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Abstract Background Spatial memory deficits and reduced neuronal survival contribute to cognitive decline seen in the aging process. Current treatments are limited, emphasizing the need for innovative therapeutic strategies. This research explored the combined effects of intranasally co-administered galanin receptor 2 (GALR2) and neuropeptide Y1 receptor (NPY1R) agonists, recognized for their neural benefits, on spatial memory, neuronal survival, and differentiation in adult rats. After intranasal co-delivery of the GALR2 agonist M1145 and a NPY1R agonist to adult rats, spatial memory was tested with the object-in-place task 3 weeks later. We examined neuronal survival and differentiation by assessing BrdU-IR profiles and doublecortin (DCX) labeled cells, respectively. We also used the GALR2 antagonist M871 to confirm GALR2's crucial role in promoting cell growth. Results Co-administration improved spatial memory and increased the survival rate of mature neurons. The positive effect of GALR2 in cell proliferation was confirmed by the nullifying effects of its antagonist. The treatment boosted DCX-labeled newborn neurons and altered dendritic morphology, increasing cells with mature dendrites. Conclusions Our results show that intranasal co-delivery of GALR2 and NPY1R agonists improves spatial memory, boosts neuronal survival, and influences neuronal differentiation in adult rats. The significant role of GALR2 is emphasized, suggesting new potential therapeutic strategies for cognitive decline.

The histochemical Falck-Hillarp method for the localization of dopamine (DA), noradrenaline (NA) and serotonin in the central nervous system (CNS) of rodents was introduced in the 1960s. It supported the existence of chemical neurotransmission in the CNS. The monoamine neurons in the lower brain stem formed monosynaptic ascending systems to the telencephalon and diencephalon and monoamine descending systems to the entire spinal cord. The monoamines were early on suggested to operate via synaptic chemical transmission in the CNS. This chemical transmission reduced the impact of electrical transmission. In 1969 and the 1970s indications were obtained that important modes of chemical monoamine communication in the CNS also took place through the extra-synaptic fluid, the extracellular fluid, and long-distance communication in the cerebrospinal fluid involving diffusion and flow of transmitters like DA, NA and serotonin. In 1986, this type of transmission was named volume transmission (VT) by Agnati and Fuxe and their colleagues, also characterized by transmitter varicosity and receptor mismatches. The short and long-distance VT pathways were characterized by volume fraction, tortuosity and clearance. Electrical transmission also exists in the mammalian CNS, but chemical transmission is in dominance. One electrical mode is represented by electrical synapses formed by gap junctions which represent low resistant passages between nerve cells. It allows for a more rapid passage of action potentials between nerve cells compared to chemical transmission. The second mode is based on the ability of synaptic currents to generate electrical fields to modulate chemical transmission. One aim is to understand how chemical transmission can be integrated with electrical transmission and how putative (aquaporin water channel, dopamine D2R and adenosine A2AR) complexes in astrocytes can significancy participate in the clearance of waste products from the glymphatic system. VT may also help accomplish the operation of the acupuncture meridians essential for Chinese medicine in view of the indicated existence of extracellular VT pathways.

IntroductionThis study may unveil novel insights into the interactions between neuropeptide Y receptor 1 (NPY1R) and galanin receptor 2 (GALR2), in the dentate gyrus of the dorsal hippocampus, shedding light on their role in neurogenesis and cognitive functions. Existing literature highlights the potential of these interactions in enhancing learning and memory, yet detailed mechanisms remain underexplored.MethodsUtilizing intracerebroventricular injections of GALR2 and NPY1R agonists in Sprague-Dawley male rats, we examined neurogenesis via markers PCNA and DCX, and memory consolidation through the object-in-place task over a three-week period.ResultsSignificant increases in NPY1R-GALR2 co-localization and neuroblast proliferation were observed, alongside enhanced memory consolidation. These findings suggest a synergistic effect of NPY1R and GALR2 activation on cognitive functions.DiscussionOur findings may foster the development of novel heterobivalent or multitargeting drugs, affecting NPY1R-GALR2 interaction, and suggest a future pharmacogical strategy for improving learning and memory found in many brain diseases. Further research is encouraged to explore these mechanisms in pathological models.

Abstract Examination of healthy and diseased human brain is essential to translational neuroscience. Protein–protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the in-situ proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in the human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer’s disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that phosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies.

Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) together with the proximity ligation method revealed the existence of G-protein-coupled receptors, Ionotropic and Receptor tyrosine kinase heterocomplexes, e.g., A2AR–D2R, GABAA–D5R, and FGFR1–5-HT1AR heterocomplexes. Molecular integration takes place through allosteric receptor–receptor interactions in heteroreceptor complexes of synaptic and extra-synaptic regions. It involves the modulation of receptor protomer recognition, signaling and trafficking, as well as the modulation of behavioral responses. Allosteric receptor–receptor interactions in hetero-complexes give rise to concepts like meta-modulation and protein modulation. The introduction of receptor–receptor interactions was the origin of the concept of meta-modulation provided by Katz and Edwards in 1999, which stood for the fine-tuning or modulation of nerve cell transmission. In 2000–2010, Ribeiro and Sebastiao, based on a series of papers, provided strong support for their view that adenosine can meta-modulate (fine-tune) synaptic transmission through adenosine receptors. However, another term should also be considered: protein modulation, which is the key feature of allosteric receptor–receptor interactions leading to learning and consolidation by novel adapter proteins to memory. Finally, it must be underlined that allosteric receptor–receptor interactions and their involvement both in brain disease and its treatment are of high interest. Their pathophysiological relevance has been obtained, especially for major depressive disorder, cocaine use disorder, and Parkinson’s disease.

