α2A adrenergic receptors highly expressed in mesoprefrontal dopamine neurons

Neuroscience 332 (2016) 130–139

a2A ADRENERGIC RECEPTORS HIGHLY EXPRESSED IN MESOPREFRONTAL DOPAMINE NEURONS M. PAOLA CASTELLI, a,by* SATURNINO SPIGA, cy ANDREA PERRA, a CAMILLA MADEDDU, a GIOVANNA MULAS, c M. GRAZIA ENNAS a AND GIAN LUIGI GESSA a,d

a2A-AR receptors control dopaminergic activity and dopamine release in the prefrontal cortex. This finding raises the question whether a2A-ARs might function as autoreceptors in the mesoprefrontal dopaminergic neurons, replacing the lack of D2 autoreceptors. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato, Italy b Center of Excellence ‘‘Neurobiology of Addiction”, University of Cagliari, 09042 Monserrato, Italy c Department of Life and Environmental Sciences, University of Cagliari, 09126 Cagliari, Italy

Keywords: a2A adrenoreceptors, meso-prefrontal, dopamine, caudate, ventral tegmental area, substantia nigra.

d

Guy Everett Laboratory” University of Cagliari, 09042 Monserrato, Italy

INTRODUCTION Abstract—a2 adrenoreceptors (a2-ARs) play a key role in the control of noradrenaline and dopamine release in the medial prefrontal cortex (mPFC). Here, using UV-laser microdissection-based quantitative mRNA expression in individual neurons we show that in hTH-GFP rats, a transgenic line exhibiting intense and specific fluorescence in dopaminergic (DA) neurons, a2A adrenoreceptor (a2A-AR) mRNA is expressed at high and low levels in DA cells in the ventral tegmental area (VTA) and substantia nigra compacta (SNc), respectively. Confocal microscopy fluorescence immunohistochemistry revealed that a2A-AR immunoreactivity colocalized with tyrosine hydroxylase (TH) in nearly all DA cells in the VTA and SNc, both in hTH-GFP rats and their wild-type Sprague–Dawley (SD) counterparts. a2A-AR immunoreactivity was also found in DA axonal projections to the mPFC and dorsal caudate in the hTH-GFP and in the anterogradely labeled DA axonal projections from VTA to mPFC in SD rats. Importantly, the a2A-AR immunoreactivity localized in the DA cells of VTA and in their fibers in the mPFC was much higher than that in DA cells of SNc and their fibers in dorsal caudate, respectively. The finding that a2A-ARs are highly expressed in the cell bodies and axons of mesoprefrontal dopaminergic neurons provides a morphological basis to the vast functional evidence that somatodendritic and nerve-terminal

Axonal projections of the Locus Coeruleus (LC) noradrenergic and ventral tegmental area (VTA) dopaminergic (DA) neurons converge at prefrontal cortex (PFC), where they play a key role in the regulation of cognitive and emotional functions (Descarries et al., 1987; Se´gue´la et al., 1990). Noradrenergic and DA dysfunctions in this region contribute to neuropsychiatric disorders ranging from depression (Dunlop and Nemeroff, 2007; El Mansari et al., 2010); schizophrenia (Masana et al., 2011; Langer, 2015), drug addiction (Koob and Nestler, 1997; Weinshenker and Schroeder, 2007; Volkow and Morales, 2015) binge eating- (Michaelides et al., 2012;), post-traumatic stress- (Arnsten et al., 2015) and attention-deficit hyperactivity disorders (Arnsten and Pliszka, 2011; Sallee et al., 2013). Somatodendritic and nerve-terminal a2-adrenergic receptors (a2-ARs) on LC-prefrontal noradrenergic neurons exert an inhibitory control on noradrenergic firing and noradrenaline release in the PFC (Starke, 2001). By contrast, meso-prefrontal DA neurons are unique among mesocorticolimbic DA neurons, as they express low mRNA levels of D2 dopamine receptors and lack functional somatodendritic autoreceptors. Accordingly, dopamine application in vitro has been shown to suppress the electrical activity of all DA neurons in the VTA except those projecting to the PFC (Lammel et al., 2008). Consistent with the paucity or lack of release inhibiting D2 autoreceptors in the PFC (Moghaddam and Bunney, 1990), results of microdialysis studies show that systemic or intra-PFC administration of D2 receptor agonists and antagonists induces a modest or has no effect on dopamine release in this region (Bannon and Roth, 1983; Hertel et al., 1999; Devoto et al., 2001). However, unlike D2 receptor ligands, a2-AR agonists and antagonists administered systemically or locally perfused into the

