# Dopamine D1-histamine H3 receptor heteromers provide a selective link to MAPK signaling in GABAergic neurons of the direct striatal pathway.

Estefanía Moreno, Hanne Hoffmann, Marta Gonzalez-Sepúlveda, Gemma Navarro, Vicent Casadó, Antoni Cortés, Josefa Mallol, Michel Vignes, Peter J Mccormick, Enric I Canela, et al.

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Estefanía Moreno, Hanne Hoffmann, Marta Gonzalez-Sepúlveda, Gemma Navarro, Vicent Casadó, et al. Dopamine D1-histamine H3 receptor heteromers provide a selective link to MAPK signaling in GABAergic neurons of the direct striatal pathway. Journal of Biological Chemistry, 2011, 286 (7), pp.5846-54. 10.1074/jbc.M110.161489. hal-00659407

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# Dopamine D₁-histamine H₃ Receptor Heteromers Provide a Selective Link to MAPK Signaling in GABAergic Neurons of the Direct Striatal Pathway*§

Received for publication, July 7, 2010, and in revised form, December 16, 2010 Published, JBC Papers in Press, December 20, 2010, DOI 10.1074/jbc.M110.161489

Estefanía Moreno‡, Hanne Hoffmann§¶, Marta Gonzalez-Sepúlveda§, Gemma Navarro‡, Vicent Casado‡, Antoni Cortés‡, Josefa Mallol‡, Michel Vignes¶, Peter J. McCormick†<fnref n="1" />, Enric I. Canela‡, Carme Lluís‡, Rosario Moratalla<fnref n="1" />, Sergi Ferré**, Jordi Ortiz§<fnref n="2" />, and Rafael Franco†⁺⁺<fnref n="2" />,<fnref n="3" />

From the ‡Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain, the §Neuroscience Institute and Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain, ¶UMR 5247 The Max Mousseron Biomolecules Institute, CNRS, University of Montpellier 1 and 2, University of Montpellier 2, 34095 Montpellier, France, ‖Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid, Spain, the **National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland 21224, and the ‡‡Centro de Investigación Médica Aplicada, Universidad de Navarra, Pio XII 55, 31008 Pamplona, Spain

Previously, using artificial cell systems, we identified receptor heteromers between the dopamine D₁ or D₂ receptors and the histamine H₃ receptor. In addition, we demonstrated two biochemical characteristics of the dopamine D₁ receptor-histamine H₃ receptor heteromer. We have now extended this work to show the dopamine D₁ receptor-histamine H₃ receptor heteromer exists in the brain and serves to provide a novel link between the MAPK pathway and the GABAergic neurons in the direct striatal efferent pathway. Using the biochemical characteristics identified previously, we found that the ability of H₃ receptor activation to stimulate p44 and p42 extracellular signal-regulated MAPK (ERK 1/2) phosphorylation was only observed in striatal slices of mice expressing D₁ receptors but not in D₁ receptor-deficient mice. On the other hand, the ability of both D₁ and H₃ receptor antagonists to block MAPK activation induced by either D₁ or H₃ receptor agonists was also found in striatal slices. Taken together, these data indicate the occurrence of D₁-H₃ receptor complexes in the striatum and, more importantly, that H₃ receptor agonist-induced ERK 1/2 phosphorylation in striatal slices is mediated by D₁-H₃ receptor heteromers. Moreover, H₃ receptor-mediated phosphorylation labeling co-distributed with D₁ receptor-containing but not with D₂ receptor-containing striatal neurons. These results indicate that D₁-H₃ receptor heteromers work as processors integrating dopamine- and histamine-related signals

involved in controlling the function of striatal neurons of the direct striatal pathway.

The striatum is the main input structure of the basal ganglia, which are subcortical structures involved in the processing of information related to the performance and learning of complex motor acts. It is widely accepted that dopamine receptor subtypes, which are fundamental for motor control and are implicated in numerous neuropsychiatric disorders, are largely segregated in the two subtypes of medium spiny neurons (MSNs).<fnref n="4" /> the most populated neuronal type in the striatum. Dopamine D₂ receptors (D₂Rs) are mostly localized in the striatopallidal MSNs, which express the peptide enkephalin and which gives rise to the indirect striatal efferent pathway, whereas dopamine D₁ receptors (D₁Rs) are mostly expressed by the striatonigral MSNs, which express substance P and dynorphin and constitute the direct striatal efferent pathway (1, 2). Dopaminergic drugs activate the ERK transduction pathway, which is involved in basic physiological processes and in synaptic plasticity (3). In the dopamine-depleted striatum, ERK signaling is implicated in the development of L-DOPA-induced dyskinesia. Thus, in dopamine-denervated mice, L-DOPA activates ERK signaling specifically in D₁Rs containing striatonigral MSNs but not in D₂Rs containing striatopallidal MSNs (4). This regulation may result in ERK-dependent changes in striatal plasticity leading to dyskinesia.

Histamine is an important regulatory transmitter in the nervous system involved in the sleep/wake cycle, attention, memory, and other functions. Four histamine receptor types (H₁R-H₄R) have been cloned. H₃Rs are expressed in abundance in the brain and high densities are particularly found in the striatum (5-7). H₃Rs were first identified as autoreceptors (8), but they were later found to act as heteroreceptors (9).

