Self-stimulation and amphetamine: Tolerance to d and l isomers and cross tolerance to cocaine and methylphenidate

The effects of the d and l isomers of amphetamine on self-stimulation responding were tested following acute and chronic administration. Tolerance and post-drug depression of responding occurred in tests with both isomers, indicating no role for p-hydroxynorephedrine (PHN) which is one of the metabolites of d-amphetamine. In the second experiment, d-amphetamine, methylphenidate and cocaine all produced quantitatively and qualitatively similar effects on self-stimulation responding following acute administration. Following chronic administration of d-amphetamine, animals showed tolerance to all three drugs, indicating cross-tolerance among them. These data are consistent with an hypothesis that tolerance and post-drug depression following chronic amphetamine treatment are the result of decreases in postsynaptic receptor sensitivity, which would lead to a decreased effectiveness of all three drugs, regardless of their pre-synaptic mechanisms.     

Effects of d-amphetamine,scopolamine, chlordiazepoxide and diphenylhydantoin on self-stimulation behavior and brain acetylcholine

The effects of d-amphetamine (0.25–8), scopolamine (0.25–8), chlordiazepoxide (2.5–40), and diphenylhydantoin (25–75), given i.p. or s.c. on a mg/kg basis, were studied on self-stimulation behavior in the male albino rat. The dose-effect relationships, the role of baseline rates of responding and their effects on brain acetylcholine (ACh) were determined in rats trained to self-stimulate for electrical reward in the lateral posterior hypothalamus. The effects of d-amphetamine were both dose and baseline-rate dependent. Low-moderate doses (0.5–2.0 mg/kg inclusive) facilitated self-stimulation and larger doses (2.0 to 8.0 mg/kg) depressed responding. Baseline rates before d-amphetamine administration were extremely important in the effect observed. Low rates of responding were facilitated and high rates were depressed by this agent. The effects of scopolamine in a wide range of dosage were less consistent. A small dose (0.5 mg/kg) facilitated only transiently self-stimulation and larger doses (1–8 mg/kg) tended to depress this behavior. Baseline rate effects were less important but high-rate responders were usually depressed by scopolamine.The effects of chlordiazepoxide were dose-dependent. A dose of (5 mg/kg) caused facilitation but larger doses (10–40 mg/kg) produced depression of selfstimulation irrespective of baseline rates. However, high-rate stimulators showed the most dramatic increases with 5 mg/kg of chlordiazepoxide. In contrast, diphenylhydantoin (25–75 mg/kg) usually depressed self-stimulation. Low rate self-stimulators showed the most marked depressant effects.Brain ACh was progressively reduced by handling of naive animals, injection of saline, and 1/2 h of self-stimulation and escape behavior. Animals not allowed to self-stimulate but given d-amphetamine (2.0 mg/kg), scopolamine (2.0 mg/kg) showed a significant decrease in brain ACh. Self-stimulation, in addition to medication with the various drugs, showed a trend for further reduction in brain ACh but the differences were not statistically significant.Supported in part by Grant MH-11846 (to EID) and MH-11627 (to JO), U.S. Public Health Service.     

Effects of catecholamine-depleting drugs and amphetamine on self-stimulation of brain following various 6-hydroxydopamine treatments

Changes in electrical self-stimulation responding were examined in rats with electrodes implanted in the lateral hypothalamus following 6-hydroxydopamine treatments which depleted brain dopamine, norepinephrine or both of these catecholamines. Acute depression of self-stimulation occurred after treatments which reduced brain dopamine, but did not occur in rats treated to deplete just brain norepinephrine. A chronic deficit in self-stimulation responding occurred in rats treated with 6-hydroxydopamine in combination with pargyline to reduce both brain amines, while responding of animals in which brain dopamine was reduced returned to levels observed prior to 6-hydroxydopamine treatment. A dose of -methyl-tyrosine (25 mg/kg), which did not affect responding of control rats, caused a significant reduction in responding of rats depleted of brain dopamine. This treatment did not affect responding of rats depleted of brain norepinephrine. Administration of the dopamine--hydroxylase inhibitor, U-14624, failed to affect self-stimulation in spite of an additional 70% reduction of brain norepinephrine content. The response to a dose of d-amphetamine (0.25 mg/kg), that increased self-stimulation of control rats, was significantly reduced in rats with brain dopamine selectively depleted. Rats in which norepinephrine was depleted responded to d-amphetamine like the control group. -Methyltyrosine antagonized the increased self-stimulation responding following administration of d-amphetamine (1 mg/kg) to reserpinized rats, while U-14624 did not. Results support the hypothesis that central dopaminergic fibers have an important involvement in the maintenance of self-stimulation of brain.     

