Given the difficulties in measuring Gαi*/*o protein activation using cAMP assays in stable clones, we employed an alternative technique. It is well-established that the key step in GPCR activation is the induction of guanine nucleotide exchange (GDP-GTP) on the G protein α-subunit. The nucleotide exchange process can be monitored by measuring the binding of non-hydrolyzable GTPγS analog, [35S]GTPγS. Because the Gαi family of G proteins has a substantially higher basal rate of guanine nucleotide exchange than other G proteins, this assay is ostensibly a measure of GPCR-mediated activation of Gαi*/*o proteins (Milligan, 2003). Indeed, the GTPγS binding assay has been widely used to measure the activation of Gαi proteins by various GPCRs. Therefore, we examined both basal and MCH-stimulated [35S]GTPγS binding using the membrane fraction of HEK293T cells transiently expressing Flag-MCHR1 and the F318K mutant (**Figure 6**). In cells expressing Flag-MCHR1, MCH dose-dependently stimulated binding of GTPγS with an EC50 value of 0*.*23 ± 0*.*07 nM, while the value for F318K was 0*.*47 ± 0*.*25 nM (mean ± S.E.M. from three-independent experiments). The values for F318K were slightly higher, but a significant difference was not observed. The maximal amount of binding for Flag-MCHR1 with 1μM MCH was 208*.*3 ± 25*.*2% of basal, while that for F318K was 190*.*6 ± 20*.*3% of basal. Overall, there was no difference in the amount of GTPγS binding between cells expressing Flag-MCHR1 and the F318K mutant, in contrast to the clear influence of the F318K mutation on Gαq-mediated signaling. There was no significant difference in basal GTPγS binding (100% for Flag-MCHR1 vs. 97*.*6 ± 0*.*4% for F318K), suggesting that the F318K mutation does not affect constitutive receptor activation.

**FIGURE 6 | MCH-induced [35S]GTPγS binding to Flag-MCHR1 and F318K.** HEK293T cells were transfected with Flag-MCHR1 or F318K. After 48 h, the cells were harvested and the membrane fractions recovered. Membrane proteins (10μg) were subsequently incubated with 0.2 nM [ 35S]GTPγS and 0.001–1000 nM MCH in GTPγS binding buffer for 30 min at 30◦C. The amounts of radioactivity bound to the membrane preparations are shown for Flag-MCHR1 (filled circles) and F318K (open circles). Results shown are representative of three-independent experiments.

#### **INTERPRETATION OF THE FUNCTIONAL IMPORTANCE OF F318K IN MCHR1**

To understand better how the signaling dynamics of F318K are related to its interaction with (and activation of) Gαq, we constructed a molecular model of F318K activation of G protein based on that for the active structure of rhodopsin in complex with a transducin peptide as a reference (Kleinau et al., 2010). Because MCHR1 belongs to the same GPCR subfamily as rhodopsin, the existing rhodopsin sequence alignment allowed us to construct a preliminary model of MCHR1 conformation. We refined this model to account for the specific properties of MCHR1 using software for protein model building and a molecular dynamics software package. Our model highlighted several putative amino acid contacts between the receptor and Gαq protein, notably at intracellular loop 1 (i1) and helix 8 (**Figure 7**, left). Lys318 is predicted to interact efficiently with Asp79 (located in i1) *via* a distinct hydrogen-bonding pattern, and these two residues create an accessible intracellular interface for the Asn357 residue in the Gαq protein. However, Asp79 in i1 also exists in human MCHR2, in which F313K had no effect on MCH potency in calcium signaling, as shown in **Table 4**. We therefore searched for alternative candidate residues that may be involved in the interaction with Asn357 by comparing sequences between rat MCHR1 and other GPCRs, including human MCHR2. The sequences we used are for GPCRs in which mutation at the position corresponding to Phe318 of MCHR1 did not produce increased signaling potency (Fritze et al., 2003; Delos Santos et al., 2006; Anavi-Goffer et al., 2007; Duvernay et al., 2009; Málaga-Diéguez et al., 2010; Kaye et al., 2011). By making multiple alignments of these sequences, we found several amino acid insertions in i1 that occurred only in rat MCHR1, and were absent in other GPCRs (**Table 5**). Notably, a sequence in i1 includes Trp73, which possesses a bulky, aromatic side chain. Hydrophobic

Homology model of rat MCHR1 mutant F318K was constructed using the crystal structure of rhodopsin E113Q mutant in complex with the C-terminal tail of transducin (PDB ID 2X72). Amino acid residues involved in coupling to Gαq or Gαi proteins in intracellular loop 1 (i1) and K318 in helix 8 are shown. **(A)** The model structure of MCHR1(F318K)-Gαq C-terminal tail. The area demarcated by the square is magnified below in panel **(C)**. **(B)** The model structure of MCHR1(F318K)-Gαi C-terminal tail. The area demarcated by the square is magnified below in panel **(D)**. **(C)** Positions of Lys318, Tyr311, Trp73, and Asp79 residues and their position relative to Asn357 of Gαq. This model depicts the hydrogen bond network between Lys318 and Asp79 in the receptor and Asn357 in Gαq C-terminal tail. Note that interactions between Trp73 in i1 loop and Asn357 in the G protein C-terminal tail. Green, red, and blue areas indicate carbon, oxygen and nitrogen atoms, respectively, within the key residues of the receptor. Carbon atoms in the Gαq protein are colored cyan. **(D)** Magnified view of Lys318, Tyr311, Trp73, and Asp79 residues in the receptor and their position relative to Gly351 of Gαi. Intramolecular hydrogen interactions predicted between Lys318 and Asp79 in the receptor are shown. Carbon atoms in the Gαi protein are colored yellow.

interaction of Trp73 with Asn357 in the Gαq C-terminal tail is predicted to stabilize the intracellular interaction between MCHR1 and Gαq. Conversely, the Gly351 residue in Gαi has no side chain to interact with Lys318 (**Figure 7**, right), which may explain the lack of enhancing effect of the F318K mutation on Gαi-mediated signaling. Collectively, a predicted model of F318K combined with sequence alignments suggests that the key residues of the MCHR1-Gαq interface are Trp73 and Asp79 in the receptor and Asn357 in the Gαq protein. We speculate that the F318K mutation somehow enhances this interface, either by promoting heightened interaction or by facilitating G protein activation, possibly by changing the binding affinity (see section "Discussion").

#### **DISCUSSION**

Recent studies have underscored that, the Phe residue is a key position in the conserved NPxxY(x)5*,*6F motif, which connects TM7 and helix 8 (Palczewski et al., 2000; Fritze et al., 2003). Indeed, of 180 class A GPCRs, this Phe residue is the most highly conserved in 135 of these receptors (Okuno et al., 2005). Mutation of the conserved Phe to Ala in rhodopsin and the M1 muscarinic acetylcholine receptor considerably reduced the potency in Gα*t*- and Gαq-mediated signaling, respectively (Fritze et al., 2003; Kaye et al., 2011). Furthermore, Phe-to-Ala substitution in this motif dramatically reduced cell surface expression of α2B-AR, resulting in extensive ER arrest (Duvernay et al., 2009). These data suggest that the highly hydrophobic Phe residue in the conserved NPxxY(x)5*,*6F motif of many GPCRs is necessary for the maintenance of proper receptor conformation, and is often involved in receptor export from the ER. In the present study, we showed that Phe-to-Ala substitution (F318A) in rat MCHR1 did not significantly affect either the level of cell surface expression or MCH-induced calcium mobilization. However, by analyzing a series of substitution mutants, Phe-to-Lys substitution led to the most increased MCH potency in stimulating calcium mobilization, a pathway largely mediated *via* Gαq. In contrast, F318K had no significant effect in the GTPγS-binding assay that measures receptor interaction with Gαi protein. These findings are surprising considering that they contradict findings in Phe substitutions of other GPCRs. Therefore, our study provides new insight into the role of the NPxxY(x)5*,*6F motif Phe residue in term of G protein activation. Although the consensus is that this residue is a key determinant of receptor structure and function, its effect may be dependent on the specific sequence of the receptor and the presence or absence of other residues that make up the receptor-G protein binding/activation interface.

We showed that MCH was the most potent in stimulating calcium mobilization in cells expressing F318K among our mutants. To date, many mutations or truncations in MCHR1 have been designed to study the roles of certain residues and sequences in the diverse functionality of the receptor (Macdonald et al., 2000; Saito et al., 2003, 2004, 2005; Tetsuka et al., 2004; Fan et al., 2005). Some caused a slight or moderate decrease in calcium mobilization (Saito et al., 2003, 2004) and the others resulted in more severe impairment or loss of function (Macdonald et al., 2000; Tetsuka et al., 2004; Fan et al., 2005; Saito et al., 2005; Aizaki et al., 2009). F318K is, therefore, the first mutation characterized by an active variant with more efficient signaling properties. We conclude that substitution of Phe318 with the moderately positively charged Lys is most responsible, at least in part, for producing increased MCH potency in stimulating calcium mobilization *via* MCHR1. Replacement with another positively charged amino acid, His, at the 318 position had no effect for signaling enhancement. Furthermore, the highly positively charged Arg could not induce equivalent effects to F318K (**Figure 2**, **Table 3**). These findings suggest that unique and strict physicochemical characteristics, including the extent of positive charge, polarity and the size of the side chain, may be required for this phenomenon to occur.

GPCRs that bind promiscuously to several Gα protein subtypes are useful tools for clarifying the determinants of G protein selectivity. To date, most studies of rhodopsin family GPCRs have emphasized the role of membrane-proximal regions in the i2 and i3 loops and/or the cytoplasmic loop of the receptor (Anavi-Goffer et al., 2007; Kunieda et al., 2007; Kleinau et al., 2010). Our present data suggest that MCHR1 couples to both Gαi and

Stars beneath sequence indicate sequence identity across the alignment, and single dot indicates conserved substitutions.

hMCHR2, human MCHR2; rmAChR1, rat muscarinic acetylcholine receptor M1; rA1AR, rat adenosine 1A receptor; ra(2B)-AR, rat α2B adrenergic receptor; hb1AR, human β1 adrenergic receptor; bovineRho, bovine rhodopsin; hCB1, human cannobinoid receptor 1.

Gαq proteins *via* overlapping intracellular regions but that Lys mutation of the 318 position in the NPxxY(x)5*,*6F motif appears to affect only the coupling of the receptor to Gαq. Phe318 is located in the juxtamembrane helix 8 in the C-terminal tail. The role of helix 8 is believed to be in regulating G protein activation by GPCRs, yet few studies have established the pivotal role of helix 8 in G protein selectivity. The position in cannabinoid receptor 1 that corresponds to the MCHR1 Phe318 residue is not a Phe but is a hydrophobic residue (Leu404), and the L404I mutation led to impaired activation of Gαi3 but not GαoA among the Gαi*/*o protein subgroup (Anavi-Goffer et al., 2007). Furthermore, the positively charged Arg687 in helix 8 of the thyrotropin receptor is important for selective interaction with Gαq (Kleinau et al., 2010). In helix 8 of MCHR1, three basic amino acid residues (Arg319, Lys320, and Arg321) are juxtaposed with Phe318. This basic region in helix 8 seems not to be involved in Gα protein coupling specificity, since alteration of the positive charge of these residues (R319Q/K320Q/R321Q) caused an equally strong inhibition of both Gαq and Gαi (Tetsuka et al., 2004). Taken together, we might conclude that each amino acid residue in helix 8 of MCHR1 may have different roles in determining receptor activity and G protein selectivity. Our clarification here of the significance of the Phe position will hopefully allow additional understanding of dual G protein coupling in other GPCRs.

Upon stimulation, GPCRs undergo a conformational change that results in G protein activation. Light-induced conformational changes in rhodopsin were elucidated by a series of biophysical studies. Evidence indicates that an important hydrophobic pairing between Tyr306 and Phe313 in the NPxxY(x)5*,*6F motif stabilizes the ground state of rhodopsin (Palczewski et al., 2000; Nygaard et al., 2009). Comparative molecular dynamics simulations of MCHR1 have also suggested that the release of intramolecular interactions between the Tyr and Phe residues in the motif is an important step in transitioning from an inactive to an active form (Vitale et al., 2004). We initially hypothesized that the Tyr311-Phe318-based switch in MCHR1, which is the homolog of a crucial relay point in rhodopsin, would be modified by substituting Phe with Lys, with disruption of an aromatic stacking interaction in the resultant ternary complex somehow affecting the efficacy of signaling. However, the activated F318K structure bound with Gαq protein (**Figure 7**) has suggested that an additional and distinct interface may exist between Phe318 and Asp79 in the i1 loop. Further analysis with multiple alignment data for other GPCRs showed that close proximity of Phe318, Asp79, and Asn357 is insufficient to induce such signaling changes and that Trp73 in the i1 loop may be important in stabilizing/maintaining the efficient binding/activation of the Gαq protein by the activated receptor. A series of combinational mutations involving F318K and several amino acid residues in the i1 loop (Trp, Cys, and Ser, as described in **Table 5**) may clarify the importance and role of each residue. A precise three-dimensional crystal structure of the MCHR1-G protein complex would also facilitate our understanding, but our present model helps to understand how a single amino acid substitution, F318K, can alter the intra-/inter-molecular interactions of the receptor-G protein complex to modulate G protein signaling *via* this receptor. At this time, we cannot exclude the possibility that F318K has an increased binding affinity, although the EC50 value of MCH in the GTPγS-binding assay showed no change between Flag-MCHR1 and F318K. The concept of modes in agonism of GPCRs has recently been expanding (Smith et al., 2011), implying that receptors can convey different signals through different pathways with varying degrees of potency or efficacy depending on the ligand used. Therefore, it may be possible that the functional selectivity observed in F318K results from enhancement of the binding affinity that favors Gαq-binding but not Gαi-binding. Overall, based on our data, we favor an interpretation that F318 is involved in receptor dynamics.

In conclusion, by analyzing a series of mutants, we report for the first time that F318K, a point mutation in the NPxxY(x)5*,*6F motif of MCHR1, most efficiently enhances the potency of MCH in stimulating calcium mobilization *via* MCHR1 without showing any increase in cell surface expression. We speculate that Phe318 might be involved in the interface between the receptor and G protein, and may regulate which G proteins the receptor can bind and activate. Because there are a limited number of GPCR mutant that significantly enhanced the signaling (Reinscheid et al., 2005; Kato et al., 2008), F318K may provide useful clues in understanding the process of intrinsic receptor dynamics. However, further research is required to demonstrate whether the role of Phe318 described here is of physiological and functional significance.

