 mg/kg i.p. dose of E E D Q , not only was the EDso value for A10 cells shifted toward higher doses than was the corresponding value for A9 cells (i.e. for A10 cells, the m e a n dose to reduce firing by 50% of the new m a x i m u m response was 5.4 + 1.3/zg/kg, an 8-fold shift), but there was also a significant reduction in the maximal response attainable (75.1 + 13% inhibition) at 80 ~g/kg, a cumulative i.v. dose 8 times greater than that needed to fully inhibit A9 cells in either vehicleor E E D Q - t r e a t e d groups (Figs. 1 and 2).

[A] + KA

where IRA] is the concentration of receptor bound by agonist and [RT] is the total concentration of receptors available to interact with agonist. Fractional occupancy at a given dose of agonist was then plotted against the percentage of maximal response obtained for that dose in the control dose-response curves for each dopamine cell group. Receptor reserves (spare receptors) were estimated by subtraction of the percent occupancy at maximal responses from 100.

Receptor occupancy-response relationships for R(-)NPA: A9/AIO differences The d o s e - r e s p o n s e curves generated above were subjected to Furchgott analysis to derive relative occup a n c y - r e s p o n s e requirements for R ( - ) - N P A - i n d u c e d slowing of d o p a m i n e cells from each region. For A9 cells,

276 Vehicle-treated

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Fig. 1. Representative rate histograms of responses of substantia nigra and ventral tegmental area dopamine cells to i.v. R(-)-NPA in vehicle(50% ethanol" in water) and EEDQ- (6 mg/kg, i.p.) treated rats. EEDQ treatment lessened maximal inhibitory responses to the agonist only in the ventral tegmental area. E E D Q t r e a t m e n t r e d u c e d the active receptor pool to give an estimated q value of 0.37. This result indicates that 37% of the original r e c e p t o r pool mediating the inhibitory response to i.v. R ( - ) - N P A r e m a i n e d functional after E E D Q t r e a t m e n t ; conversely, 63% of receptors were inactivated. The p s e u d o - K A derived for A 9 responses to N P A was 7.7 Mg/kg. The calculated q value for V T A d o p a m i n e cell responses after identical E E D Q treatments was 0.18, indicative that 18% of the receptor pool r e m a i n e d active or 82% of receptors were inactivated. The K A for R ( - ) - N P A in the A10 system was 5.5 Mg/kg. The similarities of K A values for R ( - ) - N P A in both regions suggests little, if any, difference in affinities of the receptors regulating the two d o p a m i n e cell groups under the conditions of these experiments. O c c u p a n c y - r e s p o n s e curves were generated for the effects of R ( - ) - N P A on each cell group, as described above. F r o m these o c c u p a n c y - r e s p o n s e plots (Fig. 3), we estimated that the half-maximal response of A 9 cells to i.v. N P A occurred at 3.5% receptor occupation with maximal response ( > 9 5 % inhibition) occurring at approximately 30% r e c e p t o r occupancy. For A10 cells, a 50% response was attained at 11.4% receptor occupancy, while maximal response required approximately 70% r e c e p t o r occupancy.

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R(-)NPA (d,t,g/kg i.v., cumulative dose) Fig. 2. Cumulative dose-response curves for the inhibition of nigral and VTA dopamine cell firing by i.v. R(-)-NPA in vehicle- and EEDQ-treated animals. EDso values obtained in each group are indicated in the text. Top: after EEDQ, dose-response curves of A9 dopamine cells were shifted to the right with no decline in maximal response. Bottom: dose-response curves of A10 cells were shifted further to the right after EEDQ treatments, and a 25% decline in maximal response was observed (n = 10-17 cells at each dose level).

