Behavioral evidence of depolarization block of dopamine neurons after chronic treatment with haloperidol and clozapine.

Electrophysiological studies have shown that chronic treatment with haloperidol causes depolarization block (DB) of dopamine cells in anesthetized and paralyzed rats. It has been proposed that the emergence of DB underlies the therapeutic and side effects of this drug. However, the relevance of DB to the clinical actions of haloperidol has been questioned on the grounds that chronic drug-induced DB has not yet been demonstrated in freely moving animals. In this study, responding for rewarding electrical brain stimulation was used to assess the occurrence of DB in rats chronically treated with haloperidol or clozapine. The time course of the effects of acute haloperidol (7.8-500 microg/kg) and clozapine (5-40 mg/kg) and of withdrawal from chronic drug treatment on reward and performance measures were also characterized. Haloperidol and clozapine dose-dependently attenuated reward and performance, haloperidol producing a predominant suppression of performance, and clozapine preferentially attenuating reward. Chronic (21 d) treatment with haloperidol (500 microg/kg) caused responding to cease in the six rats tested, and repeated injection with apomorphine restored the behavior in all of them; such an effect of apomorphine was observed in only two of six rats treated acutely with the same dose of haloperidol. Chronic treatment with clozapine (20 mg/kg) increased reward thresholds, an effect that was reversed by apomorphine in chronically, but not acutely, treated rats. The times at which chronic haloperidol-treated rats resumed responding was positively correlated with indices of behavioral supersensitivity after withdrawal, suggesting that the effect of apomorphine was not caused by direct stimulation of upregulated postsynaptic receptors. These findings constitute the first behavioral evidence of DB in unanesthetized, freely moving animals treated chronically with antipsychotics. They also demonstrate that the neural substrates mediating reward and performance are functionally independent and differentially sensitive to haloperidol and clozapine.

Effects of concomitant motor reactions on the measurement of rewarding efficacy of brain stimulation.

In self-stimulation behavior, the rate-frequency (R-F) function relates bar-pressing performance to the number of cathodal pulses of constant intensity, delivered in a train of fixed duration. The lateral position of the R-F function depends on the rewarding efficacy of the stimulation; a shift of the function toward larger pulse numbers after some experimental manipulation indicates a decrease in the efficacy of the stimulation. Because self-stimulation is often accompanied by stimulation-contingent motoric reactions, it is required to show that such reactions do not alter the estimates of rewarding efficacy of the stimulation. We describe an experiment in which the presence and severity of motoric reactions were controlled experimentally by simultaneous stimulation through a second electrode, located in a motoric brain region. Rats were implanted with one hypothalamic (LH) electrode (which elicited self-stimulation) and one reticular (RF) electrode (which elicited head and body movements). The rate-frequency function for each LH electrode was obtained under a single-pulse condition (LH electrode alone) and under a paired-pulse condition repeated three times, in which each LH pulse was accompanied by three different intensities of an RF pulse. Despite its severe effect on the slope and the asymptotic rate of R-F function, the interfering reaction failed to shift the R-F function to any significant degree. We concluded that these interfering reactions do not alter the estimates of neuronal density obtained through application of the curve-shift paradigm.

Psychostimulant-like effect of central microinjection of neurotensin on brain stimulation reward.

The curve shift method and the brain stimulation reward paradigm were used to dissociate reward and performance changes and determine whether unilateral ICV microinjection of neurotensin (3, 10, and 30 micrograms/10 microliters) produces neuroleptic- or psychostimulant-like effect on a dopamine-dependent behavior. At the highest dose tested, neurotensin potentiated brain stimulation reward, producing a significant time-dependent decrease in frequency threshold. Neurotensin also suppressed maximal rate of responding at every dose tested, suggesting that it was more effective at attenuating performance capability. These results suggest that a centrally acting neurotensin receptor agonist may specifically stimulate dopamine-dependent behaviors, producing psychostimulant-like effect that can be attenuated or masked by a suppression of performance capability.

A comparison of the effects of mesencephalic injections of neurotensin(1-13) and neuromedin N on brain electrical self-stimulation.

