Search

Background Selective serotonergic reuptake inhibitors, including fluoxetine (FLX), are the most commonly used for the treatment of major depression. However, they are effective for remission in only 30% of patients. Recently, we observed that Galanin (1-15) [GAL(1-15)] enhanced the antidepressant effects of FLX in naïve animals, suggesting a new augmentation strategy in depression. Methods We have analyzed in an animal model of depression, the olfactory bulbectomy (OBX) rats, the effect of GAL(1-15) on FLX-mediated responses in the forced swimming test and the sucrose preference test and the involvement of GAL receptor 2 with its antagonist, M871. We have also studied the corticosterone levels in OBX after the coadministration of GAL(1-15) with FLX. Moreover, we studied whether the effects of GAL(1-15) on FLX actions were mediated via auto- and heteroreceptor 5-HT1A (5-HT1AR), analyzing the binding characteristics, mRNA levels, and functionality of 5-HT1AR in the dorsal hippocampus. Results GAL(1-15) enhances the antidepressant-like effects induced by FLX in OBX animals in the forced swimming test and the sucrose preference test. The involvement of the GALR2 was demonstrated with M871. Importantly, the mechanism underlying the GAL(1-15)/FLX interactions in the OBX animals involves the 5-HT1AR in the hippocampus at the plasma membrane (increase of affinity and density of 5HT1AR in the DG) and transcriptional (increase of 5HT1AR mRNA levels in DG and CA1) levels. Besides, the coadministration of GAL(1-15) and FLX also reduced OBX-increased corticosterone levels. Conclusions The results open the possibility to use GAL(1-15) in combination with FLX as a novel strategy for the treatment of depression.

Alcohol Use Disorder (AUD) is among the most prevalent mental illnesses, and due to the low efficacy of the current medication, it is essential to find new biological targets that could modulate alcohol consumption. Since Galanin (1−15) [GAL(1−15)] produces a loss of motivational behaviour by an artificial reinforcer and decreases the preference an alcohol consumption in a voluntary alcohol intake, we have studied the role of GAL(1−15) in alcohol-seeking behaviour and the involvement of the corticomesolimbic system as well as the role of GAL(1−15) in context-induced alcohol relapse. In rats, we have studied GAL(1−15)-effects on alcohol-seeking in self-administration, in fixed-ratio (FR1) and progressive-ratio (PR), and the involvement of GAL receptors using siRNA GALR1 or GALR2 knockdown animals. We have analysed the transcriptional changes of C-Fos, dopamine receptors, GAL receptors and 5HT1A receptors in the corticomesolimbic system. Also, we have examined the effect of GAL(1−15) in context-induced alcohol relapse. GAL(1−15) substantially reduced alcohol-seeking behaviour in the operant self-administration model in an FR1 protocol and at the breaking point in a PR schedule. GALR1and GALR2 were involved in these effects, as indicated by the analysis by GALR2 antagonist and GALR1 and GALR2 knockdown animals. Notably, the mechanism of GAL(1−15)-mediated actions involved changes in C-Fos, Dopamine receptors and 5HT1A expression in the ventral tegmental area, accumbens nucleus and prefrontal cortex. Significantly, GAL(1−15) reduced the context-induced alcohol relapse. These results open up the possibility to use GAL(1−15) as a novel strategy in AUD. This work was supported by grants awarded by Spanish Ministry of Economy PID2020-114392RB-100, PDC2021-121566-100 and by Junta de Andalucía P20-00026-R and PI-0083-2019. Partial funding for open access charge: Universidad de Málaga

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.

Alcohol consumption is considered a major risk factor for disease and mortality worldwide. In the absence of effective treatments in alcohol use disorders, it is important to find new biological targets that could modulate alcohol consumption. We tested the role of the N-terminal galanin fragment (1-15) [GAL(1-15)] in voluntary ethanol consumption in rats using the two-bottle choice paradigm as well as compare the effects of GAL(1-15) with the whole molecule of GAL. We describe for the first time that GAL(1-15), via central mechanisms, induces a strong reduction in preference and ethanol consumption in rats. These effects were significantly different than GAL. GAL receptor (GALR) 2 was involved in these effects, because the specific GALR2 antagonist M871 blocked GAL(1-15) mediated actions in preference and ethanol intake. Importantly, the mechanism of this action involves changes in GALR expression and also in immediate-early gene C-Fos and receptors-internalization-related gene Rab5 in the striatum. The relevance of the striatum as a target for GAL(1-15) was supported by the effect of GAL(1-15) on the locomotor activity of rats after ethanol administration. These results may give the basis for the development of novel therapeutics strategies using GAL(1-15) analogues for the treatment of alcohol use disorders in humans.

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.

Background: Anhedonia is a core feature of depressive disorders. The galanin N-terminal fragment (1-15) plays a role in mood regulation since it induces depression and anxiogenic-like effects in rats. In this study, we analysed galanin N-terminal fragment (1-15) actions in anhedonic-like behaviours in rats using operant and non-operant tests and the areas involved with these effects. Methods: Galanin N-terminal fragment (1-15) effects were analysed in saccharin self-administration, sucrose preference, novelty-suppressed feeding and female urine sniffing tests. The areas involved in galanin N-terminal fragment (1-15)-mediated effects were studied with positron emission tomography for in vivo imaging, and we analysed the ventral tegmental area and nucleus accumbens. Galanin N-terminal fragment (1-15) had effects on the mRNA expression of the dopamine transporters Dat and Vmat2; the C-Fos gene; the dopamine receptors D1, D2, D3, D5; and the galanin receptors 1 and 2. Results: Galanin N-terminal fragment (1-15) at a concentration of 3 nmol induced a strong anhedonia-like phenotype in all tests. The involvement of galanin receptor 2 was demonstrated with the galanin receptor 2 antagonist M871 (3 nmol). The 18F-fluorodeoxyglucose positron emission tomography images indicated the action of galanin N-terminal fragment (1-15) over several nuclei of the limbic system. Galanin N-terminal fragment (1-15)-mediated effects also involved changes in the expression of Dat, Vmat2, D3 and galanin receptors in the ventral tegmental area as well as the expression of C-Fos, D1, D2 and D3 and TH immunoreactivity in the nucleus accumbens. Conclusions: Our results indicated that galanin N-terminal fragment (1-15) exerts strong anhedonic-like effects and that this effect was accompanied by changes in the dopaminergic mesolimbic system. These results may provide a basis for the development of novel therapeutic strategies using galanin N-terminal fragment (1-15) analogues for the treatment of depression and reward-related diseases.