277 somatic autoreceptors. The effects of iontophoretically applied dopamine agonists on A9 and A10 cells were compared for vehicle- and EEDQ-treated rats. Representative rate histogram recordings of these responses are shown in Fig. 4. Iontophoretic applications of both R ( - ) - N P A and dopamine resulted in currentdependent inhibitions of dopamine cell firing for both cell groups. In contrast to results obtained in i.v. studies, however, no regional differences in response were observed between A9 and A10 dopamine cells in terms of their responses to the iontophoretically applied agonists after either vehicle or E E D Q treatments (Fig. 5). In vehicle-treated control animals, maximal inhibitions of A9 and A10 cell firing by directly applied R ( - ) - N P A and dopamine were similar, e.g. approximately 80% inhibitions of firing at 80-100 nA ejection currents. These were the highest currents which could be attained without induction of electrical noise. The calculated 150 values (mean currents to inhibit firing by 50%) in these studies were 30 and 28 nA R ( - ) - N P A for A9 and A10 cells, respectively, and 28 and 37 nA dopamine for A9 and A10 cells, respectively. After treatment with the same dose of E E D Q used in i.v. studies (6 mg/kg i.p., 1 day before electrophysiology), the responses of cells from each group to iontophoresed R ( - ) - N P A or dopamine were shifted equally to the right. Maximal inhibitions of firing achieved by

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% OCCUPANCY (f x 1 0 0 ) Fig. 3. Receptor occupancy-response curves for R(-)-NPA on A9 (nigral) and A10 (VTA) dopamine cells. Dose-response curves (shown in Fig. 2) were subjected to Furchgon analysis to derive receptor occupancies for various levels of response in control animals. The steep, hyperbolic nature of the curve for A9 cells indicates an extremely efficient occupancy-response relationship, with an apparent receptor reserve for R(-)-NPA of about 70%. The occupancy-response relationship for the agonist in the A10 system was less favorable, with a smaller (about 30%) apparent receptor reserve.

Responses of A9 and AIO dopamine neurons to iontophoresis of dopamine agonists These studies were undertaken to assess whether apparent differences in dopamine receptor pools mediating responses of the two cell groups to i.v. dopamine agonists might be similarly apparent at the level of the

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Fig. 4. Rate histograms for the responses of substantia nigra and ventral tegmental area dopamine ceils to iontophoresis of R(-)-NPA (0.01 M in 0.02 M NaCI) and dopamine (0.2 M) in vehicle- and EEDQ-treated rats. Responses to both agonists were attenuated by EEDQ pretreatments. Drugs were applied for 1-min intervals at the currents indicated above the bars.

278

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Fig. 5. Current-response curves for inhibitions of A9 and A10 dopamine cell firing by iontophoresis of R(-)-NPA and dopamine. In control rats, responses at 100 nA did not exceed 80% inhibitions of firing for either agonist and did not differ between the regions. After EEDQ, equivalent changes in responses were noted for both cell groups. (Each point represents the average responses of 10-19 cells from 7-10 rats, except at 100 nA where n = 2-7 cells.)

R(-)-NPA or dopamine after E E D Q did not differ significantly between A9 and A10 cell groups (Fig. 5), indicating that the E E D Q treatments caused similar reductions in response to both agonists in both systems. DISCUSSION

Irreversible receptor inactivation with subsequent Furchgott analysis is a classical pharmacological approach for examining relationships between receptor occupancy and levels of response generated by an agonist 14'25. Such analyses have proven instrumental in determining differences in efficacy between agonists within a particular system, and have been used to quantitate the size of the 'spare' receptor pool for agonists in systems where such reserve exists. Recently, such methods have been successfully applied to the study of central nervous system adrenergic and dopaminergic responses under in vivo conditions using the receptor inactivator E E D Q 1"9"21"22. In our previous work, we have used this approach to determine the relative efficacies of R(-)- and S (+)-NPA for inhibition of nigral dopamine cell firing, a measure of dopaminergic agonist activity9. In that study, dopamine receptor inactivation with E E D Q revealed differences in intrinsic efficacy between the two enantiomers despite their similar, 'full' agonist profiles in normal animals. Further, the results of that study corroborated earlier reports by Meller et al. 22 and Bergstrom et al. 5, that dopamine autoreceptors in the nigrostriatal system exhibit a substantial, functional receptor reserve to potent and highly efficacious agents such as R(-)-NPA and R(-)-apomorphine. In the present investigation we have used a similar