<footnote>* This study was supported by Grants SAF2008-00146, SAF2008-03229-E, SAF2009-07276, SAF2006-08240, and SAF2009-12510 from the Spanish Ministerio de Ciencia y Tecnología, the Centre National de la Recherche Scientifique, the French Ministry of Research and Higher Education, Red de Transtornos Adictivos RD06/0001/0015, Grant 060110 from Fundació La Marató de TV3 and the Intramural Funds of the National Institute on Drug Abuse.</footnote>

<footnote>§ The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.</footnote>

<footnote n="1">¹ A Ramon y Cajal investigator.</footnote>

<footnote n="2">² Both authors contributed equally to this article.</footnote>

<footnote n="3">³ To whom correspondence should be addressed: Centro de Investigación Médica Aplicada, University of Navarra, Pio XII, 55, 31008 Pamplona, Italy. Tel.: 34-948194700; Fax: 34-948194715; E-mail: rfranco@unav.es.</footnote>

<footnote n="4">⁴ The abbreviations used are: MSN, medium spiny neurons; D₂R, dopamine D₂ receptor; H₁R, histamine H₁ receptor; D₁R, dopamine D₁ receptor; RAMH, R(−)-α-methylhistamine dihydrochloride.</footnote>
The major localization of striatal H₃Rs is postsynaptic (5, 10), and most probably in both subtypes of MSNs (6, 10). Histamine, by means of interactions with striatal H₃Rs, plays an important role in the modulation of dopamine neurotransmission (11–14). At the behavioral level, it was shown that stimulation of postsynaptic H₃R counteracts the motor activation induced by D₁R and D₂R agonists in reserpinized mice (14). These interactions may be related to the ability of H₃Rs to form heteromers with dopamine receptors. In fact, D₁R-H₃R and D₂R-H₃R heteromerization was demonstrated by biophysical techniques in mammalian cells (14, 15). However, their presence in the brain remained to be demonstrated. In addition, if H₃Rs form heteromers with both D₁R and D₂R, is there a functional difference between these two receptor heteromer pairs? One might expect that because the D₁R and D₂R receptors are found in two different neuronal pathways that the different heteromers might confer different properties. Here, we have explored this idea by taking advantage of unique properties of the D₁R-H₃R heteromers to provide evidence for their presence in rodent brain. Previously, using an *in vitro* cell system, we found an important feature of the D₁R-H₃R heteromer is that H₃R agonists only activate ERK 1/2 in a receptor heteromer context, but not in cells expressing H₃Rs without D₁R (15). Here, by taking advantage of this distinct ERK 1/2 signaling characteristic, we demonstrate the occurrence of D₁R-H₃R heteromers in rodent striatum. Despite H₃Rs being expressed in both D₁R and D₂R containing neurons, histamine-receptor-mediated phosphorylation of the ERK 1/2 kinase occurred only in neurons expressing D₁R and not in those with D₂R. Thus, D₁R-H₃R receptor heteromers confer a direct link to MAPK activation within the GABAergic neurons of the direct striatal efferent pathway.

## EXPERIMENTAL PROCEDURES

**Animals**—Sprague-Dawley male rats, 7–9 weeks old and weighing 200–250 g, were provided by the Animal Service of the Universidad Autónoma de Barcelona (Barcelona, Spain). Six-to-eight-month-old wild-type littermates and dopamine D₁ receptor knock-out C57BL6 male mice, weighing 25–30 g, were provided by Instituto Cajal (Consejo Superior de Investigaciones Científicas; Madrid, Spain) and generated by homologous recombination as described previously (16). Rats (2 per cage) or mice (five per cage) were housed in a temperature (21 ± 1 °C) and humidity-controlled (55 ± 10%) room with a 12:12 h light/dark cycle (light between 08:00 and 20:00 h) with food and water *ad libitum*. Animal procedures were conducted according to standard ethical guidelines (European Communities Council Directive 86/609/EEC) and approved by the local (Universidad Autónoma de Barcelona or Consejo Superior de Investigaciones Científicas) ethical committee.

**Cell Culture and Membrane Preparation**—SK-N-MC/H₃ cells were grown in Eagle's minimal essential medium, supplemented with 10% FBS, 50 units/ml penicillin, 50 µg/ml streptomycin, nonessential amino acids, 2 mmol/liter L-glutamine, and 50 µg/ml sodium pyruvate at 37 °C in a humidified atmosphere of 5% CO₂ to 80% confluence. The SK-N-MC cells stably expressing the human H₃R (SK-N-MC/H₃) were provided by Johnson & Johnson Pharmaceutical Research & Development,

L.L.C. Cells were disrupted with a Polytron homogenizer (PTA 20 TS rotor, setting 3; Kinematica, Basel, Switzerland) in 50 mM Tris-HCl buffer, pH 7.4, containing a protease inhibitor mixture (1/1000; Sigma). The cellular debris was removed by centrifugation at 13,000 × g for 5 min at 4 °C, and membranes were obtained by centrifugation at 105,000 × g for 1 h at 4 °C. Membranes were lysed in 50 mM Tris-HCl, pH 7.4, containing 50 mM NaF, 150 mM NaCl, 45 mM β-glycerophosphate, 1% Triton X-100, 20 µM phenylarsine oxide, 0.4 mM NaVO₄, and protease inhibitor mixture to be processed by Western blot.