Introduction: The amygdala is a limbic region of high value for understanding anxiety and its treatment. Dopamine D2 receptors (D2Rs) and oxytocin receptors (OXTRs) have both been shown to participate in modulating anxiety involving effects in the amygdala. The goal is to understand if D2R–OXTR heterocomplexes exist in the central amygdala and if, through enhancing allosteric receptor–receptor interactions, may enhance anxiolytic actions.Methods: The methods used involve the shock-probe burying test, the in situ proximity ligation assay (PLA), image acquisition and analysis, and the BRET2 assay. Bilateral cannulas were introduced into the amygdala, and the effects of the coadministration of oxytocin and the D2R-like agonist quinpirole into the amygdala were studied.Results: The combination treatment enhanced the anxiolytic effects compared to the single treatment. The D2R/D3R antagonist raclopride blocked the effects of the combination treatment of oxytocin and the D2R agonist, although oxytocin is regarded as a distinct modulator of fear-mediating anxiolytic effects. In situ PLA results indicate the existence of D2R–OXTR heteroreceptor complexes and/or the co-location of OXTR and D2R within the same cell membrane nanodomains in the central amygdala. With BRET2, evidence is given for the existence of D2R–OXTR heteromers in HEK293 cells upon co-transfection.Discussion: The enhanced behavioral effects observed upon co-treatment with OXTR and D2R agonists may reflect the existence of improved positive receptor–receptor interactions in the putative D2R–OXTR heterocomplexes in certain neuronal populations of the basolateral and central amygdala. The D2R–OXTR heterocomplex, especially upon agonist co-activation in the central amygdala, may open a new pharmacological venue for the treatment of anxiety.

Previous studies have indicated that acute treatment with the monoamine stabilizer OSU-6162 (5 mg/kg), which has a high affinity for Sigma1R, significantly increased the density of accumbal shell D2R-Sigma1R and A2AR-D2R heteroreceptor complexes following cocaine self-administration. Ex vivo studies using the A2AR agonist CGS21680 also suggested the existence of enhanced antagonistic accumbal A2AR-D2R allosteric interactions after treatment with OSU-6162 during cocaine self-administration. However, a 3-day treatment with OSU-6162 (5 mg/kg) failed to alter the behavioral effects of cocaine self-administration. To test these results and the relevance of OSU-6162 (2.5 mg/kg) and/or A2AR (0.05 mg/kg) agonist interactions, we administered low doses of receptor agonists during cocaine self-administration and assessed their neurochemical and behavioral effects. No effects were observed on cocaine self-administration; however, marked and highly significant increases using the proximity ligation assay (PLA) were induced by the co-treatment on the density of the A2AR-D2R heterocomplexes in the nucleus accumbens shell. Significant decreases in the affinity of the D2R high- and low-affinity agonist binding sites were also observed. Thus, in low doses, the highly significant neurochemical effects observed upon cotreatment with an A2AR agonist and a Sigma1R ligand on the A2AR-D2R heterocomplexes and their enhancement of allosteric inhibition of D2R high-affinity binding are not linked to the modulation of cocaine self-administration. The explanation may be related to an increased release of ATP and adenosine from astrocytes in the nucleus accumbens shell in cocaine self-administration. This can lead to increased activation of the A1R protomer in a putative A1R-A2AR-D2R complex that modulates glutamate release in the presynaptic glutamate synapse. We hypothesized that the integration of changes in presynaptic glutamate release and postjunctional heteroreceptor complex signaling, where D2R plays a key role, result in no changes in the firing of the GABA anti-reward neurons, resulting in no reduction in cocaine self-administration in the present experiments.

Different brain regions' interactions have been implicated in relevant neurological diseases, such as major depressive disorder (MDD), anxiety disorders, age-dependent cognitive decline, Alzheimer's disease (AD) and addiction. We aim to explore the role of the medial prefrontal cortex (mPFC) in the Neuropeptide Y (NPY) and Galanin (GAL) interaction since we have demonstrated specific NPY and GAL interactions in brain areas related to these brain diseases. We performed GALR2 and Y1R agonists intranasal infusion and analyzed the mPFC activation through c-Fos expression. To assess the associated cellular mechanism we studied the formation of Y1R-GALR2 heteroreceptor complexes with in situ proximity ligation assay (PLA) and the expression of the brain-derived neurotrophic factor (BDNF). Moreover, the functional outcome of the NPY and GAL interaction on the mPFC was evaluated in the novel object preference task. We demonstrated that the intranasal administration of both agonists decrease the medial prefrontal cortex activation as shown with the c-Fos expression. These effects were mediated by the decreased formation of Y1R-GALR2 heteroreceptor complexes without affecting the BDNF expression. The functional outcome of this interaction was related to an impaired performance on the novel object preference task. Our data may suggest the translational development of new heterobivalent agonist pharmacophores acting on Y1R–GALR2 heterocomplexes in the medial prefrontal cortex for the novel therapy on neurodegenerative and psychiatric diseases. Data Sharing and Data Accessibility: The data that support the findings of this study are openly available in Institutional repository of the University of Malaga (RIUMA) and from the corresponding author upon reasonable request.

In the last 10 years, it has become increasingly clear that large numbers of axon collaterals extend from the oxytocin (OXT) hypothalamic axons, especially the parvocellular components, to other brain regions. Consequently, the OXT signaling system forms, like other monoamine axons, a rich functional network across several brain regions. In this manuscript, we review the recently indicated higher order G-protein coupled heteroreceptor complexes of the oxytocin receptor (OXTR), and how these, via allosteric receptor-receptor interactions modulate the recognition, signaling, and trafficking of the participating receptor protomers and their potential impact for brain and behavior. The major focus will be on complexes of the OXTR protomer with the dopamine D2 receptor (D2R) protomer and the serotonin 2A (5-HT2AR) and 2C (5-HT2CR) receptor protomers. Specifically, the existence of D2R-OXTR heterocomplexes in the nucleus accumbens and the caudate putamen of rats has led to a postulated function for this heteromer in social behavior. Next, a physical interaction between OXTRs and the growth hormone secretagogue or ghrelin receptor (GHS-R1a) was demonstrated, which consequently was able to attenuate OXTR-mediated Gαq signaling. This highlights the potential of ghrelin-targeted therapies to modulate oxytocinergic signaling with relevance for appetite regulation, anxiety, depression, and schizophrenia. Similarly, evidence for 5-HT2AR-OXTR heteromerization in the pyramidal cell layer of CA2 and CA3 in the dorsal hippocampus and in the nucleus accumbens shell was demonstrated. This complex may offer new strategies for the treatment of both mental disease and social behavior. Finally, the 5-HT2CR-OXTR heterocomplexes were demonstrated in the CA1, CA2, and CA3 regions of the dorsal hippocampus. Future work should be done to investigate the precise functional consequence of region-specific OXTR heteromerization in the brain, as well across the periphery, and whether the integration of neuronal signals in the brain may also involve higher order OXTR-GHS-R1a heteroreceptor complexes including the dopamine (DA), noradrenaline (NA) or serotonin (5-HT) receptor protomers or other types of G-protein coupled receptors (GPCRs).