methodological and analytical approach, but instead of measuring responses to two agonists, we have extended our previous study to examine the occupancy-response relationships for two groups of dopamine neurons (nigral A9 cells and VTA A10 cells) to a single agonist, R(-)-NPA. Responses to agonists are a complex function of both tissue factors (receptor density and transduction coupling, for example) and drug properties (dose, affinity and intrinsic efficacy)19. In the experiments described in this paper, only a single agonist, R(-)-NPA, was used and, therefore, any observed differences in response are likely to be due to tissue differences between the two neuronal populations at some level of the response generating system. Since the first series of studies involved in vivo testing of a systemically administered agonist, certain departures from the traditional assumptions of Furchgott analysis should be taken into account in interpretation of the data. First, responses to the agonist were monitored in separate groups of animals which either did or did not receive prior treatment with the inactivator. Consequently, calculations of receptor reserve, q and K A may be less accurate than might be expected from in vitro experiments in which the effects of the agonist are evaluated on the same tissue both before and after treatment with the inactivator. Second, responses to the agonist were determined under non-equilibrium conditions since the drug was administered i.v. rather than by bath application, as would be typical for in vitro studies. Thus, estimates of agonist K A values are in units of mg/kg doses instead of actual tissue concentrations. Such values are valid only in making relative comparisons between groups of similarly treated animals, such as our use of

279 pseudo-KAS to compare the occupancy-response relationships for NPA acting within two parallel neuronal systems. Finally, Furchgott's method is generally considered applicable only in the theoretical situation where a drug acts on a single population of receptors to produce a single response. This criterion can be difficult to satisfy with certainty, even in in vitro studies, due to the sometimes unrecognized pharmacological complexity of the target tissue upon which the drug acts. Indeed, in our first series of experiments we began with the assumption that the nigrostriatal (A9) and mesolimbic (A10) circuitties were the sites of action of the systemically administered dopamine agonist, and that the response elicited by the agonist acting upon these 'tissues' was an inhibition of dopamine cell firing. By making no presumption about where the relevant receptors were located within the circuitry, we assumed by default the involvement of a uniform population of receptors in mediating the response. In the second series of experiments, the assumption of receptor uniformity was tested by considering the actions of dopamine agonists at just one location within these parallel circuitries, i.e. those receptors on the A9 and A10 dopamine cell bodies reachable by iontophoretically applied drugs. We reasoned that, if the dopamine receptor populations mediating the inhibition of cell firing were uniform, then sampling the responses at one subset of sites should yield the same relative differences in response capability between A9 and A10 cells that would be seen after i.v. administration of the agonist. Partial inactivation of dopamine receptors by systemic administration of E E D Q exposed clear, regional differences in the response to i.v. R(-)-NPA. Following E E D Q treatment, the mean response of A9 cells was shifted 3-fold to the right yet maximal responses were still attainable. In the nomenclature of classical receptor theory, such a finding suggests that 'spare receptors' exist in the receptor population which mediates the response to R(-)-NPA. Although a portion of that population was inactivated by E E D Q (giving a rightward shift in the dose-response curve), sufficient receptors remained after E E D Q to still allow a maximal response (complete inhibition of firing) at high doses of agonist. On the other hand, A10 responses to i.v. R(-)-NPA were differently affected by the same receptor inactivation treatment. Not only was an 8-fold rightward shift observed for the agonist after E E D Q , but relatively high doses of the agonist (up to 160/ag/kg) could not completely inhibit A10 cell firing. This significant decline in maximum response suggests that the same E E D Q treatments inactivated a greater proportion of the receptor pool needed to mediate the response in the A10 system. Indeed, too few receptors remained to elicit a full response, suggesting that the VTA system has a lesser