Galanin (1-15) [GAL(1–15)] participates in mood regulation and depression. GAL(1–15) is also able to enhance the antidepressant effects induced by Fluoxetine (FLX) in the forced swimming test through interaction between GALR1-GALR2 and 5-HT1A receptors that induced changes in the binding characteristics and mRNA of the 5-HT1AR in the hippocampus. Since the medial prefrontal cortex (mPFC) is a core region for the interaction between emotional processing and cognition with a high density of 5-HT1AR and GALR1 and GALR2, we have analyzed the binding characteristics and mRNA levels of 5-HT1AR in the mPFC after GAL(1–15)-FLX administration in the rats. GAL(1–15) increased the Kd and the Bmax of the 5HT1AR agonist binding in the mPFC as well as the mRNA levels of 5-HT1AR in mPFC. Moreover, GAL(1–15) reversed the effects of memory impairment induced by FLX(10 mg/kg) in the Novel Object Recognition task. GALR2 was involved in these effects, since the specific GALR2 antagonist M871 blocked GAL(1–15) mediated actions at behavioral level. On the contrary GAL(1–15) did not reverse the effect of FLX in the Object Location Memory task. In conclusion, our results describe an interactions between GAL(1–15) and FLX in the mPFC involving interactions at the 5-HT1AR receptor level in the plasma membrane with changes at the transcriptional level with implications also at functional level. The GALR1-GALR2-5-HT1A heteroreceptor could be postulated to be used to reverse some of the adverse effects of FLX on memory processes.

Using an immunohistochemical technique, we mapped the immunoreactive structures containing methionine-enkephalin-Arg6-Gly7-Leu8 (Met-8) (a marker for the pro-enkephalin system) in the human diencephalon. Compared with previous studies, we observed a more widespread distribution of Met-8 in the human diencephalon. Met-8-immunoreactive cell bodies and fibers exhibited a more widespread distribution in the hypothalamus than in the thalamus. We observed six populations of Met-8-immunoreactive cell bodies. These perikarya were observed in the paratenial thalamic nucleus, ventromedial and dorsomedial hypothalamic nuclei, lateral hypothalamic area, pallidohypothalamic nucleus and in the paraventricular hypothalamic nucleus (posterior part). In the thalamus, Met-8-immunoreactive fibers were primarily observed in the midline region, whereas in the hypothalamus, these fibers were widely distributed. In general, a moderate/low density of Met-8-immunoreactive fibers was observed in the diencephalic nuclei. A moderate density was observed in the paraventricular thalamic nucleus, reuniens thalamic nucleus, lateral and medial geniculate nuclei, dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus (posterior part) and ventromedial hypothalamic nucleus. The present study is the first to demonstrate the presence of clusters of Met-8-immunoreactive cell bodies in the human thalamus and hypothalamus, the distribution of fibers containing neuropeptides in the hypothalamus and the presence of these fibers in several thalamic nuclei. This neuroanatomical study will serve to elucidate the physiological roles of Met-8 in future studies of the human diencephalon.

Morphine is one of the most effective drugs used for pain management, but it is also highly addictive. Morphine elicits acute and long-term adaptive changes at cellular and molecular level in the brain, which play a critical role in the development of tolerance, dependence and addiction. Previous studies indicated that the dopamine D4 receptor (D4 R) activation counteracts morphine-induced adaptive changes of the μ opioid receptor (MOR) signaling in the striosomes of the caudate putamen (CPu), as well as the induction of several Fos family transcription factors. Thus, it has been suggested that D4 R could play an important role avoiding some of the addictive effects of morphine. Here, using different drugs administration paradigms, it is determined that the D4 R agonist PD168,077 prevents morphine-induced activation of the nigrostriatal dopamine pathway and morphological changes of substantia nigra pars compacta (SNc) dopamine neurons, leading to a restoration of dopamine levels and metabolism in the CPu. Results from receptor autoradiography indicate that D4 R activation modulates MOR function in the substantia nigra pars reticulata (SNr) and the striosomes of the CPu, suggesting that these regions are critically involved in the modulation of SNc dopamine neuronal function through a functional D4 R/MOR interaction. In addition, D4 R activation counteracts the rewarding effects of morphine, as well as the development of hyperlocomotion and physical dependence without any effect on its analgesic properties. These results provide a novel role of D4 R agonist as a pharmacological strategy to prevent the adverse effects of morphine in the treatment of pain.

