Apomorphine and haloperidol effects on striatal 3H-dopamine release in anesthetized, awake restrained and freely moving rats

The ability of apomorphine (APO) and haloperidol (HAL) to affect the spontaneous release of newly synthesized 3H-DA in the striatum was studied in halothane anesthetized, gallamine paralyzed, awake restrained and freely moving rats. The striatum was continuously superfused through a push-pull cannula with a physiological medium enriched in 3H-tyrosine. Basal levels of 3H-DA release were different in the four experimental models: highest in halothane anesthetized rats, intermediate in awake restrained and gallamine treated rats and lowest in freely moving rats. In all experimental models IV or SC injection of APO (1 mg/kg) inhibited the release of 3H-DA (30-50%) from 15 to 90 min following its administration. In awake restrained and freely moving rats, stereotyped behaviour was observed for one hour following the APO injection. In halothane anesthetized rats the inhibitory effect of APO on 3H-DA release was prevented by pretreatment with HAL (2 mg/kg IV). Injection of HAL (2 mg/kg IV or SC) failed to enhance the release of 3H-DA in anesthetized and awake restrained rats, whilst a long-lasting increase in 3H-DA release was observed in gallamine treated and freely moving animals (55% and 120% respectively). However, catalepsy was observed in both restrained and freely moving rats. It is concluded that the modifications of 3H-DA release produced by HAL but not those produced by APO are dependent on the experimental model used, a fact possibly related to the different sites of action of these two drugs.     

Apomorphine and haloperidol effects on striatal H-dopamine release in anesthetized, awake restrained and freely moving rats

The ability of apomorphine (APO) and haloperidol (HAL) to affect the spontaneous release of newly synthesized ³H-DA in the striatum was studied in halothane anesthetized, gallamine paralyzed, awake restrained and freely moving rats. The striatum was continuously superfused through a push-pull cannula with a physiological medium enriched in ³H-tyrosine. Basal levels of ³H-DA release were different in the four experimental models: highest in halothane anesthetized rats, intermediate in awake restrained and gallamine treated rats and lowest in freely moving rats. In all experimental models IV or SC injection of APO (1 mg/kg) inhibited the release of ³H-DA (30–50%) from 15 to 90 min following its administration. In awake restrained and freely moving rats, stereotyped behaviour was observed for one hour following the APO injection. In halothane anesthetized rats the inhibitory effect of APO on ³H-DA release was prevented by pretreatment with HAL (2 mg/kg IV). Injection of HAL (2 mg/kg IV or SC) failed to enhance the release of ³H-DA in anesthetized and awake restrained rats, whilst a long-lasting increase in ³H-DA release was observed in gallamine treated and freely moving animals (55% and 120% respectively). However, catalepsy was observed in both restrained and freely moving rats. It is concluded that the modifications of ³H-DA release produced by HAL but not those produced by APO are dependent on the experimental model used, a fact possibly related to the different sites of action of these two drugs.     

Autoradiographic studies on the uptake of 3H-dopamine by neurons and astrocytes in explant and primary cultures of rat CNS: effects of uptake inhibitors

The cellular localization of the uptake of ³H-dopamine was studied in explant and primary cultures from various regions of rat central nervous system by means of autoradiography. In explant cultures of substantia nigra, ³H-dopamine was taken up by cell bodies and processes of many neurons. In cultures from striatum, cerebellum and spinal cord, neuronal cell bodies were not labelled, whereas outgrowing nerve fibres revealed intense uptake of the monoamine. Uptake of ³H-dopamine by neurons was Na+- and temperature-dependent, suggesting an active uptake mechanism. In explant cultures, astrocytes did not accumulate ³H-dopamine, whereas in primary cultures, which were prepared from the same regions of rat central nervous system as the explant cultures, astrocytes also revealed uptake of this monoamine. The intensity of labelling was dependent on the incubation time. Little uptake of ³H-dopamine was observed after an incubation time of 5 min and only after 10–15 min did the astrocytes show moderate labelling. Uptake of ³H-dopamine by astrocytes was not Na+- and temperature-dependent, indicating that glial cells do not possess an active uptake mechanism for this monoamine. This is consistent with biochemical investigations by other laboratories, demonstrating that astrocytes accumulate ³H-dopamine by a facilitated diffusion system. Addition of the uptake inhibitors nomifensine or GBR 12909 to explant cultures markedly reduced or inhibited uptake of ³H-dopamine by neurons at a concentration of 10−6 M. In contrast, accumulation of ³H-dopamine by astrocytes in primary cultures was only slightly affected by nomifensine at 10−6 M. At the highest concentration used (10−5 M), nomifensine also blocked the uptake of ³H-dopamine by astrocytes. Our finding that GBR 12909 almost completely inhibited the uptake of ³H-dopamine by astrocytes already at 10−6 M suggests that this compound is a more potent inhibitor of the glial uptake of dopamine than nomifensine.     