Abstract To elucidate factors underlying the evolution of large brains in cetaceans, we examined 16 brains from 14 cetartiodactyl species, with immunohistochemical techniques, for evidence of non-shivering thermogenesis. We show that, in comparison to the 11 artiodactyl brains studied (from 11 species), the 5 cetacean brains (from 3 species), exhibit an expanded expression of uncoupling protein 1 (UCP1, UCPs being mitochondrial inner membrane proteins that dissipate the proton gradient to generate heat) in cortical neurons, immunolocalization of UCP4 within a substantial proportion of glia throughout the brain, and an increased density of noradrenergic axonal boutons (noradrenaline functioning to control concentrations of and activate UCPs). Thus, cetacean brains studied possess multiple characteristics indicative of intensified thermogenetic functionality that can be related to their current and historical obligatory aquatic niche. These findings necessitate reassessment of our concepts regarding the reasons for large brain evolution and associated functional capacities in cetaceans.

The midbrain raphe serotonin (5HT) neurons provide the main ascending serotonergic projection to the forebrain, including hippocampus, which has a role in the pathophysiology of depressive disorder. Serotonin 5HT1A receptor (R) activation at the soma-dendritic level of serotonergic raphe neurons and glutamatergic hippocampal pyramidal neurons leads to a decrease in neuronal firing by activation of G protein-coupled inwardly-rectifying potassium (GIRK) channels. In this raphe-hippocampal serotonin neuron system, the existence of 5HT1AR-FGFR1 heteroreceptor complexes has been proven, but the functional receptor–receptor interactions in the heterocomplexes have only been investigated in CA1 pyramidal neurons of control Sprague Dawley (SD) rats. In the current study, considering the impact of the receptor interplay in developing new antidepressant drugs, the effects of 5HT1AR-FGFR1 complex activation were investigated in hippocampal pyramidal neurons and in midbrain dorsal raphe serotonergic neurons of SD rats and of a genetic rat model of depression (the Flinders Sensitive Line (FSL) rats of SD origin) using an electrophysiological approach. The results showed that in the raphe-hippocampal 5HT system of SD rats, 5HT1AR-FGFR1 heteroreceptor activation by specific agonists reduced the ability of the 5HT1AR protomer to open the GIRK channels through the allosteric inhibitory interplay produced by the activation of the FGFR1 protomer, leading to increased neuronal firing. On the contrary, in FSL rats, FGFR1 agonist-induced inhibitory allosteric action at the 5HT1AR protomer was not able to induce this effect on GIRK channels, except in CA2 neurons where we demonstrated that the functional receptor–receptor interaction is needed for producing the effect on GIRK. In keeping with this evidence, hippocampal plasticity, evaluated as long-term potentiation induction ability in the CA1 field, was impaired by 5HT1AR activation both in SD and in FSL rats, which did not develop after combined 5HT1AR-FGFR1 heterocomplex activation in SD rats. It is therefore proposed that in the genetic FSL model of depression, there is a significant reduction in the allosteric inhibition exerted by the FGFR1 protomer on the 5HT1A protomer-mediated opening of the GIRK channels in the 5HT1AR-FGFR1 heterocomplex located in the raphe-hippocampal serotonin system. This may result in an enhanced inhibition of the dorsal raphe 5HT nerve cell and glutamatergic hippocampal CA1 pyramidal nerve cell firing, which we propose may have a role in depression.

A need for new therapeutic approaches are necessary for dementia conditions and memory deficits of different origins, such as Alzheimer's disease. There is complex pathophysiological mechanisms involved, affecting adult hippocampal neurogenesis, in which neuropeptides and its neurogenesis regulation seem to participate. Neuropeptide Y(NPY) Y1 receptor (Y1R) and galanin (GAL) receptor 2 (GALR2) interact in brain regions responsible for learning and memory processes, emphasizing the hippocampus. Moreover, a significant challenge for treatments involving peptide drugs is bypassing the blood-brain barrier. The current study assesses the sustained memory performance induced by GALR2 and NPYY1R agonists intranasal coadministration and their neurochemical hippocampal correlates. Memory retrieval was conducted in the object-in-place task together with in situ proximity ligation assay (PLA) to manifest the formation of GALR2/Y1R heteroreceptor complexes and their dynamics under the different treatments. We evaluated cell proliferation through a 5-Bromo-2’-deoxyuridine (BrdU) expression study within the dentate gyrus of the dorsal hippocampus. The GalR2 agonist M1145 was demonstrated to act with the Y1R agonist to improve memory retrieval at 24 hours in the object-in-place task. Our data show that the intranasal administration is a feasible technique for directly delivering Galanin or Neuropeptide Y compounds into CNS. Moreover, we observed the ability of the co-agonist treatment to enhance the cell proliferation in the DG of the dorsal hippocampus through 5- Bromo-2’-deoxyuridine (BrdU) expression analysis at 24 hours. The understanding of the cellular mechanisms was achieved by analyzing the GALR2/Y1R heteroreceptor complexes upon agonist coactivation of their two types of receptor protomers in Doublecortin-expressing neuroblasts. Our results may provide the basis for developing heterobivalent agonist pharmacophores, targeting GALR2-Y1R heterocomplexes. It involves especially the neuronal precursor cells of the dentate gyrus in the dorsal hippocampus for the novel treatment of neurodegenerative pathologies as in the Alzheimer’s disease.

Dysregulation of hippocampal neurogenesis is linked to several neurodegenereative diseases, where boosting hippocampal neurogenesis in these patients emerges as a potential therapeutic approach. Accumulating evidence for a neuropeptide Y (NPY) and galanin (GAL) interaction was shown in various limbic system regions at molecular-, cellular-, and behavioral-specific levels. The purpose of the current work was to evaluate the role of the NPY and GAL interaction in the neurogenic actions on the dorsal hippocampus. We studied the Y1R agonist and GAL effects on: hippocampal cell proliferation through the proliferating cell nuclear antigen (PCNA), the expression of neuroprotective and anti-apoptotic factors, and the survival of neurons and neurite outgrowth on hippocampal neuronal cells. The functional outcome was evaluated in the object-in-place task. We demonstrated that the Y1R agonist and GAL promote cell proliferation and the induction of neuroprotective factors. These effects were mediated by the interaction of NPYY1 (Y1R) and GAL2 (GALR2) receptors, which mediate the increased survival and neurites’ outgrowth observed on neuronal hippocampal cells. These cellular effects are linked to the improved spatial-memory effects after the Y1R agonist and GAL co-injection at 24 h in the object-in-place task. Our results suggest the development of heterobivalent agonist pharmacophores, targeting Y1R–GALR2 heterocomplexes, therefore acting on the neuronal precursor cells of the DG in the dorsal hippocampus for the novel therapy of neurodegenerative cognitive-affecting diseases.