**Brain Slice Preparation**—Rats and mice were decapitated with a guillotine, and the brains were rapidly removed and placed in ice-cold oxygenated (O₂/CO₂: 95/5%) Krebs-HCO₃⁻ buffer (124 mM NaCl, 4 mM KCl, 1.25 mM NaH₂PO₄, 1.5 mM MgCl₂, 1.5 mM CaCl₂, 10 mM glucose, and 26 mM NaHCO₃, pH 7.4). The brains were sliced at 4 °C in a brain matrix (Zivic Instruments, Pittsburgh, PA) into 0.5-mm coronal slices. Slices were kept at 4 °C in Krebs-HCO₃⁻ buffer during the dissection of the striatum. Each slice was transferred into an incubation tube containing 1 ml of ice-cold Krebs-HCO₃⁻ buffer. The temperature was raised to 23 °C and after 30 min, the medium was replaced by 2 ml Krebs-HCO₃⁻ buffer (23 °C). The slices were incubated under constant oxygenation (O₂/CO₂: 95/5%) at 30 °C for 4–5 h in an Eppendorf Thermomixer (5 Prime, Inc., Boulder, CO). The media was replaced by 200 µl of fresh Krebs-HCO₃⁻ buffer and incubated for 30 min before the addition of ligands.

**ERK Phosphorylation Assays**—Striatal slices were incubated in the presence of the indicated concentrations of histamine H₃ or dopamine D₁ receptor ligands, prepared in Krebs-HCO₃⁻ buffer. After the indicated incubation period, the solution was discarded, and slices were frozen on dry ice and stored at −80 °C. Slices were lysed by the addition of 500 µl of ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 50 mM NaF, 150 mM NaCl, 45 mM β-glycerophosphate, 1% Triton X-100, 20 µM phenylarsine oxide, 0.4 mM NaVO₄, and protease inhibitor mixture). Cellular debris was removed by centrifugation at 13,000 × g for 5 min at 4 °C, and protein was quantified by the bicinchoninic acid method using bovine serum albumin dilutions as standard. To determine the level of ERK1/2 phosphorylation, equivalent amounts of protein (10 µg) were separated by electrophoresis on a denaturing 10% SDS-polyacrylamide gel and transferred onto PVDF-FL membranes. Odyssey blocking buffer (LI-COR Biosciences, Lincoln, Nebraska) was then added, and membranes were rocked for 90 min. Membranes were then probed with a mixture of a mouse antiphospho-ERK 1/2 antibody (1:2500, Sigma) and rabbit anti-ERK 1/2 antibody (1:40,000, Sigma) for 2–3 h. The 42 and 44 kDa bands corresponding to ERK 1 and ERK 2 were visualized by the addition of a mixture of IRDye 800 (anti-mouse) antibody (1:10,000, Sigma) and IRDye 680 (anti-rabbit) antibody (1:10,000, Sigma) for 1 h and scanned by the Odyssey infrared scanner (LI-COR Biosciences). Bands densities were quantified using the scanner software and exported to Microsoft Excel. The level of phosphorylated ERK 1 and phosphorylated ERK 2 was normalized for differences in loading using the total ERK 1/2 protein band intensities.

<figure><img src="image_2.png" /></figure>
# Dopamine D₁-histamine H₃ Receptor Heteromers in Striatum

