ALX 5407: a potent, selective inhibitor of the hGlyT1 glycine transporter.

High-affinity glycine transport in neurons and glial cells is a primary means of inactivating synaptic glycine. We have synthesized a potent selective inhibitor of glycine transporter 1 (GlyT1), and characterized its activity using a quail fibroblast cell line (QT6). The glycine transporters GlyT1A, GlyT1B, GlyT1C, and GlyT2 were stably expressed in QT6 cells. The transporters expressed in these cells exhibited appropriate characteristics as described previously for these genes: Na(+)/Cl(-) dependence, appropriate K(m) values for glycine uptake, and appropriate pharmacology, as defined in part by the ability of N-methyl glycine (sarcosine) to competitively inhibit glycine transport. Furthermore, the characteristics of the transporters in the cell lines recapitulate the characteristics of glycine transporters observed in tissue preparations. We developed a sarcosine derivative, (R)-(N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (ALX 5407), and examined its activity against the cloned glycine transporters. ALX 5407 completely inhibited glycine transport in the GlyT1 cells, with an IC(50) value of 3 nM, but had little or no activity at the human GlyT2 transporter, at other binding sites for glycine, or at other neurotransmitter transporters. The inhibition of glycine transport was essentially irreversible. ALX 5407 represents a novel tool in the investigation of N-methyl-D-aspartate-receptor function. This class of drug may lead to novel therapies in the treatment of schizophrenia.

Dopamine depletion reorganizes projections from the nucleus accumbens and ventral pallidum that mediate opioid-induced motor activity.

Motor activity elicited pharmacologically from the nucleus accumbens by the mu-opioid receptor agonist D-Ala-Tyr-Gly-NMePhe-Gly-OH (DAMGO) is augmented in rats sustaining dopamine depletions. GABAergic projections from the nucleus accumbens to ventral pallidum and ventral tegmental area (VTA) are involved because stimulation of GABAB receptors in the VTA (by baclofen) or GABAA receptors in the ventral pallidum (by muscimol) inhibit the motor response induced by the microinjection of DAMGO into the nucleus accumbens. The present study was done to determine which of these projections is mediating the augmented DAMGO-induced motor activity that follows 6-hydroxydopamine lesions of the nucleus accumbens. The inhibition of DAMGO-induced activation by pallidal injections of muscimol was markedly attenuated in lesioned animals, whereas the inhibition by VTA injections with baclofen was greatly enhanced. A similar switch in emphasis from pallidal to mesencephalic efferents was not observed for dopamine-induced motor activity, because muscimol microinjections inhibited the response elicited by dopamine microinjection into the nucleus accumbens in all subjects. The stimulation of mu-opioid receptors in the ventral pallidum also elicits motor activation, and this is blocked by baclofen microinjection into the VTA. However, after dopamine depletion in the nucleus accumbens, baclofen in the VTA was ineffective in blocking the motor response by DAMGO in the ventral pallidum. These data reveal that dopamine depletion in the nucleus accumbens produces a lesion-induced plasticity that alters the effect of mu-opioid receptor stimulation on efferent projections from the nucleus accumbens and ventral pallidum.

Effects of chronic haloperidol on stress- and stimulation-induced increases in dopamine release: tests of the depolarization block hypothesis.

There is considerable neurophysiological evidence that chronically administered neuroleptics can, under certain circumstances, decrease the activity of mesencephalic dopaminergic neurons. This finding, referred to as depolarization inactivation or depolarization block, has led to the hypothesis that the delayed therapeutic effects of neuroleptic drugs are due to a graduate silencing of mesolimbic dopaminergic neurons. One prediction of depolarization inactivation is that dopamine neurons in this state should be resistant to activation by excitatory stimuli. As a test of this prediction, rats that had been treated chronically with either saline or haloperidol (0.5 mg/kg x 21 days) were exposed to either acute mild stress or electrical stimulation of the prelimbic region of the prefrontal cortex while extracellular levels of dopamine in the nucleus accumbens were monitored by in vivo microdialysis. A 10-minute exposure to acute stress via tail pinch increased dopamine release by 20% and 18% in the saline and haloperidol groups, respectively. Similarly, 20 minutes of cortical stimulation increased dopamine release by 51% and 56% in rats treated chronically with saline or haloperidol, respectively. These results indicate that contrary to a prediction of the depolarization block hypothesis, mesolimbic dopaminergic neurons can be activated in neuroleptic-treated animals.