Among mental diseases, major depressive disorder (MDD) and anxiety deserve a special place due to their high prevalence and their negative impact both on society and patients suffering from these disorders. Consequently, the development of novel strategies designed to treat them quickly and efficiently, without or at least having limited side effects, is considered a highly important goal. Growing evidence indicates that emerging properties are developed on recognition, trafficking, and signaling of G-protein coupled receptors (GPCRs) upon their heteromerization with other types of GPCRs, receptor tyrosine kinases, and ionotropic receptors such as N-methyl-D-aspartate (NMDA) receptors. Therefore, to develop new treatments for MDD and anxiety, it will be important to identify the most vulnerable heteroreceptor complexes involved in MDD and anxiety. This review focuses on how GPCRs, especially serotonin, dopamine, galanin, and opioid heteroreceptor complexes, modulate synaptic and volume transmission in the limbic networks of the brain. We attempt to provide information showing how these emerging concepts can contribute to finding new ways to treat both MDD and anxiety disorders.

The widespread distribution of heteroreceptor complexes with allosteric receptor-receptor interactions in the CNS represents a novel integrative molecular mechanism in the plasma membrane of neurons and glial cells. It was proposed that they form the molecular basis for learning and short-and long-term memories. This is also true for drug memories formed during the development of substance use disorders like morphine and cocaine use disorders. In cocaine use disorder it was found that irreversible A2AR-D2R complexes with an allosteric brake on D2R recognition and signaling are formed in increased densities in the ventral enkephalin positive striatal-pallidal GABA antireward neurons. In this perspective article we discuss and propose how an increase in opioid heteroreceptor complexes, containing MOR-DOR, MOR-MOR and MOR-D2R, and their balance with each other and A2AR-D2R complexes in the striatal-pallidal enkephalin positive GABA antireward neurons, may represent markers for development of morphine use disorders. We suggest that increased formation of MOR-DOR complexes takes place in the striatal-pallidal enkephalin positive GABA antireward neurons after chronic morphine treatment in part through recruitment of MOR from the MOR-D2R complexes due to the possibility that MOR upon morphine treatment can develop a higher affinity for DOR. As a result, increased numbers of D2R monomers/homomers in these neurons become free to interact with the A2A receptors found in high densities within such neurons. Increased numbers of A2AR-D2R heteroreceptor complexes are formed and contribute to enhanced firing of these antireward neurons due to loss of inhibitory D2R protomer signaling which finally leads to the development of morphine use disorder. Development of cocaine use disorder may instead be reduced through enkephalin induced activation of the MOR-DOR complex inhibiting the activity of the enkephalin positive GABA antireward neurons. Altogether, we propose that these altered complexes could be pharmacological targets to modulate the reward and the development of substance use disorders.

Alcohol use disorder (AUD) is highly prevalent, and over 50% of AUD patients also suffer major depressive disorders. Selective 5-HT reuptake inhibitors (SSRIs) can reduce rodent ethanol drinking but exert modest clinical efficacy in alcoholic individuals. Finding new pharmacological strategies that could modulate alcohol consumption and depression is necessary. We have analyzed the effect of Galanin (1–15) [GAL(1–15)] on escitalopram (ESC)-mediated effect in alcohol consumption using the alcohol self-administration test, the nuclei involved in the effect, and whether GAL(1–15) + ESC modulated the response in despair or anxiety tests in animals under chronic alcohol intake. GAL(1–15) + ESC combination substantially reduced alcohol intake in the alcohol self-administration test and, moreover, enhanced the reduction of reward capacity of ESC on different reinforcers such as sucrose or saccharine. GAL(1–15) + ESC coadministration significantly decreases the number of C-Fos-IR TH cell bodies in the VTA, and PCA analysis suggests that one functional network, including VTA, RMTg and DR, is involved in these effects. Significantly in rats with chronic alcohol consumption, GAL(1–15) reversed adverse ESC-mediated effects in the depression-related behavioural test and forced swimming test. The results open up the possibility of using GAL(1–15) in combination with the SSRI Escitalopram as a novel strategy in AUD comorbidity with depression.

Abstract Morphine binding to opioid receptors, mainly to μ opioid receptor (MOR), induces alterations in intracellular pathways essential to the initial development of addiction. The activation of the dopamine D4 receptor (D4R), which is expressed in the caudate putamen (CPu), mainly counteracts morphine-induced alterations in several molecular networks. These involve transcription factors, adaptive changes of MOR signaling, activation of the nigrostriatal dopamine pathway and behavioural effects, underlining functional D4R/MOR interactions. To shed light on the molecular mechanisms implicated, we evaluated the transcriptome alterations following acute administration of morphine and/or PD168,077 (D4R agonist) using whole-genome microarrays and a linear regression-based differential expression analysis. The results highlight the development of a unique transcriptional signature following the co-administration of both drugs that reflects a countereffect of PD168,077 on morphine effects. A KEGG pathway enrichment analysis using GSEA identified 3 pathways enriched positively in morphine vs control and negatively in morphine + PD168,077 vs morphine (Ribosome, Complement and Coagulation Cascades, Systemic Lupus Erythematosus) and 3 pathways with the opposite enrichment pattern (Alzheimer’s Disease, Neuroactive Ligand Receptor Interaction, Oxidative Phosphorilation). This work supports the massive D4R/MOR functional integration at the CPu and provides a gateway to further studies on the use of D4R drugs to modulate morphine-induced effects.

Selective 5-HT reuptake inhibitor antidepressants (SSRIs) are the first choice in major depressive disorder (MDD), but 50% of affected patients do not show improvement. Galanin(1-15) [GAL(1-15)] enhanced Fluoxetine antidepressant-like effects in an animal model of depression, the olfactory bulbectomy (OBX); however, further detailed analysis of GAL(1-15) effects as augmentation treatment in OBX rats are needed. In OBX rats, we analysed the effect of GAL(1–15) on Escitalopram (ESC)-mediated responses in behavioural tests related to despair. We studied whether GAL(1–15) effects involved 5-HT1AR using an in vivo model siRNA 5-HT1A knockdown rats. Moreover, we analysed by immunohistochemistry the expression of the immediate-early gene c-Fos (c-Fos IR) after the administration of GAL(1-15)+ESC in OBX rats in several nuclei involved in MDD. GAL(1-15) enhances the antidepressant-like effects of ESC, and the GALR2 antagonist M871 blocked GAL(1-15) mediated actions. The downregulation of 5-HT1AR by siRNA was sufficient to block GAL(1-15) effects. Our immunohistochemistry and principal component analysis (PCA) analysis suggest that two functional networks are involved in these effects; one includes the lateral (LHb) and medial (mHb) habenula, dorsal raphe (DR) and ventral tegmental area (VTA), and the other consists of the dentate gyrus (DG), and prefrontal cortex (PFC). The results open up the possibility of using GAL(1-15) in combination with SSRIs as a novel strategy for treating MDD.

