Apomorphine and electrical self-stimulation of rat brain

Behavioural Brain Research, 52 (1992) 73-80 9 1992 Elsevier Science Publishers B.V. All rights reserved. 0166-4328/92/$05.00

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Apomorphine and electrical self-stimulation of rat brain George Fouriezos and Stewart Francis School of Psychology, Unilersity of Ottawa, Ottawa, Ont. (Canada) (Received I 1 February 1992) (Revised version received 13 August 1992) (Accepted 24 August 1992)

Key uvrds: Apomorphine; Brain stimulation reward; Dopamine; Lateral hypothalanms; Reward; Self-stimulation

The participation of dopamine neurons in reward produced by electrical stimulation of the brain was examined by measuring self-stimulation thresholds after injections of apomorphinc, a direct agonist of dopamine receptors. Rats were trained to press a lever to obtain 0.3-s trains of electrical stimulation applied to lateral hypothalamic electrodes in a paradigm where the pulse frequency was decreased every eight stimulations by approximately 20~0. The pulse frequency interpolated at 50~0 of maximum rate was taken as threshold. In a completely within-subject design, five doses of apomorphine from 0.01 to 1.00 mg/kg and the ascorbic acid vehicle were injected in a random order and thresholds were tracked at intervals of 5 min for 2 h postinjection. Low doses from 0.01 to 0.10 mg/kg caused thresholds to increase while the two higher doses, 0.30 and 1.00 mg/kg, caused thresholds to drop; the switch in the direction of the behavioural effect is thought to parallel the shift in apomorphine's action from presynaptic to predominantly postsynaptic activation of dopamine receptors as the concentration of apomorphine increases.

INTRODUCTION

The idea that dopamine neurons help mediate the reward produced by electrical stimulation of fibers of the medial forebrain bundle has found its strongest support from pharmacological studies. Generally, drugs that interfere with dopamine transmission decrease performance or increase thresholds in self-stimulation tests, whereas drugs that facilitate dopamine transmission augment performance or decrease self-stimulation thresholds. Whereas the results obtained with receptor antagonism and with indirect agonists like amphetamine or cocaine have consistently supported a role for dopamine, the direct agonist apomorphine has resisted fitting into the scheme, partly because of its apparent inconsistency. Figs. 1 and 2 show the wide range of results reported from experiments on the effects of apomorphine on self-stimulation. Fig. 1 summarizes experiments that tested sy.stemic injections of apomorphine on self-stimulation of lateral hypothalamic or ventral tegmental placements, here grouped together as medial 9forebrain bundle sites, while Fig. 2 shows similar data for other self-stimulation loci. The vertical lines in the bodies of the figures indicate doses administered withCorrespondence:G. Fouriezos, Universityof Ottawa, 108-275 Nicholas Street, Ottawa, Ont. Canada KIN 6N5.

out effect. Lines with arrowheads show the directions of effect when significant changes were reported; in each case the arrowhead reflects the direction in inferred reward according to the usual interpretations of the different measures. For example, an up-arrowhead corresponds to an increase in rate or in total duration of stimulation consumed but to a decrease in threshold or in latency to initiate stimulation. It is difficult to discern a consistent pattern of effect across dose, even if one restricts their inspection to those measures he or she trusts. The method used to evaluate the effects of apomorphine on electrical self-stimulation is critical because there are too many a priori issues about apomorphine and the evaluation of self-stimulation that have not been pegged down. At any single dose, for example, we would not know whether it is apomorphine's presynaptic activation, postsynaptic excitation, or a mixture of both effects 19 that is responsible for the behavioural outcome, making single-dose experiments difficult to interpret. Even if one were to test a relatively large dose - - say, a dose that would produce stereotypy on its own so that there would be some assurance that its effect is postsynaptic - - then apomorphine's subjective interaction with the reward derived from electrical stimulation becomes an important factor when a performance measure (e.g. rate, duration, latency) is used. Apo-