Involvement of the ventral tegmental area in locomotion elicited from the nucleus accumbens or ventral pallidum.

This study was designed to evaluate the role of the circuit containing the nucleus accumbens, ventral pallidum (VP) and ventral tegmental area (VTA) in the motor stimulation produced by the microinjection of dopamine, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) or [D-Ala2, MePhe4,Gly-ol5]enkephalin (DAMGO) into VP or the shell and core compartments of the nucleus accumbens. Initial dose-response curves revealed that dopamine was approximately equipotent at producing motor activity after microinjection into the core and shell, AMPA was more effective in the core, whereas DAMGO was more potent in the shell. A role for the VTA in the motor responses elicited by dopamine, AMPA or DAMGO microinjection into the shell, core or VP was evaluated by microinjecting the tau-aminobutyric acidB agonist baclofen into the VTA to inhibit neuronal activity. Baclofen treatment abolished the motor responses elicited by AMPA from the shell, core and VP. The motor effect of DAMGO in the VP was abolished by baclofen, whereas the response in the shell was attenuated. The motor response to dopamine was unaltered by baclofen, regardless of the injection site. These data indicate that there exist differences between the core and shell of the nucleus accumbens in the capacity of neurotransmitter analogs to elicit motor activity, and that although AMPA-induced motor activity is dependent upon neurotransmission in the VTA after microinjection into the core, shell and VP, DAMGO-induced locomotion only requires such tone after microinjection into the VP and shell.

Substance P in the ventral pallidum: projection from the ventral striatum, and electrophysiological and behavioral consequences of pallidal substance P.

The ventral pallidum of the basal forebrain contains a high concentration of substance P and receives a massive projection from the nucleus accumbens. The present study was designed to determine whether the accumbens serves as a source for substance P-containing fibers in the ventral pallidum and characterize the function of this tachykinin peptide within the ventral pallidum. By combining in situ hybridization for messenger RNA of the substance P prohormone, beta-preprotachykinin, with Fluoro-Gold retrograde labeling from iontophoretic deposits in the ventral pallidum, a population of substance P-containing neurons was demonstrated in the shell and core components of the nucleus accumbens and the ventromedial striatum. The function of substance P within the ventral pallidum was characterized at the level of the single neuron, and the behaving animal. Electrophysiological assessment revealed that approximately 40% of the 97 ventral pallidal neurons tested were readily excited by microiontophoretic applications of substance P or a metabolically stable agonist analog, DiMeC7 [(pGlu5, MePhe8, MeGly9)-substance P5-11]. Response characteristics were distinguished from glutamate-induced excitations by a slower onset and longer duration of action. Recording sites of tachykinin-sensitive neurons were demonstrated to be located throughout the ventral pallidum and within high densities of fibers exhibiting substance P-like immunoreactivity. When behaving rats received microinjections of DiMeC7 into this same region, the animals displayed an increase in motor activity, with a response threshold of 0.1nmol per hemisphere. These results verify the existence of a substantial substance P-containing projection from the nucleus accumbens to the ventral pallidum. The projection likely serves to excite ventral pallidal neurons for these neurons readily increased firing following local exposure to tachykinins. Furthermore, an increase in motor behavior appears to be a consequence of this neuronal response.

Behavioral and neurochemical effects of opioids in the paramedian midbrain tegmentum including the median raphe nucleus and ventral tegmental area.