Comparison of the effects of morphine,pentazocine, cyclazocine and amphetamine on intracranial self-stimulation in the rat

Rats were trained to press a lever in order to stimulate their lateral hypothalamus through a chronically implanted electrode. Dose-response curves were determined for the effects of morphine (0.3–10 mg/kg), pentazocine (1.0–30 mg/kg), cyclazocine (0.03–3.0 mg/kg) and d-amphetamine (0.1–3.0 mg/kg) on responding for intracranial stimulation, and then were redetermined in the presence of one or two doses of naloxone. The three analgesics produced only dose-related decreases in responding with the following relative potencies: cyclazocine>morphine>pentazocine. The well-documented rate-increasing effects of d-amphetamine on intracranial self-stimulation were observed at 0.3 and 1.0 mg/kg of the drug; decreases in responding at 3.0 mg/kg were associated with stereotyped behavior. Naloxone, which had no effect of its own on self-stimulation, increased the dose of the analgesics required to depress response rate in a manner consistent with a competitive antagonism. In contrast, response rates were reduced at all doses of d-amphetamine tested in the presence of naloxone. Thus, the interaction between naloxone and d-amphetamine is qualitatively different from the one between naloxone and the analgesics. This finding extends to intracranial self-stimulation the generality of a previous report of interactions between d-amphetamine and naloxone on behavior in the rat.Publication No. 1303 of the Division of Basic Health Sciences of Emory University. This investigation was supported by USPHS Grant DA-00541.Recipient of Research Scientist Development Award K02-DA00008.     

Shifting of the d-amphetamine dose-response curve in rats with frontal cortical ablations

Rats trained to bar-press on a FI 15 sec schedule for water reinforcement were administered various doses of d-amphetamine (0.25–4.0 mg/kg) both before and 6–8 weeks after bilateral ablation of frontal cortex. Preoperatively, low doses (e.g. 0.25–0.5 mg/kg) of (d-amphetamine increased responding and high doses (e.g. 2.0–4.0 mg/kg) of d-amphetamine depressed responding. Postoperatively, frontal rats showed larger facilitatory effects in response to low doses of d-amphet-amine but lesser depressant effects in response to high doses of d-amphetamine; the whole dose-response curve was generally shifted higher by the frontal lesions. These results indicate that frontal lesions differentially influence mechanisms mediating two different actions of d-amphetamine.This research was supported by NIMH grant MH21156 and NIMH Research Scientist Development Award (Type 2) DA70082 to S. D. Glick.     

Drug level of d- and l-amphetamine during intravenous self-administration

Rats were allowed to self-administer dextro and levo isomers of amphetamine in doses of 0.25, 0.50, 0.75 and 1.0 mg/kg/injection for 6 h/day. Total body level of drug was calculated at the time of responding for each drug injection. Body level of amphetamine initially increased and then decreased (0–2 h), and thereafter remained relatively constant for the remainder of the experimental session (2–6 h). During 2–6 h of self-administration, calculated whole body levels of both d- and l-amphetamine remained relatively constant across injection doses. In another study, blood was removed several times during 2–6 h at the time of responding for drug injection. Again, no difference in blood level of ¹⁴C-amphetamine was found across a range of injection doses. Mean blood levels were 0.48 g/ml for l-amphetamine and 0.18 g/ml for d-amphetamine. Drug intake averaged 2.0 mg/kg/h for l-amphetamine and 0.79 mg/kg/h for d-amphetamine.     

