Long-lasting alterations of hippocampal GABAergic neurotransmission in adult rats following perinatal Δ9-THC exposure.

The long-lasting effects of gestational cannabinoids exposure on the adult brain of the offspring are still controversial. It has already been shown that pre- or perinatal cannabinoids exposure induces learning and memory disruption in rat adult offspring, associated with permanent alterations of cortical glutamatergic neurotransmission and cognitive deficits. In the present study, the risk of long-term consequences induced by perinatal exposure to cannabinoids on rat hippocampal GABAergic system of the offspring, has been explored. To this purpose, pregnant rats were treated daily with Delta9-tetrahydrocannabinol (Δ9-THC; 5mg/kg) or its vehicle. Perinatal exposure to Δ9-THC induced a significant reduction (p<0.05) in basal and K+-evoked [3H]-GABA outflow of 90-day-old rat hippocampal slices. These effects were associated with a reduction of hippocampal [3H]-GABA uptake compared to vehicle exposed group. Perinatal exposure to Δ9-THC induced a significant reduction of CB1 receptor binding (Bmax) in the hippocampus of 90-day-old rats. However, a pharmacological challenge with either Δ9-THC (0.1µM) or WIN55,212-2 (2µM), similarly reduced K+-evoked [3H]-GABA outflow in both experimental groups. These reductions were significantly blocked by adding the selective CB1 receptor antagonist SR141716A. These findings suggest that maternal exposure to cannabinoids induces long-term alterations of hippocampal GABAergic system. Interestingly, previous behavioral studies demonstrated that, under the same experimental conditions as in the present study, perinatal cannabinoids exposure induced cognitive impairments in adult rats, thus resembling some effects observed in humans. Although it is difficult and sometimes misleading to extrapolate findings obtained from animal models to humans, the possibility that an alteration of hippocampus aminoacidergic transmission might underlie, at least in part, some of the cognitive deficits affecting the offspring of marijuana users, is supported.

Functional role of striatal A2A, D2, and mGlu5 receptor interactions in regulating striatopallidal GABA neuronal transmission.

In this study, the functional role of individual striatal receptors for adenosine (A2AR), dopamine (D2R), and the metabotropic glutamate receptor mGlu5R in regulating rat basal ganglia activity was characterized in vivo using dual-probe microdialysis in freely moving rats. In particular, intrastriatal perfusion with the D2R agonist quinpirole (10 µM, 60 min) decreased ipsilateral pallidal GABA and glutamate levels, whereas intrastriatal CGS21680 (A2AR agonist; 1 µM, 60 min) was ineffective on either pallidal GABA and glutamate levels or the quinpirole-induced effects. Intrastriatal perfusion with the mGlu5R agonist (RS)-2-chloro-5-hydroxyphenylglycine (600 µM, 60 min), by itself ineffective on pallidal GABA and glutamate levels, partially counteracted the effects of quinpirole. When combined with CGS21680 (1 µM, 60 min), (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 600 µM, 60 min) fully counteracted the quinpirole (10 µM, 60 min)-induced reduction in ipsilateral pallidal GABA and glutamate levels. These effects were fully counteracted by local perfusion with the mGlu5R antagonist MPEP (300 µM) or the A2AR antagonist ZM 241385 (100 nM). These results suggest that A2ARs and mGlu5Rs interact synergistically in modulating the D2R-mediated control of striatopallidal GABA neurons. Using dual-probe microdialysis, we characterized the functional role of striatal adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R), and metabotropic glutamate receptor 5 (mGluR5) interactions in regulating rat basal ganglia activity. The results suggest the possible usefulness of using an A2AR antagonist and mGluR5 antagonist combination in the treatment of Parkinson's disease to increase the inhibitory D2 signaling on striatopallidal GABA neurons.

GET73 Prevents Ethanol-Induced Neurotoxicity in Primary Cultures of Rat Hippocampal Neurons.