Dopaminergic substrates of intracranial self-stimulation in the caudate-putamen.

An extensive mapping of the caudate-putamen in rat for intracranial self-stimulation (ICS) site was undertaken to provide addtional support for the role of dopamine in brain-stimulation reward. Eight-seven per cent of the placements in the neostriatum supported ICS, with self-stimulation rates greater than 250/15 min at 56% of these sites. Electrical stimulation also elicited rearing and clonus, and contralateral body turn, both of which varied in magnitude between animals. In a second experiment, animals were prepared with electrodes aimed at the lateral caudateputamen. Those subjects displaying ICS subsequently received 6-hydroxydopamine lesions to the dopamine cell bodies in the substantia nigra pars compacta, either ipsilateral or contralateral to the electrode. The destruction of the dopamine cell bodies attenuated ICS in both groups during the first post-lesion test sessions. However, the rates in the ipsilateral group declined to between 2 and 9% of control scores, whereas the rate in the contralateral group improved over testing to 72% of control values, 28 days after the lesion. On the basis of these data, it was concluded that unilateral destruction of the dopaminergic nigro-neostriatal bundle (NSB) has two effects on ICS behavior. First, unilateral reduction of neostriatal dopamine is accompanied by a loss of brain-stimulation reward at sites normally innervated by the NSB, specifically the caudate-putamen. Secondly, lesions of the NSB produce a general disruption in bar-pressing behavior, as evidenced by the attenuation of ICS following contralateral lesions.     

Autoradiographic studies on the uptake of H-dopamine by neurons and astrocytes in explant and primary cultures of rat CNS: effects of uptake inhibitors

The cellular localization of the uptake of ³H-dopamine was studied in explant and primary cultures from various regions of rat central nervous system by means of autoradiography. In explant cultures of substantia nigra, ³H-dopamine was taken up by cell bodies and processes of many neurons. In cultures from striatum, cerebellum and spinal cord, neuronal cell bodies were not labelled, whereas outgrowing nerve fibres revealed intense uptake of the monoamine. Uptake of ³H-dopamine by neurons was Na+- and temperature-dependent, suggesting an active uptake mechanism. In explant cultures, astrocytes did not accumulate ³H-dopamine, whereas in primary cultures, which were prepared from the same regions of rat central nervous system as the explant cultures, astrocytes also revealed uptake of this monoamine. The intensity of labelling was dependent on the incubation time. Little uptake of ³H-dopamine was observed after an incubation time of 5 min and only after 10–15 min did the astrocytes show moderate labelling. Uptake of ³H-dopamine by astrocytes was not Na+- and temperature-dependent, indicating that glial cells do not possess an active uptake mechanism for this monoamine. This is consistent with biochemical investigations by other laboratories, demonstrating that astrocytes accumulate ³H-dopamine by a facilitated diffusion system. Addition of the uptake inhibitors nomifensine or GBR 12909 to explant cultures markedly reduced or inhibited uptake of ³H-dopamine by neurons at a concentration of 10−6 M. In contrast, accumulation of ³H-dopamine by astrocytes in primary cultures was only slightly affected by nomifensine at 10−6 M. At the highest concentration used (10−5 M), nomifensine also blocked the uptake of ³H-dopamine by astrocytes. Our finding that GBR 12909 almost completely inhibited the uptake of ³H-dopamine by astrocytes already at 10−6 M suggests that this compound is a more potent inhibitor of the glial uptake of dopamine than nomifensine.     