Discriminative stimulus properties of d-amphetamine-pentobarbital combinations

Pigeons were trained to discriminate IM injections of 1.0 mg/kg d-amphetamine from water, 5.6 mg/kg pentobarbital from water, or 1.0 mg/kg d-amphetamine from 5.6 mg/kg pentobarbital by requiring them to peck different response keys depending on which substance was administered prior to the session. Excellent stimulus control was achieved under all conditions with close to 100% of the responses occurring on the injection-correlated key. In tests with doses different from those used in training, the percentage of responses on the drug key was directly related to drug dose. When d-amphetamine was given to birds trained to discriminate pentobarbital from water or when pentobarbital was given to birds trained to discriminate d-amphetamine from water, responding occurred predominately on the water-correlated key. d-Amphetamine produced a dose-related antagonism of the effects of pentobarbital for birds trained to discriminate pentobarbital from water or from d-amphetamine. Rates of responding on the drug key were generally highes after administration of the drug doses used in discrimination training; but response rates were not systematically related to the percentage of responses occurring on the drug key. All birds were subsequently trained to discriminate a combination of 1.0 mg/kg d-amphetamine and 5.6 mg/kg pentobarbital from either 1.0 mg/kg d-amphetamine or 5.6 mg/kg pentobarbital alone, demonstrating that the discriminative stimulus properties of amphetamine-pentobarbital combinations are different from either drug alone. Several of the drug effects reported were related to the drug discrimination that had been established.Portions of this paper were reported at the 49th annual meeting of the Eastern Psychological Association, Washington, D.C., 1978     

Permanent facilitation of avoidance behavior by d-amphetamine and scopolamine

Scopolamine (0.3, 0.6, 1.2, 2.4, 4.8 mg/kg) or d-amphetamine (0.25, 0.50, 1.00, 2.00, 4.00 mg/kg) was administered daily to independent groups of rats 30 min prior to training in a discriminated, Y-maze avoidance task. A dose-dependent relationship was found between amount of avoidance facilitation and drug dosage. Discontinuation of the drug following asymptotic performance resulted in a decrement in avoidance which varied as a function of the acquisition dosage. Results from a second experiment using the same task indicated that gradually reducing the dosage on consecutive training days rather than abruptly discontinuing the drug was more effective in producing permanent avoidance facilitation in the non-drug condition.     

Effects of d-amphetamine and ethanol alone and in combination on schedule-controlled responding of pigeons

Key pecking by pigeons was maintained under either a 5-min fixed-interval or a 30-response fixed-ratio schedule of food delivery. d-Amphetamine (0.1–1.0 mg/kg) either increased or did not affect overall rates of responding under the fixed-interval schedule; the lowest dose of ethanol (0.5 g/kg) did not affect or slightly decreased response rates, whereas higher doses (1.0–2.0 g/kg) substantially decreased rates. Combinations of low noneffective ethanol doses with most doses of d-amphetamine increased rates of responding under the fixed-interval schedule above those obtained with d-amphetamine alone; decreases produced by the higher doses of ethanol were attenuated by most doses of d-amphetamine. Doses of d-amphetamine (0.1–1.0 mg/kg) and ethanol (0.5–1.5 g/kg) alone generally had no effect on responding maintained under the fixed-ratio schedule; higher doses of these drugs decreased responding. The effects of dose combinations other than the highest ones generally differed little from those obtained with ethanol alone; the effects of high doses of each drug were antagonized by low to moderate doses of the other. Combinations of ethanol with d-amphetamine can result in higher rates of responding than are obtained with either drug alone. Further, effects of the drugs alone and in combination depend on the schedule under which behavior is maintained.     

Some behavioral effects of repeated d-amphetamine administrations

Effects of daily administrations of d-amphetamine were studied on key peck responses of pigeons maintained under a multiple fixed-interval 2-min, fixed-ratio 30-responseschedule. Under the fixed-interval schedule, a pause was followed by a transition to increasing rates of responding until food presentation. Under the fixed-ratio schedule, higher sustained rates of responding were maintained. Low to intermediate doses (0.3-1.0 mg/kg) of d-amphetamine changed the temporal patterns and occasionally increased rates of responding under the fixed-interval schedule. Higher doses decreased rates of responding under bothschedules. With daily injections of 1.0 mg/kg d-amphetamine prior to experimental sessions, the effects of this dose on rates and patterns of responding were attenuated, and d-anphetamine dose-effect curves were shifted to the right, primarily under the fixed-ratio schedule. Similar results were obtained with daily presession injections of 5.6 mg/kg d-amphetamine in a second group of pigeons, except that rates of responding under both schedules were decreased by this daily dose, and did not return completely to control values with repeated injections. In a third group of pigeons, 1.0 mg/kg d-amphetamine administered daily, after experimental sessions, did not alter dose-effect functions for d-amphetamine. In a second experiment, pigeons were trained to peck one response key when given 1.0 mg/kg d-amphetamine and a different key when given presession water injections. Increasing doses of d-amphetamine produced incresing percentages of d-amphetamine-key responses. Repeated administration of 5.6 mg/kg d-amphetamine shifted these dose-effect functions to the right one-half log unit. Results suggested that decreases in reinforcement frequency are not a necessary condition for the development of behavioral tolerance to d-amphetamine.     