18 p., An immunocytochemical technique has been used to study for the first time the distribution of fibers and cell bodies containing leucine-enkephalin (leu-enk), methionine-enkephalin (met-enk) or adrenocorticotropic hormone (ACTH) in the whole brainstem of the squirrel monkey Saimiri sciureus. Cell bodies containing leu-enk or met-enk were found in the superior colliculus and the formatio reticularis tegmenti mesencephali, respectively. No immunoreactive cell bodies containing ACTH were observed. Leu-enk-immunoreactive fibers were observed in 40 brainstem nuclei/tracts/regions, fibers containing met-enk were found in 38 brainstem nuclei/tracts/regions and fibers containing ACTH were found in 26 nuclei/tracts/regions. In the latter case, the density of immunoreactive fibers was always low. A high/moderate density of leu-enk- or met-enk-immunoreactive fibers were found in 18 and 16 brainstem nuclei/tracts/regions, respectively. The distribution of immunoreactive fibers containing leu-enk or met-enk was quite similar, with both leu-enk and met-enk observed in 82.5 % of the squirrel monkey brainstem nuclei/tracts/regions. This relationship is less marked for met-enk and ACTH (60.5 %) and even lower for leu-enk and ACTH (52.5 %). In 42.5 % of the nuclei/tracts/regions of the squirrel monkey brainstem (colliculus superior, substantia grisea centralis, nucleus interpeduncularis, nucleus tractus spinalis nervi trigemini, nucleus tractus solitarii, nucleus parabrachialis, formatio reticularis, substantia nigra), we observed fibers containing all three neuropeptides. The widespread distribution reported here suggests that enkephalins and ACTH can be involved in several physiological functions. The distribution of the immunoreactive fibers reported here is quite similar to that previously reported for enkephalins and ACTH in Macaca species and humans.

Galanin (GAL) and the NPYY1 agonist play a role in mood regulation and both neuropeptides interact in several central functions. The present study examined the interaction between Galanin receptor 2 (GALR2) and Neuropeptide Y Y1 receptor (NPYY1R) in the dentate gyrus (DG) of the Hippocampus in relation to depression-like behavior. Using receptor autoradiography, in situ hybridization and in situ proximity ligation assay an interaction between GALR and NPYY1R was demonstrated in the DG probably involving the formation of GALR2-NPYY1R heteroreceptor complexes. These complexes were specifically observed in the polymorphic and subgranular subregions of the DG, where both receptors were found to colocalize. Moreover, this GALR2/NPYY1R interaction was linked to an enhancement of the antidepressive-like behavior mediated by NPYY1R in the forced swimming test. Specific cells populations within DG subregions may be involved in this behavioral effect since the coactivation of GALR2 and NPYY1R enhances the NPYY1R-mediated reduction in the number of c-Fos immunoreactive nuclei in the polymorphic region. These results indicate that GALR2/NPYY1R interactions can provide a novel integrative mechanism in DG in depression-related behavior and may give the basis for the development of drugs targeting GALR2/NPYY1R heteroreceptor complexes in the DG of the hippocampus for the treatment of depression.

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibers containing somatostatin-28 (1-12) in the alpaca brainstem. Immunoreactive fibers were widely distributed throughout the whole brainstem: 34 brainstem nuclei/regions showed a high or a moderate density of these fibers. Perikarya containing the peptide were widely distributed throughout the mesencephalon, pons and medulla oblongata. Cell bodies containing somatostatin-28 (1-12) were observed in the lateral and medial divisions of the marginal nucleus of the brachium conjunctivum, reticular formation (mesencephalon, pons and medulla oblongata), inferior colliculus, periaqueductal gray, superior colliculus, pericentral division of the dorsal tegmental nucleus, interpeduncular nucleus, nucleus of the trapezoid body, vestibular nucleus, motor dorsal nucleus of the vagus, nucleus of the solitary tract, nucleus praepositus hypoglossi, and in the substantia nigra. This widespread distribution indicates that somatostatin-28 (1-12) is involved in multiple physiological actions in the alpaca brainstem. Microsc. Res. Tech. 78:363–374, 2015. © 2015 Wiley Periodicals, Inc.

Alcohol consumption is considered a major risk factor for disease and mortality worldwide. In the absence of effective treatments in alcohol use disorders, it is important to find new biological targets that could modulate alcohol consumption. We tested the role of the N-terminal galanin fragment (1-15) [GAL(1-15)] in voluntary ethanol consumption in rats using the two-bottle choice paradigm as well as compare the effects of GAL(1-15) with the whole molecule of GAL. We describe for the first time that GAL(1-15), via central mechanisms, induces a strong reduction in preference and ethanol consumption in rats. These effects were significantly different than GAL. GAL receptor (GALR) 2 was involved in these effects, because the specific GALR2 antagonist M871 blocked GAL(1-15) mediated actions in preference and ethanol intake. Importantly, the mechanism of this action involves changes in GALR expression and also in immediate-early gene C-Fos and receptors-internalization-related gene Rab5 in the striatum. The relevance of the striatum as a target for GAL(1-15) was supported by the effect of GAL(1-15) on the locomotor activity of rats after ethanol administration. These results may give the basis for the development of novel therapeutics strategies using GAL(1-15) analogues for the treatment of alcohol use disorders in humans.