# **REFERENCES**

# **ACKNOWLEDGMENTS**

We thank Yuki Kobayashi, Akiko Kojo, Yui Funakoshi and Saori Utsuda for expert technical assistance and helpful support. Source of funding: This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Kakenhi 20500337, 00215568 to Yumiko Saito).

receptors based on structurefunction studies on the type 1 angiotensin receptor. *Mol. Cell. Endocrinol.* 302, 118–127.

K. (2003). Functional role of *N*linked glycosylation on the rat melanin-concentrating hormone

modalities of GPCR ligands. *Mol. Cell. Endocrinol.* 331, 241–247.

are required for receptor function. *Endocrinology* 145, 3712–3723.

Vitale, R. M., Pedone, C., de Benedetti, P. G., and Fanelli, F. (2004). Structural features of the inactive and active states of the melaninconcentrating hormone receptors: insights from molecular simulations. *Proteins* 56, 430–448.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 August 2012; accepted: 08 November 2012; published online: 26 November 2012.*

*Citation: Hamamoto A, Horikawa M, Saho T and Saito Y (2012) Mutation of Phe318 within the NPxxY(x)*5*,*6*F motif in melanin-concentrating hormone receptor 1 results in an efficient signaling activity. Front. Endocrin. 3:147. doi: 10.3389/fendo.2012.00147*

*This article was submitted to Frontiers in Neuroendocrine Science, a specialty of Frontiers in Endocrinology.*

*Copyright © 2012 Hamamoto, Horikawa, Saho and Saito. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

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

*Kjell Fuxe1\*, Dasiel O. Borroto-Escuela1 ,Wilber Romero-Fernandez1, Alexander O. Tarakanov2, Feliciano Calvo1, Pere Garriga3, Mercé Tena3, Manuel Narvaez4, Carmelo Millón4, Concepción Parrado5, Francisco Ciruela6, Luigi F. Agnati 7,8, José A. Narvaez4 and Zaida Díaz-Cabiale4*

1 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

2 St. Petersburg Institute for Informatics and Automation, Russian Academy of Sciences, Saint Petersburg, Russia

3 Centre de Biotecnologia Molecular, Departament d´Enginyeria Química, Universitat Politécnica de Catalunya, Barcelona, Spain

4 Department of Physiology, School of Medicine, University of Málaga, Málaga, Spain

5 Department of Histology, School of Medicine, University of Málaga, Málaga, Spain

6 Unitat de Farmacologia, Departament Patologia i Terapéutica Experimental, Universitat de Barcelona, Barcelona, Spain

7 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy

8 Istituto di Ricovero e Cura a Carattere Scientifico, Lido Venice, Italy

#### *Edited by:*

Hubert Vaudry, University of Rouen, France

#### *Reviewed by:*

Wing-Ho Yung, Chinese University of Hong Kong, Hong Kong Joao C. Cardoso, University of Algarve, Portugal

#### *\*Correspondence:*

Kjell Fuxe, Department of Neuroscience, Karolinska Institutet, Retzius väg 8, 17177 Stockholm, Sweden. e-mail<EMAIL_ADDRESS>
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.

**Keywords: galanin receptors, heteromers, GPCRs, 5HT1A, NPY receptors, allosteric modulator**

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# **INTRODUCTION**

Galanin is a neuropeptide (Tatemoto et al., 1983) widely distributed in neurons within the central nervous system (CNS; Jacobowitz et al., 2004). Three Galanin receptor (GalR) subtypes, GalR1–3, have been cloned and belong to the rhodopsin subfamily of G protein-coupled receptor (GPCR; Branchek et al., 2000; Lundstrom et al., 2005; Mitsukawa et al., 2008). GalR1 and GalR2 in particular are found in many regions of the CNS as demonstrated with *in situ* hybridization, radioligand binding, and immunohistochemical studies and have all a high affinity for galanin. GalR1 and GalR3 are coupled to Gi/o leading to inhibition of adenylate cyclase (AC), increases in MAPK activity and opening of G protein-coupled inwardly rectifying K+ channels. GalR2 is coupled to Gq/11 and its activation leads to increases in phospholipase C with formation of IP3 increasing intracellular calcium levels and of diacylglycerol (DAG) with the subsequent activation of the protein kinase C. These three GalR subtypes are involved in a number of functions in the CNS modulating neuroendocrine, cardiovascular and mood regulation, pain control, food intake, and seizure threshold (Lundstrom et al., 2005; Mitsukawa et al., 2008). Galanin has also been demonstrated to exert neurotrophic and neuroprotective actions. As early as 1988 (Fuxe et al., 1988b) indications for the existence GalR–5-HT1A receptor–receptor interactions were obtained in rat limbic membranes and in 1994 (Mazarati et al., 1994) in a neuropharmacological analysis results were obtained suggesting interactions of GalRs with glutamate receptors in the dorsal striatum.

This review serves to summarize the indications that GalR subtypes may form heteromers with each other and other types of GPCRs in the CNS as a molecular mechanism to modulate the function of different types of glia–neuronal networks in the CNS.

# **RECEPTOR HETEROMERS AND THEIR ALLOSTERIC RECEPTOR–RECEPTOR INTERACTIONS VIA THE RECEPTOR INTERFACE**

We began to test the hypothesis of intramembrane receptor– receptor interactions in 1980–1981 in membrane preparations of various CNS regions and found that neuropeptides could modulate the binding characteristics, especially the affinity of the monoamine receptors, in a receptor subtype specific way (Agnati et al., 1980; Fuxe et al., 1981, 1983). Thus, intramembrane receptor–receptor interactions did exist in addition to indirect actions via phosphorylation and changes in membrane potential. However, it took around 10 years before they began to have an impact in the receptor field. But the good news were that the results were in line with earlier findings by Limbird et al. (1975), showing negative cooperativity in beta-adrenergic receptors, which could be explained by the existence of receptor homodimers leading to receptor–receptor interactions. It was also clear that adapter proteins could be involved in mediating the receptor–receptor interactions in brain membranes. A logical consequence for the indications of direct physical interactions between neuropeptide and monoamine receptors, the term heteromerization was introduced to describe a specific interaction between different types of GPCRs (see Zoli et al., 1993; Fuxe et al., 2012a).

Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) methods gave the evidence needed to demonstrate heteromers among class A GPCRs. In FRET two putative proteins can, e.g., bear a "donor" (CFP or GFP2) or an "acceptor" (YFP). If these two proteins interact then the donor and acceptor fluorophores are likely in proximity (10 nm or less) and energy transfer between donor and acceptor can occur after donor excitation by demonstration of YFP emission. In BRET two putative proteins bear a "donor" (Rluc) or an "acceptor" fluorophore (YFP or GFP2). If these proteins are in proximity they do interact. Then, a donor–acceptor energy transfer can occur after Rluc substrate (h-coelenterazine) oxidation. The bioluminescence formed can activate YFP and YFP emission develops (Fernández-Dueñas et al., 2012; Fuxe et al., 2012a).

More recently, evidence has been presented by means of *in situ* proximity ligation assay (*in situ* PLA) that in HEK293 cells D2*L*R can form heteromers with D4.7R and especially with D4.2R and D4.4R (Borroto-Escuela et al., 2011). *In situ* PLA have the potential to enable a fuller understanding of GPCR receptor–receptor and could be highly suited to investigate GPCR heteromers in tissue, providing new insights in basic biological mechanisms, heteroreceptor levels and their locations, e.g., in the brain (Borroto-Escuela et al., 2012).

It is clear that allosteric mechanisms make possible the integrative activity taking place intramolecularly in monomers and intermolecularly in homomers and heteromers (see Fuxe et al., 2010b). Allostery is a mode of long distance communication between distal sites in proteins. There may or may not exist a conformational change at the binding site by the allosteric communication. The conformational change is only one of the possible scenarios. We have preferred pathways which vary with given conditions, through which the strain energy is released from the allosteric site following a perturbation event which can pass over the receptor interface into the other protomer of the receptor homomer or heteromer (see Fuxe et al., 2010a). Kenakin (2008) rightly regards "Seven TM receptors as Nature's prototype allosteric protein: de-emphasizing the geography of binding". The allosteric receptor–receptor interactions induced by activation of one receptor protomer can influence the recognition, G proteincoupling and signaling and trafficking of the other protomers in the receptor heteromer.

A high energy strength double arginine-phosphate electrostatic interaction has been found in the A2AR–D2R heteromer by Ciruela et al. (2004) and Woods et al. (2005) which possess a covalent-like stability as demonstrated with mass spectrometry in combination with collision-induced dissociation experiments and confirmed by pull-down techniques. Based on a mathematical approach, Tarakanov and Fuxe (2010) have deduced, based on 48 pairs of receptors thatform or notform heterodimers, a set of triplet amino acid homologies that may importantly participate in receptor– receptor interactions with an origin from integrin triplets of marine sponges and toll-like receptor triplets (Tarakanov et al., 2012a,b). We show how such triplets of amino acid residues and their "teams" may be utilized to construct a kind of code that determines (and/or predicts) which receptors should or should not form heterodimers. We propose a "guide-and clasp" manner for receptor–receptor interactions where "adhesive guides" may be the triplet homologies. Ciruela et al. (2004) showed an amazing stability of epitope–epitope electrostatic interaction based on arginine phosphate. Thus, we should note that two main players contain R (Arg): IDR and AAR. Others have distinct relationships to negatively and positively charged amino acids.

The receptor heteromers due to the receptor–receptor interactions (RRI) represent a new target for drugs since they allow *inter alia* a higher selectivity in drug actions. Thus, in principle, it is possible to have not simply drugs acting as agonists, or partial agonists or antagonists on a receptor A that is assembled in a heteromer. Rather it is also possible to modulate receptor A through an action on receptor B which belongs to the same heteromer and interacts, via allosteric RRI, with receptor A (**Figure 1**). Against this background, a new field for pharmacology can be considered. Let us list the possible new opportunities to be investigated in a very simple case that of A–B-receptor heterodimers with the aim to modulate the recognition/decoding processes of receptor A. Due to the RRI in the dimer it could be possible to use: (1) receptor B-agonists, (2) receptor B-partial agonists, (3) receptor B-antagonists, and (4) receptor B allosteric modulators. These treatments could change the recognition/decoding process of:

(a) The complex endogenous ligands/receptor A.

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(b) The complexes receptor A-agonist/receptor A, receptor Apartial agonist/receptor A, or receptor A-antagonist/receptor A or receptor A allosteric modulator/receptor A.

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**integration of transmitter signals.** Three-dimensional molecular models of the seven TM regions of two putative GPCR (A and B) were built by means of the homology modeling program Accelrys Discovery Studio 2.5 (San Diego, CA, USA) to show that dimerization of GPCR can result from either covalent and non-covalent unions between receptor protomers. (Helix–helix interaction) Seen from a lateral view, representation of the A(TM-IV/)–B(TM-V) interaction in the A–B receptor heterodimer is shown. (Electrostatic interactions) Illustration of positively charged arginine-rich epitope (red) in the N-terminal part of the third intracellular loop of receptor A electrostatically interacting with the negatively charged (blue) C-terminal epitopes of the receptor B. These electrostatic interactions may represent important hot spots in the receptor interface of some receptor heteromers like A2AR–D2R,

or intermolecularly in homo/heteromers (arrow with two directions in the interface). Intermolecular allosteric mechanisms take place through the formation of different types of receptor homo/heteromers and receptor/ protein complexes which can change the function of an individual receptor protomer present in a homomer or heteromer. One example of the novel pharmacology created by heteromers is the use of heterobivalent ligands containing an A pharmacophore and a B pharmacophore linked through a spacer of variable length which may function as useful molecular probes for targeting the A–B receptor heteromer and in this way counteracting or enhancing the receptor–receptor interactions in these heteromers. Such compounds may have a potential for use in pharmacotherapy of CNS diseases.

In the case of a trimer or even higher order oligomers that is of a receptor mosaic (RM) we should consider especially

# **POTENTIAL EXISTENCE OF GALANINR–5-HT1A HETEROMERS WITH ANTAGONISTIC GalR–5-HT1A RECEPTOR–RECEPTOR INTERACTIONS IN THE ASCENDING MIDBRAIN RAPHE 5-HT NEURON SYSTEMS AND THEIR TARGET REGIONS. A NOVEL TARGET FOR ANTIDEPRESSANT DRUGS**

A substantial density of high affinity GalRs was demonstrated in the dorsal raphe by the Jacobowitz group in 1986 (Skofitsch et al., 1986) after having shown the existence of galanin IR cell bodies in the dorsal raphe (DR) after colchicine treatment (Skofitsch and Jacobowitz, 1985). This work inspired the Fuxe group to perform intraventricular (ivt) injections with galanin and evaluate its effects on regional 5-HT levels and metabolism (Fuxe et al.,1988a). In 1986 the coexistence of 5-HT and galanin IR in DR cell bodies was found (Melander et al., 1986) and in 1990 only a proportion of the 5-HT nerve cell bodies in the DR was found to costore galanin and 5-HT IR after colchicine (Fuxe et al., 1990). Intraventricular galanin reduced 5-HT metabolism in ventral limbic cortex, hippocampal formation, and fronto-parietal cortex probably via direct inhibitory actions on DR 5-HT nerve cells reducing their firing rates (Fuxe et al., 1988a). These results for the first time suggested based on the 5-HT hypothesis of depression (see Carlsson et al., 1968) that galanin, via actions on GalRs, mainly GalR in the DR, may contribute to depression by reducing firing in the ascending 5-HT neurons (see also Kehr et al., 2002). Thus, GalR antagonists may represent novel antidepressant drugs.