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Fig. 1. Apomorphine on medial forebrain bundle self-stimulation as a function ofdose. Symbols: Vertical Line, dose administered had no effect on self-stimulation; Up Arrow, self-stimulation was enhanced; Down Arrow, self-stimulation was inhibited; Double Arrow, significant changes up and down, direction usually peculiar to subject. Measures (MEAS.): THR, threshold, current (L83, SRK82) or charge (WN73) threshold estimated from graphs; RATE, number of responses per unit time; DUR., duration of stimulation consumed where rats tracked stimulation by moving to the randomly switched ON side; LAT., latency to initiate continuous stimulation in an ONOFF design. All directions of effect are shown as changes to inferred reward, so a Down Arrow corresponds to an decrease in rate or duration but to an increase in threshold or latency. Citations (REF.) are listed as first initials of authors' last names followed by the year of publication. Notes: &PAG, results from medial forebrain bundle electrodes were combined with Periaqueductal Gray placements because no site difference was detected; DA, a subset of lateral hypothalamic placements characterized by little or no difference in response to 0.5 mg/kg D- vs. L-amphetamine; NE, lateral hypothalamic sites that showed a large D- vs. L-amphetamine difference; D, dorsal lateral hypothalamic sites; V, ventral sites; C57BL/6, BALB/e, and DBA/2 are mouse strains.

morphine is self-administered by rats 2, so at some high dose it must produce a reward of its own. Herberg et al. have pointed out that direct pharmacological agonism of circuitry involved in self-stimulation should act to degrade the contingency between the lever-press (that triggers an electrical stimulation) and its central effect1 ~; whereas a re-uptake blocker like cocaine is guaranteed to enhance the impact of incoming signals derived from the electrical stimulation, the chronic, postsynaptic mimicry by apomorphine occurs without regard to input. If apomorphine does excite reward circuitry on its own to the point where it mimics the subjective effect of brain stimulation, then the effect of apomorphine on rate of response or any other performance measure cannot be predicted. If the animal attributes the chronic reward to its own action of pressing the lever, then perhaps its rate of response might rise; on the other hand, if brain stimulation produced by pressing the lever does not significantly augment the chronically elevated activity in the reward system, then rate of re-

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Fig. 2. Apomorphine on self-stimulation obtained from sites other than the medial forebrain bundle as a function of dose. Symbols: as in Fig. 1 with these additions: ?, dose was administered but omitted from the Results and graphs. SITEs: SN.R, substantia nigra zona reticulata; A9, substantia nigra zona compacta; A 10, ventral tegmental area medial to A9; SN, substantia nigra; CN, caudate nucleus; NAcb, nucleus accumbens; MPFC, medial prefrontal cortex; SPFC, sulcal prefrontal cortex; OFC, orbitofrontal cortex; PAG, periaqueductal gray; LC, locus coeruleus. Measures (},lEAS.) and citations (REF.) as in Fig. 1. Notes: R.M., Rhesus monkey; &MFB, results for PAG combined with MFB data; DBA/2 and BALB/c are mouse strains.

sponse might drop off, especially in cases where low response rates give the animal time away from the lever. Instead of using a performance measure the effects of apomorphine should be evaluated by estimating selfstimulation threshold, but even with threshold estimation it is uncertain how the animals will behave once the close of apomorphine produces its own reward. It is important, therefore, to test a range of doses so that some are just great enough to have a dominant postsynaptic effect, yet low enough to avoid the disruption in behaviour caused by the very high doses of apomorphine. In this experiment we examined the effects of apomorphine on self-stimulation over a range of doses that spanned two orders of magnitude. Using a withinsubject design, thresholds were estimated every 5 min for 2 h postinjection. Low doses of apomorphine inhibited self-stimulation while high doses facilitated it, so we concluded that presynaptic inhibition of dopamine neurons interferes with the electrical self-stimulation while the postsynaptic activation of their targets enhances the rewarding value of electrical stimulation.