The introduction of allosteric receptor-receptor interactions in G protein-coupled receptor (GPCR) heteroreceptor complexes of the central nervous system (CNS) gave a new dimension to brain integration and neuropsychopharmacology. The molecular basis of learning and memory was proposed to be based on the reorganization of the homo- and heteroreceptor complexes in the postjunctional membrane of synapses. Long-term memory may be created by the transformation of parts of the heteroreceptor complexes into unique transcription factors which can lead to the formation of specific adapter proteins. The observation of the GPCR heterodimer network (GPCR-HetNet) indicated that the allosteric receptor-receptor interactions dramatically increase GPCR diversity and biased recognition and signaling leading to enhanced specificity in signaling. Dysfunction of the GPCR heteroreceptor complexes can lead to brain disease. The findings of serotonin (5-HT) hetero and isoreceptor complexes in the brain over the last decade give new targets for drug development in major depression. Neuromodulation of neuronal networks in depression via 5-HT, galanin peptides and zinc involve a number of GPCR heteroreceptor complexes in the raphe-hippocampal system: GalR1-5-HT1A, GalR1-5-HT1A-GPR39, GalR1-GalR2, and putative GalR1-GalR2-5-HT1A heteroreceptor complexes. The 5-HT1A receptor protomer remains a receptor enhancing antidepressant actions through its participation in hetero- and homoreceptor complexes listed above in balance with each other. In depression, neuromodulation of neuronal networks in the raphe-hippocampal system and the cortical regions via 5-HT and fibroblast growth factor 2 involves either FGFR1-5-HT1A heteroreceptor complexes or the 5-HT isoreceptor complexes such as 5-HT1A-5-HT7 and 5-HT1A-5-HT2A. Neuromodulation of neuronal networks in cocaine use disorder via dopamine (DA) and adenosine signals involve A2AR-D2R and A2AR-D2R-Sigma1R heteroreceptor complexes in the dorsal and ventral striatum. The excitatory modulation by A2AR agonists of the ventral striato-pallidal GABA anti-reward system via targeting the A2AR-D2R and A2AR-D2R-Sigma1R heteroreceptor complex holds high promise as a new way to treat cocaine use disorders. Neuromodulation of neuronal networks in schizophrenia via DA, adenosine, glutamate, 5-HT and neurotensin peptides and oxytocin, involving A2AR-D2R, D2R-NMDAR, A2AR-D2R-mGluR5, D2R-5-HT2A and D2R-oxytocinR heteroreceptor complexes opens up a new world of D2R protomer targets in the listed heterocomplexes for treatment of positive, negative and cognitive symptoms of schizophrenia.

Galanin is a 29 amino acid neuropeptide widely distributed in neurons within the central nervous system. Galanin exerts its biological activities through three different G protein-receptors and participates in a number of functions, including mood regulation. Not only Galanin but also Galanin N-terminal fragments like Galanin(1-15) are active at the central level. In this work, we review the latest findings in studies on Galanin and Galanin(1-15) in depression-related behaviours. Our focus is on animal models for depression, and we pay some attention to research data obtained in human studies. Since Serotonin (5-HT), especially through 5-HT1A, and Galanin receptors interact at both pre-and postsynaptic level, the development of drugs targeting potential GAL1-GAL2-5-HT1A heteroreceptor complexes linked to the raphe-hippocampal 5-HT neurons may represent new treatment strategies in depression.

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibres containing non-opioid peptides (adrenocorticotropin hormone (ACTH), alpha-melanocyte-stimulating hormone) and opioid peptides (beta-endorphin (1-27), alpha-neo-endorphin, leucine-enkephalin) in the alpaca diencephalon. No immunoreactive cell bodies containing ACTH were found. Perikarya containing the other four peptides were observed exclusively in the hypothalamus and their distribution was restricted. Perikarya containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin showed a more widespread distribution than those containing leucine-enkephalin or beta-endorphin (1-27). Cell bodies containing pro-opiomelanocortin-derived peptides were observed in the arcuate nucleus, anterior and lateral hypothalamic areas and in the ventromedial and supraoptic hypothalamic nuclei, whereas perikarya containing alpha-neo-endorphin (a pro-dynorphin-derived peptide) were found in the arcuate nucleus, dorsal and lateral hypothalamic areas, and in the paraventricular, ventromedial and supraoptic hypothalamic nuclei. Immunoreactive cell bodies containing leucine-enkephalin were found in the lateral hypothalamic area and in the paraventricular hypothalamic nucleus. Immunoreactive fibres expressing pro-opiomelanocortin-derived peptides were more numerous than those expressing pro-dynorphin-derived peptides. A close anatomical relationship was observed: in all the diencephalic nuclei in which beta-endorphin (1-27)-immunoreactive fibres were found, fibres containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were also observed. Fibres containing beta-endorphin (1-27), alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were widely distributed throughout the diencephalon, but fibres containing ACTH or leucine-enkephalin showed a moderate distribution. The distribution of the five peptides studied here is also compared with that reported previously in other mammalian species. The widespread distribution observed indicates that both the pro-dynorphin and the pro-opiomelanocortin systems are involved in multiple physiological actions (e.g., food intake, thermoregulation, neuroendocrine and reproductive mechanisms) in the alpaca diencephalon.

The peptides dynorphin and enkephalin modulate many physiological processes, such as motor activity and the control of mood and motivation. Their expression in the caudate putamen (CPu) is regulated by dopamine and opioid receptors. The current work was designed to explore the early effects of the acute activation of D4 and/or μ opioid receptors by the agonists PD168,077 and morphine, respectively, on the regulation of the expression of these opioid peptides in the rat CPu, on transcription factors linked to them, and on the expression of μ opioid receptors. In situ hybridization experiments showed that acute treatment with morphine (10 mg/kg) decreased both enkephalin and dynorphin mRNA levels in the CPu after 30 min, but PD168,077 (1 mg/kg) did not modify their expression. Coadministration of the two agonists demonstrated that PD168,077 counteracted the morphine-induced changes and even increased enkephalin mRNA levels. The immunohistochemistry studies showed that morphine administration also increased striatal μ opioid receptor immunoreactivity but reduced P-CREB expression, effects that were blocked by the PD168,077-induced activation of D4 receptors. The current results present evidence of functional D4–μ opioid receptor interactions, with consequences for the opioid peptide mRNA levels in the rat CPu, contributing to the integration of DA and opioid peptide signaling. © 2013 Wiley Periodicals, Inc.

This article is focused on understanding the mechanisms for the interactions between the central catecholamine (CA) and oxytocin (OXY) neurons and their relevance for brain function especially social behaviour in the field of pair bonding. Such a topic is analysed under two perspectives namely the intercellular communication modes between CA and OXT neurons and the molecular integrative mechanisms at the plasma membrane level between their respective decoding systems. As a matter of fact, recent observations strongly indicate a major role of volume transmission and receptor-receptor interactions in the CA/OXT neuron interplay in the brain control of social behaviour and pair bonding. This article is part of a Special Issue entitled: Brain Integration.