*Correspondence to: M. P. Castelli, Department of Biomedical Sciences, Division of Neuroscience and Clinical Pharmacology, Cittadella Universitaria, SS 554, km. 4,500, I-09042 Monserrato (CA), Italy. Fax: +39-070-6754320. E-mail address: [email protected] (M. P. Castelli). y These authors contributed equally to the study. Abbreviations: a2-ARs, a2 adrenoreceptors; a2A-ARs, a2A adrenoreceptors; AP, anteroposterior; BSA, bovine serum albumin; Ct, threshold cycle; DA, dopaminergic; GAPDH, glyceraldehyde-3phosphate dehydrogenase; LC, Locus Coeruleus; LMD, laser microdissection; mPFC, medial prefrontal cortex; PEN, polyethylene naphthalate membrane; rtqPCR, real-time quantitative polymerase chain reaction; SD, Sprague–Dawley; SN, substantia nigra; SNc, substantia nigra compacta; TH, tyrosine hydroxylase; VTA, ventral tegmental area. http://dx.doi.org/10.1016/j.neuroscience.2016.06.037 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 130

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PFC markedly decrease and profoundly increase, respectively, dopamine release in the PFC, indicating that a2ARs play a major role in controlling dopamine release in this region (Devoto et al., 2001, 2004). Moreover, other mechanisms may participate in controlling extracellular DA levels in the medial prefrontal cortex (mPFC), namely the noradrenergic transporter (Yamamoto and Novotney, 1998; Carboni et al., 2006) and DA co-released from noradrenergic terminals (Devoto et al., 2001). Molecular cloning studies identified three distinct genes encoding the a2-AR subtypes A, B, and C (Kobilka et al., 1987; Regan et al., 1988; Weinshank et al., 1990). Previous in situ hybridization studies found that a2A adrenoreceptor (a2A-AR) mRNA is most abundant in the LC, although it is also widely distributed in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus, and amygdala (Nicholas et al., 1993; Scheinin et al., 1994). Accordingly, a2A-AR immunoreactivity is detected in almost all noradrenergic cells in the brain primarily in the LC, but also in noradrenergic axon terminals in the PFC and in different noradrenergic projection areas (Rosin et al., 1993; Talley et al., 1996). The localization of a2A-AR mRNA and immunoreactivity suggests that this receptor mediates presynaptic autoreceptor functions in noradrenergic neurons and has an important role in mediating postsynaptic effects. However, in situ hybridization studies failed to detect mRNA for the a2A- B- and C-AR subtypes in the VTA and the substantia nigra (SN), while only mRNA for subtype C was found in the caudate-putamen (McCune et al., 1993; Scheinin et al., 1994). Moreover, a2A-AR immunoreactivity was detected only in an insignificant percentage of DA cells in the VTA and SN in the rat brain (Rosin et al., 1993). Because these rather negative results are in apparent contrast to the evidence of a key role of a2A-AR in the control of mesoprefrontal dopaminergic activity, we reexamined the expression and presence of a2A-AR in mesoprefrontal DA neurons in comparison with nigrostriatal DA neurons. a2A-AR gene expression was investigated using UVlaser-microdissection-based quantitative mRNA expression profiling in individual DA neurons (Liss et al., 2005) obtained from the VTA and SNc of hTH-GFP rats, a transgenic rat line exhibiting a specific green fluorescent protein (GFP) in DA neurons that allows their anatomical visualization and microdissection under epifluorescence illumination (Iacovitti et al., 2014). a2A-AR immunoreactivity was analyzed by immunohistochemistry and confocal microscopy in DA neurons of hTH-GFP rats and their wild-type Sprague– Dawley (SD) counterparts. In SD rats DA cells were visualized by antibody immunostaining of tyrosine hydroxylase (TH), while DA axonal projections to the mPFC were examined by anterograde tract tracing and positive TH immunoreactivity.

EXPERIMENTAL PROCEDURES Animals Mice: a2A-AR knockout (KO) (/) (originally bred on C57BL6/J mice) were kindly donated by Prof Lutz Hein

