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.

Astrocytic Mechanisms Involving Kynurenic Acid Control Δ9-Tetrahydrocannabinol-Induced Increases in Glutamate Release in Brain Reward-Processing Areas.

The reinforcing effects of Δ9-tetrahydrocannabinol (THC) in rats and monkeys, and the reinforcement-related dopamine-releasing effects of THC in rats, can be attenuated by increasing endogenous levels of kynurenic acid (KYNA) through systemic administration of the kynurenine 3-monooxygenase inhibitor, Ro 61-8048. KYNA is a negative allosteric modulator of α7 nicotinic acetylcholine receptors (α7nAChRs) and is synthesized and released by astroglia, which express functional α7nAChRs and cannabinoid CB1 receptors (CB1Rs). Here, we tested whether these presumed KYNA autoreceptors (α7nAChRs) and CB1Rs regulate glutamate release. We used in vivo microdialysis and electrophysiology in rats, RNAscope in situ hybridization in brain slices, and primary culture of rat cortical astrocytes. Acute systemic administration of THC increased extracellular levels of glutamate in the nucleus accumbens shell (NAcS), ventral tegmental area (VTA), and medial prefrontal cortex (mPFC). THC also reduced extracellular levels of KYNA in the NAcS. These THC effects were prevented by administration of Ro 61-8048 or the CB1R antagonist, rimonabant. THC increased the firing activity of glutamatergic pyramidal neurons projecting from the mPFC to the NAcS or to the VTA in vivo. These effects were averted by pretreatment with Ro 61-8048. In vitro, THC elicited glutamate release from cortical astrocytes (on which we demonstrated co-localization of the CB1Rs and α7nAChR mRNAs), and this effect was prevented by KYNA and rimonabant. These results suggest a key role of astrocytes in interactions between the endocannabinoid system, kynurenine pathway, and glutamatergic neurotransmission, with ramifications for the pathophysiology and treatment of psychiatric and neurodegenerative diseases.

In Vitro Functional Characterization of GET73 as Possible Negative Allosteric Modulator of Metabotropic Glutamate Receptor 5.

The present study was aimed to further characterize the pharmacological profile of N-[4-(trifluoromethyl) benzyl]-4-methoxybutyramide (GET73), a putative negative allosteric modulator (NAM) of metabotropic glutamate subtype 5 receptor (mGluR5) under development as a novel medication for the treatment of alcohol dependence. This aim has been accomplished by means of a series of in vitro functional assays. These assays include the measure of several down-stream signaling [intracellular Ca++ levels, inositol phosphate (IP) formation and CREB phosphorylation (pCREB)] which are generally affected by mGluR5 ligands. In particular, GET73 (0.1 nM-10 µM) was explored for its ability to displace the concentration-response curve of some mGluR5 agonists/probes (glutamate, L-quisqualate, CHPG) in different native preparations. GET73 produced a rightward shift of concentration-response curves of glutamate- and CHPG-induced intracellular Ca++ levels in primary cultures of rat cortical astrocytes. The compound also induced a rightward shift of concentration response curve of glutamate- and L-quisqualate-induced increase in IP turnover in rat hippocampus slices, along with a reduction of CHPG (10 mM)-induced increase in IP formation. Moreover, GET73 produced a rightward shift of concentration-response curve of glutamate-, CHPG- and L-quisqualate-induced pCREB levels in rat cerebral cortex neurons. Although the engagement of other targets cannot be definitively ruled out, these data support the view that GET73 acts as an mGluR5 NAM and support the significance of further investigating the possible mechanism of action of the compound.

Neurotensin: A role in substance use disorder?

Neurotensin is a tridecapeptide originally identified in extracts of bovine hypothalamus. This peptide has a close anatomical and functional relationship with the mesocorticolimbic and nigrostriatal dopamine system. Neural circuits containing neurotensin were originally proposed to play a role in the mechanism of action of antipsychotic agents. Additionally, neurotensin-containing pathways were demonstrated to mediate some of the rewarding and/or sensitizing properties of drugs of abuse.This review attempts to contribute to the understanding of the role of neurotensin and its receptors in drug abuse. In particular, we will summarize the potential relevance of neurotensin, its related compounds and neurotensin receptors in substance use disorders, with a focus on the preclinical research.

Adenosine A2A-D2 receptor-receptor interactions in putative heteromers in the regulation of the striato-pallidal gaba pathway: possible relevance for parkinson's disease and its treatment.

Striatal dopamine adenosine A2A and D2 receptors interact to modulate some aspects of motor and motivational function. The demonstration of A2A/D2 receptor heteromerization in living cells constituted a progress for understanding the neurobiology of dopamine D2 and adenosine A2A receptors. In fact, the existence of putative striatalA2A/D2 receptor heteromers has been suggested to be important for striatal function under both normal and pathological conditions, such as Parkinson's disease. Consequently, the antagonistic A2A-D2 receptor interactions in a putative striatal receptor heteromer on striato-pallidal GABA neuron led to the introduction of A2A receptor antagonists as possible anti- Parkinsonian drugs. The present mini-review briefly summarizes the main findings supporting the presence of antagonistic A2A-D2 receptor interactions in putative receptor heteromers in the basal ganglia. Special emphasis is given to in vivo microdialysis findings demonstrating the functional role putative A2A/D2 heteromers on striato-pallidal GABA neurons play in the modulation of this pathway, in which A2A receptors inhibit D2 receptor signaling. The possible relevance of compounds targeting the putative striatal A2A/D2 heteromer in the Parkinson's disease pharmacological treatment is also discussed.

Neurotensin NTS1-dopamine D2 receptor-receptor interactions in putative receptor heteromers: relevance for Parkinson's disease and schizophrenia.

The tridecapeptide neurotensin (NT) acts as neurotransmitter in the central nervous system and in the periphery. NT and NT receptors are largely localized in dopamine (DA)-enriched regions of the mammalian brain. Accordingly, numerous studies indicate the presence of close functional interactions between DA neurons and the peptide. Among others mechanisms, it has been suggested that NT could modulate nigrostriatal, mesolimbic and meso-cortical DA transmission through an antagonistic receptor-receptor interaction between the NT receptor subtype 1 (NTS1) and the dopamine D2 receptor (D2R). In particular, it was originally demonstrated that the peptide reduces the D2R agonist affinity in striatal sections and in striatal membrane preparations. These effects could be a consequence of the direct allosteric NTS1/D2 receptor interactions leading to a decrease in the DA agonist affinity at the D2 receptor. Several neurochemical, biochemical and co-immunoprecipitation data have successively reinforced the indication of the presence of direct NTS1-D2 receptor interactions in the mammalian brain. The present mini-review attempts to provide a summary of current knowledge, mainly emerging from our microdialysis studies, supporting the presence of a NTS1/D2 receptor heteromer in the brain. The pre and post-synaptic mechanisms underlying the involvement of this heteromer in the striatopallidal GABA and mesocorticolimbic DA neurotransmission are discussed especially for their relevance in Parkinson's disease and schizophrenia, respectively.