**Immunohistochemistry**—Striatal slices were incubated with the indicated H₃R ligands in Krebs-HCO₃⁻ buffer for 10 min and fixed with 4% paraformaldehyde solution (Antigenfix, DiaPath) for 1 h at room temperature with gentle agitation. The slices were then washed in TBS (50 mM Tris-HCl, 0.9% NaCl, pH 7.8), treated 5 min with 1% Na₂BH₄ dissolved in TBS, followed by successive TBS washes until all Na₂BH₄ was eliminated. Finally, the slices were cryopreserved in a 30% sucrose solution overnight at 4 °C and stored at -20 °C until sectioning. 15-µm-thick coronal sections were cut on a freezing cryostat (Leica Jung CM-3000) and mounted on slide glass (three control and three treated coronal sections in each slide; STAR FROST PLUS, DELTALAB). Coronal sections were thawed at 4 °C, washed in TBS, and rocked in 7% normal donkey serum (SND, Sigma) in TBS for 1 h at 37 °C in a humidified atmosphere. Coronal sections were then incubated overnight at 4 °C in a humidified atmosphere with the primary antibodies: rabbit antiphospho-Thr²⁰²/Tyr²⁰⁴ ERK 1/2 antibody (1:300, Cell Signaling Technology, Danvers, MA), guinea pig anti-D₁ antibody (1:100, Frontier Institute, Ishikari, Hokkaido, Japan) or guinea pig anti-D₂ antibody (1:100, Frontier Institute, Ishikari, Hokkaido, Japan) alone or in combination in a solution with 0.1% TBS-Tween, 0.1% BSA-acetylated (Aurion), 7% SND (250 µl per slide). The specificity of these dopamine receptor antibodies has been previously shown by preabsorption tests with the antigen peptides and by mutually exclusive pattern and triple labeling in immunohistochemistry (17) and by Western blot (see "Results"). Coronal sections were washed in 0.05% TBS-T and left for 2 h at room temperature in a humidified atmosphere with the corresponding secondary antibodies: chicken anti-rabbit (1:200, Alexa Fluor 594, Invitrogen) and goat anti-guinea pig (1:200, Alexa Fluor 488, Invitrogen) in a solution with TBS-Tween 0.1%, BSA acetylated 0.1%, SND 7%, and then washed in TBS-T 0.05%, followed by a single wash in TBS before mounting in Mowiol medium (Calbiochem), covered with a glass, and left to dry at 4 °C for 24 h. Single and double immunostained slices were observed and imaged in a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany). Images were opened and processed with ImageJ confocal microscopy program and a Adobe Photoshop program (version 5.5; Seattle, WA). Double-labeled cells (cells stained for phospho-ERK 1/2 and D₁ or D₂ receptors) were counted in a total of two to three nonoverlapping fields of 45 coronal sections from 4 to 5 slices treated with medium (control), 1 µM RAMH, or 1 µM imetit.

Coronal sections from nontreated slices (six control coronal sections in each slide) were used for double-immunohistochemistry using rabbit anti-H₃R antibody (1:200, Chemicon, Billerica, MA) and guinea pig anti-D₁R antibody or guinea pig anti-D₂R antibody as primary antibodies and goat anti-rabbit-peroxidase (1:200, Thermo Scientific, Fremont, CA) and goat anti-guinea pig (1:200, Alexa Fluor 488, Invitrogen) as secondary antibodies by the same procedure as described above. In this case, the amplification system for the red fluorophore, TSA-cyanine 3 (1:100, Tyramide Signal Amplification, PerkinElmer Life Science) was used as described in the TSA Plus fluorescence amplification kit, before mounting in

Mowiol medium. Double-labeled cells (cells stained for H₃ and D₁ or D₂ receptors) were counted in a total of two to three nonoverlapping fields of 15 coronal sections from four to five slices. In all cases, we did not observe staining in the absence of the primary antibodies.

**Coimmunoprecipitation**—The rat striatal tissue was disrupted with a Polytron homogenizer in 50 mM Tris-HCl buffer, pH 7.4, containing a protease inhibitor mixture (1/1000, Sigma). The cellular debris was removed by centrifugation at 13,000 × g for 5 min at 4 °C, and membranes were obtained by centrifugation at 105,000 × g for 1 h at 4 °C. Membranes were washed two more times at the same conditions and were solubilized by homogenization in ice-cold immunoprecipitation buffer (phosphate-buffered saline, pH 7.4, containing 1% (v/v) Nonidet P-40) and incubated for 30 min on ice before centrifugation at 105,000 × g for 1 h at 4 °C. The supernatant (1 mg/ml of protein) was processed for immunoprecipitation as described in immunoprecipitation protocol using a Dynabeads® Protein G kit (Invitrogen). Protein was quantified by the bicinchoninic acid method (Pierce) using bovine serum albumin dilutions as standard. Immunoprecipitates were carried out with rat anti-D₁ receptor antibody (1:1000, Sigma) or rabbit anti-D₂ receptor antibody (1:1000, Milipore, Billerica, MA) As negative control anti-calnexin antibody was used (1:1000, BD Biosciences Pharmingen). Immunoprecipitates were separated on a denaturing 10% SDS-polyacrylamide gel and transferred onto PVDF membranes. Membranes were proved with the primary antibodies guinea pig anti-D₁ antibody (1:1000, Frontier Institute, Ishikari, Hokkaido, Japan), guinea pig anti-D₂ antibody (1:1000, Frontier Institute) or goat anti-H₃R antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) and the secondary antibodies goat anti-guinea pig-peroxidase (1:20,000, Sigma) and donkey anti-goat-peroxidase (1:20,000, Jackson ImmunoResearch Laboratories, West Grove, PA). Bands were visualized with a LAS-3000 (Fujifilm). Analysis of detected bands was performed by Image Gauge software (version 4.0) and Multi Gauge software (version 3.0).

## RESULTS

**D₁R and H₃R Are Functionally Coupled to MAPK Signaling Pathway in Brain Striatal Slices**—To establish whether D₁R and H₃R are functionally coupled to the MAPK pathway in rat striatum, slices were treated with a D₁R or an H₃R agonist, and ERK 1/2 phosphorylation was assayed as described under "Experimental Procedures." The time response curve obtained after treatment with 10 µM SKF 38393 (D₁R agonist) or 0.1 µM imetit (H₃R agonist) showed that phosphorylation peaked at 10 min (Fig. 1a). Therefore, all subsequent assays were analyzed at 10 min of drug treatment. Dose-response curves for different D₁R or H₃R agonists are displayed in Fig. 1b. Both SKF 81297 and SKF 38393 (full and partial D₁R agonists, respectively) were able to increase ERK 1/2 phosphorylation; SKF 81297 was more potent than SKF 39393. RAMH and imetit (H₃R agonists) also increased ERK 1/2 phosphorylation, with imetit being more potent than RAMH. The results show that striatal slices contain D₁R and H₃R functionally coupled to MAPK signaling.