AIMS: N-[(4-trifluoromethyl) benzyl] 4-methoxybutyramide (GET73) may be considered a promising therapeutic agent for the treatment of alcohol use disorders. The compound displayed anti-alcohol and anxiolytic properties in rat. In the present study, an in vitro experimental model of chronic ethanol treatment was used to investigate the ability of the compound to counteract the ethanol-induced neurotoxicity. METHODS: Primary cultures of rat hippocampal neurons were exposed to ethanol (75 mM; 4 days) and the neuroprotective effects of GET73 were assessed by evaluating cell viability, cell morphology, glutamate levels and reactive oxygen species production. RESULTS: The exposure to ethanol induced a reduction of cell viability, an alteration of cytoskeleton, a decrease in extracellular glutamate levels and an increase of reactive oxygen species production. The addiction of GET73 (1 and 10 µM) 1 h before and during chronic ethanol exposure prevented all the above ethanol-induced effects. Based on the proposed GET73 mechanism of action, the effects of mGlu5 receptor negative allosteric modulator, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), on ethanol-induced reduction of cell viability were also assessed. The results indicated that the addiction of MPEP (100 µM) 1 h before and during chronic ethanol exposure prevented the ethanol-induced cell viability reduction. CONCLUSION: The present findings provide the first evidence that GET73 shows a neuroprotective role against ethanol-induced neurotoxicity in primary cultures of rat hippocampal neurons. Together with previous findings, these results suggest that GET73 possesses multifaceted properties thus lending further support to the significance of developing GET73 as a therapeutic tool for use in the treatment of alcohol use disorders.

Understanding the Functional Plasticity in Neural Networks of the Basal Ganglia in Cocaine Use Disorder: A Role for Allosteric Receptor-Receptor Interactions in A2A-D2 Heteroreceptor Complexes.

Our hypothesis is that allosteric receptor-receptor interactions in homo- and heteroreceptor complexes may form the molecular basis of learning and memory. This principle is illustrated by showing how cocaine abuse can alter the adenosine A2AR-dopamine D2R heterocomplexes and their receptor-receptor interactions and hereby induce neural plasticity in the basal ganglia. Studies with A2AR ligands using cocaine self-administration procedures indicate that antagonistic allosteric A2AR-D2R heterocomplexes of the ventral striatopallidal GABA antireward pathway play a significant role in reducing cocaine induced reward, motivation, and cocaine seeking. Anticocaine actions of A2AR agonists can also be produced at A2AR homocomplexes in these antireward neurons, actions in which are independent of D2R signaling. At the A2AR-D2R heterocomplex, they are dependent on the strength of the antagonistic allosteric A2AR-D2R interaction and the number of A2AR-D2R and A2AR-D2R-sigma1R heterocomplexes present in the ventral striatopallidal GABA neurons. It involves a differential cocaine-induced increase in sigma1Rs in the ventral versus the dorsal striatum. In contrast, the allosteric brake on the D2R protomer signaling in the A2AR-D2R heterocomplex of the dorsal striatopallidal GABA neurons is lost upon cocaine self-administration. This is potentially due to differences in composition and allosteric plasticity of these complexes versus those in the ventral striatopallidal neurons.

Kynurenic acid, by targeting α7 nicotinic acetylcholine receptors, modulates extracellular GABA levels in the rat striatum in vivo.

Kynurenic acid (KYNA) is an astrocyte-derived non-competitive antagonist of the α7 nicotinic acetylcholine receptor (α7nAChR) and inhibits the NMDA receptor (NMDAR) competitively. The main aim of the present study was to examine the possible effects of KYNA (30 - 1000 nm), applied locally by reverse dialysis for 2 h, on extracellular GABA levels in the rat striatum. KYNA concentration-dependently reduced GABA levels, with 300 nm KYNA causing a maximal reduction to ~60% of baseline concentrations. The effect of KYNA (100 nm) was prevented by co-application of galantamine (5 µm), an agonist at a site of the α7nAChR that is very similar to that targeted by KYNA. Infusion of 7-chlorokynurenic acid (100 nm), an NMDAR antagonist acting selectively at the glycineB site of the receptor, affected neither basal GABA levels nor the KYNA-induced reduction in GABA. Inhibition of endogenous KYNA formation by reverse dialysis of (S)-4-(ethylsulfonyl)benzoylalanine (ESBA; 1 mm) increased extracellular GABA levels, reaching a peak of 156% of baseline levels after 1 h. Co-infusion of 100 nm KYNA abolished the effect of ESBA. Qualitatively and quantitatively similar, bi-directional effects of KYNA on extracellular glutamate were observed in the same microdialysis samples. Taken together, the present findings suggest that fluctuations in endogenous KYNA levels, by modulating α7nAChR function, control extracellular GABA levels in the rat striatum. This effect may be relevant for a number of physiological and pathological processes involving the basal ganglia.

