Carmelo Millón

On the existence and function of galanin receptor heteromers in the central nervous system

Galanin receptor (GalR) subtypes 1–3 linked to central galanin neurons may form heteromers with each other and other types of G protein-coupled receptors in the central nervous system (CNS). These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (gal 1-15) to modulate the function of different types of glia–neuronal networks in the CNS, especially the emotional and the cardiovascular networks. GalR–5-HT1A heteromers likely exist with antagonistic GalR–5-HT1A receptor–receptor interactions in the ascending midbrain raphe 5-HT neuron systems and their target regions. They represent a novel target for antidepressant drugs. Evidence is given for the existence of GalR1–5-HT1A heteromers in cellular models with trans-inhibition of the protomer signaling. A GalR1–GalR2 heteromer is proposed to be a galanin N-terminal fragment preferring receptor (1-15) in the CNS. Furthermore, a GalR1–GalR2–5-HT1A heterotrimer is postulated to explain why only galanin (1-15) but not galanin (1-29) can antagonistically modulate the 5-HT1A receptors in the dorsal hippocampus rich in gal fragment binding sites. The results underline a putative role of different types of GalR–5-HT1A heteroreceptor complexes in depression. GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR–NPYY1 receptor interactions in putative GalR–NPYY1 receptor heteromers in the CNS resulting in increases in NPYY1 transmission and antidepressant effects. In contrast the galanin fragment receptor (a postulated GalR1–GalR2 heteromer) appears to be linked to the NPYY2 receptor enhancing the affinity of the NPYY2 binding sites in a putative GalR1–GalR2–NPYY2 heterotrimer. Finally, putative GalR–α2-adrenoreceptor heteromers with antagonistic receptor–receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals also of likely relevance for depression.

Understanding the Role of GPCR Heteroreceptor Complexes in Modulating the Brain Networks in Health and Disease

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.

A Role for Galanin N-Terminal Fragment (1–15) in Anxiety- and Depression-Related Behaviors in Rats

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 (1-15) enhances the antidepressant effects of the 5-HT1A receptor agonist 8-OH-DPAT: involvement of the raphe-hippocampal 5-HT neuron system.

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.

Preferential activation by galanin 1-15 fragment of the GalR1 protomer of a GalR1-GalR2 heteroreceptor complex.

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 BRET(2) 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 50nM 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 (1-15) 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 (1-15) 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.

Galanin receptor 2-neuropeptide Y Y1 receptor interactions in the amygdala lead to increased anxiolytic actions

Galanin (GAL) and neuropeptide Y (NPY) are neuropeptides involved in behaviors associated with anxiety. Both neuropeptides interact in several central functions. However, the potential behavioral and cellular interactions between them in anxiety are unknown. GAL was found to act through GAL receptor 2 (GALR2) to enhance NPYY1 receptor (NPYY1R)-mediated anxiolytic behaviors in rats. Using receptor autoradiography, c-Fos expression and in situ proximity ligation assay, the medial paracapsular intercalated nuclei of the amygdala were determined to be a key area in the interaction probably involving the formation of GALR2/NPYY1R heteroreceptor complexes. In cell cultures costimulation of GALR2 and NPYY1R induced changes in the functions of these receptors. The changes involved a potentiation of the decrease in the phosphorylation of CREB induced by NPYY1R and a delay in the internalization of NPYY1R. These results indicate that GALR2/NPYY1R interactions can provide a novel integrative amygdaloid mechanism in anxiety.

Neurochemical Modulation of Central Cardiovascular Control: The Integrative Role of Galanin

Galanin (GAL) is a peptide involved in multiple functions, including central cardiovascular control. In this review, the role of GAL and its fragments in the modulation of cardiovascular neuronal networks in the nucleus of the solitary tract is presented, including its interaction with the classical neurotransmitters and other neuropeptides involved in cardiovascular responses in this nucleus. First, we describe the cardiovascular responses of GAL and the pathway involved in these responses. Then we summarize findings obtained in our laboratory on how GAL, through its receptors, interacts with two other neuropeptides--Neuropeptide Y and Angiotensin II and their receptors--as they have particularly conspicuous cardiovascular effects. All these results strengthen the role of GAL in central cardiovascular control and indicate the existence of interactions among GAL receptor subtypes and alpha2-adrenergic receptors, AT1, and Y1 receptor subtypes. These interactions are crucial for understanding the integrative mechanisms responsible for the organization of the cardiovascular responses from the NTS.