<figure><img src="image_3.png" /></figure>
Dopamine D₁-histamine H₃ Receptor Heteromers in Striatum

<figure><img src="image_4.png" /></figure>

FIGURE 1. H₃R and D₁R agonists induced ERK 1/2 phosphorylation in rat striatal slices. a, slices were treated with 10 µM SKF 38393 (black) or 1 µM imetit (white). b, slices were treated for 10 min with different SKF 81297, SKF 38393, RAMH, or imetit concentrations. ERK 1/2 phosphorylation was determined as described under "Experimental Procedures." The immunoreactive bands from five to 27 (a) or 19 to 24 (b) slices obtained from three to 14 (a) or six to nine (b) animals were quantified, and values represent the mean ± S.E. of the percentage of phosphorylation relative to basal levels of untreated slices (100%). Significant differences were calculated by one-way analysis of variance with post hoc Bonferroni's multiple tests and *, **, and *** correspond to *p* < 0.05, *p* < 0.01, and *p* < 0.001, respectively, as compared with nontreated samples (control). A representative Western blot is shown in each panel (top).

H₃R Agonist-induced ERK 1/2 Phosphorylation in Striatal Slices Is Mediated by D₁R-H₃R Heteromers—A cross-antagonism between D₁Rs and H₃Rs has been demonstrated previously in heterologous cell systems. This cross-antagonism only occurs in D₁R-H₃R-heteromer-containing cells and consists of both the ability of D₁R antagonists to block the effect of H₃R agonists and, conversely, the ability of H₃R antagonists to block the effect of D₁R agonists (15). To test whether this phenomenon also occurs *in vivo*, rat striatal slices were incubated with D₁R or H₃R agonists (SKF 81297 or RAMH, respectively) in the presence of either D₁R or H₃R antagonists (SCH 23390 or thioperamide, respectively). The results reproduced the cross-antagonism found in the heterologous cell system (Fig. 2). ERK 1/2 phosphorylation induced by RAMH (0.1 µM) was not only blocked by thioperamide (10 µM) but also by SCH 23390 (10 µM) (Fig. 2a). Similarly, ERK 1/2 phosphorylation induced by SKF 81297 (0.1 µM) was blocked by both SCH23390 and thioperamide (10 µM in both cases) (Fig. 2b). As a control, activation of striatal serotonin receptors (with 0.2 µM of serotonin) significantly induced ERK 1/2 phosphorylation, but the effect was not modified by either

SCH23390 or thioperamide (10 µM in both cases) (Fig. 2, c and d). These results provide evidence for the expression D₁R-H₃R heteromers in the striatum. Another characteristic of the D₁R-H₃R heteromer is that it allows H₃R agonists to activate MAPK signaling (15). We decided to investigate whether this heteromer characteristic persisted *in vivo* using transgenic mice lacking D₁Rs. When H₃R-mediated MAPK signaling was investigated in striatal slices from transgenic mice lacking the D₁Rs and in wild-type littermate controls displaying the same genetic background, RAMH (0.1 µM) was unable to induce ERK 1/2 phosphorylation, whereas a strong signal was obtained in slices from wild-type littermate controls displaying the same genetic background (Fig. 3). In addition in wild-type animals, RAMH-induced ERK 1/2 phosphorylation was blocked by both thioperamide (10 µM) and SCH 23390 (10 µM) (Fig. 3). These results indicate that H₃R agonist-induced ERK 1/2 phosphorylation in striatal slices is mediated by D₁R-H₃R heteromers.

To provide further insight on the function of striatal D₁R and H₃R receptors coexpressed in striatal neurons, ERK 1/2

<figure><img src="image_5.png" /></figure>
Dopamine D₁-histamine H₃ Receptor Heteromers in Striatum

<figure><img src="image_6.png" /><figcaption>FIGURE 2. Effect of H₃R and D₁R antagonists on agonist-induced ERK 1/2 phosphorylation in rat striatal slices. Slices were preincubated with medium or with 10 µM thioperamide, 10 µM SCH 23390, or both for 20 min prior to the addition of 0.1 µM RAMH (a) or 0.1 µM SKF 81297 (b) followed by a further incubation of 10 min. In c and d, slices were preincubated for 20 min with medium or with 10 µM thioperamide (c) or 10 µM SCH 23390 (d) prior to the addition of 0.2 µM serotonin followed by a further incubation of 10 min. ERK1/2 phosphorylation was determined as described under "Experimental Procedures." The immunoreactive bands from 12 to 21 (a and b) or 10 to 14 (c and d) slices obtained from 8 to 10 (a and b) or 4 to 6 (c and d) animals were quantified, and values represent the mean ± S.E. of the percentage of phosphorylation relative to basal levels found in untreated slices (100%). Significant differences were calculated by one-way analysis of variance with post hoc Bonferroni's multiple tests (***, *p* < 0.001, as compared with the first treatment in a and b, or to the basal in c and d). A representative Western blot is shown in each panel (top).</figcaption></figure>