Neuromedin N (NM-N), a hexapeptide that shares a four amino acid C-terminal homology with the tridecapeptide, neurotensin (NT), has been suggested as a potential neurotransmitter or neuromodulator that could interact with the NT-sensitive receptors. In this experiment, we compared the effects of an equimolar concentration of NM-N and NT(1-13) injected in the ventral tegmental area (VTA) on brain electrical self-stimulation (SS), a behavior previously shown to be potentiated by VTA injections of NT(1-13). Rats implanted with a stimulating electrode in the mesencephalic central gray and a guide cannula in the VTA were trained to lever press to obtain rewarding electrical stimulations. Functions relating the rate of lever pressing to the stimulation frequency were determined, on separate daily tests, before and after the injection of 3 nmol of NM-N, NT(1-13), or an equal volume of saline vehicle. At this concentration, both NM-N and NT(1-13) produced a significant facilitation of SS when compared to saline vehicle, an effect that was not seen when the peptides were injected outside the VTA. The facilitation of SS by NM-N, however, was much weaker and of a shorter duration than the one produced by NT(1-13). The shorter time course and the weaker behavioral effect of NM-N compared to NT(1-13) are consistent with its lower potency at the NT receptor and its faster rate of enzymatic degradation in the VTA, and suggest that NM-N potentiated the reward-relevant neural signal by acting on mesencephalic NT receptors.

Behavioral determination of refractory periods of the brainstem substrates of self-stimulation.

The objective of this study was to estimate the refractory periods of the brainstem neurons responsible for self-stimulation behavior in the rat. In a first experiment, we tested the robustness of the double pulse technique used to estimate the refractory periods of reward-relevant neurons. We obtained estimates of the relative T-pulse effectiveness at a wide range of stimulation frequencies. The results of this experiment suggest that the refractory period estimates obtained with the behavioral version of the double pulse technique are not dependent on the arbitrary choice of the stimulation frequency. However, the use of stimulation frequencies higher than 100 Hz should preferably be avoided. In a second experiment, we applied the double pulse technique using C-pulse intensity higher than T-pulse intensity to estimate the refractory periods of the brainstem reward-relevant neurons. Using moveable electrodes, we tested 9 metencephalic and 7 mesencephalic sites in 4 animals. In the metencephalon, the most excitable reward-relevant neurons have absolute refractory periods of less than 0.6 and 0.8 ms and have a supernormal period that occurs at least between 5 and 10 ms after the initial excitation. The mesencephalic reward-relevant neurons were found to have more heterogeneous physiological characteristics. The most excitable cells in the mesencephalon have absolute refractory periods of less than 0.4 ms and have a supernormal period occurring as soon as 2.4 ms after the initial excitation. At some mesencephalic sites, we observed first an abrupt initial recovery followed by a plateau, followed by a renewed and continuous recovery, a pattern that was never observed in the metencephalon. The hypothesis of the contribution of two distinct sub-populations of reward-relevant neurons is proposed and the implication of monoaminergic pathways in reward is discussed.

Effect of pimozide on self-stimulation threshold under a continuous and fixed-interval schedule of reinforcement.

Rats implanted with a monopolar electrode in the medial mesencephalon and trained to press a lever to self-administer trains of stimulating pulses were tested under a continuous (CRF) and a fixed interval (FI-1s) schedule of reinforcement before and after systemic pimozide injection (0.35 mg/kg). As previously shown, this dose of pimozide caused reductions in maximal response rates and in the rewarding effectiveness of the stimulation. The magnitude of the attenuation in rewarding effectiveness was significantly greater under CRF than under FI conditions, an effect that can be attributed to the decrease in the number of earned rewards concomitant to a decrease in response rates. The present results show that the tighter control over reward density provided by the use of a FI schedule of reinforcement reduces the probability of artifactually measuring increases in reward thresholds following treatments that suppress response rates.

Electrophysiological characteristics of neurons in forebrain regions implicated in self-stimulation of the medial forebrain bundle in the rat.

In an attempt to identify neurons likely to play a role in self-stimulation of the medial forebrain bundle (MFB), action potentials of single neurons in the septum and basal forebrain of anesthetized rats were recorded by means of extracellular electrodes. Refractory period estimates were obtained from cells antidromically activated by stimulation of the lateral hypothalamus or ventral tegmental area, and estimates of interelectrode conduction time were obtained from cells that were driven by stimulation of both sites. The results show that some descending MFB axons arising in the medial septum, diagonal band of Broca and neighboring forebrain structures have characteristics comparable to properties of MFB reward neurons inferred from behavioral experiments.

Pontine and mesencephalic substrates of self-stimulation.