The same year it was also discovered in limbic membrane preparations that galanin in nanomolar concentrations can reduce the affinity of postjunctional [3H]-5HT1A agonist binding sites suggesting that galanin can reduce 5-HT1A recognition and probably signaling in the limbic system (Fuxe et al., 1988b). This indicates that galanin also via such actions can contribute to development of a state of depression, since postjunctional 5-HT1A receptors likely is one of the 5-HT receptors elevating mood upon activation (see Fuxe et al., 1991). This receptor has also been implicated in the pathophysiology of depression based *inter alia* on analysis of brain samples from depressed suicides (Lowther et al., 1997). These results on GalR modulation of 5-HT1A agonist binding sites gave the first indication that brain GalR/5-HT1A heteromers may exist where GalRs antagonize postjunctional 5-HT1A recognition and signaling via intramembrane receptor–receptor interactions (Fuxe et al., 1990, 1991, 2008; Zoli et al., 1993). The antagonistic GalR/5-HT1A receptor interactions in putative receptor heteromers represented a novel integrative mechanism in 5-HT neurotransmission. The role of galanin in the modulation of 5- HT neurotransmission became, however, more complex with the observations that galanin can increase potassium-induced 5-HT release from synaptosomal preparations (Martire et al., 1991). This gave the first indication there may also exist GalRs that increase 5-HT neurotransmission and thus may be beneficial for treatment of depression. In fact, it has recently been proposed that activation of GalR2 can produce antidepressant actions (Lu et al., 2005; see Lundstrom et al., 2005; Mitsukawa et al., 2008). However, the overall inhibitory influence of GalRs was in dominance (see Fuxe et al., 1988a,b). The relevance of antagonistic GalR/5-HT1A interactions for depression were discussed especially in the frame of the 5-HT isoreceptor disbalance hypothesis of depression (see Fuxe et al., 1977, 1991; Ogren et al., 1979) and the galanin-induced reduction of activity in the ascending 5-HT neurons (Fuxe et al., 1991). GalRs counteract the postjunctional mood elevating 5-HT1A signaling while the signaling over other subtypes of 5-HT receptors like the 5-HT2-like receptors which may be blocked by classical antidepressant drugs (Fuxe et al., 1977, 1991; Ogren et al., 1979) were less affected. A behavioral correlate has been obtained to the postjunctional antagonistic GalR/5-HT1A receptor interactions (Razani et al., 2001). The activation of 5-HT1A receptors in membrane preparations and in brain sections was in contrast found to increase the affinity of the GalRs in diencephalic and telencephalic areas (Hedlund et al., 1991a). Thus, reciprocal intramembrane receptor–receptor interactions may exist in putative GalR/5-HT1A heteromers where an activation of 5-HT1A receptors increases GalR recognition and probably GalR signaling. Thus, if the 5- HT1A receptor is regarded as a hub receptor and the GalR as the accessory receptor in this receptor heteromer, it seems possible that the GalR mediates an inhibitory intramembrane feedback mechanism to dampen overactivity in 5-HT1A receptor signaling (Fuxe et al., 1988b, 1991; Hedlund and Fuxe, 1996). However, it is still unknown which GalR subtype protomers are modulated by the agonist-induced activation of the 5-HT1A protomer in the GalR– 5-HT1A heteromers. In other cases, however, the enhancement of the GalR signaling may be the major action, allowing the 5-HT1A protomer to bring down firing in the raphe nuclei further due to increased opening of the GalR regulated GIRK channels leading to increased hyperpolarization (Hedlund et al., 1991b).

 Intraventricular galanin produces first a rapid decrease of the 5-HT1A autoreceptor recognition within 10 min (reduction of affinity) in line with the findings at postjunctional 5-HT1A receptors (Fuxe et al., 1998, 2008). Thus, intramembrane antagonistic GalR/5-HT1A autoreceptor interactions in putative GalR– 5-HT1A autoreceptor heteromers can be demonstrated at the soma-dendritic level of the DR 5-HT nerve cells. This will, however, not increase their nerve cell firing since galanin itself causes hyperpolarization (Xu et al., 1998) and it may represent only an intramembrane inhibitory feedback to avoid abnormal inhibition of the DR 5-HT nerve cells.

However, this change in the 5-HT1A autoreceptor is followed after 2 h by a delayed rise in 5-HT1A autoreceptor density probably through reduced internalization of the GalR/5-HT1A receptor heteromers (Razani et al., 2000) and/or a galanin-induced increase in maturation and insertion of these heteromers into the plasma membrane. This was associated with a reduction of galanin mRNA

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levels and of 5-HT1A mRNA levels in the DR supporting this interpretation. Thus, GalR antagonists may exert antidepressant effects in the DR not only by blockade of GalR signaling but also via blockade of the galanin elicited increase in 5-HT1A autoreceptor density.

A major paper in pointing to a role of raphe GalRs in depression and for the use of GalR antagonists as novel antidepressant drugs was published by Bellido et al. (2002). The discovery was made of an increased density of GalRs in the DR of a genetic rat model of depression based on the use of Flinders Sensitive Line rats. This rise of GalR density was associated with a reduction of galanin IR in the DR and an increase in the immobility time in the forced swimming test. This may represent a primary disturbance contributing to development of human depression by reducing firing in the ascending 5-HT pathways to the limbic system and the diencephalon. These results emphasize the introduction of GalR antagonists targeting the DR in treatment of depression. However, the GalR subtypes appear to have a differential role in depression as demonstrated by Bartfai and colleagues (Bartfai et al., 2004; Lu et al., 2005; Barr et al., 2006). Thus, newly developed non-peptide GalR3 antagonists exert antidepressant like activity in various rodent models of depression (Barr et al., 2006). However, fluoxetine and electroconvulsive shock increase Gal mRNA levels in the DR accompanied by increases in GalR2 receptor binding sites (Lu et al., 2005) giving in contrast an antidepressant potential to GalR2 agonists. In fact, GalR2 may be the GalR involved in releasing 5- HT from synaptosomal preparations (Martire et al., 1991). Based on the available information it therefore seems that general GalR and GalR3 antagonists have antidepressant properties while GalR2 antagonists may reduce mood.

# **EVIDENCE FOR THE EXISTENCE OF GalR1–5-HT1A HETEROMERS IN CELLULAR MODELS**

Previous work has established homodimerization and internalization of GalR1 in living CHO cells using FRET and time lapse confocal imaging (Wirz et al., 2005). Thus, GalR1 can exist as a dimer in the plasma membrane which may undergo desensitization and internalization upon agonist activation with Gal1-29.

We have examined the possible existence of GalR–5-HT1A heteromers in HEK-293 cells co-transfected with GFP2-tagged 5- HT1A receptor and YFP-tagged GalR1 receptor using a proximitybased FRET assay (Borroto-Escuela et al., 2010). In addition, a novel bioinformatic approach to predict receptor–receptor interface interactions was used (Tarakanov and Fuxe, 2010) together with an analysis of signaling.

Upon co-expression of the 5-HT1A-GFP2 and GalR1-YFP cDNA, a significantly higher FRET signal was observed in comparison to the FRET signal obtained from a mixture of cells individually expressing one of the two receptors. It represented a constitutive heteromers since the observed FRET ratios were unaltered by agonist treatments. In cells co-expressing 5-HT1A-GFP2 and GalR1-YFP the two receptors became clearly co-distributed in the plasma membrane at 48 h. And the specificity of this heteromer is indicated by the observation that expression of GalR2 does not block the FRET signal from developing in the 5-HT1A-GFP2 and GalR1-YFP heteromer. Instead, the FRET signal is increased. The understanding of the mechanism underlying this increase may unravel whether the formation of a GalR1–GalR2–5- HT1A higher-order heteromer (RM) takes place (Borroto-Escuela et al., 2010).

Using CRE-luciferase and SRE-luciferase reporter assays it was found that signaling by either the MAPK or AC pathways by these heteromers results in a trans-inhibition phenomenon through their interacting interface via allosteric mechanisms that block the development of an excessive activation of Gi/o linked to each of the receptors and an exaggerated inhibition of AC or stimulation of MAPK activity (**Figure 2**; Borroto-Escuela et al., 2010). These receptor heteromers may exist in the ascending raphe 5-HT pathways in view of the demonstration of antagonistic GalR–5-HT1A receptor interactions in the limbic regions and in the raphe reducing the affinity of the 5-HT1A receptors (see above).

Based on a bioinformatics approach, Tarakanov and Fuxe (2010) have deduced a set of triplet homologies that may be responsible for receptor–receptor interactions. This set consists of two non-intersecting subsets: "pro-triplets" and "contra-triplets". Any pro-triplet appears as a homology in at least one heterodimer but does not appear as a homology in any non-heterodimer. The triplets SNS and LAR may have an important role in the GalR–5-HT1A receptor interface. The locations of the triplet SNS in the same transmembrane domains (TM7) of GalR1 and 5- HT1A were shown. The locations of the triplet LAR were found in the cytoplasmic (intracellular) domains of GalR1 (between TM1 and TM2) and 5-HT1A (between TM5 and TM6). These two triplets may therefore participate in the transmembrane and intracellular components of the interface of the GalR1–5-HT1A heteromer.

# **A GalR1–GalR2 HETEROMER IS PROPOSED TO BE A GALANIN N-TERMINAL FRAGMENT PREFERRING RECEPTOR (1-15) IN THE CNS**

The three cloned receptors are known to show a higher affinity for Gal than for galanin N-terminal fragments like Gal (1-15) (Branchek et al., 1998). A substantial further development of this field was the demonstration of specific N-terminal galanin fragment, galanin (1-15) binding sites in the rat brain emphasizing the powerful role of galanin fragments in galanin communication (**Figure 3**), especially in dorsal hippocampus, neocortex, and striatum having few high affinity galanin (1-29) binding sites (Hedlund et al., 1992). Our hypothesis is that these N-terminal Gal fragment preferring sites may be the result of formation of GalR1/GalR2 heteromers leading to conformational changes in their galanin recognition sites converting them into highly specific galanin fragment binding sites with markedly reduced affinity for galanin (1-29) (Fuxe et al., 2008). It is of high interest that gal (1- 15) given intraventricularly in the rat has been found to produce marked depression-like behavior in the FST and anxiogenic like effects in the open field (Millon Penuela et al., 2012).

# **POSTULATED EXISTENCE OF A GalR1–GalR2–5-HT1A HETEROTRIMER**

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In agreement with above only galanin (1-15) but not galanin (1-29) can antagonistically modulate the 5-HT1A receptors in

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**antagonistic receptor–receptor interactions in serotonin 5-HT1A and galanin receptor (GalR1) heteromers in HEK293 cells and postulated 5-HT1A–GalR1–GalR2 trimers. (A)** Schematic representation of the antagonistic interactions observed after co-treatment with 5-HT1A receptor agonist (8-OH-DPAT) and galanin (peptide 1-29) in co-transfected HEK293 cells. Signaling by either the mitogen-activated protein kinase (MAPK) or adenylyl cyclase (AC) pathways by the 5-HT1A–GalR1 heteromers indicates a trans-inhibition phenomenon through their interacting interface via allosteric mechanisms that block the development of an excessive activation of Gi/o with an exaggerated inhibition of AC or stimulation of MAPK activity. **(B)** A study of the subcellular localization of tagged 5-HT1A and tagged GalR1 in the presence of non-tagged GalR2 showed a co-distribution in the plasma membrane of tagged 5-HT1A and tagged GalR1 already at 36 h in comparison

the dorsal hippocampus and this effect may be blocked by a known GalR antagonist M35 (Hedlund et al., 1994). Thus, a RM of GalR1–GalR2/-5-HT1A receptors may exist especially in the dorsal hippocampus, neocortex, the striatum, and the raphe where galanin fragments may effectively antagonize postjunctional and autoreceptor 5-HT1A recognition and may function via activation of the postulated GalR1/GalR2 heteromer (**Figure 2**). Also a cross-inhibition of the GalR1–GalR2 heteromer by the 5-HT1A protomer in the trimer should be considered. The GalR1–GalR2–5-HT1A heterotrimer may have a higher trafficking rate from the endoplasmic reticulum to the plasma membrane than the GalR1–5-HT1A heteromer (**Figure 2**; Borroto-Escuela et al., 2010).

Thus, known GalR antagonists should be putative antidepressant drugs also by blocking galanin fragment preferring sites in addition to galanin binding sites increasing postjunctional 5- HT1A mediated 5-HT signaling and the firing of the ascending 5-HT pathways (Hedlund et al., 1994; Fuxe et al., 1998). Therefore, known GalR antagonists may have multiple targets and it would be of high interest to develop an antagonist for treatment of depression that selectively target the GalR1–GalR2 heteromer postulated to be the galanin N-terminal fragment receptor.

to cells expressing tagged 5-HT1A and tagged GalR1 only. In this case, the tagged 5-HT1A receptor showed a more reticular distribution while tagged GalR1 was mainly found in the plasma membrane. In the presence of GalR2 a co-distribution in the plasma membrane of the tagged 5-HT1A and tagged GalR1 is obtained already at 36 h and is maintained at 48 h. A facilitated interaction of the GalR1–5-HT1A heterodimer into the plasma membrane by expression of GalR2 can be explained based on the postulated receptor mosaic (5HT1A–GalR1–GalR2), where the endoplasmic reticulum (ER) trafficking rate is higher than the trafficking rate shown by the 5-HT1A–GalR1 heteromer. Available findings can be explained by postulating that a GalR1–GalR2 heteromer is a galanin fragment preferring receptor which in the postulated trimer with the 5-HT1A receptor exerts a strong antagonistic receptor–receptor interaction with the 5-HT1A protomer at the level of 5-HT1A recognition.

Evidence has been presented that N-terminal galanin fragments can more strongly and more potently reduce postjunctional 5- HT1A receptor recognition also in the ventral limbic cortex where also high affinity GalRs exist (Diaz-Cabiale et al., 2000a). These effects were also blocked by a GalRs antagonist. The galanin fragment preferring receptor may again be formed by the heteromerization of GalR1 and GalR2, since they are known to be present here like in the dorsal hippocampus (O'Donnell et al., 1999). The results underline an important role of different types of GalR–5-HT1A heteroreceptor complexes in depression.