receptor reserve than the A9 system for this physiological response. In order to quantitate this difference, the doseresponse curves to R(-)-NPA in the two dopamine cell groups were subjected to Furchgott analysis. The analysis revealed, first, that apparent dissociation constants (pseudo-KA values) for R(-)-NPA in the two regions were similar (7.7 gg/kg for the nigra and 5.5/tg/kg for the VTA). This suggests that there were no regional differences in the affinities of receptors mediating the response to this agonist. However, the calculations did reveal an apparent difference in the proportion of receptors inactivated in each system after the E E D Q treatments, as well as a difference in functional occupancy-response relationships between the two cell groups. These determinations support the qualitative assessment (above) that a larger (or more efficient) receptor population may be available to mediate rate-depressant effects of the agonist in the A9 than the A10 system. Specifically, q values derived from the plots of A versus A' were 0.37 for A9 cells and 0.18 for A10 cells, indicating a 63% inactivation of receptors in the A9 system and an 82% inactivation of receptors in the A10 system. This difference implies that there were initially fewer receptors available to interact with agonist in the A10 system. This same conclusion is derived from the occupancy-response plots for NPA on A9 and A10 cells. Nigral dopamine cells were inhibited to 50% of baseline rates at approximately 3.5% receptor occupation while the same level of response for A10 cells required 11.4% occupation. Maximal (>95%) inhibitory responses occurred at an estimated 30% and 70% receptor occupancy for A9 and A10 cells, respectively. These differences again suggest a greater receptor reserve in the A9 system, i.e. approximately 70% of the receptor population may be regarded as 'spare' for A9 cell responses, whereas only 30% of the receptor pool mediating A10 cell responses to this agonist are 'spare'. Although differing receptor reserves may, in fact, account for these results, a more appropriate way to regard the findings may be to consider that cells of the A10 system, on average, display a 'less favorable occupancy-response relationship' for the rate-slowing actions of intravenous R(-)-NPA than do dopamine cells of the nigrostriatal system. This acknowledges the possibility that differences in receptor density may not be the sole explanation for the observed A9-A10 difference. Alternatively, it may be that dopamine receptor-coupling efficiencies or response-generating mechanisms distal to receptor occupation may be less favorable in the A10 than the A9 system. In a broad sense these results were not surprising because others had proposed that subpopulations of VTA dopamine cells were less responsive than A9 cells to

280 inhibition by dopaminergic agonists. Specifically, the lower sensitivity of mesoprefrontai and mesocingulate dopamine neurons may be due to a lower density (or lack) of autoreceptors relative to mesopiriform and nigrostriatal cells 8. No attempt was made to determine projection sites in the present study, therefore, one might ascribe our results to a fortuitous sampling of VTA cells favoring those cells that were intrinsically less sensitive to agonists. This seems unlikely for several reasons. First, agonist resistant, mesocortical dopamine cells represent the minority of VTA dopamine cells 27 and are located in the medial VTA 13. The cells we recorded in this report were in the lateral parts of this region. In addition, iontophoresis studies (below), conducted at similar stereotaxic coordinates, revealed no regional differences in sensitivity to NPA or dopamine even in control animals. In the second series of experiments, we investigated whether the differences in response revealed by E E D Q treatments were similarly apparent at the level of the somatic autoreceptors of A9 and A10 dopamine neurons. Accordingly, responses to iontophoretically applied R ( - ) - N P A and dopamine were examined in each cell group after E E D Q pretreatments. Unlike the results from the i.v. studies, we did not observe regional differences in response to directly applied R ( - ) - N P A or dopamine tn either vehicle or EEDQ-treated rats. In control animals, both A9 and A10 cells were inhibited by about 80% at maximal ejection currents (80-100 nA) for each agonist. This level of response is similar to that previously reported for inhibitions of VTA neurons by these agonists 32. The 15o values from R ( - ) - N P A and dopamine current-response curves also did not differ for the two areas. This lack of a regional difference in sensitivity to iontophoresed agonists is similar to the earlier report of Aghajanian and Bunney 2 in which no differences in response to iontophoretically applied dopamine or apomorphine were noted between nigral and VTA dopamine cells. Similarly, following E E D Q pretreatments, current-response curves for both A9 and A10 cells to iontophoresed R ( - ) - N P A or dopamine were shifted to the right, but in a similar and generally parallel manner. Thus, no differences emerged between the two cell groups for this electrophysiological measure of somatic autoreceptor function. Certain technical aspects of iontophoresis technique (see ref. 18) argue against the appropriateness of performing the same quantitative assessments of occupancy and response that were done for i.v. studies. For instance, application of currents exceeding 80-100 nA was difficult to achieve so that it was not possible to be certain that maximal inhibitory responses had been attained even in control animals. Thus, it was not possible to determine whether maximal attainable re-