Junta de Andalucia CVI6476.Universidad de Malaga. Campus de Excelencia Internacional Andalucia Tech.

Ponencia Invitada Galanin (GAL) is involved in several functions including mood regulation. The GAL N-terminal fragment (1-15) [GAL(1-15)] also participates at central level and a differential role of GAL(1-15) compared with GAL has been proposed. In this work we have analysed if GAL(1-15) contributes to depression- and anxiety -related behaviours using the forced swimming test, tail suspension test, open field and light/dark test. We tested the involvement of the GAL receptor 2 (GALR2) in GAL(1-15) effects with the GAL receptor antagonist M871 and with an in vivo model of siRNA GALR2 knockdown rats. The proximity of GALR1 and GALR1 was also examined with the proximity ligation assay (PLA). GAL(1-15) induced strong depression-like and anxiogenic-like effects in all the tests. The involvement of the GALR2 was demonstrated with M871 and with the siRNA GALR2 knockdown rats. The PLA indicated the existence of GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. Our results indicate that GAL(1-15) exerts strong depression-related and anxiogenic-like effects and may give the basis for the development of drugs targeting GALR1-GALR2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety. This study was supported by Junta de Andalucía CVI6476. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Using an immunocytochemical technique, we report for the first time the distribution of immunoreactive cell bodies and fibers containing somatostatin-28 (1–12) in the alpaca diencephalon. Somatostatin-28 (1–12)-immunoreactive cell bodies were only observed in the hypothalamus (lateral hypothalamic area, arcuate nucleus and ventromedial hypothalamic nucleus). However, immunoreactive fibers were widely distributed throughout the thalamus and hypothalamus. A high density of such fibers was observed in the central medial thalamic nucleus, laterodorsal thalamic nucleus, lateral habenular nucleus, mediodorsal thalamic nucleus, paraventricular thalamic nucleus, reuniens thalamic nucleus, rhomboid thalamic nucleus, subparafascicular thalamic nucleus, anterior hypothalamic area, arcuate nucleus, dorsal hypothalamic area, around the fornix, lateral hypothalamic area, lateral mammilary nucleus, posterior hypothalamic nucleus, paraventricular hypothalamic nucleus, suprachiasmatic nucleus, supraoptic hypothalamic nucleus, and in the ventromedial hypothalamic nucleus. The widespread distribution of somatostatin-28 (1–12) in the thalamus and hypothalamus of the alpaca suggests that the neuropeptide could be involved in many physiological actions.

The interactions between neuropeptide Y (NPY), specifically through NPY Y1 and Y2 receptor subtypes and the Galanin N-terminal fragment (1-15) [GAL(1-15)] were analyzed at the cardiovascular level. The cardiovascular effects of intracisternal coinjections of GAL(1-15) and NPY, NPY Y1 or Y2 agonist have been investigated as well as quantitative receptor autoradiography of the binding characteristics of NPY Y1 and Y2 receptor subtypes in the nucleus of the solitary tract (NTS) in the presence or absence of GAL(1-15). The coinjection of NPY with GAL(1-15) induces a significant vasopressor action. The coinjection of the NPY Y2 agonist and GAL(1-15) induced a similar increase of mean arterial pressure as induced by NPY+GAL(1-15), actions that were not observed with the NPY Y1 agonist+GAL(1-29). No interactions were observed at heart rate level. GAL(1-15) 3 nM significantly and substantially increased NPY-Y2 agonist binding in the NTS by about 50%. This effect was significantly blocked (p0.01) in the presence of the specific Galanin antagonist M40. The NPY-Y1 agonist binding was not modified in the presence of GAL(1-15). The present findings suggest the existence of a facilitatory effect of GAL(1-15) mediated via Galanin receptors on the NPY Y2 receptor subtype and its cardiovascular function within the NTS.

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.

Previously we reported that basal neuropeptide Y (NPY)-like immunoreactivity-(LI) in hippocampus of the "depressed" Flinders Sensitive Line (FSL) rats was lower compared to the control Flinders Resistant Line (FRL) and that electroconvulsive stimuli (ECS) raise NPY-LI in discrete brain regions. Here we studied NPY mRNA expression, NPY Y(1) receptor (Y(1)) mRNA expression and binding sites, and behavior under basal conditions (Sham) and after repeated ECS. Baseline NPY and Y(1) mRNAs in the CA1-2 regions and dentate gyrus were lower while the Y(1) binding was higher in the FSL. ECS had larger effects on both NPY and behavior in the FSL rats. ECS increased NPY mRNA in the CA1-2, dentate gyrus and hypothalamus in FSL, but only in the dentate gyrus in FRL. ECS also increased Y(1) mRNA in the CA1-2, dentate gyrus and the parietal cortex in both strains, while in the hypothalamus the increase was observed only in the FSL rats. Consistently with Y(1) mRNA increase, Y(1) binding was downregulated in the corresponding regions. ECS decreased FSL immobility in the Porsolt swim test. These findings suggest that NPY is involved in depressive disorder and that antidepressant effects of ECS may in part be mediated through NPY.

Morphine is one of the most effective drugs used for pain management, but it is also highly addictive. Morphine elicits acute and long-term adaptive changes at cellular and molecular level in the brain, which play a critical role in the development of tolerance, dependence and addiction. Previous studies indicated that the dopamine D

Galanin N-terminal fragment (1-15) [GAL(1-15)] is associated with depression-related and anxiogenic-like effects in rats. In this study, we analyzed the ability of GAL(1-15) to modulate 5-HT1A receptors (5-HT1AR), a key receptor in depression. GAL(1-15) enhanced the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT in the forced swimming test. These effects were stronger than the ones induced by Galanin (GAL). This action involved interactions at receptor level since GAL(1-15) affected the binding characteristics and the mRNA levels of 5-HT1AR in the dorsal hippocampus and dorsal raphe. The involvement of the GALR2 was demonstrated with the GALR2 antagonist M871. Proximity ligation assay experiments indicated that 5-HT1AR are in close proximity with GALR1 and GALR2 in both regions and in raphe RN33B cells. The current results indicate that GAL(1-15) enhances the antidepressant effects induced by 8-OH-DPAT acting on 5-HT1AR operating as postjunctional or as autoreceptors. These results may give the basis for the development of drugs targeting potential GALR1-GALR2-5-HT1AR heteroreceptor complexes linked to the raphe-hippocampal 5-HT neurons for the treatment of depression.