Long-term l-Dopa pretreatment of mice: Central receptor subsensitivity or supersensitivity?

The effect of d-amphetamine added to the drinking water on the rate of conditioned lever pressing by rats was determined using fixed-ratio 30 (FR-30) and fixed-interval 2-min (FI-2) schedules of food presentation. After 32 days of gradual increase in drug concentration the average drug ingestion was 13 mg/kg/day. In tests with various doses of d-amphetamine injected before and after the chronic ingestion regimen, the rate-decreasing effects of d-amphetamine on FR responding were attenuated after chronic treatment, indicating development of a two- to three-fold tolerance. However, the rate-decreasing effect of d-amphetamine on FI responding was not altered by chronic ingestion. Since acute amphetamine treatment reduced the reinforcement frequency under the FR but not the FI schedule, these results are consistent with the hypothesis that a behavioral tolerance will develop most readily to drug effects that decrease the frequency of reinforcement. Upon removal of d-amphetamine from the drinking water there was some increase in the rate of FR responding, but no change in FI responding.     

Alterations by centrally acting drugs of the suppression of self-stimulation behavior in the rat by tetrabenazine,physostigmine, chlorpromazine and pentobarbital

The effects of pre-treatment (32 min) with d-amphetamine (2 mg/kg), scopolamine (0.5 mg/kg), and chlordiazepoxide (5 mg/kg) were studied on the suppression of self-stimulation behavior in the male albino rat by central depressants. The antagonism of each compound was determined against the suppressant action of tetrabenazine (2 mg/kg), physostigmine (0.1 mg/kg), chlorpromazine (2.5 mg/kg) and pentobarbital sodium (10 mg/kg).Against the suppression produced by tetrabenazine, only d-amphetamine gave partial protection throughout the test. Scopolamine and chlordiazepoxide had a transient delaying action. Against the suppressant effect of chlorpromazine, protection was given by d-amphetamine, scopolamine and chlordiazepoxide.Against physostigmine, scopolamine gave full protection, d-amphetamine partial protection, and chlordiazepoxide was without effect.Against the effect of pentobarbital on self-stimulation behavior, there was no protection by d-amphetamine and scopolamine when the animal showed motor deficits, and a stimulant action when these had worn off, but the rates of responding were still depressed. Chlordiazepoxide potentiated the action of pentobarbital.These results are interpreted in terms of a short-run stimulant action on depressed rates of responding, and a longer-run protective action against changes produced by the compounds suppressing self-stimulation behavior in levels of transmitter-like substances.Supported by grant MH-16978, U. S. Public Health Service.The author is indebted to Mrs. S. Foster and Mrs. H. Cevallos for technical assistance.     

Effects of morphine alone and in combination with naloxone or d-amphetamine on shock-maintained behavior in the squirrel monkey

Key-pressing behavior in the squirrel monkey was maintained under an 8-min fixed-interval (FI) schedule of electric-shock delivery. The acute i.m. administration of morphine prior to a daily session decreased response rates at doses of 1.0–3.0 mg/kg but had little systematic effect on rate at doses of 0.03–0.3 mg/kg. When naloxone was administered concomitantly with morphine prior to a session, 0.01 mg/kg naloxone required a three-fold increase in the dose of morphine necessary to obtain decreased response rates, 0.1 mg/kg naloxone required a 30-fold increase in morphine, and 1.0 mg/kg required more than a 30-fold increase in morphine. Moreover, the administration of naloxone with morphine resulted in increased rates of responding at certain combinations of doses of the two drugs. The administration of d-amphetamine (0.03 or 0.1 mg/kg) alone increased mean response rates under the FI schedule; when combined with 0.03–0.3 mg/kg morphine the increases in responding were greater than obtained with d-amphetamine alone. The negative slope of the linear regression lines relating the effects of morphine to control rates of responding engendered under the FI schedule was decreased when morphine was combined with naloxone, but not with d-amphetamine. These results show that naloxone, but not d-amphetamine, can antagonize the response-rate decreasing effect of morphine when responding in the squirrel monkey is maintained by response-produced electric shock.     