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(Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Germany) (Altman et al., 1999). Littermates (+/), originating after crossing male a2A-AR (/) mice with female C57BL6/ J mice, were intercrossed to obtain the a2A-AR (/) (KO) and a2A-AR wild-type (+/+) (WT) mice. Male a2A-AR KO (n = 3) and their counterpart WT mice (n = 3), weighing 17–20 g, were used for experiments to verify the specificity of our a2A-AR antibody. Rats: The hTH-GFP transgenic rats (Iacovitti et al., 2014) were acquired from Taconic Inc, USA. The hTH-GFP transgene is located on the X chromosome. Thus, as reported by Taconic, hemizygous hTH-GFP females were mated to WT SD males (Harlan); hTHGFP carrier males (n = 10) and their counterpart male WT SD rats (n = 6), weighing 250–300 g were used. Rats and mice were housed 4 and 10 per cage, respectively, in standard plastic cages with wood chip bedding, at a temperature of 22 ± 2 °C and 60% humidity, under a 12-h light/dark cycle (lights on from 7.00 am). Tap water and standard laboratory rodent chow (Mucedola, Settimo Milanese, Italy) were provided ad libitum in the home cage. All experiments were performed in accordance with the guidelines of the European Committee Directive of 24 November 1986 (86/609/EEC) and the Italian Legislation (D.P.R. 116/92). Animal genotyping Rat and mouse littermate genotyping was performed using genomic DNA isolated from tail biopsies and the polymerase chain reaction (PCR) as follows: The forward (F) and reverse (R) primer sequences for a2A-AR KO and a2A-AR WT mice were (F) 50 -GGTGA CACTGACGCTGGTTT-30 , (R) 50 -CGAGATCCAC 0 0 TAGTTCTAGC-3 , and (F) 5 -GCACGTCGAGAGCCAAA TAG-30 , (R) 50 -GTTCATGTTCCGCCAGGAG-30 , respectively. After a 2-min hot start at 94 °C, PCR amplification was performed under the following conditions: (i) a2A-AR KO: 30 cycles at 94 °C for 15 s, 56 °C for 20 s, 72 °C for 25 s and a final 7-min extension at 72 °C; (ii) a2A-AR WT: 35 cycles at 94 °C for 15 s, 61 °C for 30 s, 68 °C for 30 s and a final 1-min extension at 72 °C. The PCR products were 410 bp and 260 bp for a2A-AR WT and a2A-AR KO, respectively. Transgenic hTH-GFP rat pups were identified by PCR using the following primers: F 50 -CAGCACGACTTCTT CAAGTCC-30 and R 50 -GATCTTGAAGTTCACCTT GATGC-30 and DNA fragments were amplified by a 15-min hot-start, followed by 35 cycles of 4500 at 94 °C, 6000 at 60 °C, 72” at 60 °C and a final 5-min extension at 72 °C. The resulting PCR product was 264 bp for transgenic hTH-GFP rats. Laser microdissection (LMD) of single neurons Two 6-lm-thick coronal sections (bregma: +6.15) were cut with a Leica CM 1950 cryostat and mounted on SuperFrost Ultra Plus microscope glass slides. Other serial cryosections (6–8), 16 lm thick, were mounted on special glass slides covered with a nucleic acid and nuclease-free, 1-mm thick, polyethylene naphthalate

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membrane (PEN) and stored at 80 °C until use. Of the two 6-lm sections, the first one was used to check the presence of GFP-labeled neurons in the VTA and SNc. The second one was stained with hematoxylin-eosin and used to obtain histological landmarks for LMD. Briefly, the section was digitalized; three reference points were identified (third ventricle and lateral ventricles), recorded, and used as landmarks to align each PEN-membrane mounted section, allowing rapid identification of the VTA and SNc. Before LMD, PEN-membrane sections were quickly thawed dehydrated/fixed in two changes of 100% nuclease-free ethanol and mounted on a Leica LMD 6500 laser microdissection system. GFP-positive neurons in the VTA and SNc were detected under epifluorescence illumination with a 63 objective, and cellular profiles were microdissected (laser setting: power 55; aperture 10; speed 9). Dissected neurons were collected by gravity in the cap of a 200 ll nuclease-free centrifuge tube, containing 30 ll of the RNA extraction buffer. For each sample, real-time quantitative PCR (rtqPCR) was performed in a pool of three DA neurons each from the VTA of three rats. Pools of 30, 20, 10 and 3 or 2 neurons were collected and used to set up the rtqPCR technique for very low amounts of starting material (data not shown). The entire procedure from thawing to sample collection lasted at most 20 min, ensuring mRNA integrity. A total of 110 single neurons were microdissected. To compare a2A-AR expression in VTA and SNc the same procedure was used; 3 pools of 12 DA neurons from VTA and 3 pools of 12 DA neurons from SNc, were collected and analyzed from three rats.