Dopamine D2 receptor signaling dynamics of dopamine D2-neurotensin 1 receptor heteromers.

Biochemical, histochemical and coimmunoprecipitation experiments have indicated the existence of antagonistic dopamine D2 (D2R) and neurotensin 1 (NTS1R) receptor-receptor interactions in the dorsal and ventral striatum indicating a potential role of these receptor-receptor interactions in Parkinson's disease and schizophrenia. By means of Bioluminiscence Resonance energy transfer (BRET(2)) evidence has for the first time been obtained in the current study for the existence of both D2LR/NTS1R and D2SR/NTS1R heteromers in living HEK293T cells. Through confocal laser microscopy the NTS1R(GFP2) and D2R(YFP) were also shown to be colocated in the plasma membrane of these cells. A bioinformatic analysis suggests the existence of a basic set of three homology protriplets (TVM, DLL and/or LRA) in the two participating receptors which may contribute to the formation of the D2R/NTS1R heteromers by participating in guide-clasp interactions in the receptor interface. The CREB reporter gene assay indicated that the neurotensin receptor agonist JMV 449 markedly reduced the potency of the D2R like agonist quinpirole to inhibit the forskolin induced increase of the CREB signal. In contrast, the neurotensin agonist was found to markedly increase the quinpirole potency to activate the MAPK pathway as also studied with luciferase reporter gene assay measuring the degree of SRE activity as well as with ERK1/2 phosphorylation assays. These dynamic changes in D2R signaling produced by the neurotensin receptor agonist may involve antagonistic allosteric receptor-receptor interactions in the D2LR-NTS1R heteromers at the plasma membrane level (CREB pathway) and synergistic interactions in PKC activation at the cytoplasmatic level (MAPK pathway).

GET73 increases rat extracellular hippocampal CA1 GABA levels through a possible involvement of local mGlu5 receptor.

N-[(4-trifluoromethyl) benzyl] 4-methoxybutyramide (GET73) is a newly synthesized compound displaying anti-alcohol and anxiolytic properties. In light of the importance of the hippocampal CA1 subregion in alcohol addiction and anxiety-like behaviors-this in vivo microdialysis study characterized the effect of GET73 on extracellular GABA levels in the hippocampal CA1 region of the freely moving rat-including a possible role for mGlu5 receptor in mediating this effect. Both intraperitoneal administration (2-10 mg/kg) and local intra-hippocampal CA1 perfusion with GET73 (50-1000 nM) were associated with a transient, step-wise increase in dialysate hippocampal CA1 GABA levels. The GET73 (10 mg/kg)-induced increase in GABA levels was not affected by intra-CA1 perfusion with either the GABA reuptake inhibitor SKF89976A (0.5 mM) or by local GABAA (bicuculline; 1µM) and GABAB (CGP35348; 500 µM) receptor antagonists. On the contrary, the GET73-induced increase in GABA levels was partially counteracted by the intra-CA1 perfusion with the mGlu5 receptor negative allosteric modulator MPEP (300 µM). Interestingly, GET73 at the lowest (2 mg/kg) dose tested, by itself ineffective, fully counteracted the increase in GABA levels induced by the mGlu5 receptor agonist CHPG (1000 µM). Taken together, these findings suggest that the GET73-induced increase in hippocampal CA1 GABA levels operates independently of local GABA reuptake and/or GABAA or GABAB receptors. Furthermore, the present data lead to hypothesize a possible interaction between GET73 and mGluR5-mediated regulation of hippocampal CA1 GABA transmission, an effect which may be relevant to the ability of GET73 to reduce alcohol intake in an alcohol-preferring rat strain.

Unbalance of CB1 receptors expressed in GABAergic and glutamatergic neurons in a transgenic mouse model of Huntington's disease.

Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntington's disease (HD). However, the functional meaning of this reduction, if any, is still unclear. Here, the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN) were investigated on striatal synaptic transmission and on glutamate and GABA release in symptomatic R6/2 mice, a genetic model of HD. The expression levels of CB1Rs in glutamatergic and GABAergic synapses were also evaluated. We found that in R6/2 mice, WIN effects on synaptic transmission and glutamate release were significantly increased with respect to wild type mice. On the contrary, a decrease in WIN-induced reduction of GABA release was found in R6/2 versus WT mice. The expression of CB1Rs in GABAergic neurons was drastically reduced, while CB1Rs levels in glutamatergic neurons were unchanged. These results demonstrate that the expression and functionality of CB1Rs are differentially affected in GABAergic and glutamatergic neurons in R6/2 mice. As a result, the balance between CB1Rs expressed by the two neuronal populations and, thus, the net effect of CB1R stimulation, is profoundly altered in HD mice.

Striatal NTS1 , dopamine D2 and NMDA receptor regulation of pallidal GABA and glutamate release--a dual-probe microdialysis study in the intranigral 6-hydroxydopamine unilaterally lesioned rat.

The current microdialysis study elucidates a functional interaction between the striatal neurotensin NTS(1) receptor and the striatal dopamine D(2) and N-methyl-d-aspartic acid (NMDA) receptors in the regulation of striatopallidal gamma-aminobutyric acid (GABA) and glutamate levels after an ipsilateral intranigral 6-hydroxydopamine-induced lesion of the ascending dopamine pathways to the striatum. Lateral globus pallidus GABA levels were higher in the lesioned group while no change was observed in striatal GABA and glutamate levels. The 6-hydroxydopamine-induced lesion did not alter the ability of intrastriatal NT (10 nm) to counteract the decrease in pallidal GABA and glutamate levels induced by the dopamine D(2) -like receptor agonist quinpirole (10 µm). A more pronounced increase in the intrastriatal NMDA- (10 µm) induced increase in pallidal GABA levels was observed in the lesioned group while it attenuated the increase in striatal glutamate levels and amplified the increase in pallidal glutamate levels compared with that observed in the controls. NT enhanced the NMDA-induced increase in pallidal GABA and glutamate and striatal glutamate levels; these effects were counteracted by the NTS(1) antagonist SR48692 (100 nm) in both groups. These findings demonstrate an inhibitory striatal dopamine D(2) and an excitatory striatal NMDA receptor regulation of striatopallidal GABA transmission in both groups. These actions are modulated by NT via antagonistic NTS(1) /D(2) and facilitatory NTS(1) /NMDA receptor-receptor interactions, leading to enhanced glutamate drive of the striatopallidal GABA neurons associated with motor inhibition, effects which all are counteracted by SR48692. Thus, NTS(1) antagonists in combination with conventional treatments may provide a novel therapeutic strategy in Parkinson's disease.

Neurotensin regulates cortical glutamate transmission by modulating N-methyl-D-aspartate receptor functional activity: an in vivo microdialysis study.

The aim of the present in vivo microdialysis study was to investigate whether the tridecapeptide neurotensin (NT) influences the N-methyl-D-aspartate (NMDA) receptor-mediated increase of cortical glutamate transmission in freely moving rats. Intracortical perfusion with NT influenced local extracellular glutamate levels in a bell-shaped, concentration-dependent manner. One hundred and three hundred nanomolar NT concentrations increased glutamate levels (151% ± 7% and 124% ± 3% of basal values, respectively). Higher (1,000 nM) and lower (10 nM) NT concentrations did not alter extracellular glutamate levels. The NT receptor antagonist SR48692 (100 nM) prevented the NT (100 nM)-induced increase in glutamate levels. NMDA (100 and 500 µM) perfusion induced a concentration-dependent increase in extracellular glutamate levels, the lower 10 µM NMDA concentration being ineffective. When NT (10 nM, a concentration by itself ineffective) was added in combination with NMDA (100 µM) to the perfusion medium, a significant greater increase in extracellular glutamate levels (169% ± 7%) was observed with respect to the increase induced by NMDA (100 µM) alone (139% ± 4%). SR48692 (100 nM) counteracted the increase in glutamate levels induced by the treatment with NT (10 nM) plus NMDA (100 µM). The enhancement of cortical glutamate levels induced by NMDA (100 and 500 µM) was partially antagonized by the presence of SR48692, at a concentration (100 nM) that by itself was ineffective in modulating glutamate release. These findings indicate that NT plays a relevant role in the regulation of cortical glutamatergic transmission, especially by modulating the functional activity of cortical NMDA receptors. A possible role in glutamate-mediated neurotoxicity is suggested.