# **POTENTIAL EXISTENCE OF GalR–NPYY1 RECEPTOR HETEROMERS IN THE NUCLEUS TRACTUS SOLITARIUS (NTS), THE HYPOTHALAMUS AND THE DORSAL RAPHE WITH ANTAGONISTIC GalR–NPYY1 RECEPTOR INTERACTIONS**

In a series of papers by Diaz-Cabiale and colleagues (Diaz-Cabiale et al., 2006, 2011; Parrado et al., 2007) evidence has been obtained for an antagonistic GalR modulation of NPY receptor mechanisms suggesting the existence of GALR–NPYY1 interactions involving a likely reduction of NPYY1 receptor agonist affinity probably taking place in GalR/NPYY1 receptor heteromers (**Figure 4A**) in NTS, hypothalamus, and DR (Diaz-Cabiale et al., 2006, 2011; Parrado

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et al., 2007). The results open up the possibility that GalR/NPYY1 receptor–receptor interactions in putative heteromers is a frequent phenomenon in CNS with implications for the integrative functions of galanin and NPY in these regions. Thus, the GalR subtype involved has not been determined. This interaction may be based on a GalR/NPYY1 receptor heteromerization where the galanin induced conformational change in the GalR can cause a conformational change in the NPYY1 receptor via the GalR/NPYY1 interface leading to reduced NPYY1 recognition and G protein coupling and thus to reduced NPYY1 receptor signaling.

In the NTS (Diaz-Cabiale et al., 2006) the results suggest the existence of antagonistic GalR–NPYY1 receptor interactions in cardiovascular regions of this nucleus, reducing NPYY1 signaling and thus vasodepressor activity leading to enhanced vasopressor and tachycardic actions of GalR activation in the NTS.

In the hypothalamus (Parrado et al., 2007) evidence was, for the first time, obtained that GalR activation significantly reduced the NPYY1 receptor agonist binding in the hypothalamus without effects on NPYY2 receptor agonist binding. These GalR–NPYY1 receptor interactions have physiological implications since the food intake induced by the NPYY1 receptor agonist is blocked by galanin. The changes observed on c-Fos expression support the hypothesis that GalR activation modulates the response elicited by the NPYY1 agonist.

In the DR we may postulate, on the basis of the galanininduced decrease in NPYY1 binding, an inhibitory GalR/NPYY1 receptor–receptor interaction that modulates behavioral functions associated with mood and motivation. Behavioral and neurochemical studies support a role of galanin and NPY in mood disorders and GalR1-3 and NPYY1 receptors have been the receptors implicated in depression with GalR subtype specific antagonists and NPYY1 agonists having an antidepressant role (Lu et al., 2005; Lundstrom et al., 2005; Ishida et al., 2007; Jimenez-Vasquez et al., 2007; Fuxe et al., 2008). The decrease of NPYY1 agonist binding induced by galanin and the demonstrated crossinhibition of c-Fos expression in the dorsal raphe upon GalR and NPYY1 agonist co-activation provides one possible basis for the use of synergistic interactions of GalR subtype specific antagonists and NPYY1 receptor agonists as a strategy for treatment of depression. Thus, GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR–NPYY1 receptor interactions resulting in increases in NPYY1 transmission and antidepressant effects.

In the amygdala (Parrado et al., 2007) we may instead on the basis of the galanin-induced increase in NPYY1 binding postulate a facilitatory GalR/NPYY1 receptor interaction (**Figure 4B**) that could be expected to produce anxiolytic actions. Behavioral studies support a role of galanin and NPY in reducing anxiety in the amygdala and GalRs, NPYY1, and NPYY5 have been the receptors implicated in this effect (see Moller et al., 1999; Heilig, 2004). The increase of NPYY1 binding induced by galanin in this study provides a possible basis for synergistic interactions of GalR agonists and NPYY1 receptor agonists in counteracting anxiety behavior. The differential modulation of NPYY1 binding by galanin in the hypothalamus and in the amygdala could be explained by *inter alia* the involvement of different GalR subtypes in the interaction with the NPYY1 receptor in the hypothalamus and amygdala, respectively. It may also be that the same GalR subtype and NPY receptor subtypes are involved in the two areas but they may be part of different RMs (cluster of multiple receptors; higher order heteromers) leading to altered GalR/NPYY1 interactions in the two areas due to differences in the multiple receptor–receptor interactions (Rimondini et al., 1999) in discrete RMs (Agnati et al., 1982, 2003; Fuxe et al., 2010a,b). Thus, in this way the GalR may

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subtypes1-3 linked to central galanin neurons may form heteromers with each other and other types of GPCRs in the CNS. These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (galanin 1-15) to modulate the function of different types of glia–neuronal networks in the CNS, especially the emotional and the cardiovascular networks. The allosteric receptor–receptor interactions induced constitutively or by activation of one receptor protomer can influence the recognition, G protein coupling, signaling and trafficking of the other protomers in the receptor heteromer. **(A)** NPYY1–GalRs heteromers likely exist with antagonistic NPYY1–GalR receptor interactions involving a reduction of NPYY1 receptor agonist affinity probably taking place in postulated GalR/NPYY1 receptor heteromers in NTS, hypothalamus, and dorsal raphe. 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

NPYY1 binding induced by GalR activation in this region expected to produce anxiolytic actions. **(C)** Putative GalR–α2-adrenoreceptor heteromers with antagonistic receptor–receptor interactions reducing α2 function may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals, also of likely relevance for depression. **(D)** Galanin fragment preferring receptor (a postulated GalR1–GalR2 heteromer) appears to be linked to the NPYY2 receptor enhancing the affinity and signaling of the NPYY2 receptor via unknown signaling pathways in a putative GalR1–GalR2–NPYY2 heterotrimer; a reduction of Gi/o signaling may contribute. **(E)** Furthermore, the postulated GalR1–GalR2 heteromer, a postulated galanin N-terminal fragment preferring receptor (1-15), appears to be linked also to the AT1R in a postulated AT1R–GalR1– GalR2 receptor mosaic enhancing the signaling of the AT1R within the NTS via unknown signaling pathways; a reduction of Gi/o signaling may contribute.

reduce NPYY1 signaling in the hypothalamus and increase it in the amygdala.

# **POSTULATED EXISTENCE OF A GalR1–GalR2–NPY Y2 HETEROTRIMER IN THE NTS**

The presence of specific binding sites for the galanin fragment 1-15 in the nuclei involved in central cardiovascular regulation has been described (Hedlund et al., 1992). In the brainstem a high density of these galanin (1-15) binding sites appears within the NTS, supporting the hypothesis of the existence of a receptor with a higher affinity for the N-terminal fragment than for galanin (Diaz-Cabiale et al., 2005b). We have proposed (see above) that these N-terminal galanin fragment preferring sites may be the result of formation of GalR1/GalR2 heteromers leading to conformational changes in their galanin recognition sites converting them into highly specific galanin fragment binding sites with markedly reduced affinity for galanin (Fuxe et al., 2008). As a matter of fact galanin and N-terminal fragment galanin (1- 15) have specific and different roles (hypotensive and vasopressor responses, respectively) in cardiovascular regulation (Diaz-Cabiale et al., 2005b). The N-terminal fragment galanin (1-15) antagonized the cardiovascular effects of galanin (Narvaez et al., 1994) and galanin fragment (1-15) but not galanin decreases baroreceptor reflex sensitivity (Diaz et al., 1996). Galanin and galanin (1-15) also stimulate the expression of c-Fos with different temporal and spatial profiles, especially in the NTS and in the ventrolateral medulla (Marcos et al., 2001). The GalR antagonist M40 is able to block the cardiovascular responses elicited by the N-terminal fragment galanin (1-15) (Narvaez et al., 1994) giving evidence that regular GalR antagonists also can block the galanin fragment preferring receptors (postulated GalR1–GalR2 heteromer).

NPY operates in central cardiovascular regulation through the NPYY1 and Y2 receptor subtypes. Leu31Pro34NPY, a specific NPYY1 receptor agonist, microinjected into the NTS elicits vasodepressor and bradycardic responses (Yang et al., 1993), whereas the injection of the NPY C-terminal fragment (13-36), a specific NPYY2 receptor agonist, leads to vasopressor responses at low doses (Aguirre et al., 1990; Yang et al., 1993). In the study of Diaz-Cabiale et al. (2010) the co-injection of threshold doses of galanin (1-15) and of the NPYY2 agonist resulted in an increase of MAPK of the same magnitude as observed with threshold doses of NPY and galanin (1-15). Galanin (1-15) was also found to specifically increase the NPYY2 agonist binding in the NTS without inducing any effect on NPYY1 agonist binding (Diaz-Cabiale et al., 2010). The increase by galanin (1-15) of the NPYY2 agonist binding may indicate a galanin (1-15)-induced increase of Y2 receptor affinity in the NTS, since the concentration of the NPYY2 agonist used (25 pM) is in the range of the *Kd* value, where mainly affinity changes affect the binding level. This interaction may be based on GalR1–GalR2–NPYY2 receptor heteromerization (**Figure 4D**) where the galanin (1-15) induced conformational change in the GalR1–GalR2 heteromer via the interface with NPYY2 can cause a conformational change in the NPYY2 receptor leading to increased NPYY2 recognition and switching of G protein coupling likely to Gq and thus to increased novel NPYY2 receptor signaling producing the vasopressor activity (**Figure 4D**; Diaz-Cabiale et al., 2010). These results illustrate the high impact of the allosteric receptor–receptor interactions in heteromers in the integrative mechanisms responsible for the organization of the cardiovascular responses from the local circuit level of the NTS.

# **POSTULATED EXISTENCE OF GalR1–GalR2–AT1 HETEROTRIMER IN THE NTS**

Galanin (1-15)/angiotensin II (Ang II) interactions have also been observed in central cardiovascular control (Diaz-Cabiale et al., 2005a). Thus, intracisternal co-injections of threshold doses of Ang II with galanin (1-15) induce a significant vasopressor response that was maintained during the whole recording period, without any significant effect on heart rate. This response was blocked by the AT1 specific antagonist DuP753 (Diaz-Cabiale et al., 2005a). These data suggest the existence of a synergistic interaction between Ang II and galanin (1-15), in which the AT1 and Gal receptor subtypes participate. It may involve allosteric receptor–receptor interactions in a GalR1–GalR2–AT1 heterotrimer (**Figure 4E**) within the NTS and switching of the AT1R to Gq/11 mediated signaling.

# **POSTULATED EXISTENCE OF GalR–**α**2 ADRENORECEPTOR HETEROMERS IN THE CNS**

This proposal is *inter alia* based on the effects of galanin on α2-adrenoreceptor activation were evaluated on central cardiovascular regulation in the NTS using also quantitative receptor autoradiography (Diaz-Cabiale et al., 2000b). Central administration of threshold doses of galanin together with an effective vasodepressor dose of the α2-adrenoreceptor agonist clonidine was found to induce a rapid vasopressor and tachycardic response. On the contrary, the co-injection of threshold doses of clonidine and N-terminal galanin fragment (1-15) did not result in any significant cardiovascular change (Diaz-Cabiale et al., 2000b). These functional findings suggest that galanin, but not galanin fragment (1-15), antagonizes α2-adrenoreceptor signaling via a GalR–α2 adrenoreceptor interaction in a postulated heteromer built up of these receptor protomers (**Figure 4C**). The GalR subtypes involved in these heteromers are presently unknown.

Quantitative receptor autoradiography supported this view since galanin decreased the affinity of the α2-adrenoreceptor agonist [3H]p-aminoclonidine in the NTS and also increased significantly the density of the α2-adrenoreceptor agonist binding sites. These effects disappeared in presence of the specific GalR antagonist M35, demonstrating that this action is a direct consequence of GalR activation by galanin (Diaz-Cabiale et al., 2000b, 2005b). Galanin also reduced the affinity of the α2-adrenoreceptor agonist [3H]p-aminoclonidine in the tel- and diencephalon which was also blocked by the specific receptor antagonist M35 (Diaz-Cabiale et al., 2001). Thus, GalR–α2 adrenoreceptor heteromers with antagonistic receptor–receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline (NA) signals.

These antagonistic receptor–receptor interactions participate in the regulation of pre- and postjunctional central NA transmission and many antidepressant drugs exert part of their therapeutic effects by increasing NA transmission via blockade of the NA

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transporter (Carlsson et al., 1966, 1969). Thus, these antagonistic GalR/α2-adrenoreceptor interactions are of relevance for depression and its treatment.

In the NA cell bodies, dendrites and terminal networks the α2 adrenoreceptor function as NA autoreceptors reducing NA cell firing and NA release from the terminal networks. At this level this interaction will therefore favor increases in NA release and thus NA transmission. At the postjunctional level this antagonistic interaction found all over the tel- and diencephalon will change the balance of the pattern of NA isoreceptor activation and favor the increased activation of beta-adrenergic receptor and α1-adrenoreceptor subtypes. In view of the calming influence of α2-adrenoreceptors at the behavioral level (see Fuxe et al., 2012b) the preferential activation of the other adrenergic receptor subtypes by galanin through the antagonistic GalR/α2-adrenoreceptor interaction may result in increased arousal. It will therefore be interesting to explore how this interaction may be altered in the locus coeruleus and in the limbic networks in models of depression.

# **EXISTENCE OF DopamineR–galaninR HETEROMERS IN THE VENTRAL HIPPOCAMPUS**

There exists evidence for the existence of D1R–GalR1 and D5R– GalR1 heteromers in cellular models and D1-like receptors upon agonist-induced activation were shown to enhance the GalR1 induced MAPK signaling (Moreno et al., 2011). D1-like receptor antagonists blocked galanin-induced MAPK activation in the ventral hippocampus and in synaptosomes from this region galanin facilitated acetylcholine release upon co-activation of the D1-like receptors. Thus facilitatory allosteric D1-like-GalR interactions in heteromers may exist in control of hippocampal acetylcholine

#### **REFERENCES**

A novel, systemically active, selective galanin receptor type-3 ligand exhibits antidepressant-like activity in preclinical tests. *Neurosci. Lett.* 405, 111–115.

release and electrophysiological experiments in hippocampal slices using field EPSP recordings suggest a modulatory role of the dopamineR–galaninR heteromers in cholinergic neurotransmission (Moreno et al., 2011).