Single or twin, moveable monopolar stimulating electrodes were implanted in male adult rats in order to map the medial pons and mesencephalon for self-stimulation behaviour. The electrodes were implanted 6 mm below the surface of the skull and subsequently moved down by steps of 0.13 or 0.16 mm. Each bar press in a Skinner box delivered a train (0.4 s in duration) of cathodal rectangular pulses of fixed intensity (200 microA) and width (0.1 ms). Self-stimulation was recorded from zero to the maximum performance by varying the number of pulses per stimulating train. The rewarding efficacy of the stimulation at each electrode location was inferred from determination of the pulse period corresponding to the threshold and half-maximal performance. Out of 361 mesencephalic and pontine sites sampled, 289 supported self-stimulation. Within the metencephalon, the study revealed a continuous band of positive sites, extending over a dorso-ventral distance of 4 mm, between the floor of the aqueduct and the pontine nuclei. Hence, all electrode locations in the central grey, dorsal raphe and median raphe supported self-stimulation. Within the mesencephalon, the positive band was restricted between the floor of the central grey and the middle part of the interpeduncular nucleus. At the rostral mesencephalon, it shifted laterally towards the substantia nigra. The overlap between the self-stimulation sites and some of the best known ascending and descending pathways is discussed.

Opioid-neuroleptic interaction in brainstem self-stimulation.

Injection of morphine into the ventral tegmental area (but not dorsal to it) induced a dose-dependent decrease in the frequency threshold for midline metencephalic brain stimulation reward. Facilitating doses of ventral tegmental morphine also reversed, in 4 of 6 animals, the threshold-increasing effects of pimozide (0.35 mg/kg, i.p.). This reversal was itself reversed by naloxone (2 mg/kg, i.p.), suggesting a direct action of morphine at ventral tegmental opiate receptors. These data fit with electrophysiological evidence that ventral tegmental morphine stimulates or disinhibits dopamine impulse flow, which would result in increased synaptic dopamine concentrations and decreased synaptic pimozide effectiveness. In the remaining two animals, the combined neuroleptic-opiate treatment resulted in a complete cessation of responding that was not reversed by a 5-fold increase in stimulation frequency. This finding suggested a complete inactivation of the reward mechanism, which might be expected from the interaction of high doses of two drugs that are each known to be capable of producing depolarization inactivation of dopaminergic neurons. These data confirm that brainstem self-stimulation, like medial forebrain bundle self-stimulation, depends critically on the function of the mesocorticolimbic dopamine system.

Behavioral evidence for midbrain dopamine depolarization inactivation.

High (2.5-5 micrograms) doses of ventral tegmental morphine, which normally facilitate brain stimulation reward, were found to cause a complete cessation of bar pressing for brainstem stimulation in animals pretreated with systemic pimozide (0.175-0.35 mg/kg). It was hypothesized that the behavioral failure was due to depolarization inactivation of the dopamine system. Electrophysiological evidence indicates that sufficient doses of morphine or neuroleptics can each cause inactivation by themselves. The behavior was reinstated by ventral tegmental muscimol, which normally suppresses both the behavior and dopamine cell firing but which reinstates dopamine cell firing in depolarization-inactivated cells. This behavioral reinstatement appears to confirm the hypothesis that depolarization inactivation of the dopamine system caused the behavioral failure, and appears to establish depolarization inactivation as a phenomenon of behavioral, and thus potential clinical, importance.

Mesencephalic microinjections of neurotensin-(1-13) and its C-terminal fragment, neurotensin-(8-13), potentiate brain stimulation reward.

Using the curve shift method, we assessed the effects of ventromedial mesencephalic tegmental (VMT) microinjections of an equimolar concentration of neurotensin-(1-13) (NT-(1-13)) and of its C-terminal fragment, neurotensin-(8-13) (NT-(8-13)), on operant responding for rewarding electrical stimulation of the caudal mesencephalic central gray. The effects of NT-(1-13) and NT-(8-13) on brain stimulation reward (BSR) were also compared to those of systemically administered quinpirole (0.1 and 0.2 mg/kg, s.c.), a direct dopamine agonist, and GBR-12909 (10 and 20 mg/kg, i.p.), a selective dopamine uptake blocker. At the concentration injected, NT-(8-13) was as effective as NT-(1-13) at facilitating BSR, producing significant leftward shifts of the function relating the rate of responding to the stimulation frequency (R/F function); neither form of the peptide was effective when injected in regions dorsal to the VMT. Similarly, GBR-12909 produced a parallel leftward shift of the R/F function, but, unlike NT-(1-13), also significantly increased the asymptotic rates of responding. In contrast, the high dose of quinpirole produced non-parallel leftward shifts of the R/F function and suppressed the asymptote. The similarity between the effects of neurotensin and GBR-12909 on one hand, and the differences between those of neurotensin and quinpirole on the other, suggest that activation VMT neurotensin receptors potentiate BSR by enhancing increases in dopamine neurotransmission that are contingent upon operant responding or rewarding brain stimulation, or both.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological evidence that a subset of midbrain dopamine neurons integrate the reward signal induced by electrical stimulation of the posterior mesencephalon.