MATERIALS AND METHODS

Rats, surgery, and drugs Four male Long-Evans rats were individually housed in clear plastic 'shoe-box' cages with unrestricted access to Purina Rat Chow and water in a

75 temperature and humidity controlled room with 12 h of light daily beginning at 07.00 h. The rats weighed between 400 and 500 g at surgery. They were anaesthetized with an intraperitoneal (i.p.) injection of 60 mg/kg of Na pentobarbital and were subcutaneously administered 0.08 mg of atropine sulphate. To reduce the discomfort at the points of contact with the stereotaxic apparatus, a 2 ~ solution of xylocaine was applied topically to the external auditory canals and to the upper palate just posterior to the incisors. The rats were stereotaxically implanted with single 0.25-mm-diam., stainless-steel electrodes which were insulated with Formvar to their fiat tips. With the incisor bar set 5 mm above the interaural line 17, the coordinates used to guide the monopolar electrodes to the lateral hypothalamus were 0.5 mm posterior to b r e g m a , 1.7 mm left of the midsagittal suture, and 8.0 mm below dura. A wire wrapped around each of four skull screws was used as the current return. Apomorphine HCI (Sigma Chemical Co.) was dissolved in a vehicle of 0.5 mg/ml ascorbic acid in saline at concentrations of 0.01, 0.03, 0.1, 0.3, and 1.0 mg/ml. The solutions were prepared between 1 and 2 hours prior to administration.

Apparatus, protocol, and threshoM esthnation The behavioural testing was conducted in two identical wooden test chambers measuring 36 • 30 • 38 cm. Except for the front wall, which was made of clear Plexiglas, the chambers were painted white. A Lehigh Valley rodent lever was mounted on the right wall 4 cm above the 13 mm grid floor. A second, but inactive, lever was mounted on the left wall. S elf-stimulation was conducted using equipment that was specially designed for the rapid collection of response-rate vs. pulse-frequency data, from which estimates of the stimulation strength needed to sustain criterion performance could be calculated. The apparatus issued a tone at the beginning of l-min cycles, indicating that the stimulation strength had been reset to its highest value, and it rewarded rats with 0.3-s trains of rectangular, cathodal pulses of 100/~s duration when the active response lever was pressed, but the strength of the stimulation was quickly swept from low pulse periods (i.e. interpulse intervals or reciprocols of pulse frequency) that supported vigorous responding to high periods that supported little or no responding at all. The period was incremented to the next (weaker) value after every eight self-administered trains thus: the period of the first set was 10.0 ms, the next period was 12.8 ms, then 16.0, 20.0, 25.2 ms, etc. The sequence followed approximately 0.I log.increments, or 2 0 ~ drops in strength, through to a total of 15 available

steps. By carefully adjusting the test current, which was held constant after the training sessions, it was possible to get the rats to self-stimulate for about 20-30 s; this usually corresponded to traversing 4 or 5 steps in the sequence before they would quit responding, presumably because of having arrived at a non-rewarding level of stimulation. The rats themselves could not reset the stimulation to its strongest value; the stimulation was reset by a timer at 60-s intervals, and as mentioned above, the reset was signalled by the sounding of a tone. The equipment automatically accumulated the number of lever presses made and the time spent in obtaining each cluster of eight, same-strength stimulations. These data could be read out manually without disturbing the testing. They were converted to rates of response and used to interpolate pulse periods that corresponded to half-maximum rates of responding. In this paper we will refer to the periods that sustain half maximum rates as thresholds. A description of this protocol which focusses on the methodology and which contrasts this method to the 'autotitration' technique zl has been published elsewhere9.