In vivo and in vitro effects of acrylamide on synaptosomal neurotransmitter uptake and release

Evidence suggests acrylamide (ACR) neurotoxicity is mediated by impaired presynaptic transmission. To assess the effects of ACR on nerve terminal function, [3H] glutamate release and uptake were determined in brain synaptosomes isolated from intoxicated rats (50mg/kg per day x 8 days, i.p. or 21 mg/kg per day x 21 days, p.o.). Regardless of ACR dose-rate, a significant reduction in synaptosomal K(+)-stimulated, Ca(2+)-dependent release was detected, whereas kinetic analysis of Na(+)-dependent uptake did not reveal consistent changes. Immunoblot analysis showed normal protein levels (e.g. SNAP-25) in dysfunctional synaptosomes isolated from ACR-intoxicated rats. This suggests that defective release does not involve changes in protein synthesis and/or anterograde delivery of presynaptic constituents. To identify potential targets, synaptosomes were exposed in vitro to [ 14C ]-ACR and radiolabeled proteins were separated by gel electrophoresis and detected by autoradiography. [14C]-ACR labeling of distinct synaptosomal protein bands (10.5-154000 kDa) was blocked by the sulfhydryl alkylating agent, N-ethylmaleimide (NEM; 4mM) but not by the non-neurotoxic structural analog propionamide (10mM). In vitro characterization of synaptosomal [3H] glutamate uptake and release showed that ACR, NEM and iodoacetic acid (IAA) produced concentration-dependent decreases in each parameter that were highly correlated to reductions in free sulfhydryl content. All three chemicals were equiefficacious with respect to reducing sulfhydryl content and neurotransmitter uptake/release, although the relative potencies differed; [3H]. Kinetic analysis of uptake showed that in vitro exposure to ACR, IAA or NEM at their respective IC(50)'s caused similar reductions in V(max). These data suggest that ACR-induced synaptic dysfunction involves adduction of presynaptic protein thiol groups and subsequent reduction in neurotransmitter release.     

Improvement of shuttle-box learning with pre- and post-trial intracranial self-stimulation in rats

The effects of intracranial self-stimulation (ICSS) in the lateral hypothalamus upon the acquisition and long-term retention (LTR) of shuttle box avoidance conditioning were studied in Wistar rats. Two groups of subjects learned the avoidance task in 5 daily training sessions and were allowed to self-stimulate either before (Pre-ICSS group), or after (Post-ICSS group) each training session. A control group received training but no ICSS. Ten days following the last training session, LTR of the task was determined in one avoidance session without ICSS. A fourth group was added post-hoc which was allowed to self-stimulate before the training sessions as well as before the LTR test. Both the Post-ICSS and Pre-ICSS groups improved in acquisition of the learned response over the successive training sessions, as compared with Controls. In the LTR test, the animals of the Post-ICSS group maintained the response level achieved in the last acquisition session. In contrast, the subjects of the Pre-ICSS group showed a significant decrease of the same response, unless they were given ICSS treatment prior to the LTR test. This may indicate a 'state-dependent learning' effect being responsible for the decrease in the LTR observed in Pre-ICSS group. Because both pre- and post-training ICSS treatments improved the acquisition and the LTR of the learned response, it is suggested that the contingency of the treatment with training (that is, ICSS treatment immediately after the training sessions) is not a necessary condition to facilitate the acquisition and the consolidation of two-way active avoidance learning.     

Reinforcing versus anticonvulsant drugs: effects on intracranial self-stimulation rate-frequency M50 indices

Drugs of abuse, such as amphetamine and morphine, produce reward-related shifts on intracranial self-stimulation (ICSS) thresholds. The facilitatory effects on ICSS thresholds of drugs that act through the GABAergic system, however, are reported to be attributed to their antiseizure and anticonvulsant effects, rather than their reinforcing effects. Using a rate-frequency ICSS paradigm, we examined the effects of amphetamine (a reinforcing drug of abuse that acts via the catecholaminergic system), pentobarbital (a GABA(A) receptor agonist and reinforcing barbiturate with anticonvulsant properties), and gabapentin (a nonspecific GABAergic agonist and anticonvulsant with low abuse potential) on ICSS M(50) indices. All three doses of amphetamine (0.5, 1.0, and 2.0 mg/kg) and pentobarbital (2.5, 5.0, and 10.0 mg/kg) significantly lowered rate-frequency M(50) values. Gabapentin, on the other hand, significantly raised rate-frequency M(50) values, albeit only at the highest dose administered (30 mg/kg). Our results indicate that shifts in ICSS M(50) values produced by pentobarbital are associated with the reinforcing, not the anticonvulsant, effect of pentobarbital. These results are consistent with the view that there is a common system underlying the reinforcing effects of drugs and ICSS reinforcement, and suggest that the reinforcing and anticonvulsant effects of GABA agonists are dissociable.     