This study was aimed at determining whether midbrain dopamine (DA) neurons are trans-synaptically activated by rewarding electrical stimulation applied near the midline in the posterior mesencephalon (PM), and in the affirmative, whether the increase in firing was proportional to the rewarding effectiveness of the stimulation. Experiments were performed on male Long-Evans rats trained to lever press to obtain 400 ms trains of cathodal rectangular pulses. Following the training period, curves relating the rates of responding to the stimulation frequencies were determined at two current intensities and reward thresholds were calculated for each animal. Each animal was then anesthetized with urethane (1.2 g/kg, i.p.) and firing rate of DA neurons were recorded before, during, and after each of 50 trains (1 train/3 s) of stimulation to the PM using stimulation parameters that either sustained near threshold responding (rewarding), or failed to sustain responding (non-rewarding), in the behavioral tests. A total of 24 DA cells were recorded from 13 behaviorally trained animals, and of these, 17 (71%) responded to rewarding stimulation by an increase in firing, five (21%) were unresponsive and two (8%) were inhibited. In 12 of the 17 cells that were activated, the increase in firing was proportional to the rewarding effectiveness of the stimulation rather than the total strength of the stimulation. These results provide evidence that a subset of midbrain DA neurons are trans-synaptically activated by rewarding PM stimulation and constitute a second, or subsequent, stage of the reward-relevant pathway that integrates the PM reward signal.

Localization of reward-relevant neurons in the pontine tegmentum: a moveable electrode mapping study.

Monopolar moveable stimulation electrodes were implanted in male adult rats in order to map the reward substrate in the pontine tegmentum. Electrodes were implanted 6 mm below the surface of the skull and subsequently lowered by steps of 0.16 or 0.32 mm. Each bar press in a Skinner box delivered a train (0.4 s in duration) of cathodal rectangular pulses of fixed intensity (200 microA) and width (0.1 ms). Self-stimulation was recorded from zero to maximum performance by varying the number of pulses per train. The rewarding effectiveness of the stimulation at each positive site was inferred by determining the frequency threshold. Out of 476 sites that were sampled, 137 supported self-stimulation. Eighty-one percent of the positive sites (111 out of 137) were located within 1 mm of the midline. Of the 181 sites that were sampled in the region posterior to the caudal end of the dorsal raphe, only 9 sites (less than 5%) supported self-stimulation. These results suggest that the majority of neurons that constitute the brainstem reward substrate either originate from and/or terminate in the rostral pons.

Glutamate injection into the suprachiasmatic nucleus stimulates brown fat thermogenesis in the rat.

Injection of glutamate (100 mM to 1 M, in 0.25 micrograms saline) into the hypothalamic suprachiasmatic nucleus (SCN) dose-dependently increased interscapular brown adipose tissue (IBAT) and core temperatures in the urethane-anaesthetized rat. This effect was more pronounced in rats tested during the light-off period than in animals tested during the light-on period. Prior injection of the local anaesthetic, procaine (5% in 0.5 microliter saline), into the ipsilateral ventromedial hypothalamic nucleus (VMH) attenuated the increases in IBAT and core temperatures induced by intra-SCN glutamate. The VMH has previously been implicated in the central regulation of BAT thermogenesis; the present results suggest the pathway arising in the SCN exerts an excitatory influence on VMH neurons involved in the control of BAT function.

Forebrain neurons driven by rewarding stimulation of the medial forebrain bundle in the rat: comparison of psychophysical and electrophysiological estimates of refractory periods.

Psychophysically derived estimates of recovery from refractoriness were obtained at self-stimulation sites in the lateral hypothalamus and ventral tegmental area. The refractory periods of single units driven by the same stimulation electrodes and stimulation fields were then measured electrophysiologically. Antidromically driven units with refractory periods longer than those of the neurons responsible for the rewarding effect were concentrated in the septal complex. Units with refractory periods that overlapped the estimates for the reward-related neurons were found in this region as well but were also encountered in neighboring structures lateral, ventral, and/or caudal to the septal nuclei. It is argued that this latter class of units should be considered as possible constituents of the directly stimulated substrate for the rewarding effect because they are driven by rewarding stimulation, have refractory periods similar to those of the reward-related neurons and arise in or near regions in which lesions have been effective in decreasing the rewarding effect of stimulating the medial forebrain bundle.

Estimates of the axonal refractory period of midbrain dopamine neurons: their relevance to brain stimulation reward.