Procedttre Trainhlg and stability. The rats were given a minimum of 4 days to recover from surgery before training began. They were connected, placed into the test chamber, and given a few minutes to explore the apparatus. With the stimulation parameters adjusted to a combination of values that was expected to support self-stimulation, the rats were shaped to bar-press by manually reinforcing successive approximations to the lever-press response. The training took place using the test protocol, that is, with the programmed progression through weaker rewards activated. Accordingly, shaping was restricted to times soon after the tone-signalled reset to the start of the sequence. Early in training, the rats were manually primed with a train or two of stimulation about 1-3 s after the reset but, as training progressed, primes and reinforcement for incomplete lever presses were omitted. The rats were considered to be trained when they reliably and rapidly returned to self-stimulate after resets with no prompting other than the programmed tone. The rats were then given 30-min sessions daily and they were deemed to be stable when period thresholds fluctuated within a + 10% corridor about the mean over 3 consecutive days. Test sessions. A total of six test sessions were held for each rat to evaluate the effects of i.p. injections of the ascorbic acid vehicle and of these 5 doses of apomorphine: 0.01, 0.03, 0.1, 0.3, and 1.0mg/kg. All doses including the vehicle were administered to each rat following pseudorandomly generated sequences and a

76 minimum of 4 days separated the tests. The test sessions lasted 150 rain, comprising 30 threshold estimates taken 5 min apart. From the rats' perspective, the sessions comprised 150 1-min cycles through the descending sequence of pulse frequency. From the vantage of data acquisition, however, each block of 5 cycles was split into two portions; data were accumulated over two cycles (2min) and read out during the next 3 cycles (3 min). Although no data could be accumulated during readout, switching from acquisition to readout interfered in no way with the self-stimulation paradigm. The 5 thresholds collected in the first 25 min defined baseline performance. During the readout period following the last of the baseline thresholds, the experimenter waited until the rat had quit lever-pressing and then picked up the animal to inject it. As far as we could tell, the rats always reacted normally to the next tone signalling the beginning of another pass through the sequence of rewards. Postinjection performance was then monitored for 25 more threshold estimates (125 min).

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RESULTS

Fig. 3 depicts the raw data collected 20 min after each injection for all four rats. Each panel contains plots of rate of bar-pressing against the pulse period on the logarithmic abscissa for one rat. Within each panel, the six curves are labeled with the dose of apomorphine used or with 'AA' which designates the ascorbic acid vehicle. Generally, asymptotically high rates of response were observed in the first few pulse periods. Note that the rates collected in the first bin, when the rats were offered a period of 10.0 ms, have been omitted from the figure; rates during the first stimulus were usually lower than those for subsequent stimuli, possibly because of a decay in the priming effect1~ that would occur during the half rain or so since the last response. The relatively high rates of response, rates in the vicinity of 2 responses per second, are sustained over three or four pulse periods before dropping to operant levels. It is apparent from Fig. 3 that the higher doses of apomorphine 20 min postinjection tended to shift to the right the point at which response rates fell to low levels. In con-

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Fig. 3. Raw data collected 20 min postinjection for each of the four rats (separate panels) at each of the six doses of apomorphine (individual curves). Before injection we obtained five similar sets of data to serve as baseline; the set shown here taken 20 min postinjection is one of 25 postinjection sets taken from t = 0 to t = 120 min at 5-min intervals. Each point is the response rate in presses per second in obtaining stimulation of progressively decreasing pulse frequency (shown here against an abscissa of increasing pulse period). The characters AA designate the ascorbic acid vehicle while each dose is indicated beside its curve. Generally, the lower doses of apomorpbine caused rats to quit earlier, at relatively higher strengths of stimulation, while greater doses often caused rats to quit at weaker stimuli.