The heteroreceptor complexes present a novel biological principle for signal integration. These complexes and their allosteric receptor–receptor interactions are bidirectional and novel targets for treatment of CNS diseases including mental diseases. The existence of D2R-5-HT2AR heterocomplexes can help explain the anti-schizophrenic effects of atypical antipsychotic drugs not only based on blockade of 5-HT2AR and of D2R in higher doses but also based on blocking the allosteric enhancement of D2R protomer signaling by 5-HT2AR protomer activation. This research opens a new understanding of the integration of DA and 5-HT signals released from DA and 5-HT nerve terminal networks. The biological principle of forming 5-HT and other heteroreceptor complexes in the brain also help understand the mechanism of action for especially the 5-HT hallucinogens, including putative positive effects of e.g., psilocybin and the indicated prosocial and anti-stress actions of MDMA (ecstasy). The GalR1-GalR2 heterodimer and the putative GalR1-GalR2-5-HT1 heteroreceptor complexes are targets for Galanin N-terminal fragment Gal (1–15), a major modulator of emotional networks in models of mental disease. GPCR-receptor tyrosine kinase (RTK) heteroreceptor complexes can operate through transactivation of FGFR1 via allosteric mechanisms and indirect interactions over GPCR intracellular pathways involving protein kinase Src which produces tyrosine phosphorylation of the RTK. The exciting discovery was made that several antidepressant drugs such as TCAs and SSRIs as well as the fast-acting antidepressant drug ketamine can directly bind to the TrkB receptor and provide a novel mechanism for their antidepressant actions. Understanding the role of astrocytes and their allosteric receptor–receptor interactions in modulating forebrain glutamate synapses with impact on dorsal raphe-forebrain serotonin neurons is also of high relevance for research on major depressive disorder.

Volume transmission is a new major communication signaling via extracellular fluid (interstitial fluid) pathways. It was proposed by the current authors that such pathways can explain the meridian phenomena and acupuncture effects. To investigate whether meridian-like structures exist in fish body and operate via volume transmission in extracellular fluid pathways, we injected alcian blue (AB) under anesthesia into Gephyrocharax melanocheir, which has a translucent body. The migration of AB could be seen directly and was recorded by a digital camera. The fish was then embedded and cut transversely to observe the position of tracks in three dimensions. Eight longitudinal threadlike blue tracks were recognized on the fish. The positions of these threadlike tracks were similar to meridians on the human body. Transverse sections showed that these tracks distributed to different layers of distinct subcutaneous loose connective tissues and intermuscular septa. Lymphatic vessels were sometimes associated with the extracellular blue tracks where the migration of AB occurred. Extracellular fluid pathways were found on fish through their transport of AB. These pathways operating via volume transmission appeared to be similar in positions and functions to the acupuncture meridians in Chinese medicine.

G protein-coupled receptors (GPCRs) not only exist as monomers but also as homomers and heteromers in which allosteric receptor-receptor interactions take place, modulating the functions of the participating GPCR protomers. GPCRs can also form heteroreceptor complexes with ionotropic receptors and receptor tyrosine kinases modulating their function. Furthermore, adaptor proteins interact with receptor protomers and modulate their interactions. The state of the art is that the allosteric receptor-receptor interactions are reciprocal, highly dynamic and substantially alter the signaling, trafficking, recognition and pharmacology of the participating protomers. The pattern of changes appears to be unique for each heteromer and can favor antagonistic or facilitatory interactions or switch the G protein coupling from e.g., Gi/o to Gq or to beta-arrestin signaling. It lends a new dimension to molecular integration in the nervous system. Future direction should be aimed at determining the receptor interface involving building models of selected heterodimers. This will make design of interface-interfering peptides that specifically disrupt the heterodimer possible. This will help to determine the functional role of the allosteric receptor-receptor interactions as well as the integration of signals at the plasma membrane by the heteroreceptor complexes, vs. integration of the intracellular signaling pathways. Integration of signals also at the plasma membrane seems crucial in view of the hypothesis that learning and memory at a molecular level takes place by reorganization of homo and heteroreceptor complexes in the postsynaptic membrane. Homo and heteroreceptor complexes are in balance with each other, and their disbalance is linked to disease. Targeting heteroreceptor complexes represents a novel strategy for the treatment of brain disorders.

Based on the work in the Central Nervous System with discoveries of allosteric receptor-receptor interactions in homo- and heteroreceptor complexes representing a major integrative mechanism in synapses and extrasynaptic regions, it is proposed that a similar mechanism may exist in the immunological synapses. We discuss a putative additional molecular mechanism for the ability of the inhibitory T cell signaling proteins CTLA-4 and PD-1 and the adenosine A2AR to diminish T cell activation leading to enhancement of cancer development. We suggest that in the same immunological synapse involving T cells and antigen presenting cells multiple heteroreceptor complexes may participate and be in balance with each other. Their composition can vary between functional states and among different types of T cells. The T cell receptor (TCR) and its accelerators, strongly enhancing T cell activation, can be under inhibitory control by T cell signaling proteins CTLA4 and PD-1 and also the adenosine A2AR through inhibitory allosteric receptor-receptor interactions in different types of heteroreceptor complexes. As a result, inhibitory tumor induced immunosuppression can develop due to a dominance of the inhibitory signaling causing a brake on the TCR and/or its accelerator and the cancer immunotherapy becomes blocked.