Psychophysical studies have shown that the directly activated neurons subserving the rewarding effect produced by electrical stimulation of the medial forebrain bundle (MFB) have refractory periods (RPs) shorter than those of dopaminergic (DA) neurons: this suggests that the directly stimulated substrate for the rewarding effect does not include DA neurons. Comparison of RP estimates. however, is difficult since those of DA neurons were obtained with the standard electrophysiological technique that characterizes cell body/initial segment rather than the directly stimulated axonal segment. Using electrophysiological recording techniques in urethane anesthetized rats, we re-estimated and compared the RP of DA neurons that project to the MFB and the ventral striatum (VST) with two stimulation procedures: one that characterizes the axonal segment near the stimulation electrode and the standard one that characterizes the cell body/initial segment near the recording electrode. Results showed that DA axonal RPs range from 1.0 to 2.2 ms, whereas cell body/initial segment RPs range from 1.0 to 3.0 ms. The axonal RP was equal to or shorter (mean difference = 0.22 ms, n = 15) than the cell body/initial segment RP. Axonal RP estimates for MFB sites range from 1.3 to 2.1 ms. values that slightly overlap with the late recovery from refractoriness reported in psychophysical studies for reward-relevant neurons. Axonal RP estimates obtained for VST sites were very similar (mean = 1.66, LH and 1.62 ms, VST) suggesting that the excitability of DA axons does not differ along their path. These results further support the hypothesis that DA axons are unlikely to constitute a major component of the directly-stimulated reward-relevant axons in the MFB. They also suggest that the direct contribution of DA axons to reward produced by VST stimulation is more important than by the MFB.

Thermogenesis in brown adipose tissue is activated by electrical stimulation of the rat dorsal raphe nucleus.

Brainstem mechanisms involved in the central control of thermogenesis in brown adipose tissue (BAT) were investigated, in urethane-anaesthetized rats, by observing changes in the temperature of interscapular BAT following electrical stimulation of sites in the dorsal raphe nucleus (DRN). Large increases in temperature could be produced by single 30 s stimulation trains. The magnitude of the temperature increase grew as a function of both current and frequency. Non-linear temporal summation was observed when near-threshold trains were delivered in close succession. These results are consistent with the view that ascending projections from the DRN relay thermal information to the basal forebrain and hypothalamus.

Acute depolarization block of A10 dopamine neurons: interactions of morphine with dopamine antagonists.

Extracellular single-unit recording techniques were used to determine the effects of morphine, administered either systematically (intravenous) or locally (microiontophoresis), on ventral tegmental area (A10) dopamine (DA) neuronal activity in animals pretreated with D1 (SCH 23390) or D2 (pimozide) DA receptor antagonists. In rats pretreated with the D2 antagonist pimozide, A10 DA neurons readily entered a state of apparent depolarization block in response to either i.v. or iontophoretically applied morphine. Whether the inactivation of DA neurons was induced by systemic or local morphine, it was reversed in 22 of 27 cases by iontophoretic administration of the inhibitory amino acid transmitter gamma-aminobutyric acid (GABA), suggesting depolarization block as the underlying mechanism. Pretreatment of rats with the D1 antagonist SCH 23390 did not significantly alter the tendency of A10 DA neurons to enter apparent depolarization block in response to morphine. These data support recent behavioral evidence suggesting that the combination of systemic pimozide and ventral tegmental area morphine can result in depolarization inactivation of the mesoaccumbens DA reward system.

Repeated activation of neurotensin receptors sensitizes to the stimulant effect of amphetamine.

Effects of repeated intracerebroventricular microinjections of 18 nmol/10 microl of neurotensin, [D-Tyr11]neurotensin, or saline were tested on motor activity in different groups of rats. One week after the fourth central injection, sensitivity to the behavioral stimulant effect of amphetamine (1 mg/kg, i.p.) was tested. As previously reported, neurotensin attenuated motor activity while [D-Tyr11]neurotensin when compared to saline produced an initial suppression followed by an excitation. Despite such different behavioral effects, both peptides produced sensitization to the stimulant effect of amphetamine. These results show that repeated activation of neurotensin receptors produces long-lasting changes in responsiveness to a psychostimulant drug.

GBR 12909 reverses the SCH 23390 inhibition of rewarding effects of brain stimulation.

Curve-shift was used to test the effect of SCH 23390, a dopamine D1 receptor antagonist, and GBR-12909, a selective dopamine uptake blocker, on self-stimulation behavior. GBR 12909 (8 mg/kg) produced a significant decrease in self-stimulation threshold when administered alone, and reversed the increase in threshold produced by SCH 23390 (0.08 mg/kg). The reversal of the SCH 23390 effect by GBR 12909 suggests that the inhibition of self-stimulation produced by SCH 23390 is due to blockade of dopamine receptors.