Injections of morphine into the median raphe nucleus (MR) of rats produced a dose-dependent, naloxone sensitive increase in locomotor activity. Dose-dependent increases in activity also could be produced by intra-MR injections of the mu-opioid agonist Tyr-D-Ala-Gly-MePhe-Gly(ol)-enkephalin (DAMGO) and the delta-opioid agonist D-Pen2,D-Pen5-enkephalin (DPDPE), but not by the kappa-opioid agonist Dynorphin A (1-13). Mapping studies demonstrated that DPDPE produced larger responses when injected into the MR than into a number of adjacent structures, whereas the effective zone for obtaining responses with DAMGO appeared to extend forward into the caudal portion of the ventral tegmental area. The induction of hyperactivity by DPDPE and DAMGO was unaltered in animals with large depletions of forebrain serotonin produced by injections of 5,7-dihydroxytryptamine, suggesting that these effects were not mediated through serotonergic mechanisms. Post-mortem assays indicated that serotonin turnover in the hippocampus was reduced slightly after intra-MR injections of DPDPE, but no effects were observed after injections of DAMGO or Dynorphin A (1-13). Injections of either DPDPE or DAMGO into the MR resulted in a large increase in dopamine turnover in the nucleus accumbens. Finally, intra-MR injections of DAMGO or Dynorphin A(1-13), but not DPDPE, stimulated ingestive behavior in nondeprived animals, although the effects were substantially smaller than those seen after injections of muscimol. These results demonstrate that pronounced behavioral and neurochemical effects can be produced by stimulation of opioid receptors within the MR and that the pattern of these effects depends upon which opioid receptor subtype is stimulated.

Receptor mechanisms mediating clozapine-induced c-fos expression in the forebrain.

The atypical antipsychotic clozapine produces distinctly different regional patterns of c-fos expression in rat forebrain than does the prototypical neuroleptic haloperidol. While haloperidol-induced c-fos expression appears to be mediated by its D2 dopamine receptor antagonist properties, the mechanisms by which clozapine increases c-fos expression remain uncertain. Using a combination of brain lesion, pharmacological and immunohistochemical techniques, the present study sought to determine the receptor mechanisms by which clozapine increases the number of Fos-like immunoreactive neurons in various regions of the forebrain. To test whether serotonergic and/or noradrenergic systems are involved in clozapine-induced c-fos expression, rats received either 5,7-dihydroxytryptamine lesions of the medial forebrain bundle or 6-hydroxydopamine lesions of the dorsal noradrenergic bundle two weeks prior to clozapine (20 mg/kg) injections. Neither type of lesion affected clozapine-induced c-fos expression in the rat forebrain, suggesting that neither serotonergic nor noradrenergic mechanisms are involved in this action of clozapine. In another experiment, the 5-hydroxytryptamine2 receptor antagonist ritanserin (5 mg/kg), either alone or in combination with haloperidol (1 mg/kg), failed to mimic the pattern of c-fos expression produced by clozapine. This suggests that clozapine's antagonist actions at 5-hydroxytryptamine2 receptors cannot explain the unique pattern of regional c-fos expression produced by this compound. To determine whether the blockade of subtypes of the D2 dopamine receptor family may contribute to clozapine's effects, the dopamine receptor agonists quinpirole and 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) were injected 15 min prior to clozapine. Quinpirole produced a small but significant decrease in clozapine-induced c-fos expression in the medial prefrontal cortex, had larger effects in the lateral septum, and blocked clozapine's actions in the nucleus accumbens and major island of Calleja. Pretreatment with 7-OH-DPAT attenuated clozapine-induced c-fos expression in the nucleus accumbens and lateral septum, completely blocked the expression in the major island of Calleja, but was without effect in the medial prefrontal cortex. Given the different affinities of quinpirole and 7-OH-DPAT for D2, D3 and D4 receptors, these data suggest that clozapine-induced increases in c-fos expression in the nucleus accumbens, major island of Cajella and lateral septal nucleus are due to antagonist actions of this antipsychotic at D3 dopamine receptors. They also indicate that while antagonist actions at D4 receptors may contribute, the primary mechanisms by which clozapine increases c-fos expression in the medial prefrontal cortex remain to be determined.

Cocaine and d-amphetamine increase c-fos expression in the rat cerebellum.