# **CONCLUSIONS**

GalR subtypes may have a major role in modulating the emotional networks of the brain through heteromerization with 5-HT1A, NPYY1, and α2-adrenoreceptors leading to antagonistic allosteric receptor–receptor interactions producing reductions in 5-HT1A, NPYY1, and α2-adrenoreceptor pre and especially postsynaptic signaling in the central 5-HT and NA neurons. This may be one way in which the activity at certain GalR subtypes and at galanin fragment preferring receptors may contribute to a reduction of mood, which may lead to depression. The GalR heteromers also participate in cardiovascular functions, food intake and regulation of fear and anxiety. The hypothesis is introduced that the galanin fragment preferring receptor is formed by the GalR1–GalR2 heteromer which can mediate the strong depressant actions of Gal 1-15 upon intraventricular injections. Its postulated formation of a trimer with 5-HT1A receptors may represent a novel target for antidepressant drugs.

# **ACKNOWLEDGMENTS**

This work has been supported by grants from *Fundacio la Marato* de TV3, Generalitat de Catalunya, Barcelona, Spain and from the Swedish Research Council (04X-715) to Kjell Fuxe. Karolinska Institutets Forskningsstiftelser 2010 and 2011 to Dasiel O. Borroto-Escuela. Alexander O. Tarakanov has not received any support for this work.

et al. (2012). The existence of FGFR1- 5-HT1A receptor heterocomplexes in midbrain 5-HT neurons of the rat: relevance for neuroplasticity. *J. Neurosci.* 32, 6295–6303.

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antidepressant-like effect via Y1 receptor. *Hippocampus* 17, 271–280.

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of the novel galanin receptor subtype GALR2 in the adult rat CNS: distinct distribution from GALR1. *J. Comp. Neurol.* 409, 469–481.

binding sites in the rat central nervous system. *Peptides* 7, 1029–1042.

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into the nucleus tractus solitarius of the rat counteract the vasodepressor responses of NPY(1-36) and of a NPY Y1 receptor agonist. *Brain Res.* 621, 126–132.

Zoli, M., Agnati, L. F., Hedlund, P. B., Li, X. M., Ferre, S., and Fuxe, K. (1993). Receptor–receptor interactions as an integrative mechanism in nerve cells. *Mol. Neurobiol.* 7, 293–334.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 August 2012; paper pending published: 16 September 2012; accepted: 05 October 2012; published online: 26 October 2012.*

*Citation: Fuxe K, Borroto-Escuela DO, Romero-Fernandez W, Tarakanov AO, Calvo F, Garriga P, Tena M, Narvaez M, Millón C, Parrado C, Ciruela F, Agnati LF, Narvaez JA and Díaz-Cabiale Z (2012) On the existence and function of galanin receptor heteromers in the central nervous system. Front. Endocrin. 3:127. doi: 10.3389/fendo.2012.00127*

*This article was submitted to Frontiers in Neuroendocrine Science, a specialty of Frontiers in Endocrinology.*

*Copyright © 2012 Fuxe, Borroto-Escuela, Romero-Fernandez, Tarakanov, Calvo, Garriga, Tena, Narvaez, Millón, Parrado, Ciruela, Agnati, Narvaez and Díaz-Cabiale. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# Differential roles of orexin receptors in the regulation of sleep/wakefulness

# **Michihiro Mieda\*, Natsuko Tsujino and Takeshi Sakurai \***

Department of Molecular Neuroscience and Integrative Physiology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa, Japan

#### **Edited by:**

Hubert Vaudry, University of Rouen, France

#### **Reviewed by:**

Etienne Challet, Centre National de la Recherche Scientifique, France Christelle Peyron, INSERM U1028, France

#### **\*Correspondence:**

Michihiro Mieda and Takeshi Sakurai, Department of Molecular Neuroscience and Integrative Physiology, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan. e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
**INTRODUCTION**

The neuropeptides orexin A and orexin B (also known as hypocretin 1 and hypocretin 2), were originally identified as endogenous ligands for two orphan G-protein-coupled receptors (Sakurai et al., 1998). Both orexin A and orexin B are derived from a common precursor peptide, prepro-orexin. An mRNA encoding the same precursor peptide of hypocretin 1 (corresponding to orexin A) and hypocretin 2 (orexin B) was independently identified by de Lecea et al. (1998) as a hypothalamus-specific transcript.

The actions of orexins are mediated by two receptors, orexin 1 (OX1R) and orexin 2 (OX2R) receptors (also known as HCRTR1 and HCRTR2) (Sakurai et al., 1998). OX1R shows a higher affinity for orexin A than orexin B by one-order, while OX2R binds orexin A and orexin B with similar affinities. Both receptors are coupled to Gq/11 subclass of G proteins and caused strong excitatory effects on neurons examined thus far (Sakurai and Mieda, 2011). OX2R has also been reported to couple to Gi/o in a neuronal cell line when overexpressed (Zhu et al., 2003).

Orexins are produced in a specific population of neurons (orexin neurons) that are located exclusively in the hypothalamus, including the lateral hypothalamic area (LHA), perifornical area, and posterior hypothalamus (PH) (de Lecea et al., 1998; Peyron et al., 1998; Sakurai et al., 1998; Date et al., 1999; Nambu et al., 1999). Orexin neurons project in almost all brain areas with especially dense projections to monoaminergic neurons, which play important roles in the regulation of sleep/wake states. Orexins are thought to be a critical regulator of sleep/wake states. Intracerebroventricular (ICV) administration of orexin A and orexin B increases wakefulness and suppresses both rapid-eye-movement (REM) sleep and non-REM (NREM) sleep (Hagan et al., 1999). More importantly, orexin deficiency causes sleep disorder narcolepsy in humans and animals (Chemelli et al., 1999; Lin et al.,

Orexin A and orexin B are hypothalamic neuropeptides that play critical roles in the regulation of sleep/wakefulness, as well as in a variety of physiological functions such as emotion, reward, and energy homeostasis. The actions of orexins are mediated by two receptors, orexin 1 (OX1R) and orexin 2 (OX2R) receptors. OX1R and OX2R show partly overlapping but distinct distributions throughout the central nervous system, suggesting their differential roles.This review presents and discusses the current knowledge concerning the physiological roles of each orexin receptor subtype, focusing on the regulation of sleep/wakefulness.

**Keywords: orexin, hypothalamus, sleep, monoamine, narcolepsy, arousal**

1999; Peyron et al., 2000; Thannickal et al., 2000; Hara et al., 2001). At present, various reports suggest that orexins are involved not only in the sleep/wake regulation but also in other physiological and behavioral processes such as food intake, emotion, stress response, and reward via activation of OX1R and OX2R (Yamanaka et al., 2003; Akiyama et al., 2004; Mieda et al., 2004; Boutrel et al., 2005; Harris et al., 2005; Sakurai et al., 2005; Narita et al., 2006; Yoshida et al., 2006). Recent reports revealed that each orexin receptor has a different function in these processes. Thus, understanding such a difference is important for understanding the function of the orexin system.

This review will discuss the physiological roles of the orexin system in the regulation of sleep/wakefulness, especially focusing on the functions of each orexin receptor.

# **DISTRIBUTIONS OF OREXIN RECEPTOR mRNA**

According to data obtained by *in situ* hybridization histochemistry, many brain regions have been shown to differentially express *OX1R* and *OX2R* mRNAs (Trivedi et al., 1998; Lu et al., 2000; Greco and Shiromani, 2001; Marcus et al., 2001). Consistent with the broad projections of orexin neurons, *OX1R* and *OX2R* show wide distributions within the brain with partly overlapping but distinct and complementary distributions. For example, *OX2R* is expressed in layers 2 and 6 throughout the cerebral cortex, while *OX1R* is expressed in layers 5 and 6, except the cingulate cortex where *OX1R* mRNA is found in layer 3. In the ammon's horn of the hippocampus, CA1 and CA2 express *OX1R*, while CA3 expresses *OX2R*.

In the arcuate nucleus and paraventricular nucleus of the hypothalamus, which are involved in the regulation of feeding, energy homeostasis, autonomic and endocrine systems, *OX2R* is almost exclusively observed. Concerning the nuclei implicated

in sleep/wake regulation, the locus ceruleus (LC), laterodorsal tegmental nucleus (LDT), and pedunculopontine tegmental nucleus (PPT) mainly express *OX1R* mRNA, while the tuberomammillary nucleus (TMN) almost exclusively expresses *OX2R* mRNA. The dorsal raphe (DR) and median raphe (MnR) express both subtypes. These distributions suggest partly overlapping and partly distinct roles of these two receptors.

Monoaminergic (i.e., histaminergic, noradrenergic, and serotonergic) and cholinergic neurons in these nuclei have been considered critical for sleep/wakefulness regulation (Pace-Schott and Hobson, 2002; Zeitzer et al., 2006). Our recent work determined precise cellular localization of two orexin receptors in these nuclei (**Figure 1**) (Mieda et al., 2011). All histaminergic neurons in the TMN exclusively expressed *OX2R*, while all noradrenergic neurons in the LC exclusively expressed *OX1R*. In the DR and MnR, approximately 90% of serotonergic neurons expressed *OX1R* and/or *OX2R*. In addition, many non-serotonergic cells in the DR/MnR also expressed *OX1R* or *OX2R* mRNA. At least some populations of these cells were likely to be GABAergic, since a population of *Gad1*-positive cells also expressed detectable *OX1R* or *OX2R* mRNA. In the LDT and PPT, all cholinergic neurons expressed *OX1R* but not *OX2R* mRNA, while many *OX1R*-positive and/or *OX2R*-positive non-cholinergic neurons were intermingled with cholinergic neurons in the area. *Gad1* mRNA staining further revealed that *OX1R*- or *OX2R*-expressing cells included both GABAergic and non-GABAergic cells.

Orexin receptors-positive GABAergic neurons in the DR/MnR and LDT/PPT, which are intermingled with serotonergic and cholinergic neurons, respectively, are intriguing. Since GABAergic neurons are likely to function as local inhibitory interneurons and orexin receptors usually cause excitatory effects on neurons, simultaneous activation of serotonergic/cholinergic neurons with GABAergic interneurons by orexins within a nucleus may inhibit serotonergic/cholinergic neurons. Thus, balance of orexinergic activation between principal neurons and interneurons may be of importance. This point will be further discussed in a later section.

# **OREXIN AND MONOAMINERGIC/CHOLINERGIC SYSTEMS**

Orexin neurons project almost all brain regions with especially dense projections being seen in monoaminergic and cholinergic nuclei involved in the regulation of sleep/wakefulness. Various reports suggested that orexin excites these neurons directly and/or indirectly *in vivo* and *in vitro* (**Figure 1**). This orexinergic regulation may be important to control sleep/wakefulness. Here, we will review *in vitro* and *in vivo* electrophysiological studies of orexin receptors in monoaminergic and cholinergic neurons.

#### **SEROTONERGIC NEURONS OF THE DORSAL RAPHE**

In 2001, Brown et al. reported that orexin A strongly excited DR serotonergic neurons in brain slices of rats under the whole-cell patch clamp recording. The depolarization persisted in the presence of the voltage dependent Na+ channel blocker, tetrodotoxin,

**FIGURE 1 | Schematic illustration of presumed pathways underlying orexin actions on NREM and REM sleep (Mieda et al., 2011)**. Orexins activate histaminergic (His)/GABAergic (GA), serotonergic (5HT), noradrenergic (NA), and cholinergic (ACh) neurons as well as GABAergic putative interneurons in wake-promoting nuclei, including the TMN, DR/MnR, LDT/PPT, and LC. These neurons differentially express OX1R and/or OX2R, and regulate wakefulness/NREM sleep and NREM/REM sleep transitions. OX1R and OX2R may be expressed in the same populations of GABAergic neurons, as shown in the figure, or may be expressed in distinct populations of these neurons in each area.

Wake/REM-on cholinergic neurons (ACh/W) are likely to suppress NREM sleep, but REM-on cholinergic neurons (ACh/R), are likely to induce REM sleep. Wake-active serotonergic and noradrenergic neurons in the DR/MnR and LC, respectively, counteract activation of REM-on cholinergic neurons in the LDT/PPT, as well as REM-on neurons in the brainstem reticular formation (Pace-Schott and Hobson, 2002; Sakurai, 2007). Previous reports have suggested contributions of GABAergic interneurons inhibiting PPT cholinergic and raphe serotonergic neurons (Liu et al., 2002; Takakusaki et al., 2005). LHA, lateral hypothalamic area; PH, posterior hypothalamus. Reproduced from Sakurai and Mieda (2011) with permission.

indicating a direct postsynaptic action on the serotonin cells (Brown et al., 2001). Moreover, orexin A and orexin B induced inward currents with similar amplitude and dose-dependency in most serotonergic neurons (Brown et al., 2001; Liu et al., 2002; Soffin et al., 2004) via activation of Na+/K+ non-selective cation channels (Liu et al., 2002). These results may suggest that OX2R is mainly involved in the activation of serotonergic neurons, although single-cell PCR, *in situ* hybridization, and immunohistochemical studies revealed that serotonergic neurons express both *OX1R* and *OX2R* (Brown et al., 2001; Liu et al., 2002; Wang et al., 2005; Mieda et al., 2011).

A recent report revealed that orexin A depolarized DR neurons by activating a noisy inward current (Kohlmeier et al., 2004, 2008). This current was through non-selective cation channels that did not contribute to the somatic Ca2+ influx. These reports suggested that orexin A has two independent actions: activation of non-selective cation channels and activation of a protein kinase C-dependent enhancement of Ca2+ transients mediated by L-type Ca2+ channels.

At higher concentrations, orexin also increased spontaneous inhibitory postsynaptic currents in serotonergic neurons indicating orexin excited GABAergic interneurons that project to serotonergic neurons in the DR (Liu et al., 2002). Such a structure of intra-DR circuit may execute a negative feedforward regulation of the activation of serotonergic neurons by orexin neurons.

Regulation of DR neurons by orexin seems to be even more complex. Haj-Dahmane and Shen found that orexin B depresses the evoked glutamate-mediated synaptic currents in DR serotonergic neurons. This effect was mediated by retrograde endocannabinoid release, which depended on the stimulation of postsynaptic orexin receptors and subsequent activation of phospholipase C and DAG lipase enzymatic pathways but not on a rise in postsynaptic intracellular calcium (Haj-Dahmane and Shen, 2005).

*In vivo* extracellular recording also revealed that application of orexin A increased firing frequency of serotonergic DR neurons in unanesthetized rats during REM sleep or slow-wave sleep, while during wakefulness, a similar amount of orexin A did not increase the firing rate (Takahashi et al., 2005). These reports suggest that orexin excited DR serotonergic neurons in a behavioral state-dependent manner.