Serotonin communication operates mainly in the extracellular space and cerebrospinal fluid (CSF), using volume transmission with serotonin moving from source to target cells (neurons and astroglia) via energy gradients, leading to the diffusion and convection (flow) of serotonin. One emerging concept in depression is that disturbances in the integrative allosteric receptor–receptor interactions in highly vulnerable 5-HT1A heteroreceptor complexes can contribute to causing major depression and become novel targets for the treatment of major depression (MD) and anxiety. For instance, a disruption and/or dysfunction in the 5-HT1A-FGFR1 heteroreceptor complexes in the raphe-hippocampal serotonin neuron systems can contribute to the development of MD. It leads inter alia to reduced neuroplasticity and potential atrophy in the raphe-cortical and raphe-striatal 5-HT pathways and in all its forebrain networks. Reduced 5-HT1A auto-receptor function, increased plasticity and trophic activity in the midbrain raphe 5-HT neurons can develop via agonist activation of allosteric receptor–receptor interactions in the 5-HT1A-FGFR1 heterocomplex. Additionally, the inhibitory allosteric receptor–receptor interactions in the 5-HT1AR-5-HT2AR isoreceptor complex therefore likely have a significant role in modulating mood, involving a reduction of postjunctional 5-HT1AR protomer signaling in the forebrain upon activation of the 5-HT2AR protomer. In addition, oxytocin receptors (OXTRs) play a significant and impressive role in modulating social and cognitive related behaviors like bonding and attachment, reward and motivation. Pathological blunting of the OXTR protomers in 5-HT2AR and especially in 5-HT2CR heteroreceptor complexes can contribute to the development of depression and other types of psychiatric diseases involving disturbances in social behaviors. The 5-HTR heterocomplexes are novel targets for the treatment of MD.

The discovery of the central monoamine neurons not only demonstrated novel types of brain stem neurons forming global terminal networks all over the brain and the spinal cord, but also to a novel type of communication called volume transmission. It is a major mode of communication in the central nervous system that takes places in the extracellular fluid and the cerebral spinal fluid through diffusion and flow of molecules, like neurotransmitters and extracellular vesicles. The integration of synaptic and volume transmission takes place through allosteric receptor-receptor interactions in heteroreceptor complexes. These heterocomplexes represent major integrator centres in the plasma membrane and their protomers act as moonlighting proteins undergoing dynamic changes and their structure and function. In fact, we propose that the molecular bases of learning and memory can be based on the reorganization of multiples homo and heteroreceptor complexes into novel assembles in the post-junctional membranes of synapses.

Several psychoactive drugs can evoke substance use disorders (SUD) in humans and animals, and these include psychostimulants, opioids, cannabinoids (CB), nicotine, and alcohol. The etiology, mechanistic processes, and the therapeutic options to deal with SUD are not well understood. The common feature of all abused drugs is that they increase dopamine (DA) neurotransmission within the mesocorticolimbic circuitry of the brain followed by the activation of DA receptors. D2 receptors were proposed as important molecular targets for SUD. The findings showed that D2 receptors formed heteromeric complexes with other GPCRs, which forced the addiction research area in new directions. In this review, we updated the view on the brain D2 receptor complexes with adenosine (A)2A receptors (A2AR) and discussed the role of A2AR in different aspects of addiction phenotypes in laboratory animal procedures that permit the highly complex syndrome of human drug addiction. We presented the current knowledge on the neurochemical in vivo and ex vivo mechanisms related to cocaine use disorder (CUD) and discussed future research directions for A2AR heteromeric complexes in SUD.

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.

The A2A adenosine (A2AR) and D2 dopamine (D2R) receptors form oligomers in the cell membrane and allosteric interactions across the A2AR–D2R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A2AR–D2R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A2AR–D2R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A2AR–D2R receptor–receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A2AR blocked heterodimer interactions and disrupted the allosteric effect of A2AR activation on D2R agonist binding. Protein–protein docking was used to construct a model of the A2AR–D2R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A2AR–D2R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A2AR–D2R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.

This perspective article provides observations supporting the view that nigro-striatal dopamine neurons and meso-limbic dopamine neurons mainly communicate through short distance volume transmission in the um range with dopamine diffusing into extrasynaptic and synaptic regions of glutamate and GABA synapses. Based on this communication it is discussed how volume transmission modulates synaptic glutamate transmission onto the D1R modulated direct and D2R modulated indirect GABA pathways of the dorsal striatum. Each nigro-striatal dopamine neuron was first calculated to form large numbers of neostriatal DA nerve terminals and then found to give rise to dense axonal arborizations spread over the neostriatum, from which dopamine is released. These neurons can through DA volume transmission directly influence not only the striatal GABA projection neurons but all the striatal cell types in parallel. It includes the GABA nerve cells forming the island-/striosome GABA pathway to the nigral dopamine cells, the striatal cholinergic interneurons and the striatal GABA interneurons. The dopamine modulation of the different striatal nerve cell types involves the five dopamine receptor subtypes, D1R to D5R receptors, and their formation of multiple extrasynaptic and synaptic dopamine homo and heteroreceptor complexes. These features of the nigro-striatal dopamine neuron to modulate in parallel the activity of practically all the striatal nerve cell types in the dorsal striatum, through the dopamine receptor complexes allows us to understand its unique and crucial fine-tuning of movements, which is lost in Parkinson's disease. Integration of striatal dopamine signals with other transmitter systems in the striatum mainly takes place via the receptor-receptor interactions in dopamine heteroreceptor complexes. Such molecular events also participate in the integration of volume transmission and synaptic transmission. Dopamine modulation of the glutamate synapses on the dorsal striato-pallidal GABA pathway involves D2R heteroreceptor complexes such as D2R-NMDAR, A2AR-D2R, and NTSR1-D2R heteroreceptor complexes. The dopamine modulation of glutamate synapses on the striato-entopeduncular/nigral pathway takes place mainly via D1R heteroreceptor complexes such as D1R-NMDAR, A2R-D1R, and D1R-D3R heteroreceptor complexes. Dopamine modulation of the island/striosome compartment of the dorsal striatum projecting to the nigral dopamine cells involve D4R-MOR heteroreceptor complexes. All these receptor-receptor interactions have relevance for Parkinson's disease and its treatment.