77 trast, the very low doses seemed to shift the curves to the left; rats quit responding earlier in the sequence of gradually weaker stimuli. To analyze the effects of apomorphine on threshold, each rate-period curve was cut at a criterion of half of its maximum rate and the interpolated abscissa was taken as the period threshold. These period thresholds were normalized against the mean of the five preinjection thresholds, averaged across rats, and plotted in Fig. 4. The thresholds derived from Fig. 3 now appear as the fourth point postinjection (to the right of the arrow) in each curve of Fig. 4. Each symbol represents the mean and S.E.M. for the four rats. Some of the thresholds following the highest dose, 1.0 mg/kg, could not be calculated because the animals sometimes continued responding without stopping, earning progressively weaker trains of stimulation until the programmed reset to the initial stimulus. In these instances of persistent responding, thresholds could not be estimated if none of the rates fell below the halfmaximum criterion. The second postinjection point is therefore based on three animals instead of all four while the third through seventh points are based on only two rats. Because of the large number of missing values in the 1.0 mg/kg dose (11/100), the data from this dose were left out of the statistical treatment. The thresholds collected after injections of vehicle and all doses except the highest were assessed using a two factor (Dose by Time) analysis of variance with Subjects crossed with Dose and Time. The analysis revealed a significant Dose by Time interaction (F96,288 = 2.03, P < 0.01) indicating that, overall, injections of apomorphine caused departures from baseline at different times in the sessions. To help us to decide where the departures from baseline occurred, a linear regression of the postinjection, vehicle means was calculated and the resulting regression line, along with lines representing two standard errors of estimate above and below it, was drawn in Fig. 4 over the postinjection data at each dose. Period thresholds were stable, tending to 100~o even 2 h following control injections of the ascorbic acid vehicle. The three lowest doses of apomorphine reduced period thresholds (that is, they increased frequency thresholds) to approximately 80~o of vehicle levels. Thresholds returned to baseline 25 min after injections of the 0.01 mg/kg dose, appeared to return.to baseline about 1 h after the 0.03 mg/kg dose, and they seemed to remain below control levels for the duration of the session after 0.1 mg/kg. The two highest doses, 0.3 and 1.0 mg/kg, resulted in elevated period thresholds for approximately 1/2 and 1 hour, respectively, after which they returned to levels just below baseline for the remainder of the tests.

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TIME POST INJECTION (Min) Fig. 4. Time- and dose-response data on self-stimulation thresholds. The data of Fig. 3 along with 29 similar sets of data were converted to period thresholds, expressed as percentages of the mean of the five preinjection estimates, and plotted here as functions of time (abscissa) and dose (different curves). The calculation thus expresses an inhibition of the rewarding value of the stimulation as a downward excursion. Each point represents the ratio of drug-topredrug period threshold averaged across the four rats along with error bars showing one standard error wherever it would have exceeded the radius of the symbol. Note that the five preinjection thresholds are included in the graph; like the rest of the data, each of these was divided by the average of the five preinjection thresholds. In the graph for the ascorbic acid control, a horizontal line was drawn at the 100~ (no effect) level. In the other graphs, there are three lines drawn over the data postinjection to aid comparison to control thresholds: The center line represents the regression equation for postvehicle thresholds; it is bounded by lines two standard errors above and below it. The three lowest doses produced shallow, but progressively longer, decreases in the rewarding value of the brain stimulation while the two highest doses potentiated the reward.

The variability of the data at the two high doses is pronounced. At 0.30 mg/kg, three of the rats showed substantial increases in period threshold in the second through sixth postinjection measures whereas the fourth rat's threshold dropped below control levels through-

78 out, much as it had done at the lower doses. At the highest dose, Points 3 through 7 postinjection were based on only two rats because thresholds could not be estimated for the other two, so the error estimates are somewhat inflated; where thresholds were obtained for the other two (beginning with the 8th postinjection measure), they too demonstrated elevated thresholds. By then, at 40 min postinjection, two rats had returned to baseline while the other two maintained elevated thresholds until about 1 h postinjection. The higherthan-normal thresholds were, therefore, invariant consequences of high doses of apomorphine, despite their appearance at doses or at times that were out of phase across the four rats; individual peaks in threshold ranged from a factor of 2.9 to 3.8 above baseline.