sponses were depressed by E E D Q treatments. Furthermore, because iontophoretic 'doses' are indirect functions of applied ejection currents, calculations of K A and receptor occupancy would have given values that would be difficult to interpret. However, several important conclusions can be drawn from a general comparison of the iontophoresis data from both cell groups. First, current-response curves to R ( - ) - N P A and dopamine were shifted to the right by E E D Q treatments for both A9 and A10 cells. This finding provides further functional evidence that systemically administered E E D Q indeed inactivates nigral and VTA dopamine autoreceptors. Secondly, the failure of E E D Q treatments to reveal regional differences in response to directly applied R ( - ) - N P A or dopamine suggests that the dopamine receptor populations which mediate the inhibition of A9 and A10 cell firing by systemically administered agonists are apparently not uniform, as was first supposed. If the receptor pools for each cell group had been uniform in their influences over cell firing, the responses involving a subset of the total receptor pool should have been reflective of the pattern seen after systemic administration of the agonist, i.e. a greater loss of response would be seen for A10 than A9 cells after E E D Q pretreatments. Since treatments with the inactivator did not differentially reduce A10 cell responses to iontophoresed agonists, it can be concluded that the differences between A9 and A10 cells observed in our earlier studies with i.v. R ( - ) - N P A were probably not due to regional differences in the pool of somatic autoreceptors reached by iontophoresis. This opens the possibility that these differences were more likely due to differences in receptor density or coupling efficiency at other sites that influence dopamine cell firing rates. First among these might be the distal dendritic autoreceptors which lie beyond the sphere of iontophoretically applied drugs and, thus, would not have contributed to the inhibitory response to applied NPA or dopamine. The relative sizes and coupling efficiencies of these dendritic pools of receptors for A9 and A10 dopamine neurons are largely unknown, and these could be the basis for the different levels of receptor reserve between the two systems. Alternatively, forebrain inhibitory feedback circuits may also be a source of these differences. Long-loop inhibitory circuits are believed to contribute to some of the actions of i.v. dopamine agonists on A9 firing rates 6'7'15, a role that may not be equally important for inhibition of A10 cells z93°. The exact contributions of remote dopamine receptor pools and somatodendritic autoreceptors to the responses of midbrain dopamine cells to i.v. dopamine agonists are unclear. These roles should be better defined by studies using selective, regional inactivation of dopamine receptors. For in-

