Effects of Long-Term Alcohol Drinking on the Dopamine D2 Receptor: Gene Expression and Heteroreceptor Complexes in the Striatum in Rats.

BACKGROUND: Reduced dopamine D2 receptor (D2R) ligand binding has repeatedly been demonstrated in the striatum of humans with alcohol use disorder (AUD). The attenuated D2R binding has been suggested to reflect a reduced D2R density, which in turn has been proposed to drive craving and relapse. However, results from rodent studies addressing the effects of alcohol drinking on D2R density have been inconsistent. METHODS: A validated alcohol drinking model (intermittent access to 20% alcohol) in Wistar rats was used to study the effects of voluntary alcohol drinking (at least 12 weeks) on the D2R in the striatum compared to age-matched alcohol-naïve control rats. Reverse transcriptase quantitative PCR was used to quantify isoform-specific Drd2 gene expression levels. Using bisulfite pyrosequencing, DNA methylation levels of a regulatory region of the Drd2 gene were determined. In situ proximity ligation assay was used to measure densities of D2R receptor complexes: D2R-D2R, adenosine A2A receptor (A2AR)-D2R, and sigma1 receptor (sigma1R)-D2R. RESULTS: Long-term voluntary alcohol drinking significantly reduced mRNA levels of the long D2R isoform in the nucleus accumbens (NAc) but did not alter CpG methylation levels in the analyzed sequence of the Drd2 gene. Alcohol drinking also reduced the striatal density of D2R-D2R homoreceptor complexes, increased the density of A2AR-D2R heteroreceptor complexes in the NAc shell and the dorsal striatum, and decreased the density of sigma1R-D2R heteroreceptor complexes in the dorsal striatum. CONCLUSIONS: The present results on long-term alcohol drinking might reflect reduced D2R levels through reductions in D2R-D2R homoreceptor complexes and gene expression. Furthermore, based on antagonistic interactions between A2AR and D2R, an increased density of A2AR-D2R heteroreceptor complexes might indicate a reduced affinity and signaling of the D2R population within the complex. Hence, both reduced striatal D2R levels and reduced D2R protomer affinity within the striatal A2AR-D2R complex might underlie reduced D2R radioligand binding in humans with AUD. This supports the hypothesis of a hypodopaminergic system in AUD and suggests the A2AR-D2R heteroreceptor complex as a potential novel treatment target.

Existence of Brain 5-HT1A-5-HT2A Isoreceptor Complexes with Antagonistic Allosteric Receptor-Receptor Interactions Regulating 5-HT1A Receptor Recognition.

Studies on serotonin-selective reuptake inhibitors have established that disturbances in the ascending 5-HT neuron systems and their 5-HT receptor subtypes and collateral networks to the forebrain contribute to the etiology of major depression and are targets for treatment. The therapeutic action of serotonin-selective reuptake inhibitors is of proven effectiveness, but the mechanisms underlying their effect are still unclear. There are many 5-HT subtypes involved; some need to be blocked (e.g., 5-HT2A, 5-HT3, and 5-HT7), whereas others need to be activated (e.g., postjunctional 5-HT1A and 5-HT4). These state-of-the-art developments are in line with the hypothesis that the development of major depression can involve an imbalance of the activity between different types of 5-HT isoreceptors. In the current study, using in situ proximity ligation assay (PLA), we report evidence for the existence of brain 5-HT1A-5-HT2A isoreceptor complexes validated in cellular models with bioluminescence resonance energy transfer (BRET2) assay. A high density of PLA-positive clusters visualizing 5-HT1A-5-HT2A isoreceptor complexes was demonstrated in the pyramidal cell layer of the CA1-CA3 regions of the dorsal hippocampus. A marked reduction in the density of PLA-positive clusters was observed in the CA1 and CA2 regions 24 h after a forced swim test session, indicating the dynamics of this 5-HT isoreceptor complex. Using a bioinformatic approach, previous work indicates that receptors forming heterodimers demonstrate triplet amino acid homologies. The receptor interface of the 5-HT1A-5-HT2A isoreceptor dimer was shown to contain the LLG and QNA protriplets in the transmembrane and intracellular domain, respectively. The 5-HT2A agonist TCB2 markedly reduced the affinity of the 5-HT1A agonist ipsapirone for the 5-HT1A agonist binding sites in the frontal lobe using the 5-HT1A radioligand binding assay. This action was blocked by the 5-HT2A antagonist ketanserin. It is proposed that the demonstrated 5-HT1A-5-HT2A isoreceptor complexes may play a role in depression through integration of 5-HT recognition, signaling and trafficking in the plasma membrane in two major 5-HT receptor subtypes known to be involved in depression. Antagonistic allosteric receptor-receptor interactions appear to be involved in this integrative process.