DISCUSSION

The lowest dose of apomorphine 0.01 mg/kg, produced a small reduction in the rewarding value of brain stimulation that lasted approximately 20 rain. Progressively higher doses through to 0.10 mg/kg prolonged, but did not appear to deepen, the inhibition, whereas the two highest doses, 0.30 and 1.0 mg/kg, resulted in the opposite, a facilitation of the stimulation's reinforcement. The switch in the direction of the behavioural effect is in accord with the high affinity for apomorphine of presynaptic receptors compared to the lower affinity demonstrated by postsynaptic receptorslg; we think the inhibition of reward at the low doses of apomorphine was caused by the activation of presynaptic autoreceptors, which would decrease firing rates in dopamine neurons, while the high-dose facilitation of self-stimulation reflected the direct agonism of postsynaptic receptors. The autoreceptor mediated inhibition of dopamine neurons would continue to occur at high concentrations of apomorphine but, because the activation of postsynaptic receptors is functionally downstream of the presynaptic inhibition, the facilitation would take precedence at the higher concentrations of apomorphine. That the shift from inhibition to facilitation of reward as dose increases does correspond to the p r e - t o postsynaptic shift is, in some measure, quantitatively supported by relative doses over which the two directions of effect occur; Skirboll et al. 19 have shown apomorphine's presynaptic inhibition of firing rate to grow from 10 to 90~0 with cumulative intravenous doses from 0.5 to 25 ls while the postsynaptic rise occurs between 10 and 256/~g/kg. The presynaptic and postsynaptic effects on neuron firing rate are thus separated by about one common log unit of apomorpbine dose, which is the about the same separation in

dose between inhibition and facilitation of selfstimulation seen here. Perhaps our results go some o f the way towards explaining why apomorphine's effects on reward have been poorly understood. The inhibitory effect on reward obtained at the lower doses shows up over a wide range; it is there from 0.01 to 0.1 mg/kg as the sole effect and it appears to just lead and to trail after the facilitation seen at the greater doses, so, to some degree, there may be a time dependence to the direction of effect at the greater doses. As robust as the inhibition is across dose, this presynaptic effect is small, producing a reduction of the rewarding effect of stimulation to 80~o of control at its deepest. Many of the studies summarized in Fig. 1 used performance measures like rate of response or duration of stimulation accepted by the rat, so mapping the correspondence of a 20?/0 reduction in the potency of the stimulation in our data to effects on performance measures is chancy. A small drop in stimulus strength may cause anything from a modest increase in performance to its complete obliteration depending upon where in the performance vs. stimulus-strength function the measure is taken. Nevertheless, in many of the test protocols used, the investigators carefully selected stimuli that supported moderate responding, at levels where performance functions were thought to be sensitive to changes in stimulus strength. Many of the reported effects of apomorphine are decreases in performance, and this outcome appears to dominate the left half of Fig. 1, that is, at doses less than 0.2 mg/kg. In contrast to the presynaptic, the postsynaptic effect of apomorphine in our study was large in magnitude, with period thresholds boosted by peak factors of 2 or 3, but reliable measurement of the facilitation was possible only within a narrow dose range before the behavioural disruption of stimulant stereotypy intruded. The right half of Fig. 1 (0.2 mg/kg and greater) does contain more instances of increased behavioural avidity than does the left half, but they are nested in a field of observed decreases in performance. As explained earlier in the Introduction, if apomorphine were administered at doses great enough to produce its own reward, then one might predict decreases in performance as readily as increases, so the mixture of both effects seen when performance measures were taken, we think, says more about the intricacies of scaling techniques than about apomorphine's functional effect. It is possible that by measuring threshold, we were able to widen the postsynaptic dose range over which reliable data could be collected because the strength of the electrical stimulation was traded against the drug effect; if the disruptive effects on behaviour of stimulant stereotypy are augmented by electrical stimulation of