Nanomolar concentrations of cocaine enhance D2-like agonist-induced inhibition of the K+-evoked [3H]-dopamine efflux from rat striatal synaptosomes: a novel action of cocaine.

Previous studies have indicated that cocaine binding sites contain both high- and low-affinity binding components and have actions not related to dopamine uptake inhibition. Therefore, it has been studied if concentrations of cocaine in the range of 0.1-100 nM can affect not only dopamine uptake but also the quinpirole-induced inhibition of the K(+)-evoked [(3)H]-dopamine efflux from rat striatal synaptosomes. It was found that quinpirole-induced inhibition of K(+)-evoked [(3)H]-dopamine efflux was significantly enhanced by cocaine at 1 and 10 nM but not at 0.1 nM with cocaine alone being inactive and 1 nM cocaine lacking effects on [(3)H]-dopamine uptake in rat striatal synaptosomes. The results indicate the existence of a novel allosteric agonist action of cocaine in low concentrations, not affecting dopamine uptake, at striatal D(2) autoreceptors modulating striatal dopamine transmission.

Multiple Adenosine-Dopamine (A2A-D2 Like) Heteroreceptor Complexes in the Brain and Their Role in Schizophrenia.

In the 1980s and 1990s, the concept was introduced that molecular integration in the Central Nervous System could develop through allosteric receptor-receptor interactions in heteroreceptor complexes presents in neurons. A number of adenosine-dopamine heteroreceptor complexes were identified that lead to the A2A-D2 heteromer hypothesis of schizophrenia. The hypothesis is based on strong antagonistic A2A-D2 receptor-receptor interactions and their presence in the ventral striato-pallidal GABA anti-reward neurons leading to reduction of positive symptoms. Other types of adenosine A2A heteroreceptor complexes are also discussed in relation to this disease, such as A2A-D3 and A2A-D4 heteroreceptor complexes as well as higher order A2A-D2-mGluR5 and A2A-D2-Sigma1R heteroreceptor complexes. The A2A receptor protomer can likely modulate the function of the D4 receptors of relevance for understanding cognitive dysfunction in schizophrenia. A2A-D2-mGluR5 complex is of interest since upon A2A/mGluR5 coactivation they appear to synergize in producing strong inhibition of the D2 receptor protomer. For understanding the future of the schizophrenia treatment, the vulnerability of the current A2A-D2like receptor complexes will be tested in animal models of schizophrenia. A2A-D2-Simag1R complexes hold the highest promise through Sigma1R enhancement of inhibition of D2R function. In line with this work, Lara proposed a highly relevant role of adenosine for neurobiology of schizophrenia.

Acute cocaine treatment enhances the antagonistic allosteric adenosine A2A-dopamine D2 receptor-receptor interactions in rat dorsal striatum without increasing significantly extracellular dopamine levels.

BACKGROUND: Antagonistic adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) receptor-receptor interactions have previously been demonstrated in A2AR-D2R heteroreceptor complexes in the rat dorsal striatum. They mainly involve a reduction of affinity in the high-affinity component of the D2R agonist binding site upon activation in vivo of the A2AR by an A2AR agonist. Upon cocaine self-administration, this antagonistic A2AR-D2R interaction disappeared in the dorsal striatum. METHODS: In the current experiments, it was tested whether such modifications in the antagonistic A2AR-D2R receptor-receptor interactions can develop also after an acute systemic injection of a low cocaine dose (1 mg/kg; sc). RESULTS: Microdialysis experiments indicated that acute cocaine did not significantly alter the extracellular dopamine levels in the dorsal striatum of the awake Wistar rats. Competition dopamine receptor binding experiments demonstrated that in the acute cocaine group, the A2AR agonist CGS-21680 produced significantly larger increases in the D2R Ki, High values (reduction of high-affinity) versus the saline-injected (i.e. control) group. Furthermore, in the dorsal striatum membrane preparation from acute cocaine-injected rats, CGS-21680 also produced significant increases in the D2R Ki, Low values (reduction of low-affinity) and in the proportion of D2Rs in the high-affinity state (RH). Such significant effects were not observed with CGS-21680 in the control group. CONCLUSIONS: The molecular mechanism involved in the acute cocaine-induced increase in the antagonistic allosteric A2AR-D2R receptor-receptor interactions may be an increased formation of higher-order complexes A2AR-D2R-sigma1R in which cocaine by binding to the sigma1R protomer also allosterically enhances the inhibitory A2AR-D2R interaction in this receptor complex.