281 stance, microinjections of r e c e p t o r inactivating agents such as E E D Q into d o p a m i n e cell b o d y and terminal areas could help to identify the relative roles of d o p a m i n e receptors at these locations in mediating the responses to systemically a d m i n i s t e r e d agonists. In fact, this laboratory is presently pursuing such studies in o r d e r to m o r e conclusively address this issue. In s u m m a r y , these results reveal that d o p a m i n e cells of the A 9 system exhibit an efficient o c c u p a n c y - r e s p o n s e relationship such that low levels of r e c e p t o r occupation p r o d u c e high degrees of response. Conversely, V T A d o p a m i n e cells display a less favorable o c c u p a n c y response relationship insofar as higher levels of r e c e p t o r

occupation are r e q u i r e d for all levels of response relative to A 9 cells. These differences suggest that a greater density or coupling efficiency exists a m o n g d o p a m i n e receptors involved in regulating the activities of A 9 than for A10 d o p a m i n e neurons. T h e source of this regional variability appears not to reside at the level of the somatic autoreceptors, but m a y be due to differences at m o r e distal dendritic a u t o r e c e p t o r s or at o t h e r sites which indirectly mediate responses of d o p a m i n e cells to systemic agonists.

REFERENCES

Research, 405 (1987) 46-55. 14 Furchgott, R.E and Burstyn, P., Comparison of dissociation constants and of relative efficacies of selected agonists acting on parasympathetic receptors, Ann. N.Y. Acad. Sci., 144 (1967) 882-899. 15 Grace, A.A. and Bunney, B.S., Opposing effects of striatonigral feedback pathways on midbrain dopamine cell activity, Brain Research, 33 (1985) 271-284. 16 Hand, T.H., Kasser, R.J. and Wang, R., Effects of acute thioridazine, metoclopramide and SCH23390 on the basal activity of A9 and A10 dopamine cells, Eur. J. Pharmacol., 137 (1987) 251-255. 17 Hamblin, M.W. and Creese, I., Behavioral and radioligand binding evidence for irreversible dopamine receptor blockade by N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline, Life Sci., 32 (1983) 2247-2255. 18 Hicks, T.P., The history and development of microiontophoresis in experimental neurobiology, Prog. Neurobiol., 22 (1984) 185-240. 19 Kenakin, T.E, Challenges for receptor theory as a tool for drug and drug receptor classification, Trends Pharmacol. Sci., 10 (1989) 18-22. 20 Koob, G.E and Bloom, EE., Cellular and molecular mechanisms of drug dependence, Science, 242 (1988) 715-723. 21 Meller, E., Bohmaker, K., Namba, Y., Friedhoff, A.J. and Goldstein, M., Relationship between receptor occupancy and response at striatal dopamine autoreceptors, Mol. Pharmacol., 31 (1987) 592-598. 22 Meller, E., Helmer-Matyjek, E., Bohmaker, K., Adler, C.H., Friedhoff, A.J. and Goldstein, M., Receptor reserve at striatal dopamine autoreceptors: implications for selectivity of dopamine agonists, Eur. J. Pharmacol., 123 (1986) 311-314. 23 Oades, R.D. and Halliday, G.M., Ventral tegmental (A10) system: neurobiology. 1. Anatomy and connectivity, Brain Res. Rev., 12 (1987) 117-165. 24 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, Orlando, FL, 1986. 25 Ruffolo, R.R., Important concepts of receptor theory, J. Autonom. Pharmacol., 2 (1982) 277-295. 26 Seeman, P., Dopamine receptors and the dopamine hypothesis of schizophrenia, Synapse, 1 (1987) 133-152. 27 Swanson, L.W., The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat, Brain Res. Bull., 9 (1982) 321-353. 28 Wang, R.Y., Dopaminergic neurons in the rat ventral tegmental area. I. Indentification and characterization, Brain Res. Rev., 3 (1981) 123-140. 29 Wang, R.Y., Dopaminergic neurons in the rat ventral tegmental area. II. Evidence for autoregulation, Brain Res. Rev., 3 (1981) 141-151.