Introduction. The distribution of the immunoreactive cell bodies and fibers containing neurotensin in the alpaca diencephalon was determined by an immunohistochemical technique. Material and methods. The study was carried out in four male alpacas that lived at sea level. Brains of deeply anesthetized animals were fixed by perfusion with 4% paraformaldehyde. Cryostat sections were stained by a standard immunohistochemical method. Results. Cell bodies containing neurotensin were observed in the zona incerta and hypothalamus. A low/moder-ate density of these cell bodies was observed in the lateral hypothalamic area, anterior and dorsal hypothalamic areas, suprachiasmatic nucleus, periventricular region of the hypothalamus and in the ventromedial hypothalamic nucleus. In both thalamus and hypothalamus, immunoreactive fibers showed a widespread distribution. In the thalamus, a high density of these fibers was mainly found in the midline nuclei, whereas in the hypothalamus a high density was in general observed in the whole structure. Conclusions. In comparison with other mammals, the thalamus of the alpaca showed the most widespread distribution of neurotensin-immunoreactive fibers. The widespread distribution of neurotensin through the alpaca diencephalon suggests that the peptide can be involved in many physiological actions. © Polish Society for Histochemistry and Cytochemistry Folia Histochem Cytobiol. 2018.

Background: Galanin (GAL) plays a role in mood regulation. In this study we analyzed the action of the active N-terminal fragment [GAL(1–15)] in anxiety- and depression-related behavioral tests in rats. Methods: The effect of GAL(1–15) was analyzed in the forced swimming test, tail suspension test, open field test, and light/dark test. The proximity of GAL1 and GAL2 receptors was examined with the proximity ligation assay (PLA). We tested the GAL receptors involved in GAL(1–15) effects with the GAL2 receptor antagonist M871 and with an in vivo model of siRNA GAL2 receptor knockdown or siRNA GAL1 receptor knockdown rats. The effects of GAL(1–15) were also studied in the cell line RN33B. Results: GAL(1–15) induced strong depression-like and anxiogenic-like effects in all the tests. These effects were stronger than the ones induced by GAL. The involvement of the GAL2 receptor was demonstrated with M871 and with the siRNA GAL2 receptor knockdown rats. The PLA indicated the possible existence of GAL1 and GAL2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. In the siRNA GAL1 receptor knockdown rats the behavioral actions of GAL(1–15) disappeared, and in the siRNA GAL2 receptor knockdown rats the reductions of the behavioral actions of GAL(1–15) was linked to a disappearance of PLA. In the cell line RN33B, GAL(1–15) decreased 5-HT immunoreactivity more strongly than GAL. Conclusions: Our results indicate that GAL(1–15) exerts strong depression-related and anxiogenic-like effects and may give the basis for the development of drugs targeting GAL1 and GAL2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety.

Galanin N-terminal fragment (1-15) [Gal(1-15)] is involved in mood regulation. The intracerebroventricular (icv) administration of Gal(1-15) produces a depressive-like behaviour in the forced swim test (FST) and an anxiety-like behaviour in the open field test (OF) in rats. In this work we analyze the effect of Gal(1-15) in two more behavioural tests, the tail suspension test (TLT) and the light/dark test. Gal(1-15) (3nmol), effective dose in FST and OF, or artificial cerebrospinal were injected in animals (n=5-8) 15 minutes before the test. Behavioural assessment were conducted with at least one week between tests. Student’s t-test was used for comparision between experimental groups. In the light/dark test we analyzed during 5 min three parametres as indicators of anxiety-like behaviour. Gal(1-15) significantly reduced the time spent in the light compartiment by 52% (p

In 1980/81 Agnati and Fuxe introduced the concept of intramembrane receptor-receptor interactions and presented the first experimental observations for their existence in crude membrane preparations. The second step was their introduction of the receptor mosaic hypothesis of the engram in 1982. The third step was their proposal that the existence of intramembrane receptor-receptor interactions made possible the integration of synaptic (WT) and extrasynaptic (VT) signals. With the discovery of the intramembrane receptor-receptor interactions with the likely formation of receptor aggregates of multiple receptors, so called receptor mosaics, the entire decoding process becomes a branched process already at the receptor level in the surface membrane. Recent developments indicate the relevance of cooperativity in intramembrane receptor-receptor interactions namely the presence of regulated cooperativity via receptor-receptor interactions in receptor mosaics (RM) built up of the same type of receptor (homo-oligomers) or of subtypes of the same receptor (RM type1). The receptor-receptor interactions will to a large extent determine the various conformational states of the receptors and their operation will be dependent on the receptor composition (stoichiometry), the spatial organization (topography) and order of receptor activation in the RM. The biochemical and functional integrative implications of the receptor-receptor interactions are outlined and long-lived heteromeric receptor complexes with frozen RM in various nerve cell systems may play an essential role in learning, memory and retrieval processes. Intramembrane receptor-receptor interactions in the brain have given rise to novel strategies for treatment of Parkinson's disease (A2A and mGluR5 receptor antagonists), schizophrenia (A2A and mGluR5 agonists) and depression (galanin receptor antagonists). The A2A/D2, A2A/D3 and A2A/mGluR5 heteromers and heteromeric complexes with their possible participation in different types of RM are described in detail, especially in the cortico-striatal glutamate synapse and its extrasynaptic components, together with a postulated existence of A2A/D4 heteromers. Finally, the impact of intramembrane receptor-receptor interactions in molecular medicine is discussed outside the brain with focus on the endocrine, the cardiovascular and the immune systems.