Interactions of naloxone with morphine,amphetamine and phencyclidine on fixed interval responding for intracranial self-stimulation in rats

Rats were implanted with stimulating electrodes aimed at the medial forebrain bundle-lateral hypothalamus (MFB-LH) and were trained to lever-press for brain self-stimulation on a fixed interval: 60 s schedule of reinforcement. The effects of graded doses of naloxone (0.1–30 mg/kg), morphine (0.3–5.6 mg/kg), naloxone plus morphine,d-amphetamine (0.03–1.0 mg/kg), naloxone plusd-amphetamine, phencyclidine (0.3–5.6 mg/kg), and naloxone plus phencyclidine were tested. Naloxone produced a significant decrease in rates at 30 mg/kg. Naloxone (0.1–1.0 mg/kg) plus morphine blocked the dose-dependent decrease produced by morphine alone. In contrast, naloxone (1.0–10 mg/kg) plusd-amphetamine attenuated the graded increase in response rates produced byd-amphetamine. Naloxone (1.0–10 mg/kg) plus phencyclidine did not reliably change the increase in response rates produced by phencyclidine alone. The use of the fixed interval schedule of brain self-stimulation to study these drug interactions is novel, and further demonstrates that the highly reinforcing aspects of brain stimulation, known to be influenced by dopamine, may also be modulated by the endogenous opiate system.     

An analysis of some discriminative properties of d-amphetamine

Male albino rats were trained and tested on a two-lever discrimination task based upon the presence or absence of d-amphetamine (1.0 mg/kg). This compound was found to produce strong discriminative cues (i.e., 90% correct choice behavior). A dose-effect function was then ascertained and the discriminative ED₅₀ (following training with 1.0 mg/kg) was found to be 0.23 mg/kg d-amphetamine.In order to determine the effective duration of d-amphetamine action, the interval between injection and testing was varied; it was found that the discriminative effects of the drug began to dissipate between 60 and 90 min post-injection.In an attempt to compare the discriminative cues of other drugs with those of d-amphetamine, injections of LSD (0.04 and 0.08 mg/kg), psilocybin (0.50 and 1.0 mg/kg), THC (0.50 and 1.0 mg/kg), mescaline (5.0 and 10.0 mg/kg), and caffeine (6.0 and 20.0 mg/kg) were given during extinction. In all cases, the rats responded predominantly on the saline-related lever. Only methamphetamine (1.0 mg/kg) produced d-amphetamine-like responding.Finally, alpha-methyl-para-tyrosine (AMPT), a compound which depletes brain catecholamines (CA), was found to disrupt the d-amphetamine-saline discrimination.     

Antagonism of the behavioral effects of cocaine and d-amphetamine by prazosin

Key pecking by pigeons was maintained under a 30-response fixed-ratio schedule of food delivery; lever pressing by squirrel monkeys was maintained under a 3-min fixed-interval schedule of food delivery. Administered alone, d-amphetamine (0.1–3.0 mg/kg), cocaine (1.0–3.0 mg/kg) and bupropion (1.0–30 mg/kg) either did not affect or decreased fixed-ratio responding of pigeons, whereas d-amphetamine (0.056–0.3 mg/kg) either increased or decreased (0.56 mg/kg) responding of monkeys maintained under the fixed-interval schedule. Prazosin, a selective centrally-active alpha₁ antagonist, produced a dose-dependent reversal of the rate-decreasing effects of d-amphetamine and cocaine but not of bupropion on fixed-ratio responding in pigeons. Prazosin also reversed both the rate-increasing and rate-decreasing effects of d-amphetamine on fixed-interval responding of squirrel monkeys. In contrast, the non-selective alpha-antagonist phentolamine enhanced d-amphetamine-induced decreases in fixed-ratio responding. These findings suggest that the behavioral effects of d-amphetamine and cocaine are produced at least in part by activation of central alpha₁ receptors. Prazosin may be a useful tool for better understanding the mechanisms through which cocaine, amphetamine, and other abused stimulant drugs exert their potent behavioral effects.     