Cannabinoid CB1 and cholecystokinin CCK2 receptors modulate, in an opposing way, electrically evoked [3H]GABA efflux from rat cerebral cortex cell cultures: possible relevance for cortical GABA transmission and anxiety.

The effects of treatments with cannabinoid (CB)(1) and cholecystokinin (CCK)(2) receptor agonists and antagonists, as well as compounds that enhance endocannabinoid signaling by inhibiting degradation, e.g., the fatty acid amide hydrolase inhibitor 3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) or the endocannabinoid reuptake inhibitor (5Z,8Z,11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707), were studied both on spontaneous and electrically evoked [(3)H]GABA efflux from rat cerebral cortex cell cultures. The CCK(2) receptor agonist CCK-8S, the CB(1) receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2), URB597, UCM707, the CB(1) receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A), and the CCK(2) receptor antagonist 2-[2-(5-Br-1H-indol-3-yl)ethyl]-3-[3-(1-methylethoxy)phenyl]-4-(3H)-quinazolinone (LY225910) did not affect spontaneous [(3)H]GABA efflux. CCK-8S concentration-dependently increased electrically evoked [(3)H]GABA overflow, and this effect was prevented by LY225910. WIN55,212-2, URB597, and UCM707 induced a reduction of electrically evoked [(3)H]GABA overflow. This reduction was counteracted by SR141716A. When CCK-8S and one of cannabinoid-interfering compounds were simultaneously added, at concentrations by themselves ineffective, to the superfusion medium, an enhancement in electrically evoked [(3)H]GABA efflux was observed. This increase was counteracted by either SR141716A or LY225910 as well as by the inhibitor of protein kinase C, (1R)-2-[12-[(2R)-2-(benzoyloxy)propyl]-3,10-dihydro-4,9-dihydroxy-2,6,7,11-tetramethoxy-3,10-dioxo-1-perylenyl]-1-methylethylcarbonic acid 4-hydroxyphenyl ester (calphostin C). These results indicate that CB(1) and CCK(2) receptors modulate, in an opposing way, electrically evoked [(3)H]GABA efflux from rat cerebral cortex cell cultures. The existence of a CB(1)/CCK(2) receptor heteromer on cortical GABA terminals, with a possible relevance for cortical GABA transmission and anxiety, is postulated.

Integrated signaling in heterodimers and receptor mosaics of different types of GPCRs of the forebrain: relevance for schizophrenia.