Psychostimulant drugs have been reported to increase the expression of some immediate-early genes in the cerebellum. In the present study, immunohistochemical techniques were used to assess the pattern of c-fos expression in the cerebellum produced by d-amphetamine or cocaine. Systemic administration of d-amphetamine (1.5, 6 mg/kg) or cocaine (10, 20 mg/kg) increased locomotor activity, which at low doses was blocked by pretreatment with the dopamine D1 receptor antagonist SCH 23390 (1 mg/kg). Within the cerebellum, basal levels of c-fos expression were abolished by SCH 23390, with the exception of lobule VI. Dose-dependent increases in Fos-like immunoreactivity were elicited by d-amphetamine and cocaine. Pretreatment with SCH 23390 greatly reduced the extent to which either stimulant increased c-fos expression. Psychostimulant-induced Fos-like immunoreactive nuclei were generally restricted to the granule cell layer within each of the midvermal cerebellar lobules (I-X), although occasional nuclei were found in the Purkinje cell layer. In addition, a homogeneous pattern of Fos-like immunoreactive nuclei, of sparse density, was also found near the pial surface of the molecular layer following d-amphetamine but not cocaine. Within the granule cell layer dense clusters of Fos-like immunoreactive neurons extended from the molecular layer to the Purkinje cell layer and were found at both the pial surface as well as in the deep portions of individual folia. These data add to a growing body of evidence indicating that the induction of regionally specific alterations in c-fos expression by psychostimulants is mediated via a D1 receptor mechanism.

Behavioral and neurochemical studies of opioid effects in the pedunculopontine nucleus and mediodorsal thalamus.

The brain circuitry mediating spontaneous and psychostimulant-induced locomotion comprises, in part, connections between the ventral tegmental area, nucleus accumbens and ventral pallidum. Two primary efferent projections from the ventral pallidum are to the mediodorsal thalamic nucleus (MD) and the pedunculopontine nucleus (PPN), including the mesencephalic motor area. To assess the functional role of the PPN and MD in this motor circuit, the behavioral and neurochemical effects of intra-PPN and intra-MD administration of the mu opioid receptor agonist Tyr-D-Ala-Gly-MePhe-Gly(ol) (DAMGO) were examined. Bilateral microinjections of DAMGO into either the PPN or MD elicited a dose-dependent increase in motor activity which was blocked by pretreatment with naloxone (2.0 mg/kg i.p.). Three studies were conducted to evaluate a role for mesoaccumbens dopamine transmission in DAMGO-induced motor activity. Systemic administration of the dopamine antagonist, heloperidol (0.1 mg/kg i.p.) produced a partial antagonism of the motor effect elicited by DAMGO in the MD, but abolished the response to DAMGO in the PPN. Inhibition of dopamine neurons by microinjecting the gamma-aminobutyric acidB agonist, baclofen (0.15 nmol/side), into the ventral tegmental area attenuated the motor activity elicited by DAMGO in the PPN but was without effect on DAMGO in the MD. Finally, microdialysis revealed that DAMGO microinjection into either the PPN or MD elicited a dose-related increase in extracellular dopamine content in the nucleus accumbens. However, only after DAMGO in the PPN were extracellular levels of dopamine metabolites increased. These results demonstrate that the motor stimulant response to DAMGO in the PPN is dopamine dependent and involves stimulation of mesoaccumbens dopamine neurons. In contrast, the motor response by DAMGO in the MD only partly involves dopaminergic mechanisms, perhaps via a presynaptic action because the effect was not altered by inhibiting impulse flow in mesoaccumbens dopamine neurons with baclofen.

GABA and enkephalin projection from the nucleus accumbens and ventral pallidum to the ventral tegmental area.