1 Adler, C.H., MeUer, E. and Goldstein, M., Receptor reserve at the alpha-2 adrenergic receptor in the rat cerebral cortex, J. Pharmacol. Exp. Ther., 240 (1986) 508-515. 2 Aghajanian, G.K. and Bunny, B.S., Dopamine 'autoreceptors': pharmacological characterization by microiontophoretic single cell recording studies, Naunyn-Schmiedeberg's Arch. Pharmacol., 297 (1977) 1-7. 3 Bannon, M.J., Wolf, M.E. and Roth, R.H., Pharmacology of dopamine neurons innervating the prefrontal, cingulate and piriform cortices, Eur. J. Pharmacol., 91 (1983) 119-125. 4 Beckstead, R.M., Domesick, V.B. and Nauta, W.J.H., Efferent connections of the substantia nigra and ventral tegmental area in the rat, Brain Research, 175 (1979) 191-217. 5 Bergstrom, D.A., Carlson, J.H., Pan, H.S. and Waiters, J.R., Dopamine autoreceptor reserve on substantia nigra dopamine neurons can mask partial agonist properties of putative autoreceptor agonists, Soc. Neurosci. Abstr., 13 (1987) 490. 6 Bunney, B.S. and Aghajanian, G.K., D-Amphetamine-induced inhibition of central dopaminergic neurons: mediation by a striatonigral feedback pathway, Science, 192 (1976) 391-393. 7 Bunney, B.S. and Aghajanian, G.K., D-Amphetamine-induced depression of central dopamine neurons: evidence for mediation by both autoreceptors and a striatonigral feedback pathway, Naunyn-Schmiedeberg's Arch. PharmacoL, 304 (1978) 255-266. 8 Chiodo, L.A., Bannon, M.J., Grace, A.A., Roth, R.H. and Bunney, B.S., Evidence for the absence of impulse regulating somatodendritic and synthesis modulating autoreeeptors on subpopulations of mesocortical dopamine neurons, Neuroscience, 12 (1984) 1-16. 9 Cox, R.E and Waszczak, B.L., Differences in dopamine receptor reserve for N-n-propylnorapomorphine enantiomers: single unit recording studies after partial inactivation of receptors by EEDQ, Mol. Pharmacol., 34 (1989) 125-131. 10 Cox, R.E, Neumeyer, J.L. and Waszczak, B.L., Effects of N-n-propylnorapomorphine enantiomers on single unit activity of substantia nigra pars compaeta and ventral tegmental area dopamine neurons, J. Pharmacol. Exp. Ther., 247 (1988) 355-362. 11 Dahlstrtm, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the central nervous system, Acta Physiol. Scand., Suppl., 62 (1964) 232. 12 DeLean, A., Munson, P. and Rodbard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay and physiological dose-response curves, Am. J. Physiol., 235 (1978) E97-E102. 13 Freeman, A.S. and Bunney, B.S., Activity of A9 and A10 dopaminergic neurons in unrestrained rats: further characterization and effects of apomorphine and cholecystokinin, Brain

Acknowledgements. The authors gratefully acknowledge the skillful secretarial assistance of Nancy Weston. This work was supported by NIH Grant NS23541.

282 30 Wang, R.Y., Dopaminergic neurons in the rat ventral tegmental area. III. Effects of D- and L-amphetamine, Brain Res. Rev., 3 (1981) 153-165. 31 Waszczak, B.L., Eng, N. and Waiters, J.R., Effects of muscimol and picrotoxin on single unit activity of substantia nigra neurons, Brain Research, 188 (1980) 185-197. 32 White, EJ. and Wang, R.Y., Pharmacological characterization

of dopamine autoreceptors in the rat ventral tegmental area: microiontophoretic studies, J. Pharmacol. Exp. Ther., 231 (1984) 275-280. 33 Yokoo, H., Goldstein, M. and Meller, E., Receptor reserve at striatal dopamine receptors modulating the release of [3H]dopamine, Eur. J. Pharrnacol., 155 (1988) 323-327.