delta reporter-normalized (dRn), which was calculated by the formula dRn = Rn – baseline. A relative quantification strategy was preferred to the end point-PCR approach because it allowed a good estimation of abundance (relative to the housekeeping gene) of the transcript in each microdissected pool. In this quantification strategy, to determine the expression levels of the target gene, the differences (delta) between the threshold cycle (Ct) for target and housekeeping gene were measured. This method, summarized as the delta Ct method (dCt), allowed estimating the gene expression relative to the endogenous control. Thus, if the abundance of the housekeeping is already known from literature, dCt is a reliable method to estimate the relative abundance of the target (Wittwer et al., 2001). Results were analyzed using the Sequence Detection System (SDS) software and given as threshold cycle (Ct) or -deltaCt (-dCt). In a TaqMan assay, Ct is the fractional cycle number at which the dRn passes a threshold level that is set in the exponential phase of the amplification curve. The -dCt of each sample is calculated as the difference between the Ct value of the housekeeping (GAPDH) gene and that of the target (a2A-AR). Values were expressed as the mean of three replicates ± standard deviation (SD). For quantitative comparison of a2A-AR expression in VTA, relative to SN, a delta–delta Ct method was applied (Livak and Schmittgen, 2001). Accordingly, the difference between the dCt from VTA and SN was calculated. The results were expressed as fold change in alpha 2AR expression in VTA relative to SN. The mean ddCt value of a2A-AR expression in SN was set as a reference sample for relative quantification.

RNA extraction and rtqPCR The tubes containing the samples were centrifuged, the extraction buffer volume was increased to 50 ll, and RNA was extracted using the PicoPure RNA Isolation Kit as previously described (Perra et al., 2014). RNA was eluted in 10 ll of RNase and nuclease-free water. One microliter of total RNA was used to evaluate its concentration with NanoDrop ND1000, and another 8 ll were retro transcribed to complementary DNA (cDNA) with the SuperScript III First-Strand Synthesis System (Life Technologies), according to the manufacturer’s instructions, using oligo(dt) to hybridize to 30 poly(A) tails, with a final reaction volume of 20 ll. Amplification of cDNA was performed using the Gene Expression Master Mix (Life Technologies). The TaqMan primer/ probes for a2A-AR and for the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Applied Biosystems (a2A-AR, Rn00562488_s1; GAPDH, Rn01775763_g1). The rtqPCR was performed with the 7300 Real-Time PCR System (Applied Biosystems) under the following thermal conditions: 50 °C for 2 min, 95 °C for 10 min, 50 cycles at 95 °C for 15 s and 60 °C for 1 min. All reactions were performed in triplicate and no-template and no-reverse-transcriptase negative controls were included. The magnitude of the fluorescence signal generated during cDNA amplification was indicated as

HISTOLOGY Anterograde tract-tracing Dextran-tetramethylrhodamine (Fluoro-Ruby, FR; MW 10,000; 10% in 0.1 M PBS, pH 7.4, Invitrogen) was bilaterally injected by iontophoresis (5 lA, 7 s on/off cycles for 30 min) into the ventral tegmental area (VTA) (anteroposterior (AP): 6.0 from bregma; L: 0.4 mm lateral from midline, V: 7.4 from the cortical surface). One week after tracer injection, rats (n = 6) were deeply anaesthetized with equitesin (containing per 100 ml, 0.97 g pentobarbital, 2.1 g MgSO4, 4.25 g chloral hydrate, 42.8 ml propylene glycol, and 11.5 ml 90% ethanol; 5 ml/kg, i.p.) and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). Brains were rapidly removed and post-fixed in the same fixative overnight. After repeated washing in 0.1 M PBS, brains were cryoprotected in 30% sucrose in PBS for 48 h. Coronal sections (thickness: 40 lm) were obtained using a cryostat and immunostaining was performed on free-floating sections. Alpha 2A-AR antibody The a2A-AR subtype rabbit polyclonal antibody raised against a peptide mapping at the third intracellular loop