In vitro effects of D-2-hydroxyglutaric acid on glutamate binding, uptake and release in cerebral cortex of rats

Neurological dysfunction is common in patients with D-2-hydroxyglutaric aciduria (DHGA). However, the mechanisms underlying the neuropathology of this disorder are far from understood. In the present study, we investigated the in vitro effects of D-2-hydroxyglutaric acid (DGA) at various concentrations (0.1-1.0 mM) on various parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomal preparations, Na(+)-dependent L-[3H]glutamate uptake by synaptosomal preparations and Na(+)-independent L-[3H]glutamate uptake by synaptic vesicles, as well as of Na(+)-independent and dependent L-[3H]glutamate binding to synaptic plasma membranes from cerebral cortex of male adult Wistar rats. We observed that DGA significantly increased synaptosomal L-[3H]glutamate uptake, without altering the other parameters. Although these findings do not support a direct excitotoxic action for DGA since the metabolite did not affect important parameters of the main neurotransmission system, they do not exclude a direct action of DGA on NMDA or other glutamate receptors. More comprehensive studies are therefore necessary to evaluate the exact role of DGA on neurotransmission.     

Reduced 3H-imipramine binding but unaltered 3H-serotonin uptake in platelets of adolescent enuretics

High affinity 3H-imipramine binding and 3H-serotonin uptake to platelets was evaluated in nine untreated adolescent enuretics (ages 13-18) and nine age- and sex-matched controls. A significant decrease in the maximal binding of 3H-imipramine (Bmax) was observed in the enuretics as compared to the controls. No alteration in the affinity of 3H-imipramine to its binding sites (Kd) or in serotonin uptake kinetic parameters (Vmax, Km) was detected. The lack of correlation between Bmax and Vmax values might indicate that the binding sites for imipramine and the sites for serotonin uptake are not identical.     

Morphine and intracranial self-stimulation in the hypothalamus and dorsal brainstem: differential effects of dose, time and site.

The purpose of this study was to examine if morphine, a drug of abuse, exerts site-specific effects on intracranial self-stimulation (ICSS). Rats, implanted with dorsal brainstem (DB) and hypothalamic (HYP) electrodes, bar-pressed for ICSS at two current intensities eight hours a day during six days each of predrug saline, morphine (2.5, 5.0, 7.5 or 10.0 mg/kg) and postdrug saline conditions. There were three patterns of drug effects: "pure" depressions, "pure" facilitations and a biphasic pattern (depressions followed by facilitations). Repeated morphine administration modified the temporal patterning of these effects: shortened duration of depressions and produced earlier onsets of facilitations. Within an animal, DB electrodes displayed more depressions than the HYP electrodes. Tolerance to the depressant effects, observed frequently, occurred occasionally to the facilitative effects of morphine. The drug effects on ICSS were dissociated from those observed on other behavioral measures, and thus are not artifacts of concomitant changes in activity levels.     

Region-specific reductions of intracranial self-stimulation after uncontrollable stress: Possible effects on reward processes

Rates of responding for intracranial self-stimulation from the medial forebrain bundle, nucleus accumbens and substantia nigra were evaluated in mice that had been exposed to either escapable shock, yoked inescapable shock or no shock treatment. Whereas performance was unaffected by escapable shock, marked reductions of responding from the medial forebrain bundle and nucleus accumbens were evident following the uncontrollable shock treatment. Responding from the substantia nigra was unaffected by the stress treatment. Uncontrollable shock is thought to reduce the rewarding value of responding for electrical brain stimulation from those brain regions in which stressors are known to influence dopamine activity.     

Involvement of the parafascicular nucleus in the facilitative effect of intracranial self-stimulation on active avoidance in rats

To evaluate whether parafascicular nucleus (PF) is involved in the facilitative effect of lateral hypothalamic intracranial self-stimulation (LH-ICSS) on two-way active avoidance acquisition (5 sessions, 10 trials each, one daily) and long-term retention (10 days), rats were lesioned bilaterally at the PF and implanted with an electrode aimed at the LH to obtain ICSS behavior. After each acquisition session rats were allowed to self-administer 2500 trains of LH-ICSS. The main results were: (1) LH-ICSS facilitated the acquisition and retention of conditioning; (2) PF lesions impaired both acquisition and retention of two-way active avoidance; (3) there was a positive relationship between PF lesions size and learning disruption, and (4) LH-ICSS failed to facilitate learning when PF was lesioned. We concluded that the lesion size is a critical variable to evaluate the effects of PF lesions on learning and memory, and that LH-ICSS treatment may exert their effects through the PF nucleus or, at least, the integrity of PF is required for LH-ICSS to improve clearly the task.     