The nucleus tractus solitarii (NTS) is a key nucleus in central cardiovascular control. In this mechanism it is well known the role of the alpha(2)-adrenoreceptors for the modulation of the autonomic pathways. Moreover a number of neuropeptides described in the NTS, including Neuropeptide Y (NPY), Galanin (GAL) and Angiotensin II (Ang II), have different roles in regulating the cardiovascular function within this nucleus. We show in this review several data which help to understand how these neuropeptides (NPY, GAL and Ang II) could modulate the cardiovascular responses mediated through alpha(2)-adrenoreceptors in the NTS. Also we show for the first time the interactions between neuropeptides in the brain, specifically the interactions between NPY, GAL, and Ang II, and its functional relevance for central cardiovascular regulation. These data strength the role of neuropeptides on central autonomic control and provide some evidences to understand the neurochemical mechanisms involved in the cardiovascular responses from the NTS.

The interactions between neuropeptide Y (NPY), specifically through NPY Y1 and Y2 receptor subtypes, and galanin [GAL(1–29)] have been analysed at the cardiovascular level. The cardiovascular effects of intracisternal coinjections of GAL(1–29) with NPY or NPY Y1 or Y2 agonists, as well as quantitative receptor autoradiography of the binding characteristics of NPY Y1 and Y2 receptor subtypes in the nucleus of the solitary tract (NTS), in the presence or absence of GAL(1–29), have been investigated. The effects of coinjections of GAL(1–29) and the NPY Y1 agonist on the expression of c-FOS immunoreactivity in the NTS were also studied. The coinjection of NPY with GAL(1–29) induced a significant vasopressor and tachycardic action with a maximum 40% increase ( P< 0.001). The coinjection of the NPY Y1 agonist and GAL(1–29) induced a similar increase in mean arterial pressure and heart rate as did NPY plus GAL(1–29), actions that were not observed with the NPY Y2 agonist plus GAL(1–29). GAL(1–29), 3 nm, significantly and substantially (by � 40%) decreased NPY Y1 agonist binding in the NTS. This effect was significantly blocked ( P< 0.01) in the presence of the specific galanin antagonist M35. The NPY Y2 agonist binding was not modified in the presence of GAL(1–29). At the c-FOS level, the coinjection of NPY Y1 and GAL(1–29) significantly reduced the c-FOS-immunoreactive response induced by either of the two peptides. The present findings suggest the existence of a modulatory antagonistic effect of GAL(1–29) mediated via galanin receptors on the NPY Y1 receptor subtype and its signalling within the NTS.

The aim of this work was to investigate the interactions between angiotensin II (Ang II) and galanin(1–29) [GAL(1–29)] or its N-terminal fragment galanin(1–15) [GAL(1–15)] on central cardiovascular control. The involvement of angiotensin type1 (AT1) receptor subtype was analyzed by the AT1 antagonist, DuP 753. Anesthesized male Sprague–Dawley rats received intracisternal microinjections of Ang II (3 nmol) with GAL(1–29) (3 nmol) or GAL(1–15) (0.1 nmol) alone or in combination. The changes in mean arterial pressure (MAP) and heart rate (HR) recorded from the femoral artery were analyzed. The injection of Ang II and GAL(1–15) alone did not produce any change in MAP. However, coinjections of both Ang II and GAL(1–15) elicited a significant vasopressor response. This response was blocked by DuP 753. Ang II and GAL(1–15) alone produced an increase in HR. The coinjections of Ang II with GAL(1–15) induced an increase in HR not significantly different from the tachycardia produced by each peptide. The presence of DuP 753 counteracted this response. GAL(1–29) alone elicited a transient vasopressor response that disappeared in the presence of Ang II. The coinjections of Ang II with GAL(1–29) and with DuP 753 restored the transient vasopressor effect produced by GAL(1–29). GAL(1–29) produced a slight but significant tachycardic effect that was not modified in the presence of Ang II. The presence of DuP 753 did not modify the tachycardic response produced by Ang II and GAL(1–29). These results give indications for the existence of a differential modulatory effect of Ang II with GAL(1–15) and GAL(1-29) on central blood pressure response that might be dependent on the activity of the angiotensin AT1 receptor subtype.

The aim of this work was to evaluate whether oxytocin administered in male rats subcutaneously early in life in the absence or presence of food restriction during pregnancy has life-long effects on the alpha(2)-agonist binding sites in the nucleus of the solitarii tract (NTS), in the hypothalamus and the amygdala, as evaluated by quantitative receptor autoradiography. Maternal food restriction alone increased the affinity of the alpha(2)-agonist [(3)H]UK14.304 binding sites exclusively in the NTS. In offspring from ad libitum fed dams, oxytocin treatment significantly increased the density of alpha(2)-agonist binding sites in the NTS and in the hypothalamus. The K(d) value of the alpha(2)-agonist binding sites in the hypothalamus of these rats, but not in the other regions studied, was also significantly increased. In offspring from food-restricted dams, oxytocin treatment produced a significant increase of the B(max) values in the hypothalamus and the amygdala and the K(d) value of the alpha(2)-agonist binding sites in the NTS of these rats also was selectively and significantly increased. These results suggest that a postnatal, oxytocin-induced increase of regional alpha(2)-adrenoceptor function can be seen in adulthood by a persistent, regionally selective increase in the density of central alpha(2)-adrenoceptor agonist binding sites, in the absence of an affinity change in the NTS. Such a regional increase of alpha(2)-adrenoceptor signalling in adulthood may contribute to the anti-stress action of postnatal oxytocin. By contrast, after prenatal stress, the potential increase in alpha(2)-adrenoceptor signalling takes place via selective increases of density with no changes of affinity of the alpha(2)-agonist binding sites in the hypothalamus and the amygdala.