Receptor-receptor interactions within receptor heterodimers and receptor mosaics formed by different types of GPCRs represent an important integrative mechanism for signaling in brain networks at the level of the plasma membrane. The malfunction of special heterodimers and receptor mosaics in the ventral striatum containing D(2) receptors and 5-HT(2A) receptors in cortical networks may contribute to disturbances of key pathways involving ventral striato-pallidal GABA neurons and mediodorsal thalamic prefrontal glutamate neurons that may lead to the development of schizophrenia. The ventral striatum transmits emotional information to the cerebral cortex through a D(2) regulated accumbal-ventral pallidal-mediodorsal-prefrontal circuit which is of special interest to schizophrenia in view of the reduced number of glutamate mediodorsal-prefrontal projections associated with this disease. This circuit is especially vulnerable to D(2) receptor activity in the nucleus accumbens, since it produces a reduction in the prefrontal glutamate drive from the mediodorsal nucleus. The following D(2) receptor containing heterodimers/receptor mosaics are of special interest to schizophrenia: A(2A)-D(2), mGluR5-D(2), CB(1)-D(2), NTS(1)-D(2) and D(2)-D(3) and are discussed in this review. They may have a differential distribution pattern in the local circuits of the ventral striato-pallidal GABA pathway, predominantly located extrasynaptically. Specifically, trimeric receptor mosaics consisting of A(2A)-D(2)-mGluR5 and CB(1)-D(2)-A(2A) may also exist in these local circuits and are discussed. The integration of receptor signaling within assembled heterodimers/receptor mosaics is brought about by agonists and allosteric modulators. These cause the intramembrane receptor-receptor interactions, via allosteric mechanisms, to produce conformational changes that pass over the receptor interfaces. Exogenous and endogenous cooperativity is discussed as well as the role of the cortical mGluR2-5-HT(2A) heterodimer/receptor mosaic in schizophrenia (Gonzalez-Maeso et al. 2008). Receptor-receptor interactions within receptor heterodimer/receptor mosaics of different receptors in the ventral striatum and cerebral cortex give novel strategies for treatment of schizophrenia involving, e.g., monotherapy with either A(2A), mGluR5, CB(1) or NTS(1) agonists or combined therapies with some of these agonists combined with D(2)-like antagonists that specifically target the ventral striatum. In addition, a combined targeting of receptor mosaics in the ventral striatum and in the cerebral cortex should also be considered.

Neurotensin receptor involvement in the rise of extracellular glutamate levels and apoptotic nerve cell death in primary cortical cultures after oxygen and glucose deprivation.

In view of the ability of neurotensin (NT) to increase glutamate release, the role of NT receptor mechanisms in oxygen-glucose deprivation (OGD)-induced neuronal degeneration in cortical cultures has been evaluated by measuring lactate dehydrogenase (LDH) levels, mitochondrial dehydrogenase activity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide levels, and microtubule-associated protein 2 (MAP2) immunoreactivity. Apoptotic nerve cell death was analyzed measuring chromatin condensation with Hoechst 33258, annexin V staining, and caspase-3 activity. Furthermore, the involvement of glutamate excitotoxicity in the neurodegeneration-enhancing actions of NT was analyzed by measurement of extracellular glutamate levels. NT enhanced the OGD-induced increase of LDH, endogenous extracellular glutamate levels, and apoptotic nerve cell death. In addition, the peptide enhanced the OGD-induced loss of mitochondrial functionality and increase of MAP2 aggregations. These effects were blocked by the neurotensin receptor 1 (NTR1) antagonist SR48692. Unexpectedly, the antagonist at 100 nM counteracted not only the NT effects but also some OGD-induced biochemical and morphological alterations. These results suggest that NTR1 receptors may participate in neurodegenerative events induced by OGD in cortical cultures, used as an in vitro model of cortical ischemia. The NTR1 receptor antagonists could provide a new tool to explore the clinical possibilities and thus to move from chemical compound to effective drug.

Neurotensin receptors as modulators of glutamatergic transmission.

Functional studies have provided evidence supporting the concept that the tridecapeptide neurotensin (NT) acts in the central nervous system as a classical neurotransmitter and/or as an important modulator of neuronal signalling. The role of NT in the regulation of the striatal amino acidergic transmission, mainly by antagonising D2 receptor function, will be analysed. In addition, in different rat brain regions, including the basal ganglia, the contribution of NT receptors in modulating and reinforcing glutamate signalling will be shown including the involvement of interactions between NT and NMDA receptors. Since the enhancement of glutamate transmission and in particular the excessive activation of NMDA receptors, has been postulated to be an important factor in the induction of glutamate-mediated neuronal damage, the involvement of NT in the glutamate-induced neurodegenerative effects will be discussed. Moving from these observations and in order to further investigate this issue, results from preliminary behavioural, functional and biochemical experiments will be presented on the putative neuroprotective effect obtained by the blockade of NT receptor 1 (NTS1) via the systemic administration of the selective NTS1 antagonist SR48692 in an in vivo animal model of Parkinson's disease [unilateral nigral 6-hydroxydopamine (6-OHDA) induced lesion of the nigrostriatal pathway].

Acute Cocaine Enhances Dopamine D2R Recognition and Signaling and Counteracts D2R Internalization in Sigma1R-D2R Heteroreceptor Complexes.