Effect of D-fenfluramine on serotonin release in brains of anaesthetized rats

Using in vivo microdialysis of brains of anaesthetized rats, we have examined the acute and chronic effects of D-fenfluramine on the release of serotonin (5-HT) and 5-HIAA within the frontal cortex, the lateral hypothalamus and the nucleus accumbens. A single dose of the drug (10 mg/kg) stimulated 5-HT release by 331-810% and decreased 5-hydroxyindoleacetic acid (5-HIAA) release by 30%, within all 3 brain areas. These changes were maximal 30 min after drug administration, and values returned to baseline after 120 min. Among animals receiving D-fenfluramine (3 or 10 mg/kg, i.p.) daily for 8 days and examined 24 h after the last dose, the basal release of 5-HT from frontal cortex was unaffected. However, the levels of 5-HT in this region, and its evoked release after a subsequent dose of D-fenfluramine (10 mg/kg), were significantly reduced in animals that had received the larger chronic dose. 5-HT release was restored to normal if such rats were given tryptophan (100 mg/kg, i.p.) 1 h prior to the acute D-fenfluramine dose; moreover, 5-HT release from, and levels in, frontal cortex also returned to normal without additional treatment after a 28-day washout period. These observations suggest that the chronic administration of D-fenfluramine fails to affect spontaneous 5-HT release in rat brain, and reduces the release evoked by acute D-fenfluramine only when very high doses are given. Moreover, this reduction is reversible with time or with administration of 5-HT's circulating precursor, tryptophan.     

Application of in vivo electrochemistry to the measurement of changes in dopamine release during intracranial self-stimulation

Stearate-modified graphite paste recording electrodes were acutely or chronically implanted into the nucleus accumbens along with bipolar stimulating electrodes in the ipsilateral ventral tegmental area (VTA). Chronoamperometry was used to monitor changes in electrochemical signals that may correspond to the oxidation of dopamine (DA) during experimenter-administered stimulation (EAS) and intracranial self-stimulation (ICS). Application of EAS to stimulating electrodes in the VTA produced increases in the electrochemical signal in both the anesthetized and conscious preparation. The magnitude of both effects increased as a function of current intensity. Initiation of ICS was also accompanied by an immediate increase in the electrochemical signal. Rate-intensity experiments revealed a corresponding increase in both the ICS rates and the electrochemical signal with successive increases or decreases in current intensity. In subsequent experiments, intraperitoneal injections of DA uptake blockers nomifensine and GBR-12909 produced significant increases in the amplitude of the chronoamperometric signal which corresponded to drug-induced increases in bar press rates. The noradrenergic uptake blocker desipramine had no significant effect on either ICS rates or oxidation current. These data indicate that ICS of the VTA may produce concurrent increases in DA neurotransmission in the nucleus accumbens. The pharmacological studies are consistent with a dopaminergic substrate of brain stimulation reward at electrode sites in the VTA.     

Sensitization to the effects of repeated amphetamine administration on intracranial self-stimulation: Evidence for changes in reward processes

The effects of daily administration of 1.0 mg/kg of d-amphetamine for 20 consecutive days on self-stimulation responding from the substantia nigra, and nucleus accumbens were evaluated at several current intensities. Raising current intensity increased rates of responding when electrodes were situated in these areas, and amphetamine significantly enhanced rates of responding from both brain regions. Moreover, the drug-induced response enhancements were facilitated further after repeated drug/test pairings. Although response sensitization was observed at several current intensities, it developed sooner at the lower current levels indicating that the sensitizing effect of repeated drug administration on self stimulation responding was not due to variations in locomotor activity or arousal levels induced by amphetamine treatment. Furthermore, sensitization was observed at current levels that engendered both high and low levels of responding, suggesting that the sensitization was unrelated to the rate dependent effects of the drug. Rather, it was argued that repeated amphetamine treatment sensitized animals to the rewarding properties of electrical brain stimulation. Possible neurochemical and behavioral mechanisms that may be involved in the development of reverse tolerance after repeated amphetamine treatment were discussed.