Effects of d-amphetamine on F-I shock presentation with Sδ probe

The functional relationship was tested in 4 squirrel monkeys (Saimiri sciurea) between multiple S interruptions of fixed-interval performance maintained by electric-shock presentation and graded doses of d-amphetamine. Small doses (0.10–0.30 mg/kg) increased responding relative to baseline rates and a larger dose (1.0 mg/kg) depressed responding during S^D segments of the fixed-interval ; amphetamine generally produced smaller effects on S rates. Some of the effects got larger (e.g., relative increase by R7) and some effects changed in direction following repeated, intermittent daily injections of d-amphetamine, suggesting some manifestation of behavioral tolerance, and interpretation of results solely in terms of the rate-dependent effects of amphetamine is incomplete.     

Effects of chlorpromazine and d-amphetamine on schedule-controlled and schedule-related behavior of rabbits

A lever-lifting response by Dutch Belted and New Zealand White rabbits was maintained in water-deprived animals by 0.25% saccharin solution and in food-deprived animals by food pellets under a multiple 3-min fixed-interval (FI) 30-response fixed-ratio (FR) schedule. Rabbits responding for the saccharin solution had food freely available during the session and in the home cage, whereas those responding for pellets had water continuously available during the session as well as in the home cage. Under nondrug conditions the FR and FI schedules controlled different rates and patterns of responding in the rabbit that were characteristic of those found with other species. In addition, eating or drinking occurred during the inital portion of the FI under the saccharin solution and initial food presentation schedules, respectively. Doses of d-amphetamine (0.1–10.0 mg/kg) increased responding under the FI and FR schedules of food delivery, but increased only FI responding maintained by the saccharin solution. Doses of 3.0–10.0 mg/kg d-amphetamine produced extremely high (300–800% of control) rates of stereotyped perseverative lever responding. Schedule-related eating or drinking were unaffected or decreased at doses of d-amphetamine that increased schedule-controlled responding. Chlorpromazine (0.03–0.3 mg/kg) increased FI responding maintained both by saccharin and food, whereas FR responding generally was unaffected at these dose levels; eating but not drinking was increased with chlorpromazine. Since the behavioral effects of drugs such as amphetamine and chlorpromazine differ somewhat in the rabbit from those found with other typically studied nonhuman mammals, further studies with the rabbit may yield useful information for comparative behavioral pharmacology.     

Fear-dependent variations in continuous avoidance behavior of pigs

Rats performing on a progressive-ratio schedule that required five additional responses for each successive food reinforcement were administered d-amphetamine. The number of responses required increased until the next ratio was not completed within 15 min. The number of reinforcements obtained during a session increased with increasing doses of d-amphetamine from 0.25 mg/kg to a maximum behavioral effect at 1.0 to 2.0 mg/kg followed by a decline with higher doses. To assess the effects of hyperbaric air on the established d-amphetamine performance, the same doses were administered to rats breathing compressed air at seven times normal atmospheric pressure (7.1 ATA). In the hyperbaric condition the dose-effect function was displaced toward smaller doses such that the maximum behavioral effect was obtained at lower doses than under normal atmospheric pressure; doses that produced maximum behavioral effects under normal pressure produced a decline in reinforcements obtained.Supported by Naval Medical Research and Development Command, Navy Department, Research Subtask MPN 10.03.3021. The opinions and statements contained herein are the privates ones of the writer and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large.     

Effects of d-amphetamine and naloxone on brain stimulation reward

Self-stimulation thresholds were determined in rats by means of a modification of the psychophysical method of limits. Reinforcement values were determined after the administration of d-amphetamine alone, naloxone alone, and naloxone administered concurrently with d-amphetamine. d-Amphetamine yielded dose-related decreases in the threshold (0.25–2.00 mg/kg IP), while naloxone alone (2.0–16 mg/kg IP) caused no consistent changes. For each animal, a dose of d-amphetamine that substantially lowered the threshold was then selected to be administered with varying doses of naloxone. The threshold-lowering effect of d-amphetamine was blocked by naloxone at doses as low as 2.0 or 4.0 mg/kg. This finding suggests the possible involvement of an opiate receptor in the mediation of the enhancement by d-amphetamine of brain stimulation reward.