#### **NORADRENERGIC SYSTEM**

Orexin A and orexin B increased the firing frequency of all LC neurons in the presence of tetrodotoxin in rat brain slices by intracellular recording (Horvath et al., 1999; Soffin et al., 2002). The effects of orexin A are fivefold more potent than orexin B. Moreover, the effect was inhibited by selective orexin 1 receptor antagonist, SB-334867 (Soffin et al., 2002, 2004). These data suggest that orexins directly excited LC noradrenergic neurons via OX1R, which is consistent with exclusive expression of *OX1R* in these neurons demonstrated by *in situ* hybridization (Mieda et al., 2011). The orexin-induced depolarization of LC noradrenergic neurons may be produced by augmentation of the non-selective cationic conductance and suppression of G-protein-coupled inward rectifier (GIRK) channel activity (Hoang et al., 2003; Murai and Akaike, 2005).

In addition to the action of orexins within the LC, orexin A and B also evoked noradrenaline release in the rat cerebrocortical slice, suggesting that orexins act on OX1R on the orexin fibers projecting from the LC in the cerebral cortex (Hirota et al., 2001). Orexin-evoked noradrenaline release was time- and concentration-dependent and partially extracellular Ca2+-dependent (Hirota et al., 2001).

Another report suggested that the orexin modulates LC functions via OX1R by regulating noradrenaline release not only from the axonal terminals, but also from the somatodendritic region of LC noradrenergic neurons (Chen et al., 2008). Application of orexin alone dose-dependently induced somatodendritic noradrenaline release. Orexin A also potentiated *N*-methyld-aspartate (NMDA) receptor-mediated somatodendritic noradrenaline release from LC neurons, which was blocked by SB-334867, an OX1R antagonist, or a PKC inhibitor, indicating the involvement of OX1R and PKC. Orexin A enhanced NMDAinduced intracellular Ca2+ elevation as well (Chen et al., 2008). Taken together, activation of OX1R of LC noradrenergic neurons regulates not only noradrenergic input to their targets, but also noradrenergic communication in the soma and dendrites.

Excitatory effects of orexin A on LC neurons have been demonstrated also *in vivo* by measuring their firing rates (Bourgin et al., 2000) or noradrenaline release into the hippocampus (Walling et al., 2004). In addition, local administration of orexin A but not orexin B in the LC of rats suppressed REM sleep in a dosedependent manner and increased wakefulness at the expense of REM sleep and deep slow-wave sleep (Bourgin et al., 2000).

 Orexin A and B showed almost the same potency, indicating that orexin-induced excitation of histaminergic neurons is mediated via OX2R. Consistent with electrophysiological data, *OX2R* was shown to be the principle subtype of orexin receptors by single-cell RT-PCR study, although both receptor mRNAs were detected in most tuberomammillary neurons (Eriksson et al., 2001). Histological studies have demonstrated that TMN histaminergic neurons express *OX2R* almost exclusively (Yamanaka et al., 2002; Mieda et al., 2011).

Orexin-induced depolarization was associated with a small decrease in input resistance. The mechanisms of depolarization of histaminergic neurons are likely to be related to the activation of both the electrogenic Na+/Ca2+ exchanger and Ca2+ current (Eriksson et al., 2001), as well as suppression of GIRK channels (Hoang et al., 2003).

On the other hand, a recent *in vitro* optogenetic study demonstrated that postsynaptic currents and rapid increase of firing rates in TMN histaminergic neurons evoked by stimulation of orexin neurons were mediated by glutamate rather than by orexins (orexin neurons are also glutamatergic) (Schone et al., 2012). This suggests a possibility that orexin might play as a modulator of the glutamatergic transmission in these cells.

*In vivo* application of orexin A to the TMN increased histamine release in both the medial preoptic area and the frontal cortex in a dose-dependent manner (Huang et al., 2001). Moreover, perfusion of orexin A into the TMN of rats through a microdialysis probe promptly increased wakefulness, concomitant with a reduction in REM and NREM sleep (Huang et al., 2001; Yamanaka et al., 2002). These findings indicate that orexins excite TMN histaminergic neurons mainly via OX2R and enhance histamine release from the TMN to maintain wakefulness.

#### **CHOLINERGIC SYSTEM**

#### **Laterodorsal tegmental nucleus**

Whole-cell recordings revealed that orexin exhibited actions both presynaptically and postsynaptically on LDT cholinergic neurons, as well as in non-cholinergic neurons in the same area. Orexin increased frequency and amplitude of spontaneous EPSCs in these neurons. Postsynaptically, orexin produced an inward current and an increase in membrane current noise, which were accompanied by a conductance increase (Burlet et al., 2002). Being similar to DR serotonergic neurons, orexin A has two independent actions on LDT cholinergic neurons: activation of non-selective cation channels and activation of a protein kinase C-dependent enhancement of Ca2+ transients mediated by L-type Ca2+ channels (Kohlmeier et al., 2004, 2008).

In a study of *in vivo* extracellular recordings of LDT neurons in rats, the application of orexin A induced long-lasting excitation in five of seven cholinergic neurons (Takahashi et al., 2002). Furthermore, when orexin A was microinjected in the LDT of cats, time spent in wakefulness was significantly increased due to an increase in the duration of wake episodes, and time spent in REM sleep was significantly reduced due to a decrease in the frequency of active sleep episodes (Xi et al., 2001).

#### **Pedunculopontine tegmental nucleus**

Whole-cell patch clamp recordings revealed that orexin A and orexin B depolarized PPT cholinergic and non-cholinergic neurons dose-dependently in the presence of tetrodotoxin (Kim et al., 2009a). Approximately 80% of PPT neurons were depolarized by orexin A, and 20% of PPT neurons did not respond to orexin A (Kim et al., 2009b). SB-334867, a selective inhibitor for OX1R, significantly suppressed the orexin A-induced depolarization. These results suggest that orexin depolarized PPT neurons directly through OX1R. This OX1R-mediated depolarization was caused by a decrease of K+ conductance and an increase of non-selective cationic conductance (Kim et al., 2009a). It was also indicated that it was blocked by D609, a phosphatidylcholine-specific PLC inhibitor, suggesting that the excitatory effects of orexin on PPT neurons are mediated by PLC-dependent pathway (Kim et al., 2009b).

An *in vivo* study demonstrates that orexin inhibited PPT neurons via GABAergic neurons (Takakusaki et al., 2005). In decerebrated cats, an injection of orexin into the PPT increased the intensity of electrical stimulation required at the PPT to induce muscle atonia. The effect of orexin on PPT was abolished by simultaneous injection of bicuculline, a GABAA receptor antagonist, into the PPT. These results suggest that orexin enhances GABAergic effects on presumably cholinergic PPT neurons *in vivo* to control postural muscle tone.

Cholinergic neurons in these areas include two functionally distinct types of neurons. Some are active during wakefulness and REM sleep (W/REM-on neurons) and others are specifically active during REM sleep (REM-on neurons) (Pace-Schott and Hobson, 2002; Sakurai, 2007). The latter population is likely to play a critical role in REM sleep-related physiological phenomena, including muscle atonia. Orexin A excites both cholinergic and non-cholinergic neurons of the LDT in slice preparations, as described previously (Burlet et al., 2002). Furthermore, orexin A microinjection in the cat LDT increases wakefulness and reduces REM sleep. In addition, as mentioned previously, GABAergic neurons in the PPT mediate suppression of REM sleep and muscle atonia following local injection of orexin A into this area (Takakusaki et al., 2005). Consistent with the actions of orexins on both cholinergic and non-cholinergic neurons, orexin receptors are differentially expressed in cholinergic and GABAergic neurons of the LDT/PPT, shown by a histological study (Mieda et al., 2011). Taken together, these results indicate that in the LDT/PPT, orexin may activate W/REM-on cholinergic neurons through OX1R to facilitate wakefulness. Simultaneously, orexin might activate GABAergic interneurons to inhibit REM-on cholinergic neurons in these nuclei (**Figure 1**).

#### **Basal forebrain**

Electrophysiological studies were performed on cultured nucleus basalis neurons from the basal forebrain and on rat brain slice (Eggermann et al., 2001; Hoang et al., 2004). In cultured neurons, orexin A induced a depolarization and increased firing accompanied by a decrease of whole-cell conductance in cholinergic neurons. The mechanism of this neuronal excitation was shown to be accompanied by inhibition of inward rectifier K+ channel (KirNB) activity (Hoang et al., 2004). In rat brain slices, orexin has a direct excitatory effect on the cholinergic neurons of the contiguous basal forebrain (Eggermann et al., 2001). All cholinergic neurons were excited by orexin A and even more potently by orexin B. This result suggests that the action depends on OX2R. Thus, orexins may excite cholinergic neurons in the basal forebrain via OX2R and contribute to the cortical activation.

The medial septum-diagonal band of Broca (MSDB), via its cholinergic and GABAergic projections to the hippocampus, controls the hippocampal theta rhythm and associated learning and memory functions. MSDB receives a dense innervation of orexin neurons, and neurons of the MSDB express very high levels of OX2R. Septohippocampal cholinergic neurons were excited by orexin A and B with similar EC50 in a concentrationdependent manner, mediated via a direct postsynaptic mechanism (Wu et al., 2004). The orexin effect is likely to be mediated by inhibition of a K+ current, presumably an inward rectifier, and activation of the Na+–Ca2+ exchanger. Thus orexin effects within the septum should increase hippocampal acetylcholine release and thereby promote hippocampal arousal (Wu et al., 2004).

# **DIFFERENTIAL ROLES OF EACH OREXIN RECEPTOR SUBTYPE IN THE REGULATION OF SLEEP/WAKEFULNESS**

#### **MOLECULAR GENETIC STUDIES**

Recent studies have established that the orexin (hypocretin) system is one of the most important regulators of sleep/wake states and that its deficiency results in the human sleep disorder narcolepsy (Zeitzer et al., 2006; Sakurai and Mieda, 2011). Narcolepsy is characterized by excessive daytime sleepiness that often results in "sleep attacks" (sudden onset of NREM sleep), cataplexy (sudden bilateral skeletal muscle weakening triggered by emotions without loss of consciousness), hypnagogic hallucinations, and sleep paralysis. These symptoms can be divided into two independent pathological phenomena. One is the inability to maintain a consolidated awake period characterized by abrupt transitions from wakefulness to NREM sleep (i.e., dysregulation of NREM sleep onset). This phenomenon manifests clinically as excessive daytime sleepiness or sleep attacks. The other key phenomenon is the pathological intrusion of REM sleep into wakefulness or at sleep onset (i.e., dysregulation of REM sleep onset). It is during these periods that patients experience cataplexy, hypnagogic hallucinations, and sleep paralysis.

Mice with targeted deletion of the *prepro-orexin* gene (*orexin*−*/*− mice) display a phenotype strikingly similar to narcolepsy: abrupt behavioral arrests with muscle atonia (cataplexy), fragmented wakefulness (inability to maintain consolidated wakefulness episodes), and direct transitions from wakefulness to REM sleep (Chemelli et al., 1999). In addition, functionally null mutations in the *OX2R* gene were found in two independent lines of familial narcoleptic dogs (Lin et al., 1999).

Based on studies on orexin receptor-deficient mice (*OX1R*−*/*− and *OX2R*−*/*− mice), it has been suggested that deletion of *OX1R* produces no measurable effect on sleep/wakefulness states (Sakurai, 2007; Hondo et al., 2010). However, *OX2R*−*/*− mice have clear characteristics of narcolepsy, although their behavioral and EEG phenotype is less severe than that found in *orexin*−*/*− mice (Willie et al., 2003). In infrared videophotographic studies in the dark phase, *OX2R*−*/*− mice showed abrupt behavioral arrests. However, its frequency was much less than in *orexin*−*/*− mice (31-fold lower frequency in *OX2R*−*/*− mice as compared to *orexin*−*/*− mice). Instead, *OX2R*−*/*− mice showed a distinct variety of behavioral arrests with onsets that were more gradual in nature (gradual arrests). Moreover, *orexin*−*/*− mice also exhibit gradual arrests with a frequency similar to *OX2R*−*/*− mice in addition to plenty of abrupt arrests.

In contrast to abrupt arrests, gradual arrests typically began during quiet wakefulness and could be easily distinguished from the normal onset of resting behavior by (i) the absence of stereotypic preparation for sleep (e.g., nesting and/or assumption of a curled or hunched posture with limbs drawn under the body) and (ii) a characteristic ratchet-like"nodding"of the head over a period of several seconds with a transition to a collapsed posture (Willie et al., 2003).

Detailed observations of behaviors during EEG/EMG recordings found that abrupt arrests in *orexin*−*/*− and *OX2R*−*/*− mice occurred during direct transitions from wakefulness to REM sleep, whereas EEG/EMG correlates of gradual arrests in both *orexin*−*/*−

and *OX2R*−*/*− mice invariably revealed the onset of attenuated muscle tone, but not atonia, and an EEG transition from wakefulness to NREM sleep (Chemelli et al., 1999;Willie et al., 2003). Pharmacologically, abrupt arrests in *orexin*−*/*− mice were suppressed by systemic administration of clomipramine, an anticataplectic agent used for treatment of human narcolepsy. Whereas, administration of caffeine, a psychostimulant used to treat excessive sleepiness in human narcolepsy, tended to produce a mild exacerbation of abrupt arrest frequency. In clear contrast, systemic administration of caffeine dose-dependently suppressed gradual arrests, while administration of an anticataplectic agent clomipramine did not affect the frequency of gradual arrests in both *orexin*−*/*− and *OX2R*−*/*− mice. These observations showed strong similarity with those observed in human narcolepsy patients; psychostimulant drugs are effective for the sleepiness, but exacerbate cataplexy, which is treatable with antidepressants. This detailed characterization of behavioral, pharmacological, and electrophysiological features of *orexin*−*/*− and *OX2R*−*/*− mice defined abrupt and gradual arrests as the presumptive mouse correlates of cataplexy and sleep attack in human narcolepsy, respectively.