79 the medial forebrain bundle, then threshold estimation would minimize the disruption because as the drug effect grows, the electrical stimulation is reduced. The exchange of one stimulus for another, electrical for chemical, inherent to psychophysical scaling itself may account for the clarity of our results. In all of the studies listed in Figs. 1 and 2, the effects of apomorphine were evaluated by changes rate of responding or similar measures of performance, but three of these studies allow a more direct comparison to the present results because performance data were collected over a range of stimulation current or pulse frequency. Leith's ~3 rate-current curves were displaced horizontally towards greater currents at doses of 0.02, 0.1, and 0.2 mg/kg of apomorphine, results which agree with ours over the same range of doses. Wauquier and Niemegeers 24 reported the effects on response rate of doses from 0.08 progressively doubled through to 1.25 mg/kg. The pulse frequency and current were both varied in the testing, but the stimulation followed an orderly progression of total charge, so, to examine the effects on threshold charge, we replotted their ratedose curves at six stimulus combinations as rate-charge functions at five different doses. The greatest effects were 6-7~o facilitations that just attained statistical reliability (using two-tailed, 95~o confidence limits) at a dose of 0.32 mg/kg, but just failed to attain significance at 0.63 mg/kg. The next dose, 1.25 mg/kg, could not be analysed because response rates were erratic or too low. None of their doses increased charge thresholds. Some of the discrepancy between our finding and theirs may be due to the different times at which the data were collected; their rates were accumulated during 1-h periods beginning 1/2 h postinjection whereas o u r thresholds were obtained every 5 min beginning 25 min before injection. If one were to average thresholds from 35 to 90 min postinjection in Fig. 4, then the bulk of the facilitation seen after the two highest doses would be washed away, so we think the tests of Wauquier and Niemegeers 24 began too late and lasted too long to capture apomorphine's effects on reward. Strecker et al. 23 found no effect of a subcutaneous dose of 0.1 mg/kg on current thresholds for lateral hypothalamic self-stimulation in normal rats, but rats lesioned with 6-hydroxydopamine infusions to nucleus accumbens shifted their rate-current functions to the left without altering maximum rates of response. (Because Fig. 1 summarizes data only from intact animals, the data from lesioned rats were not included in the figure.) The lack of effect on controls and the presence of one on lesioned rats might be explained thus: the subcutaneous route of administration initially bypasses liver catabolism, so their subcutaneously administered dose

of 0.1 mg/kg would correspond to greater doses administered by us intraperitoneally; perhaps it would correspond to doses in our study where inhibitory and facilitative effects of apomorphine are equally likely. As a group, therefore, intact rats injected with 0.1 mg/kg subcutaneously may have shown no consistent facilitation or inhibition; their average rate-current results would be the same as controls. In 6-hydroxydopaminelesioned rats tested 2-3 weeks postlesion, however, denervation supersensitivity would potentiate the apomorphine effect, making it potent enough to consistently produce a facilitation. If the explanation is correct, then the results of Strecker et al. 23 are entirely consistent with our own. Our findings provide strong support for a role for dopamine neurons in reward, but parametric studies in self-stimulation suggest that dopamine neurons are not directly activated by electrical pulses. The self-stimulation neurons that are directly excited recover from refractoriness too quickly 25, conduct action potentials too quickly 3, and conduct them in the wrong direction 4 for the directly activated neurons to be considered members of the ascending dopamine systems. If it is the ascending dopamine fibers arising from the ventral tegemental area or substantia nigra that mediate the pharmacological effects of apomorphine, then these dopamine cells must be acting either as modulators to the descending, directly activated, substrate or as second (or third, etc.) links in a chain of reward neurons 4. The results of this study do not help us to decide whether the participation of dopamine neurons is modulatory or in series to the main self-stimulation pathway; because psychophysical scaling estimates the stimulation needed to just support consistent responding, both the modulatory and series models would predict the postsynaptic effects of a direct agonist to be facilitative. Nevertheless, the dose-dependence and time-dependence of apomorphine's effect on self-stimulation thresholds do resolve the confusion over how apomorphine influences self-stimulation, and make it clear that dopamine neurons play a central role in reward. ACKNOWLEDGEM ENTS

The support of the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. REFERENCES I Atrcns, D.M., Beckcr, F.T. and Hunt, G.E., Apomorphine: selective inhibition of lateral hypothalamic self-stimulation, Psychopharmacology, 71 (1980) 97-99.

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