Cocaine self-administration specifically increases A2AR-D2R and D2R-sigma1R heteroreceptor complexes in the rat nucleus accumbens shell. Relevance for cocaine use disorder.

Adenosine 2A receptor (A2AR) agonists were indicated to reduce cocaine reward and cocaine seeking mainly through activation of antagonistic allosteric A2AR-dopamine D2R (D2R) interactions in A2AR-D2R heteroreceptor complexes. Furthermore, it was shown that modulation of cocaine reward involves antagonistic A2AR-D2R interactions in the ventral but not the dorsal striatum in rats. In the current work the proximity ligation assay (PLA) was used to further study the A2AR-D2R heteroreceptor complexes in the nucleus accumbens shell and core as well as the dorsal striatum under the influence of cocaine self-administration in rats. A significant increase in the A2AR-D2R PLA positive clusters was observed in the nucleus accumbens shell but not in the other regions vs yoked saline controls using the duolink software. Additionally, cocaine self-administration evoked a selective and significant increase in the density of D2R-sigma1R positive clusters in the nucleus accumbens shell vs yoked saline controls, while a significant reduction of the density of the D2R-sigma1R positive clusters was found in the dorsal part of the dorsal striatum. The results suggest that cocaine self-administration can reorganize A2AR and D2R into increased A2AR-D2R heteroreceptor complexes in the nucleus accumbens shell associated with increases in the D2R-sigma1R heteroreceptor complexes in this region. This reorganization can contribute to the demonstrated anti-cocaine actions of A2A receptor agonists and the putative formation of A2AR-D2R-sigma1R heterocomplexes.

Enhancement of the FGFR1 signaling in the FGFR1-5-HT1A heteroreceptor complex in midbrain raphe 5-HT neuron systems. Relevance for neuroplasticity and depression.

New findings show existence of FGFR1-5-HT1A heteroreceptor complexes in 5-HT nerve cells of the dorsal and median raphe nuclei of the rat midbrain and hippocampus. Synergistic receptor-receptor interactions in these receptor complexes indicated their enhancing role in hippocampal plasticity. The existence of FGFR1-5-HT1A heteroreceptor complexes also in midbrain raphe 5-HT nerve cells open up the possibility that antidepressant drugs by increasing extracellular 5-HT levels can cause an activation of the FGF-2/FGFR1 mechanism in these nerve cells as well. Therefore, the agonist modulation of the FGFR1-5-HT1A heteroreceptor complexes and their specific role is now determined in rat medullary raphe RN33B cells and in the caudal midline raphe area of the midbrain rich in 5-HT nerve cells. The combined i.c.v. treatment with FGF-2 and the 5-HT1A agonist 8-OHDPAT synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and in the RN33B cells. Cotreatment with FGF2 and the 5-HT1A agonist induced RN33B cell differentiation as seen from development of an increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TMV but not by TMII of the 5-HT1A receptor. Taken together, the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A heteroreceptor complex in the midbrain raphe 5-HT nerve cells appears to have also a trophic role in the central 5-HT neuron systems besides playing a key role in reducing the firing of these neurons.

Evidence for the existence of FGFR1-5-HT1A heteroreceptor complexes in the midbrain raphe 5-HT system.

The ascending midbrain 5-HT neurons known to contain 5-HT1A autoreceptors may be dysregulated in depression due to a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by co-location of the FGFR1 and 5-HT1A immunoreactivities in midbrain raphe 5-HT cells, evidence for the existence of FGFR1-5-HT1A heteroreceptor complexes were obtained in the dorsal and median raphe nuclei of the Sprague-Dawley rat. Their existence in the rat medullary raphe RN33B cell cultures was also established. After combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA positive clusters was found in the RN33B cells. Similar results were reached upon coactivation by agonists in HEK293T cells using the Fluorescent Resonance Energy Transfer (FRET) technique resulting in increased FRETmax and reduced FRET50 values. The heteroreceptor complex formation was dependent on TMV of the 5-HT1A receptor since it was blocked by incubation with TMV but not with TMII. Taken together, the 5-HT1A autoreceptors by being recruited into a FGFR1-5-HT1A heteroreceptor complex in the midbrain raphe 5-HT nerve cells may develop a novel function, namely a trophic role in many midbrain 5-HT neuron systems originating from the dorsal and medianus raphe nuclei.

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons.

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.