The current study was performed to establish the actions of nanomolar concentrations of cocaine, not blocking the dopamine transporter, on dopamine D2 receptor (D2R)-sigma 1 receptor (δ1R) heteroreceptor complexes and the D2R protomer recognition, signaling and internalization in cellular models. We report the existence of D2R-δ1R heteroreceptor complexes in subcortical limbic areas as well as the dorsal striatum, with different distribution patterns using the in situ proximity ligation assay. Also, through BRET, these heteromers were demonstrated in HEK293 cells. Furthermore, saturation binding assay demonstrated that in membrane preparations of HEK293 cells coexpressing D2R and δ1R, cocaine (1 nM) significantly increased the D2R Bmax values over cells singly expressing D2R. CREB reporter luc-gene assay indicated that coexpressed δ1R significantly reduced the potency of the D2R-like agonist quinpirole to inhibit via D2R activation the forskolin induced increase of the CREB signal. In contrast, the addition of 100 nM cocaine was found to markedly increase the quinpirole potency to inhibit the forskolin-induced increase of the CREB signal in the D2R-δ1R cells. These events were associated with a marked reduction of cocaine-induced internalization of D2R protomers in D2R-δ1R heteromer-containing cells vs D2R singly expressing cells as studied by means of confocal analysis of D2R-δ1R trafficking and internalization. Overall, the formation of D2R-δ1R heteromers enhanced the ability of cocaine to increase the D2R protomer function associated with a marked reduction of its internalization. The existence of D2R-δ1R heteromers opens up a new understanding of the acute actions of cocaine.

Antagonistic cannabinoid CB1/dopamine D2 receptor interactions in striatal CB1/D2 heteromers. A combined neurochemical and behavioral analysis.

In vitro results show the ability of the CB(1) receptor agonist CP 55,940 to reduce the affinity of D(2) receptor agonist binding sites in both the dorsal and ventral striatum including the nucleus accumbens shell. This antagonistic modulation of D(2) receptor agonist affinity was found to remain and even be enhanced after G-protein activation by Gpp(NH)p. Using the FRET technique in living HEK-293T cells, the formation of CB(1)-D(2) receptor heteromers, independent of receptor occupancy, was demonstrated. These data thereby indicate that the antagonistic intramembrane CB(1)/D(2) receptor-receptor interactions may occur in CB(1)/D(2) formed heteromers. Antagonistic CB(1)/D(2) interactions were also discovered at the behavioral level through an analysis of quinpirole-induced locomotor hyperactivity in rats. The CB(1) receptor agonist CP 55,940 at a dose that did not change basal locomotion was able to block quinpirole-induced increases in locomotor activity. In addition, not only the CB(1) receptor antagonist rimonobant but also the specific A(2A) receptor antagonist MSX-3 blocked the inhibitory effect of CB(1) receptor agonist on D(2)-like receptor agonist-induced hyperlocomotion. Taken together, these results give evidence for the existence of antagonistic CB(1)/D(2) receptor-receptor interactions within CB(1)/D(2) heteromers in which A(2A) receptors may also participate.

Enhanced striatal glutamate release after the administration of rimonabant to 6-hydroxydopamine-lesioned rats.

While recent studies have shown that the blockade of cannabinoid CB(1) receptors might be beneficial to alleviate the motor inhibition typical of Parkinson's disease (PD), the neurochemical substrates for this effect remain elusive. Here we have carried out microdialysis experiments to determine whether the effects of rimonabant, a selective antagonist of CB(1) receptors, might be associated with changes in striatal glutamate release in a rat model of PD generated by intracerebroventricular injection of 6-hydroxydopamine. Our data demonstrate that the treatment with rimonabant slightly increased striatal glutamate release in control rats, although this effect was only evident with the highest dose of rimonabant tested (1mg/kg). However, the increase in glutamate release was much more marked in the parkinsonian rats where similar changes were observed at a dose of 1 and 0.1mg/kg, exactly the same dose that relieved motor inhibition in previous behavioral studies. In summary, the potential of rimonabant to act as a possible antihypokinetic agent in parkinsonian rats seems to be related to enhanced glutamate release from excitatory afferents to the striatum. This observation is of potential clinical interest, particularly for those parkinsonian patients that exhibit a poor response to classic levodopa treatment.