Anxiety is evoked by a threatening situation and display adaptive or defensive behaviors, found similarly in animals and humans. Neuropeptide Y (NPY) Y1 receptor (NPYY1R) and Galanin (GAL) receptor 2 (GALR2) interact in several regions of the limbic system, including the amygdala. In a previous study, GALR2 enhanced NPYY1R mediated anxiolytic actions on spatiotemporal parameters in the open field and elevated plus maze, involving the formation of GALR2/NPYY1R heteroreceptor complexes in the amygdala. Moreover, the inclusion of complementary ethological parameters provides a more comprehensive profile on the anxiolytic effects of a treatment. The purpose of the current study is to evaluate the anxiolytic effects and circuit activity modifications caused by coactivation of GALR2 and NPYY1R. Ethological measurements were performed in the open field, the elevated plus-maze and the light-dark box, together with immediate early gene expression analysis within the amygdala-hypothalamus-periaqueductal gray (PAG) axis, as well as in situ proximity ligation assay (PLA) to demonstrate the formation of GALR2/NPYY1R heteroreceptor complexes. GALR2 and NPYY1R coactivation resulted in anxiolytic behaviors such as increased rearing and head-dipping, reduced stretch attend postures and freezing compared to single agonist or aCSF injection. Neuronal activity indicated by cFos expression was decreased in the dorsolateral paracapsular intercalated (ITCp-dl) subregion of the amygdala, ventromedial hypothalamic (VMH) nucleus and ventrolateral part of the periaqueductal gray (vlPAG), while increased in the perifornical nucleus of the hypothalamus (PFX) following coactivation of GALR2 and NPYY1R. Moreover, an increased density of GALR2/NPYY1R heteroreceptor complexes was explicitly observed in ITCp-dl, following GALR2 and NPYY1R coactivation. Besides, knockdown of GALR2 was found to reduce the density of complexes in ITCp-dl. Taken together, these results open up the possibility that the increased anxiolytic activity demonstrated upon coactivation of NPYY1R and GALR2 receptor was related to actions on the ITCp-dl. GALR2-NPYY1R heteroreceptor complexes may inhibit neuronal activity, by also modifying the neuronal networks of the hypothalamus and the PAG. These results indicate that GALR2/NPYY1R interactions in medial paracapsular intercalated amygdala can provide a novel integrative mechanism in anxiolytic behavior and the basis for the development of heterobivalent agonist drugs targeting GALR2/NPYY1R heteromers, especially in the ITCp-dl of the amygdala for the treatment of anxiety.

Adenosine is a nucleoside mainly formed by degradation of ATP, located intracellularly or extracellularly, and acts as a neuromodulator. It operates as a volume transmission signal through diffusion and flow in the extracellular space to modulate the activity of both glial cells and neurons. The effects of adenosine are mediated via four adenosine receptor subtypes: A1R, A2AR, A2BR, A3R. The A2AR has a wide-spread distribution but it is especially enriched in the ventral and dorsal striatum where it is mainly located in the striato-pallidal GABA neurons at a synaptic and extrasynaptic location. A number of A2AR heteroreceptor complexes exist in the striatum. The existence of A2AR-D2R heteroreceptor complexes with antagonistic A2AR-D2R interactions in the striato-pallidal GABA neurons is well-known with A2AR activation inhibiting Gi/o mediated signaling of D2Rs. A2AR-mGluR5 heteroreceptor complexes were also found in with synergistic receptor-receptor interactions enhancing the inhibition of the D2R protomer signaling. They are located mainly in extrasynaptic regions of the striato-pallidal GABA neurons. Results recently demonstrated the existence of brain A2AR-A2BR heteroreceptor complexes, in which A2BR protomer constitutively inhibited the function of the A2AR protomer. These adenosine A2AR heteroreceptor complexes may modulate alpha-synuclein aggregation and toxicity through postulated bidirectional direct interactions leading to marked increases in A2AR signaling both in nerve cells and microglia. It is of high interest that formation of A2AR-A2ABR heteroreceptor complexes provides a brake on A2AR recognition and signaling opening up a novel strategy for treatment of A2AR mediated neurodegeneration.

The FGFR1-5-HT1A heteroreceptor complexes are involved in neuroplasticity in the rat hippocampus and in the mesencephalic raphe 5-HT nerve cells. There exists a 5-HT1A protomer enhancement of FGFR1 protomer signaling. Acute and 10 day treatment with intracerebroventricular (i.c.v.) FGF-2 and the 5-HT1A agonist 8-OH-DPAT produced enhanced antidepressant effects in the forced swim test (FST). We studied in the current work the disturbances in the FGFR1-5-HT1A heterocomplexes in a genetic rat model of depression, the Flinders sensitive line (FSL) rats of Sprague-Dawley (SD) origin, by means of neurochemical, neurophysiological and behavioral techniques. In control SD rats, the FGFR1 agonist SUN11602 and FGF2 produced a significant reduction of G protein-coupled inwardly rectifying K+ channel (GIRK) currents induced by 8-OH-DPAT in the CA1 area of the hippocampus. In FSL rats, only i.c.v. 8-OH-DPAT alone treatment produced a significant reduction in the immobility time. The combined i.c.v. treatment (FGF2 + 8-OH-DPAT) in FSL rats did not cause a significant decrease in immobility time in the FST. However, in the SD rats this combined treatment produced a significant reduction. Furthermore, in the FSL rat a significant increase in the density of FGFR1-5-HT1A proximity ligation assay (PLA) positive clusters was only found after i.c.v. 8-OH-DPAT treatment alone in the CA2 and CA3 areas. In the SD rat a significant increase in the density of specific PLA clusters was only observed in the CA2 area of the i.c.v. combined treatment (FGF2 + 8-OH-DPAT) group. No treatment led to significant changes in the PLA clusters of the dorsal raphe in the FSL rat. However, significant changes in the density of specific PLA clusters were only found in the dorsal raphe of SD rats after combined treatment and treatment with 8-OH-DPAT alone. The results indicate that in FSL rats compared with SD rats alterations may develop in the ability of 8-OH-DPAT and combined FGFR1 and 5-HT1A agonist treatment to increase the density of FGFR1-5-HT1A heteroreceptor complexes of the dorsal raphe. It is proposed that such deficits in FSL rats may possibly reflect a failure of the combined agonist treatment to uncouple the 5-HT1A autoreceptors from the GIRK channels. This may contribute to the failure of producing antidepressant-like effects in the FSL rat by combined agonist treatment as seen in the SD rat. The antidepressant-like effects seen with the 5-HT1A agonist alone treatment in FSL but not in SD rats may instead involve significant increases in the FGFR1-5-HT1A complexes of the CA2 and CA3 areas of the hippocampus.

G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html.