GABAergic and enkephalinergic afferents to the ventral tegmental area were investigated in the rat using retrograde tracing techniques combined with in situ hybridization. Following iontophoretic deposit of Fluoro-Gold in the ventral tegmental area labeling in the forebrain was most dense in the shell of the nucleus accumbens, rostral ventromedial ventral pallidum and diagonal band of Broca. A smaller density was also observed in the lateral septum. In these forebrain regions, the portion of retrogradely labeled cells that contained mRNA for glutamate decarboxylase ranged from 25% to 50%, whereas only 5% to 15% were double-labeled for preproenkephalin mRNA. Cells double-labeled with either glutamate decarboxylase or preproenkephalin mRNA were most numerous in the lateral septum, shell of the nucleus accumbens, rostral ventral pallidum and diagonal band of Broca. Large Fluoro-Gold deposits which invaded the medial substantia nigra resulted in a significant number of retrogradely labeled cells in the core of the nucleus accumbens, and a portion of these neurons also contained mRNA for glutamate decarboxylase or preproenkephalin. These data demonstrate the presence of GABAergic and enkephalinergic neurons projecting from the nucleus accumbens, ventral pallidum and diagonal band of Broca to the ventral tegmental area.

Topography and functional role of dopaminergic projections from the ventral mesencephalic tegmentum to the ventral pallidum.

A dopaminergic projection from the ventral tegmental area to the ventral pallidum was identified in the rat using anterograde tract tracing and combined retrograde tracing-immunocytochemistry. The projection was found to be topographically organized such that fibers innervating the ventromedial ventral pallidum arose from neurons located along the midline nuclei of the ventral mesencephalon, including the nucleus interfascicularis and nucleus linearis caudalis. Ventral tegmental neurons situated more laterally, in the nucleus parabrachialis pigmentosus and nucleus paranigralis, projected to the ventromedial and dorsolateral ventral pallidum. The substantia nigra did not supply a major contribution to this projection. The proportion of ventral tegmental area dopaminergic neurons projecting to the ventral pallidum ranged from approximately 30% to 60%. The functional significance of the projection is indicated since intra-ventral pallidum microinjections of dopamine elicited a dose-dependent increase in locomotor activity. Furthermore, whereas pretreatment of the ventral pallidum with the GABAA agonist muscimol has been shown to attenuate opioid-induced locomotor activity elicited from the ventral pallidum, it did not attenuate the dopamine-induced motor response. Thus, while mu-opioids in the ventral pallidum may presynaptically regulate GABAergic efferents from the nucleus accumbens, it appears that the dopaminergic input directly influences the ventral pallidal output neuron which is involved in locomotion.

Chronic neuroleptic administration decreases extracellular GABA in the nucleus accumbens but not in the caudate-putamen of rats.

The extracellular levels of gamma-aminobutyric acid (GABA) in the caudate-putamen and the nucleus accumbens of rats following administration of haloperidol decanoate, fluphenazine decanoate, or vehicle for 8 months were assessed using intracranial microdialysis. Basal levels of extracellular GABA were significantly decreased in the nucleus accumbens of both neuroleptic-treated groups while levels of GABA in the caudate-putamen were not significantly different between groups. These results provide evidence for selective chronic neuroleptic-induced effects on in vivo GABA function in different terminal regions containing dopamine receptors.

Regulation of somatodendritic dopamine release in the ventral tegmental area by opioids and GABA: an in vivo microdialysis study.

Microdialysis of the ventral tegmental area in conscious rats was used to evaluate the influence of opioids and GABA agonists on extracellular levels of GABA and somatodendritically released dopamine. The administration of morphine through the dialysis probe elicited significant, dose-dependent increases in the levels of extracellular dopamine and significantly reduced the extracellular concentration of GABA. In contrast, a dose-dependent decrease in somatodendritic extracellular dopamine was produced following the administration of the GABAB agonist baclofen. The increase in dopamine levels elicited by morphine (100 microM) was completely blocked by either baclofen (100 microM) coadministration or peripheral injection of naloxone (2 mg/kg, i.p.). Application of the GABAA agonist muscimol produced a significant increase in both extracellular levels of dopamine and locomotor activity. The present results, together with other electrophysiological, neurochemical, and behavioral data, support a hypothesis that stimulation of mu-opioid or GABAA receptors inhibits the activity of GABAergic afferents to dopamine neurons, thereby removing tonic inhibitory regulation, whereas stimulation of GABAB receptors directly inhibits dopamine neurons.