The aim of this work was to investigate the modulation of the cardiovascular effects of neuropeptide Y (NPY) in the nucleus tractus solitarii (NTS) by angiotensin II (Ang II) and to determine the NPY receptor subtype involved in this modulation. Anesthesized Sprague-Dawley rats received microinjections in the NTS of Ang II (threshold and ED(50) doses) with NPY Y(1) agonist Leu(31)Pro(34)NPY and NPY Y(2) agonist NPY(13-36) (threshold and ED(50) doses). The changes in mean arterial pressure (MAP) and heart rate (HR) recorded in the femoral artery were analyzed during 60 min after the microinjections. The injection of threshold doses of Ang II, Y(1) agonist or Y(2) agonist alone did not produce any change in cardiovascular parameters. However, the co-injections into the NTS of threshold doses of both Ang II and the Y(1) agonist elicited significant increases of MAP and HR of about 12 and 10%, respectively. The co-administration of threshold doses of Ang II with the Y(2) agonist also induced a significant vasopressor response. The vasodepressor and bradycardiac effect of an ED(50) dose of the Y(1) agonist was significantly counteracted (P

Interactions between subtypes of dopamine, glutamate and adenosine receptors seem to play an important integrative role in the function of striatal gamma-aminobutyric acid (GABA)ergic efferent neurons. Recent behavioral and biochemical studies suggest the existence of specific interactions between adenosine A2A receptors (A(2A)R), dopamine D2 receptors (D2R) and the group I metabotropic mGlu5 receptors (mGlu5R) in the dorsal striatum. The dual-probe approach in vivo microdialysis technique in freely moving rats was used to study the role of mGlu5R/A2AR/D2R interactions in the modulation of the ventral striopallidal GABA pathway. Perfusion of a selective mGlu5R agonist (CHPG) in the nucleus accumbens facilitated GABA release in the ipsilateral ventral pallidum. This effect was strongly potentiated by co-perfusion with the A2AR agonist CGS 21680. Co-perfusion with the D2R agonist quinpirole counteracted the increase in pallidal GABA levels induced by CGS 21680 and by CGS 21680 plus CHPG. These results demonstrate that mGlu5R/A2AR/D2R interactions play an important modulatory role in the function of the ventral striopallidal GABA pathway, which might have implications for the treatment of schizophrenia and drug addiction.

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibers containing somatostatin-28 (1-12) in the alpaca brainstem. Immunoreactive fibers were widely distributed throughout the whole brainstem: 34 brainstem nuclei/regions showed a high or a moderate density of these fibers. Perikarya containing the peptide were widely distributed throughout the mesencephalon, pons and medulla oblongata. Cell bodies containing somatostatin-28 (1-12) were observed in the lateral and medial divisions of the marginal nucleus of the brachium conjunctivum, reticular formation (mesencephalon, pons and medulla oblongata), inferior colliculus, periaqueductal gray, superior colliculus, pericentral division of the dorsal tegmental nucleus, interpeduncular nucleus, nucleus of the trapezoid body, vestibular nucleus, motor dorsal nucleus of the vagus, nucleus of the solitary tract, nucleus praepositus hypoglossi, and in the substantia nigra. This widespread distribution indicates that somatostatin-28 (1-12) is involved in multiple physiological actions in the alpaca brainstem.

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1-15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1-GalR2 heteromers which may play a significant role in mediating galanin fragment (1-15) signaling. In HEK293T cells evidence for the existence of GalR1-GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET2 assays. PLA positive blobs representing GalR1-GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1-15) was more potent than galanin (1-29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1-GalR2 transfected cells. The inhibition of CREB by 50 nM of galanin (1-15) and of galanin (1-29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (115) vs galanin (1-29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1-29). In contrast, in NFAT reporter gene assays galanin (1-29) shows a higher efficacy than galanin (115) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1-GalR2 heteroreceptor complexes induced by galanin (1-15) may contribute to depression-like actions since GalR1 agonists produce such effects. (C) 2014 Elsevier Inc. All rights reserved.

Allosteric receptor–receptor interactions in GPCR heteromers appeared to introduce an intermolecular allosteric mechanism contributing to the diversity and bias in the protomers. Examples of dopamine D2R heteromerization are given to show how such allosteric mechanisms significantly change the receptor protomer repertoire leading to diversity and biased recognition and signaling. In 1980s and 1990s, it was shown that neurotensin (NT) through selective antagonistic NTR–D2 like receptor interactions increased the diversity of DA signaling by reducing D2R-mediated dopamine signaling over D1R-mediated dopamine signaling. Furthermore, D2R protomer appeared to bias the specificity of the NTR orthosteric binding site toward neuromedin N vs. NT in the heteroreceptor complex. Complex CCK2R–D1R–D2R interactions in possible heteroreceptor complexes were also demonstrated further increasing receptor diversity. In D2R–5-HT2AR heteroreceptor complexes, the hallucinogenic 5-HT2AR agonists LSD and DOI were recently found to exert a biased agonist action on the orthosteric site of the 5-HT2AR protomer leading to the development of an active conformational state different from the one produced by 5-HT. Furthermore, as recently demonstrated allosteric A2A–D2R receptor–receptor interaction brought about not only a reduced affinity of the D2R agonist binding site but also a biased modulation of the D2R protomer signaling in A2A–D2R heteroreceptor complexes. A conformational state of the D2R was induced, which moved away from Gi/o signaling and instead favored ß-arrestin2-mediated signaling. These examples on allosteric receptor–receptor interactions obtained over several decades serve to illustrate the significant increase in diversity and biased recognition and signaling that develop through such mechanisms