The mu opioid receptor (MOR) is critical in mediating morphine analgesia. However, prolonged exposure to morphine induces adaptive changes in this receptor leading to the development of tolerance and addiction. In the present work we have studied whether the continuous administration of morphine induces changes in MOR protein levels, its pharmacological profile, and MOR-mediated G-protein activation in the striosomal compartment of the rat CPu, by using immunohistochemistry and receptor and DAMGO-stimulated [35S]GTPγS autoradiography. MOR immunoreactivity, agonist binding density and its coupling to G proteins are up-regulated in the striosomes by continuous morphine treatment in the absence of changes in enkephalin and dynorphin mRNA levels. In addition, co-treatment of morphine with the dopamine D4 receptor (D4R) agonist PD168,077 fully counteracts these adaptive changes in MOR, in spite of the fact that continuous PD168,077 treatment increases the [3H]DAMGO Bmax values to the same degree as seen after continuous morphine treatment. Thus, in spite of the fact that both receptors can be coupled to Gi/0 protein, the present results give support for the existence of antagonistic functional D4R-MOR receptor-receptor interactions in the adaptive changes occurring in MOR of striosomes on continuous administration of morphine.

The arginine-vasopressin (AVP)-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs) are known for their role in hydro-electrolytic balance control via their projections to neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula, and other brain regions, in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA). The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS), consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptors mRNA were not detected, using the same method. Water-deprivation for 24 hrs, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze test, and this effect was mimicked by bilateral microinfusion of VP into the CeA. Anxious behavior induced by either water deprivation or VP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of central amygdala inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala.

The interplay between the dopamine (DA) and opioid systems in the brain is known to modulate the additive effects of substances of abuse. On one hand, opioids serve mankind by their analgesic properties, which are mediated via the mu opioid receptor (MOR), a Class A G protein-coupled receptor (GPCR), but on the other hand, they pose a potential threat by causing undesired side effects such as tolerance and dependence, for which the exact molecular mechanism is still unknown. Using human embryonic kidney 293T (HEK 293T) and HeLa cells transfected with MOR and the dopamine D2 receptor (D2R), we demonstrate that these receptors heterodimerize, using an array of biochemical and biophysical techniques such as coimmunoprecipitation (co-IP), bioluminescence resonance energy transfer (BRET1), Fӧrster resonance energy transfer (FRET), and functional complementation of a split luciferase. Furthermore, live cell imaging revealed that D2LR, when coexpressed with MOR, slowed down internalization of MOR, following activation with the MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO).

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.

Adenosine A2A receptor (A2AR) antagonists are being investigated as promising treatment strategy for Parkinson's disease (PD). To test whether A2AR antagonists are beneficial in early PD stages we used MitoPark mice, a genetic model with gradual degeneration of DA cells. Daily treatment of young MitoPark mice for eight weeks with the A2AR antagonist MSX-3 prevented the reduction of spontaneous locomotor activity observed in saline or L-DOPA treated animals. Chronic A2AR antagonist treatment neither induced desensitization of receptors nor accumulation of the drug in brain tissue. Despite beneficial effects on behavior, which are not improved upon addition of a low dose of L-DOPA, the characteristic decline of dopamine levels was not changed. Our results indicate that effective dosing with A2AR antagonists should be tested as monotherapy in early PD, and serves to remind us that positive behavioral effects of such treatment need not be reflected in rescue of striatal dopamine levels.

Due to the binding to a number of proteins to the receptor protomers in receptor heteromers in the brain, the term “heteroreceptor complexes” was introduced. A number of serotonin 5-HT1A heteroreceptor complexes were recently found to be linked to the ascending 5-HT pathways known to have a significant role in depression. The 5-HT1A–FGFR1 heteroreceptor complexes were involved in synergistically enhancing neuroplasticity in the hippocampus and in the dorsal raphe 5-HT nerve cells. The 5-HT1A protomer significantly increased FGFR1 protomer signaling in wild-type rats. Disturbances in the 5-HT1A–FGFR1 heteroreceptor complexes in the raphe-hippocampal 5-HT system were found in a genetic rat model of depression (Flinders sensitive line (FSL) rats). Deficits in FSL rats were observed in the ability of combined FGFR1 and 5-HT1A agonist cotreatment to produce antidepressant-like effects. It may in part reflect a failure of FGFR1 treatment to uncouple the 5-HT1A postjunctional receptors and autoreceptors from the hippocampal and dorsal raphe GIRK channels, respectively. This may result in maintained inhibition of hippocampal pyramidal nerve cell and dorsal raphe 5-HT nerve cell firing. Also, 5-HT1A–5-HT2A isoreceptor complexes were recently demonstrated to exist in the hippocampus and limbic cortex. They may play a role in depression through an ability of 5-HT2A protomer signaling to inhibit the 5-HT1A protomer recognition and signaling. Finally, galanin (1–15) was reported to enhance the antidepressant effects of fluoxetine through the putative formation of GalR1–GalR2–5-HT1A heteroreceptor complexes. Taken together, these novel 5-HT1A receptor complexes offer new targets for treatment of depression.

It has been demonstrated that some viruses, such as the cytomegalovirus, code for G-protein coupled receptors not only to elude the immune system, but also to redirect cellular signaling in the receptor networks of the host cells. In view of the existence of receptor-receptor interactions, the hypothesis is introduced that these viral-coded receptors not only operate as constitutively active monomers, but also can affect other receptor function by interacting with receptors of the host cell. Furthermore, it is suggested that viruses could also insert not single receptors (monomers), but clusters of receptors (receptor mosaics), altering the cell metabolism in a profound way. The prevention of viral receptor-induced changes in host receptor networks may give rise to novel antiviral drugs that counteract viral-induced disease.

By analyzing the functional role of adenosine receptor heteromers, we review a series of new concepts that should modify our classical views of neurotransmission in the central nervous system (CNS). Neurotransmitter receptors cannot be considered as single functional units anymore. Heteromerization of neurotransmitter receptors confers functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Some of these characteristics can be used as a “biochemical fingerprint” to identify neurotransmitter receptor heteromers in the CNS. This is exemplified by changes in binding characteristics that are dependent on coactivation of the receptor units of different adenosine receptor heteromers. Neurotransmitter receptor heteromers can act as “processors” of computations that modulate cell signaling, sometimes critically involved in the control of pre- and postsynaptic neurotransmission. For instance, the adenosine A1-A2A receptor heteromer acts as a concentration-dependent switch that controls striatal glutamatergic neurotransmission. Neurotransmitter receptor heteromers play a particularly important integrative role in the “local module” (the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit), where they act as processors mediating computations that convey information from diverse volume-transmitted signals. For instance, the adenosine A2A-dopamine D2 receptor heteromers work as integrators of two different neurotransmitters in the striatal spine module.