activation was studied in rat striatal slices in the presence of agonists for the two receptors. This would mimic the situation when the two neurotransmitters histamine and dopamine are simultaneously impacting a given GABAergic neuron. Interestingly, the effect of the D₁R agonist SKF 81297 (10 µM) was significantly counteracted by the H₃R agonist, RAMH (1 µM). Furthermore, the combination of RAMH (10 µM) and SKF 81297 (1 µM) produced a significantly weaker effect than that of either drug alone (Fig. 4), indicating the existence in striatal neural circuits of an agonist-induced D₁R-H₃R reciprocal negative cross-talk.

Selective D₁R-H₃R Heteromer-mediated Effects only in Striatal Neurons of Direct Pathway—Dopamine receptors are segregated in the two main types of GABAergic striatal effent neurons: dynorphinergic neurons of the direct pathway expressing D₁Rs and enkephalinergic neurons of the indirect pathway expressing dopamine D₂Rs. Evidence supporting the

presence of H₃R in both types of neurons had been obtained previously by autoradiography and lesion studies (5) and by *in situ* hybridization (10). Accordingly, by double immunohistochemistry using H₃R and either D₁R or D₂R antibodies, we found H₃R immunostaining in cells labeled with either D₁R or D₂R antibodies (Fig. 5). In fact, 95 ± 12% of D₁R stained neurons or 89 ± 15% of D₂R stained neurons showed H₃R staining (Fig. 6a). Thus, co-expression of D₁R and H₃R in GABAergic neurons of the direct pathway and co-expression of D₂R and H₃R in GABAergic neurons of the indirect pathway was found. We have described previously that both D₁R and D₂R may form heteromers with H₃R in living cells (14, 15). To test D₁R-H₃R and D₂R-H₃R heteromer expression in the rat striatum, co-immunoprecipitation experiments were carried out. The immunoprecipitates with the anti-D₁R antibody (Fig. 7a) or with the anti-D₂R antibody (Fig. 7b) were not stained in a Western blot using anti-D₂R or anti-D₁R anti-

<figure><img src="image_7.png" /></figure>
bodies respectively, showing the specificity of the antibodies. Interestingly, specific H₃R staining was detected by Western blot in both immunoprecipitates using anti-D₁R or anti-D₂R antibodies but not with an irrelevant antibody (Fig. 7c). These

results corroborate the expression of D₁R-H₃R heteromers in the neurons of the direct pathway and suggest the expression of D₂R-H₃R heteromers in the neurons of the indirect pathway.

In striatal slices incubated with 1 µM imetit and subjected to immunohistochemistry, we observed that imetit-induced ERK 1/2 phosphorylation occurs in a high number of neurons stained using the anti-D₁R antibody, but only in a small number of neurons stained using the anti-dopamine D₂R antibody (Fig. 8). In fact, 85 ± 7% of phospho-ERK 1/2-positive neurons displayed specific D₁ receptor immunostaining, whereas only 23 ± 5% of phospho-ERK 1/2-positive neurons were positive for D₂ receptor labeling (Fig. 6b). It should be noted that despite D₂R-H₃R heteromers may play a role in this signaling pathway, neurons containing both the D₁R and D₂R may exist in the striatum (18). Similar results were obtained in striatal slices incubated with 1 µM RAMH (results not shown). Furthermore, the effect of H₃R agonists in striatal slices was independent of changes in presynaptic neurotransmitter release (e.g. dopamine or histamine), which could potentially contribute to trigger ERK 1/2 phosphorylation in D₁R-expressing cells. In fact, the presence of 1 µM tetrodotoxin affected neither the D₁R agonist nor the H₃R agonist-induced ERK 1/2 phosphorylation (supplemental Fig. 1). Collectively, these results demonstrate that histamine-induced MAPK pathway activation in striatal slices is specifically mediated by the D₁R and H₃R heteromers present in neurons of the direct pathway, but not by the H₃Rs localized in the indirect pathway or as autoreceptors or heteroceptors in neighboring nerve terminals.

<figure><img src="image_8.png" /></figure>

<table><tr><td>RAMH</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>THIOPERAMIDE</td><td>-</td><td>-</td><td>+</td><td>-</td><td>+</td></tr><tr><td>SCH 23390</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td></tr></table>

FIGURE 3. **H₃R agonist-induced ERK 1/2 phosphorylation in striatal slices from wild-type and dopamine D₁R knock-out mice.** Wild-type (white) or D₁R knock-out mice (black) slices were treated for 10 min with 0.1 µM RAMH or for 10 min with 10 µM thioperamide and/or 10 µM SCH 23390 prior to the addition of 0.1 µM RAMH and incubation for further 10 min. ERK 1/2 phosphorylation was determined as described under "Experimental Procedures." For each treatment, the immunoreactive bands from four to six slices from a total six wild-type and nine knock-out animals were quantified, and values represent the mean ± S.E. of the percentage of phosphorylation relative to basal levels found in untreated slices (100%). No significant differences were obtained between the basal levels of the wild-type and the D₁R knock-out mice, and no significant differences were observed between basal and slices treated (20 min) with 10 µM thioperamide or 10 µM SCH 23390. Significant treatment and genotype effects were analyzed by a bifactorial analysis of variance followed by post hoc Bonferroni's tests. There were significant genotype, treatment, and interaction effects, explained by the ability of RAMH to strongly and selectively induce ERK 1/2 phosphorylation in wild-type mice (***, *p* < 0.001, as compared with knock-out mice). A representative Western blot is also displayed (top).