In addition to the gradual behavioral arrests, *OX2R*−*/*− mice exhibit fragmentation of wakefulness, another sign of sleepiness, to the extent similar to that in *orexin*−*/*− mice (Willie et al., 2003). *OX1R*−*/*−; *OX2R*−*/*− mice appear to have the same phenotype with *orexin*−*/*− mice, implying that these two receptors are sufficient to mediate regulation of sleep/wakefulness by orexins (Sakurai, 2007; Hondo et al., 2010). These results of mouse reverse genetic studies suggest that normal regulation of wake/NREM sleep transitions depends critically on OX2R activation, whereas the profound dysregulation of REM sleep control are unique to narcolepsy from loss of signaling through both OX1R- and OX2R-dependent pathways.

Substantially lower frequency of cataplexy in *OX2R*−*/*− mice as compared to *orexin*−*/*− mice appears to be inconsistent with the fact that mutations of *OX2R* gene are solely responsible for an inherited canine model of narcolepsy, which demonstrate frequent occurrence of cataplexy as well as excessive sleepiness (Lin et al., 1999). This may result from species difference (e.g., the precise expression patterns of two orexin receptors). However, even in dogs, the absence of orexin peptides may cause severe narcoleptic symptoms as compared to *OX2R* mutation. Early studies of narcoleptic Dobermans and Labradors found these dogs to be 30 to 80-fold less severely affected with cataplexy than poodles with sporadic narcolepsy that showed literally hundreds of attacks a day (Baker et al., 1982), an effect previously attributed solely to differences in breed and breed size.

As an experiment complementary to the behavioral studies and baseline sleep/wakefulness recordings of *OX1R*−*/*− and *OX2R*−*/*− mice, we elucidated that two orexin receptors play distinct and differential roles in the regulation of sleep and wakefulness states by comparing the effects of ICV orexin A administration in wild-type, *OX1R*−*/*− and *OX2R*−*/*− mice (Mieda et al., 2011). The effects of orexin A on wakefulness and NREM sleep were significantly attenuated in both knockout mice as compared to wild-type mice, with substantially larger attenuation in *OX2R*−*/*− mice than in *OX1R*−*/*− mice. These results suggest that although the OX2R-mediated pathway has a pivotal role in the promotion of wakefulness, OX1R also plays additional roles in promoting arousal.

In contrast, suppression of REM sleep by orexin A administration was slightly and similarly attenuated in both *OX1R*−*/*− and *OX2R*−*/*− mice, suggesting a comparable contribution of the two receptors to REM sleep suppression (Mieda et al., 2011). In addition, our observations further suggest that orexin A directly suppresses transitions from NREM sleep to REM sleep, and that activation of OX1R is sufficient for this effect. Supplementary role of OX1R in the suppression of NREM sleep is consistent with the fact that *OX2R*−*/*− mice on a C57BL/6J genetic background show less fragmented wakefulness when compared to *orexin*−*/*− mice and *OX1R*−*/*−; *OX2R*−*/*− mice (but show similarly fragmented wakefulness on a C57BL/6J-129/SvEv mixed background) (Sakurai, 2007; Mochizuki et al., 2011), suggesting that OX1R is indispensable for the maintenance of wakefulness in the absence of OX2R.

Although application of exogenous orexins has been shown to excite many types of neurons (Sakurai and Mieda, 2011), neurons directly downstream to orexin neurons in physiological conditions (i.e., neurons influenced by endogenous orexins that mediate their wake-promoting and REM-suppressing effects), have remained uncertain. Several reports suggested that histaminergic neurons in the TMN play an important role in the arousal-promoting effect of orexin, supported by the facts that the effect of ICV orexin A administration is markedly attenuated by the histamine H1 receptor antagonist pyrilamine (Yamanaka et al., 2002) and is absent in *H1 histamine receptor* knockout mice (Huang et al., 2001) and that the TMN expresses OX2R abundantly (Marcus et al., 2001; Mieda et al., 2011), the subtype whose absence causes narcoleptic phenotype in mice and dogs (Lin et al., 1999; Willie et al., 2003). Mochizuki et al. (2011) produced a mouse model in which a *loxP*-flanked gene cassette disrupts production of the OX2R, but normal OX2R expression can be restored by Cre recombinase. They showed that targeted Cre expression (i.e., focal restoration of OX2R expression), in the TMN and adjacent regions rescues fragmentation of wakefulness in this mouse model, suggesting that the orexin signaling mediated by OX2R in the TMN (and possibly its surrounding area in the PH) is sufficient to prevent sleepiness caused by systemic OX2R deficiency.

On the other hand, this hypothesis is still controversial. Mice lacking both OX1R and histamine H1 receptors demonstrate no abnormality in sleep/wakefulness (Hondo et al.,2010). In addition, a recent optogenetic study showed that orexin-mediated sleepto-wake transitions do not depend on the TMN histaminergic neurons (Carter et al., 2009). Rather, another optogenetic study suggested the role of LC noradrenergic neurons as a direct downstream pathway of orexin neurons by demonstrating that optogenetic inhibition of LC noradrenergic neurons blocked arousal effects of optogenetic stimulation of orexin neurons (Carter et al., 2012).

#### **REFERENCES**

Akiyama, M., Yuasa, T., Hayasaka, N., Horikawa, K., Sakurai, T., and Shibata, S. (2004). Reduced food anticipatory activity in genetically orexin (hypocretin) neuron-ablated

mice. *Eur. J. Neurosci.* 20, 3054–3062.

Baker, T. L., Foutz, A. S., McNerney, V., Mitler, M. M., and Dement, W. C. (1982). Canine model of narcolepsy: genetic and

#### **PHARMACOLOGICAL STUDIES**

Antagonists for orexin receptors are drawing people's attention as novel medications for insomnia (Scammell and Winrow, 2011; Mieda and Sakurai, 2013). Several non-selective (dual) antagonists for orexin receptors, as well as subtype-selective antagonists, have been developed. These drugs are indeed useful also for studying the roles of each subtype in the regulation of sleep/wakefulness without considering any chronic compensatory changes that might complicate the phenotypes of genetically engineered models.

A study reported that an OX2R-selective antagonist JNJ10397049 has a better ability to promote NREM sleep than the dual antagonist almorexant in rats (Dugovic et al., 2009). They suggested that simultaneous inhibition of OX1R attenuates the sleep-promoting effects mediated by selective OX2R blockade, possibly correlated with dopaminergic neurotransmission. In contrast, another recent study led to a different conclusion (Morairty et al., 2012). It reported that an OX1R-selective antagonist SB-334867 produces small increases in REM and NREM sleep, and an OX2R-selective antagonist EMPA produces a significant increase in NREM sleep. But administration of almorexant increases NREM sleep more than these subtype-selective antagonists, leading to the conclusion that dual orexin receptor antagonism is more effective for sleep promotion than subtype-selective antagonism. The difference in these drugs' capabilities of crossing the blood-brain barrier, as well as their pharmacokinetic/pharmacodynamic characters, might influence the effectiveness. The latter view is also consistent with the conclusion derived from studies using subtypespecific knockout mice that OX2R plays a pivotal role, but OX1R has additional effects on promotion of wakefulness (Sakurai, 2007; Mieda et al., 2011).

#### **CONCLUSION**

The orexin system is a necessary component for the normal regulation of sleep/wakefulness. Nevertheless, broad expression of orexin receptors throughout the brain makes it difficult to identify neurons and orexin receptor subtypes that are directly regulated by endogenous orexins and mediate their effects on the physiology of interest in a natural context. Future studies using molecular genetic strategies such as brain region/cell type-specific deletions and brain region/cell type-specific rescues of orexin receptors, as well as pharmacological studies of focal administration of subtype-specific orexin agonists/antagonists, would further dissect the differential roles of orexin receptors in the regulation of sleep/wakefulness. Furthermore, the orexin system is also important for the regulation of a variety of physiological functions, such as feeding, reward, and emotions. Thus, precise information of the different roles of the two orexin receptors is beneficial not only for understanding the mechanisms underlying orexinergic regulations of physiology, but also for application of orexin agonists/antagonists as medications for various diseases.

developmental determinants. *Exp. Neurol.* 75, 729–742.

Bayer, L., Eggermann, E., Serafin, M., Saint-Mleux, B., Machard, D., Jones, B., et al. (2001). Orexins (hypocretins) directly excite

tuberomammillary neurons. *Eur. J. Neurosci.* 14, 1571–1575.

Bourgin, P., Huitron-Resendiz, S., Spier, A. D., Fabre, V., Morte, B., Criado, J. R., et al. (2000). Hypocretin-1 modulates rapid eye movement

sleep through activation of locus coeruleus neurons. *J. Neurosci.* 20, 7760–7765.

Orexins/hypocretins excite basal forebrain cholinergic neurones. *Neuroscience* 108, 177–181.

innervation of the locus coeruleus noradrenergic system. *J. Comp. Neurol.* 415, 145–159.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 10 January 2013; accepted: 25 April 2013; published online: 16 May 2013.*

*Citation: Mieda M, Tsujino N and Sakurai T (2013) Differential roles of orexin receptors in the regulation of sleep/wakefulness. Front. Endocrinol. 4:57. doi: 10.3389/fendo.2013.00057*

*This article was submitted to Frontiers in Neuroendocrine Science, a specialty of Frontiers in Endocrinology.*

*Copyright © 2013 Mieda, Tsujino and Sakurai. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, providedthe original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# Control of ventricular ciliary beating by the melanin concentrating hormone-expressing neurons of the lateral hypothalamus: a functional imaging survey

#### **Grégory Conductier 1,2† , Agnès O. Martin3,4,5† , Pierre-Yves Risold6† , Sonia Jego7 , Raphaël Lavoie7 , Chrystel Lafont 3,4,5, Patrice Mollard3,4,5‡ , Antoine Adamantidis 7‡ and Jean-Louis Nahon1,2,8\* ‡**

1 UMR7275, Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Valbonne, France

3 UMR5203, Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Montpellier, France

4 U661, INSERM, Montpellier, France

#### **Edited by:**

Hubert Vaudry, University of Rouen, France

#### **Reviewed by:**

Gert Jansen, Erasmus Medical Centre, Netherlands Serge H. Luquet, University Paris Diderot, France

#### **\*Correspondence:**

Jean-Louis Nahon, UMR7275, Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, 660 Route des Lucioles, Sophia Antipolis, Valbonne, France e-mail<EMAIL_ADDRESS>
†Co-Authors ‡Co-Directors

#### **INTRODUCTION**

First identified in the early 80s from chum salmon pituitaries, the melanin concentrating hormone (MCH) draw its name from its capability to induced the concentration of melanin in the skin melanophores (2). However, this function seems to be restricted to teleosts [reviewed in Ref. (3)]. In contrast with high MCH structural conservation, the neuronal distribution appears quite different, reflecting evolutionary changes in the prosencephalon across vertebrate species (4). In mammals, this cyclic peptide is mainly expressed in neurons of the lateral hypothalamic area (LHA), projecting widely throughout the brain (5); reviewed in Ref. (6). Accordingly, MCH is involved in a broad spectrum of cerebral functions [for recent reviews, see Ref. (7, 8)]. Nevertheless, all of these seem to converge to the adaptation of global physiologic state to metabolic needs by promoting memory processes and reward pathways activation on one hand and by decreasing arousal and thermogenesis on the other hand. Activation of these cognitive and neuroendocrine networks leads to an increase in food intake and energy storage, respectively [reviewed in Ref. (9, 10)].

The structure of the *Pmch* gene locus appears to be complex and sense/antisense transcripts could generate different proteinderivatives. Indeed, the precursor ppMCH may be processed mainly, but not exclusively, in two different peptides (MCH and

The cyclic peptide Melanin Concentrating Hormone (MCH) is known to control a large number of brain functions in mammals such as food intake and metabolism, stress response, anxiety, sleep/wake cycle, memory, and reward. Based on neuro-anatomical and electrophysiological studies these functions were attributed to neuronal circuits expressing MCHR1, the single MCH receptor in rodents. In complement to our recently published work (1) we provided here new data regarding the action of MCH on ependymocytes in the mouse brain. First, we establish that MCHR1 mRNA is expressed in the ependymal cells of the third ventricle epithelium. Second, we demonstrated a tonic control of MCH-expressing neurons on ependymal cilia beat frequency using in vitro optogenics. Finally, we performed in vivo measurements of CSF flow using fluorescent micro-beads in wild-type and MCHR1-knockout mice. Collectively, our results demonstrated that MCHexpressing neurons modulate ciliary beating of ependymal cells at the third ventricle and could contribute to maintain cerebro-spinal fluid homeostasis.

**Keywords: MCH, MCHR1, non-neuronal function, cilia, CSF flow**

NEI) in the brain and in several intermediates, including the dipeptide MCH-NEI, in peripheral organs (11–14). An additional protein, named MGOP, may be produced by an alternative splicing of the *Pmch* gene primary transcript in all cells producing MCH (15, 16). Finally a set of proteins, involved in DNA repair, may be synthesized by expression of the *AROM/PARI* gene located on the complementary strand overlapping the *Pmch* gene (8, 17). Based on this disparity in gene-products expression, it is quite difficult to associate a single molecular substrate responsible to the wide phenotypic changes observed in *Pmch* gene KO mice in which the full exon-intron sequences of the *Pmch* gene as well as the 30UTR region of spliced AROM/PARI gene transcripts were deleted. Meanwhile, the issue of developmental compensation (or adaptation) in these genetic models of *Pmch* gene inactivation should also be considered [see Ref. (9) for discussion of this point].

Efforts to identify the MCH receptor initially led to the discovery of a spliced variant of the seven-transmembrane G-coupled protein named SLC-1 (18) as a cognate MCH receptor and thereafter referred to as MCHR1 (19–23). MCHR1 is widely localized in brain regions involved in the control of neuroendocrine, reward, motivational, and cognitive aspects of feeding behavior (9, 10, 24– 26). Interestingly, MCHR1-deficient mice are lean due to hyperactivity and increased metabolism (27). A second MCH receptor,

2 University of Nice Sophia Antipolis, Nice, France

named here MCHR2, was identified and characterized in human tissues and cell lines (27–33). This MCH receptor displayed a brain distribution that overlapped partially with that of MCHR1 in the primate and fish brain (32, 34). However, MCHR2 is lacking in rat and mouse genomes (35). Furthermore, in contrast to MCHR1 that signals to either Gai or Gaq, depending on the transfected or native cell systems, MCHR2 signaling operates apparently exclusively through Gaq protein [our unpublished data; reviewed in Ref. (35–37)].