Long-lasting memory deficits in mice withdrawn from cocaine are concomitant with neuroadaptations in hippocampal basal activity, GABAergic interneurons and adult neurogenesis

ABSTRACT Cocaine addiction disorder is notably aggravated by concomitant cognitive and emotional pathology that impedes recovery. We studied whether a persistent cognitive/emotional dysregulation in mice withdrawn from cocaine holds a neurobiological correlate within the hippocampus, a limbic region with a key role in anxiety and memory but that has been scarcely investigated in cocaine addiction research. Mice were submitted to a chronic cocaine (20 mg/kg/day for 12 days) or vehicle treatment followed by 44 drug-free days. Some mice were then assessed on a battery of emotional (elevated plus-maze, light/dark box, open field, forced swimming) and cognitive (object and place recognition memory, cocaine-induced conditioned place preference, continuous spontaneous alternation) behavioral tests, while other mice remained in their home cage. Relevant hippocampal features [basal c-Fos activity, GABA+, parvalbumin (PV)+ and neuropeptide Y (NPY)+ interneurons and adult neurogenesis (cell proliferation and immature neurons)] were immunohistochemically assessed 73 days after the chronic cocaine or vehicle protocol. The cocaine-withdrawn mice showed no remarkable exploratory or emotional alterations but were consistently impaired in all the cognitive tasks. All the cocaine-withdrawn groups, independent of whether they were submitted to behavioral assessment or not, showed enhanced basal c-Fos expression and an increased number of GABA+ cells in the dentate gyrus. Moreover, the cocaine-withdrawn mice previously submitted to behavioral training displayed a blunted experience-dependent regulation of PV+ and NPY+ neurons in the dentate gyrus, and neurogenesis in the hippocampus. Results highlight the importance of hippocampal neuroplasticity for the ingrained cognitive deficits present during chronic cocaine withdrawal. Summary: Hippocampal functional activity and neuroplasticity are altered in mice long withdrawn from cocaine, with concomitant memory deficits, emphasizing the role of the hippocampus in persistent cognitive manifestation of cocaine withdrawal.

Galanin (1-15) enhancement of the behavioral effects of Fluoxetine in the forced swimming test gives a new therapeutic strategy against depression

ABSTRACT The pharmacological treatment of major depression is mainly based on drugs elevating serotonergic (5‐HT) activity. Specifically, selective 5‐HT reuptake inhibitors, including Fluoxetine (FLX), are the most commonly used for treatment of major depression. However, the understanding of the mechanism of action of FLX beyond its effect of elevating 5‐HT is limited. The interaction between serotoninergic system and neuropeptides signaling could be a key aspect. We examined the ability of the neuropeptide Galanin(1–15) [GAL(1–15)] to modulate the behavioral effects of FLX in the forced swimming test (FST) and studied feasible molecular mechanisms. The data show that GAL(1–15) enhances the antidepressant‐like effects induced by FLX in the FST, and we demonstrate the involvement of GALR1/GALR2 heteroreceptor complex in the GAL(1–15)‐mediated effect using in vivo rat models for siRNA GALR1 or GALR2 knockdown. Importantly, 5‐HT1A receptors (5HT1A‐R) also participate in the GAL(1–15)/FLX interactions since the 5HT1AR antagonist WAY100635 blocked the behavioral effects in the FST induced by the coadministration of GAL(1–15) and FLX. The mechanism underlying GAL(1–15)/FLX interactions affected the binding characteristics as well as the mRNA levels of 5‐HT1A‐R specifically in the dorsal hippocampus while leaving unaffected mRNA levels and affinity and binding sites of this receptor in the dorsal raphe. The results open up the possibility to use GAL(1–15) as for a combination therapy with FLX as a novel strategy for treatment of depression. HIGHLIGHTSGAL(1–15) enhances the antidepressant‐like effects induced by Fluoxetine.5‐HT1A receptors participate in the GAL(1–15)/FLX interactions.GAL(1–15)/FLX interactions affected binding and 5‐HT1AR mRNA levels in hippocampus.

Galanin receptor 2-neuropeptide Y Y1 receptor interactions in the dentate gyrus are related with antidepressant-like effects

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.