DISCUSSION

We have previously described that not only D₁R but also D₂R may form heteromers with H₃R in living cells (14, 15). Here, it is demonstrated that both D₁R and D₂R co-immunoprecipitate H₃R from rat striatum supporting the expression

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

FIGURE 4. **Negative cross-talk between D₁Rs and H₃R receptors on ERK 1/2 phosphorylation in rat striatal slices.** Slices were treated for 10 min with 10 µM SKF 81297 and/or 1 µM RAMH (a) or 10 µM RAMH and/or 1 µM SKF 81297 (b). ERK 1/2 phosphorylation was determined as described under "Experimental Procedures." The immunoreactive bands from 10 to 24 (a) or eight to 23 (b) slices obtained from four to six animals were quantified, and values represent the mean ± S.E. of the percentage of phosphorylation relative to basal levels found in untreated slices (100%). Significant differences were calculated by one-way analysis of variance with post hoc Bonferroni's multiple tests. (**) and (**, *p* < 0.01 and *p* < 0.001, respectively, as compared with 10 µM SKF 81297 in (a) or 10 µM RAMH in (b)).

<figure><img src="image_11.png" /></figure>
Dopamine D₁-histamine H₃ Receptor Heteromers in Striatum

<figure><img src="image_12.png" /><figcaption>FIGURE 5. **Co-localization between H₃R and D₁R or D₂R in striatal MSNs.** Confocal microscope representative images of coronal sections from striatal slices are shown. Slices were labeled with anti-H₃R antibody (red). Labeling (green) using an anti-D₁R antibody (a) or an anti-D₂R antibody (b) is also shown. In a and b, colocalization is shown in yellow. Scale bars, 60 µm.</figcaption></figure>

of D₁R-H₃R and D₂R-H₃R heteromers in the neurons of the direct and indirect striatal efferent pathways, respectively. From our earlier work, it was unclear whether D₁R-H₃R and D₂R-H₃R heteromers were engaging similar signaling pathways in the two different neuronal populations or whether there was a functional difference that might help delineate the direct and indirect pathways of the striatum via the existence of these heteromers. The data presented in this paper indicate that D₁R-H₃R heteromers in the striatonigral GABAergic neurons of the direct pathway, but not the H₃R receptors in the indirect pathway, allow direct histaminergic activation of the MAPK pathway.

Biophysical techniques can provide strong support for the existence of receptor heteromers in artificial cell systems (19, 20), but, as these techniques are difficult to perform in intact tissues, obtaining evidence for naturally occurring heteromer expression remains a significant challenge. For many receptor heteromers, we depend on an indirect approach for their identification in native tissues, which relies on the discovery of a characteristic signature of the heteromer. This characteristic, which is usually identified in a heterologous cell system, may be then used as a “fingerprint” to demonstrate the presence of the heteromer in the native tissue (21–24). A specific characteristic of the D₁R-H₃R heteromer, previously identified in transfected cells is cross-antagonism (15), i.e. the ability of both D₁R and H₃R antagonists to block the effect of either D₁R or H₃R agonists. This phenomenon, in which an antagonist of one of the receptor units in the receptor heteromer blocks signaling originated by ligand binding to the other receptor unit in the heteromer, has also been observed with other receptor heteromers, such as the cannabinoid CB₁-orexin OX₁ receptor heteromer (25). Significantly, the same D₁R-H₃R cross-antagonism on MAPK signaling, which was described in transfected cells (15), was observed in rat striatal slices (Fig. 2), strongly supporting the occurrence of D₁R-H₃R

heteromers in the rodent striatum. Of note, a further characteristic of the D₁R-H₃R heteromer is its ability to allow the activation of the MAPK cascade by H₃R-selective agonists, which otherwise cannot drive this signaling pathway (15). In fact, H₃R agonist-induced ERK 1/2 phosphorylation was demonstrated in striatal slices of wild-type but not of D₁R knock-out mice, indicating the occurrence of D₁R-H₃R heteromers in the rodent striatum. As the H₃R agonist was unable to activate MAPK signaling in slices from D₁R-deficient mice (Fig. 3) it is likely that only neurons containing both H₃R and D₁R are able to link histaminergic neurotransmission to the MAPK cascade. Interestingly, although H₃R were found to be co-expressed with D₁R- and D₂R-containing neurons, the H₃R-mediated phospho-ERK labeling only co-distributed with D₁R- but not with D₂R-containing neurons (Figs. 5 and 8) and was not dependent on neurotransmitter release from neighboring cells.