Based on neuro-anatomical mapping and electrophysiological data, it was assumed that synaptic transmission represents the main mode of action of MCH in the brain. However, non-neuronal intercellular communication or "volume" transmission may also be involved but evidence were lacking. In a recently published study (1), we mapped numerous MCH fibers in close vicinity to MCHR1 expressed into ependymocytes of the ventral part of the third ventricle (3V). Developing new techniques to measure and analyze the ependymal cilia beat frequency (CBF) in acute mouse brain slice preparations, we also showed that the CBF is increased by MCH application or LHA stimulation, an effect blocked by a selective MCHR1 antagonist and absent in MCHR1-knockout (MCHR1-KO) mice. In addition, using *in vivo* brain MRI, we demonstrated that the volume of both the lateral and third ventricles is increased in MCHR1-KO mice compared to their wild-type (WT) littermates. Thus, our study revealed a previously unknown function of the MCH/MCHR1 signaling system in non-neuronal cells. Here, we first demonstrated MCH mRNA expression in the ventral 3V ependymal cells isolated by laser-capture and *in situ* hybridization. We then extended our previous work, by using *in vitro* optogenetic activation or inhibition of MCH neurons. Finally, we investigated *in vivo* tracking of fluorescent micro-beads through the 3V in WT and MCHR1-KO mice. Collectively, we demonstrate a dynamic control of MCH neurons on spontaneous CBF of MCHR1 mRNA-expressing ependymal cells and discuss the current strategies for measuring CSF flows in small animal models.

# **MATERIALS AND METHODS**

#### **ANIMALS**

The experiments were conducted with male C57BL/6J mice (for laser-captured cell mapping, *in situ* hybridization and cellular optogenetic measurements) and female KO MCHR1 mice (*in vivo* CSF flow experiments) of 10–12 weeks of age. The animals were obtained from heterozygous breeding in the local animal facilities and maintained on a 12-h dark/light cycle (7 a.m./7 p.m.) with food and water *ad libitum*. The MCHR1-KO mice were established as previously described (38).

All the protocols were carried out in accordance with French ethical guidelines for laboratory animals (Agreement N°75–178, 05/16/2000) and were approved by the IPMC care committee. Attention was paid to use only the number of animals requested and necessary to generate reproducible results.

#### **LASER MICRO-DISSECTION OF THIRD VENTRICLE EPITHELIUM**

After decapitation, each brain (*n* = 2) was dissected out in <2 min and immediately frozen at −80°C using a Snapfrost (Alphelys, France). Sections (10µm thick) were cut on a cryostat (Microm

HM 560; object holder and chamber were kept at −21°C). Eight sections passing through the posterior hypothalamus were collected on pen membrane slides. Slides, continuously maintained on dry ice, were dehydrated in three baths of increasing ethanol baths (70, 95, and 100%) and two baths of fresh xylene (Roth, France) for 5 min each. Sections were air dried and kept in the vacuum of a dessicator until dissection.

Dissections were performed using a PixCell® (Arcturus Engineering) with CapSure® HS LCM caps. The dissection time never exceeded 20 min/slide, starting from when the slide was removed from the dessicator. Laser parameters were calibrated for each dissection by measuring the impact of shots on the membrane of the slide adjacent to the tissue. The area of interest was then dissected and laser-captured using UV laser to cut the tissue and IR laser to capture the sample. Four samples were collected per cap (micro-dissection of two slides in <40 min in total) and only one cap per brain. As soon as the fourth sample was obtained, the cap was examined under the microscope to ensure the absence of unwanted debris. The sample lysis and the RNA extraction were performed using the RNAqueous®-Micro Kit (Ambion, France) following the manufacturer's instructions. The quality of the samples was finally evaluated with the Agilent 2100 Bioanalyzer (Agilent Technology). A rin of 6.1 was twice obtained.

After reverse transcription (Superscript III, Invitrogen), cDNA corresponding to 1 ng of RNA was used as input in a PCR reaction (GoTaq Green MasterMix, Promega, Charbonnières, France) for MCHR1 and HPRT as positive control (MCHR1 F: 50 -GCTCTATGCCAGGCTTATCC-30 , MCHR1 R: CAGCTGTCTGAGCATTGCTG-30 , amplicon size: 494 bp; HPRT F: CTCCGGAAAGCAGTGAGGTAAG, HPRT R: GGAGGGA-GAAAAATGCGGAGTG, amplicon size: 306 bp). Sample for which reverse transcriptase was omitted was used as negative control. PCR protocol used was designed as follow: initial denaturation: 95°C, 5 min follow by 40 cycles composed of 95°C, 30 s, 58°C, 30 s, 72°C, 1 min, and a final elongation for 7 min at 72°C.

#### **IN SITU HYBRIDIZATION**

Frozen sections were post-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer and digested with proteinase K (1µg/mL,Roche) for 30 min at 37°C. Slides were incubated for 8 min in 0.1 M triethanolamine (TEA), pH 8.0, and then for 5 min at room temperature in 100 mL 0.1 M TEA + 500µL acetic anhydride followed by a decarboxylation in active diethyl pyrocarbonate (DEPC).

Sections were then rinsed briefly with 5× standard citrate sodium (SSC) buffer then incubated for 2 h in prehybridization buffer at 56°C. After rinsing in 0.2× SSC, the sections were incubated overnight at 56°C, in humid chambers, with 50µL hybridization buffer containing 5% Denhardt's and 50 ng labeled RNA probes. After rinsing with 5× SSC, sections were incubated successively in 2× SSC at 56°C (1 h 30 min) and 0.2× at room temperature (5 min). They were incubated in anti-DIG Fab fragments conjugated to alkaline phosphatase (1/1300, overnight) and revealed with enzyme substrate NBT-BCIP (overnight, at room temperature).

Two MCHR1 RNA probes were used; one probe was kindly provided by Drs Civelli and Chung (University of California, Irvine, CA, USA) and one made by reverse transcription/polymerase chain reaction from mouse genomic DNA. Control hybridization, including hybridization with sense DIG-labeled riboprobes was realized and did not reveal any signal.

#### **MEASURES OF CILIA BEAT FREQUENCY USING MCH NEURON-SPECIFIC OPTOGENETIC EXCITATION OR INHIBITION**

We have previously shown that electrical stimulation of the LHA induced an increase in the CBF in the 3V (1). To further extend and improve the specificity of the response, we used new models allowing the optogenetic control of MCH neurons activity. All procedures and controls were previously published in (39). Briefly, using cre-dependent Ef1a-DIO-ChETA-EYFP AAV mediated transduction, the fast mutant of the light-activated Channel rhodopsin-2 (ChETA) or the chloride pump halorhodopsin (NpHR) was specifically expressed in MCH neurons of two different groups of 3 week-old mice. Four weeks after stereotactic injection of the AAV vectors in the LHA (AP: −1.45 mm, ML: ±1 mm, DV: −5.5 mm), brain slices were made and recordings were made in CBF as described elsewhere (1) before, during, and after stimulation of ChETA-expressing MCH neurons (473 nm, stimulation frequency: 1, 5, 10, 20, and 40 Hz, pulse length: 10 ms, total stimulation duration: 3 min) or NpHR (590 nm, continuous stimulation during 8.5 s each 10 s, 5 min). For each slice and area of recording, the instantaneous CBF was calculated. All samples in which basal CBF was out of a range comprised between 5 and 20 Hz (considered as the natural basal frequency in healthy slices) were not included. The mean of the basal frequency during the first 5 min of recordings were used as a baseline for normalization of the experimental values. Results are expressed as the percentile variation of this baseline.

#### **MEASURE OF CSF FLOW INDEX USING FLUORESCENT MICRO-BEADS**

Littermate controls and female MCHR1-KO mice (aged 12 weeks old) were anesthetized by intraperitoneal injection of ketamine hydrochloride 50 mg/kg and xylazine 10 mg/kg and placed in a stereotaxic frame tip of a 26 gage needle was brought to the following coordinates relative to the bregma: 1.75 mm posterior, 2.5 mm ventral, and 0 mm right and left. About 10µL of polystyrene beads (diameter 3µm (sigma L4530) dilution 1:4 in 0.09% NaCl) was injected in the third ventricle.

Fibered confocal fluorescence microscopy (FCFM) (CellviZio; Mauna Kea Technologies, Paris, France) imaging was used to visualize the *in vivo* movement of the polystyrene beads in the CSF flux. FCFM provides an easy access to these regions of interest with low disturbance of brain structure (40, 41). Small-diameter fiber-optic probe consisting of tens of thousands of fibers was implanted in the brain of the mice and connected to a Laser scanning unit LSU-488 (FibroScan) that uses a laser source with a wavelength of 488 nm. We used a MiniZ probe of 300µm diameter with a working distance of 70µm. The probe was stereotaxically lowered in the third ventricle at 2.5 mm ventral. Sensitive, single-pixel detection of fluorescence stimulated by the photodiode laser pulse through each fiber element, combined with the high scan rate allows the visualization of beads movements. Four acquisition sessions of at least 10 min was recorded for each animal at a frame rate of 11 frames/s.

# **STATISTICS**

#### **Variation in the CBF**

Statistics were performed using Prism software (Graphpad Inc., La Jolla, CA, USA). The global mean for grouped time points (baseline, stimulation, and recovery) were compared Using One way ANOVA followed by Bonferroni's multiple comparison test (BMCT). N = number of mice, n = number of slices, *n* = number of cells considered. *p* Value <0.05 were considered significant.

#### **Measure of CSF flow index using fluorescent micro-beads**

Movies were visualized on ImageCell™ viewer. The speed of the beads was analyzed by tracking 10 beads/10 min films. The mean speed for an animal was the mean of the four films speeds. The movies displaying significant modifications of the speed over time were excluded as probably corresponding to pressure due to probe positioning or blood clot.

#### **RESULTS**

#### **LASER MICRO-DISSECTION OF THIRD VENTRICLE EPITHELIUM AND IN SITU HYBRIDIZATION**

As illustrated in **Figures 1A,B**; ependymal cell layer was carefully dissected and used for RNA extraction. RT-PCR results indicate that mRNA coding for MCHR1 were present in the 3V epithelium (**Figure 1C**). This was further confirmed by *in situ* hybridization with two specific probes recognizing MCHR1 mRNA. Indeed, numerous (but not all; see open arrowhead) ependymocytes were labeled with antisense probe (**Figure 1D**), within the cytoplasm (**Figure 1E**), while sense probe did not stain any cell types (**Figure 1F**). These results are in agreement with our immunohistochemical study (1) and recent data from Maratos-Flier's lab using a *MCHR1-cre/tdTomato* mouse strain (26).

#### **MCH NEURON-SPECIFIC OPTOGENETIC TOOLS**

The ChETA-NpHR system was used to dissect MCH neuronal circuitry reaching 3V ependymal cells and controlling CBF.

The stimulation of ChETA-expressing MCH neurons in the LHA induces an increase in the CBF reaching 134% of the basal value (**Figures 2A,B**; 1 Hz, N = 5, n = 7, *n* = 10, *F*5.069, 29 = 0.0131, BMCT: baseline vs. ChETA *t* = 2.896, *p* < 0.05). After 10 min recovery, the subsequent stimulation at 5 Hz tended to increase the CBF but did not reach the significance level (**Figure 2B**; 5 Hz, N = 5, n = 7, *n* = 10). For higher frequencies, no effect was observed (not shown, see Discussion). On the other hand, the stimulation of NpHR induced a marked decrease in the CBF reaching 76% of the basal (**Figures 2C,D**; N = 4, n = 8, *n* = 15, *F*4.616, 44 = 0.0154, BMCT: baseline vs. NpHR *t* = 3.027, *p* < 0.05).

#### **MEASURE OF CSF FLOW INDEX USING FLUORESCENT MICRO-BEADS**

In order to address the physiological relevance of MCH-driven ciliary beating regulation, we conducted *in vivo* flow measure. FCFM imaging was used to visualize the movement of the polystyrene beads in the CSF flux *in vivo* in groups of WT and KO MCHR1 mice (*n* = 5 each). The visualization of fluorescent beads movements in the third ventricle allow to approximate the speed of the CSF flux using *in vivo* brain imagery (**Figure 3**).

Overall, no significant statistical difference in the mean speed between the two groups was found. However, 3/5 animals in the

: after reverse transcription,

KO group displayed a twofold increase in the mean speed by comparison with the WT group (that shows consistently low speed). However, it should be stressed that we are measuring the bulk flow driven by the arterio-venous pressure gradients and arterial pulsations and that the laminar flow (close to the ventricle wall and therefore dependent upon cilia beating) remains too small to be measured. Furthermore, we believe that the technical caveat of the implantation of the probe and beads injection *in vivo* (blood clot, exact position, size of the probe . . .) may induce a methodological bias (see Discussion).

: negative control of the reverse transcription. HPRT: positive control. **(D)** Photomicrograph to illustrate the distribution of the MCHR1 in situ

MCHR1 mRNA in the ventricular epithelium. RT+

#### **DISCUSSION**

RT−

The MCH system is involved in a broad spectrum of function through mainly the synaptic release of the peptide(s) and neuronal activity modulation in mammalian brain. However, a growing body of data indicates that MCH may also have non-neuronal function, especially by regulating the activity of more or less specialized peripheral blood mononuclear cells [PBMCs (42)]. The expression of ppMCH and/or MCHR1 genes in pancreatic islets or in adipocytes (43–45) may highlight a metabolism-related function of MCH at the periphery. Nevertheless, such action may not be totally independent of the intracerebral and/or spinal MCH pathway (46). Based on our previous study (1) and the present set of data, we demonstrate a new role for the hypothalamic peptide MCH in modulation of CBF in the ventral part of the third ventricle through activation of ependymal cells.

In combining the laser micro-dissection of ependymal epithelium of the 3V followed by RNA extraction and RT-PCR experiments and *in situ* hybridization data, we demonstrate the presence of MCHR1 transcripts within the epithelium. This is consistent with the data obtained by immunohistochemistry (1) or in a

expression of MCHR1 mRNA in discrete ependymal cells. **(F)** A negative control using sense MCHR1 gene probe. Scale bar = 25µm in **(A)**; Scale

bar = 20µm in (D); Scale bar = 10µm in **(E)** and **(F)**.