Elicitation of feeding, drinking, and gnawing following microinjections of muscimol into the median raphe nucleus of rats.

Previous studies have demonstrated that injections of muscimol into the median raphe nucleus (MR) result in large increases in locomotor activity and food intake. The current experiment extends these results by showing that intra-MR muscimol injections in nondeprived rats also elicit nonprandial drinking and gnawing of wooden blocks. These findings indicate that stimulation of GABA receptors within the MR is able to energize a wide range of oral behaviors and is compatible with the view that the MR may be part of a "nonspecific" behavioral activation system.

Comparative studies of locomotor behavior following microinjections of muscimol into various sites in the paramedian tegmentum.

Microinjections of various doses of muscimol into the median raphe nucleus, the dorsal raphe nucleus or the caudal portion of the ventral tegmental area elicited dose-dependent increases in locomotor activity. In contrast, injections into the rostral portion of the ventral tegmental area or the midline pontine tegmentum caudal to the median raphe were ineffective. Lower doses of muscimol were required to produce hyperactivity after injections into the median raphe than after injections into any of the other sites. These findings suggest that the median raphe nucleus is the most sensitive site in the paramedian tegmentum for the elicitation of hyperactivity by muscimol.

Is dopamine involved in the hyperactivity produced by injections of muscimol into the median raphe nucleus?

Many studies have shown that experimental manipulations of the median raphe nucleus are able to produce profound effects on locomotor activity. Other data indicate that the raphe nuclei may exert an inhibitory influence over dopamine systems projecting to the forebrain. These results raise the possibility that the median raphe's influence over locomotion may be mediated through alterations in forebrain dopamine release. We examined this possibility in the current report by studying the role of dopamine in the hyperactivity produced by microinjections of the GABA agonist muscimol into the median raphe. Muscimol injections resulted in pronounced hyperactivity which was accompanied by a decrease in serotonin metabolism within the hippocampus and an increase in dopamine metabolism within the nucleus accumbens. Systemic injections of high doses of the dopamine antagonist haloperidol, however, were not able to attenuate muscimol's effect on activity. These results suggest that dopaminergic mechanisms do not play an essential role in mediating the effects of intraraphe muscimol on locomotor activity.

Comparative studies of the ingestive behaviors produced by microinjections of muscimol into the midbrain raphe nuclei of the ventral tegmental area of the rat.

Microinjection of the GABA-A agonist muscimol into the median (MR) or dorsal (DR) raphe nuclei or the ventral tegmental area (VTA) of non-deprived rats induced intense feeding and drinking in a dose-dependent and site-specific manner. Lower doses of muscimol were required to increase food intake, spillage and water intake with injections into the MR than with injections into the other two sites. These data demonstrate that the MR is a more sensitive site for the elicitation of ingestive behavior than either the DR or the VTA.

Evidence against serotonin involvement in the hyperactivity produced by injections of muscimol into the median raphe nucleus.

Microinjections of muscimol into the median raphe nucleus were found to result in pronounced hyperactivity which could not be attenuated by the serotonin depletion produced either by systemic treatment with p-chlorophenylalanine or by intra-raphe injections of 5,7-dihydroxytryptamine. Furthermore, hyperactivity could not be produced by intra-median raphe injections of serotonin or of fenfluramine, compounds which would be expected to inhibit serotonergic raphe cells. These results argue strongly against an essential involvement of serotonin in mediating the effects of intra-median raphe muscimol injections. Muscimol failed to produce hyperactivity, however, when injected into rats who had previously received an electrolytic median raphe lesion. This finding suggests that muscimol injected into the median raphe produces hyperactivity as a result of an action on local cell bodies, rather than by diffusion to a distant site. The simplest explanation of the current results is that muscimol injected into the median raphe produces hyperactivity as a result of an inhibition of nonserotonergic cells within the median raphe nucleus.