The results obtained with co-administration of D₁R and H₃R agonists suggest that the D₁R-H₃R heteromer works as a processor that integrates dopamine and histamine-related signals, and its output consists of quantitatively different activation of the MAPK pathway. Strong MAPK signaling was obtained with either D₁R or H₃R activation, but a significantly weaker MAPK signaling was obtained upon co-activation of both receptors. Thus, at very low dopamine concentrations, histamine can foster MAPK signaling by activating H₃Rs in D₁R-H₃R-coexpressing neurons. In contrast, when the two neurotransmitters are present, the MAPK activation in the striatonigral MSN would be repressed. Because the MAPK pathway is considered critical to activity-dependent changes underlying synaptic strengthening (26), our results predict that not only dopamine but also histamine plays an important role in MAPK-dependent neuroplasticity in the striatonigral MSN.
Dopamine D₁-histamine H₃ Receptor Heteromers in Striatum

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

**FIGURE 6.** **Quantification of colocalization in confocal microscope images.** Quantification of H₃R expression (a) or 1 µM imetit-induced ERK 1/2 phosphorylation (b) in neurons expressing D₁R (D₁ neurons) or D₂R (D₂ neurons). Values are mean ± S.E. of the percentage of double-labeled cells (cells stained for H₃Rand D₁Ror D₂R in a or cells stained for imetit-induced phospho-ERK 1/2 and D₁Ror D₂R in b) were counted in a total of two to three nonoverlapping fields of 15 (a) or 45 (b) coronal sections from four to five slices.

A negative cross-talk between striatal D₁R and H₃R has also been described for the adenyllyl cyclase-induced signaling pathway, as histamine H₃R activation inhibits D₁R-mediated cAMP accumulation in striatal slices (27). Additional examples of H₃R-mediated responses able to inhibit D₁R-mediated effects are the ability of H₃R agonists to inhibit the effects of D₁R agonists on GABA release in striatal slices (12) and motor activation in reserpinized mice (14). Overall, these results are consistent with an antagonism at the level of adenyllyl cyclase between H₃R and D₁R that would not require heteromer formation. In fact, it is known that H₃R and D₁R couple to Gᵢ and Gₛ, respectively (9, 28–30). Although it is difficult to confirm these results in living animals, studies in transfected cells indicate that D₁R-H₃R heteromers couple to Gᵢ, but not to Gₛ, to direct histaminergic input toward the MAPK pathway.

Taken together, it appears that histamine and dopamine antagonism mediated by D₁Rs and H₃Rs may rely on balancing ERK activation in GABAergic neurons where D₁R and H₃R are co-expressed and where D₁R-H₃R heteromerization is likely occurring. Heteromers not only allow neu-

<figure><img src="image_15.png" /></figure>

**FIGURE 7.** **Co-immunoprecipitation of H₃R and D₁R or D₂R.** Rat striatal membranes were solubilized and processed for immunoprecipitation as described under "Experimental Procedures" using rat anti-D₁R antibody, rabbit anti-D₂R antibody, or rabbit anti-calnexin antibody as negative control. As positive controls and to test the specificity of dopamine receptors antibodies, immunoprecipitates were analyzed by SDS-PAGE and immunoblotted using guinea pig anti-D₁R antibody (a) or guinea pig anti-D₂R antibody (b). To test the co-immunoprecipitation, immunoprecipitates were blotted with goat anti-H₃R antibody (c). The right panel in c corresponds to solubilized membranes from SK-N-MC and SK-N-MC/H₃R cells analyzed by SDS-PAGE and blotted with anti-H₃R antibody to test the specificity of the antibody. IP, immunoprecipitation; MW, molecular mass.

<figure><img src="image_16.png" /></figure>

**FIGURE 8.** **Imetit-induced ERK 1/2 phosphorylation in rat striatal GABAergic neurons.** Confocal microscopy images of coronal sections from striatal slices were treated with medium (a) or treated with 1 µM imetit (b–d). Slices were labeled with antiphospho-ERK 1/2 antibody (red). Labeling (green) using an anti-D₁ receptor antibody (c), or an anti-D₂ receptor antibody (d) is also shown. Insets in c and d are 2× magnification of the indicated parts of the figure. Scale bars, 100 µm (a and b) or 80 µm (c and d). Representative images of coronal sections are displayed.

rons to differentially “sense” a given neurotransmitter, but they serve to process the different signals impacting them at a given time frame (31, 32). Therefore D₁R-H₃R receptor heteromers would be actively involved in controlling the response of striatal neurons of the direct striatal efferent pathway. The qualitative and quantitative output on ERK 1/2 phosphorylation would largely depend on the concentrations of histamine and dopamine impacting neurons expressing D₁R-H₃R complexes.

<figure><img src="image_17.png" /></figure>
*Acknowledgments—We acknowledge the technical help obtained from Jasmina Jiménez (Molecular Neurobiology Laboratory, Barcelona University) and Mar Castillo (Neuroscience Institute, Universidad Autónoma de Barcelona).*

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