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portion of the rat a2A-AR was custom-made by GeneCust service. Protein sequence alignment and comparison software (FASTA) (Pearson and Lipman, 1988) was used to find an immunogenic portion of a2A-AR without similarity to either a2B-AR or a2C-AR. Then, rabbits were immunized by GeneCust service with a peptide of 47 aa (KGKTKASQVKPGDSLRRRGP GAAGPGASGSGQGEERAGGAKASRWRG, aa 318–364 of the rat a2A-AR). The specificity and the effectiveness of this antibody were extensively validated by GeneCust service using immunoblotting, enzymelinked immunosorbent assay, and preabsorption with immunogenic peptides. As previously reported by Rosin et al. (1993), these antibodies do not cross react with either a2B-AR or a2C-AR. In addition, while the antibodies produced a2A-AR typical staining in the LC of WT mice, no immunostaining was detected in the LC of a2A-AR-KO mice (Fig. 2B). Immunofluorescence Preblocking of tissue sections was performed with 10% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.2% Triton X-100 in PBS for 1 h at room temperature (RT). For multiple labeling in FR-treated rats (n = 6), three sections from each animal were incubated for 48 h at 4 °C with rabbit polyclonal anti-a2A-AR (1:500; 7.2 lg/ml) and with a mouse monoclonal anti-TH antibody (1:400, Millipore) in PBS containing 0.2% Triton X-100, 0.1% BSA, and 1 % NGS. Then, sections were incubated with biotinylated goat anti-rabbit IgG (1:200, Vector Laboratories, Burlingame, CA, USA) and Cy5-labeled goat anti-mouse IgG (1:250; Alexa Fluor, USA) for 1 h in the dark at RT. Subsequently, sections were incubated with Avidin Alexa FluorÒ 488 for 1 h in the dark at RT. For a2A-AR-immunofluorescence single-labeling in hTH-GFP transgenic rats (n = 4), three sections from each animal were incubated for 48 h at 4 °C with a rabbit anti-a2A-AR (1:500; 7.2 lg/ml) polyclonal antibody in PBS containing 0.2% Triton X-100, 0.1% BSA, and 1% NGS. After washing, sections were incubated with biotinylated goat anti-rabbit IgG (1:200, Vector Laboratories) for 1 h in the dark at room temperature. Sections were then incubated with Streptavidin Alexa FluorÒ 594 (1:1000) for 1 h in the dark at RT. For double labeling in a2A-AR KO (n = 3) and WT mice (n = 3), three tissue sections from each animal were incubated in a working solution of mouse immunoglobulin-blocking reagent prepared as indicated by the manufacturer (Vector Laboratories) for 1 h at 20 °C. Then, sections were incubated for 48 h at 4 °C with rabbit polyclonal anti-a2A-AR (1:500; 7.2 lg/ml) and mouse monoclonal anti-TH (1:400, Millipore) in PBS containing 0.2% Triton X-100, 0.1% BSA, and 1% NGS. Then, sections were incubated with biotinylated goat anti-rabbit IgG (1:200, Vector Laboratories) and 594-labeled goat anti-mouse IgG (1:500; Alexa Fluor, USA) for 1 h in the dark at room temperature.

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Subsequently, sections were incubated with Avidin Alexa FluorÒ 488 for 1 h in the dark at RT. Finally, all sections were rinsed and mounted on slides using VectaShield anti-fade mounting media (Vector Inc.). We performed standard control experiments by omitting either the primary or secondary antibody; no cellular labeling was detected. Laser scanning confocal microscopy and image processing The following rat brain regions, defined by a stereotaxic atlas (Paxinos and Watson, 2007), were analyzed for confocal analysis: mPFC (AP + 3.7), dorsal caudate nucleus (AP + 1.0), and VTA (AP  6.0). The LC (AP  5.3) in the mouse was identified using the mouse stereotaxic atlas (Paxinos and Franklin, 2013). A Leica TCS SP5 laser scanning etg microscope equipped with white light laser super continuum was used to analyze the fluorescent material. Images were generated using PL Fluotar 100 (na. 1.3), 40  oil (na. 1.0), and (e) 63 oil (na. 1.2) objectives. Scans were performed using excitation channels for fluorescein, rhodamine, and CY5 wavelengths separately. The resulting confocal images (40–50 images, z step of 0.5 lm) were combined, frame by frame, for simultaneous rendering. For 3D reconstructions, maximum intensity was used (Imaris 7.6.1). The co-localization analysis was performed on confocal images using specialized software (Imaris 7.6.1). The same settings were used for all images captured and consisted of identical detector gain, amplifier gain, amplifier offset, pinhole diameter (0.5 Airy unit), laser power excitation, scan mode and speed, and frame size (512  512 pixels, 8 bit). Confocal settings were chosen to ensure that the signal intensities obtained from the co-localization of TH/a2A-AR, GFP/a2A-AR, and TH/FR/a2A-AR were fully resolved within the dynamic range of detection (8 bit, 0–255 gray values) and no saturation of signal occurred. The threshold, defined as the gray value below which the signal is considered background, was set at 40–60 on a scale of 0–255 of gray values. For quantitative co-localization in the collated confocal images, four regions of interest (ROI) (x = 40 lm; y = 40 lm; z = 10 lm) were randomly chosen, and in each one, fluorescent volumes were calculated using specialized software (Imaris 7.6.1). Specifically, in hTH-GFP rats (n = 3), the fluorescent volume of a2A-AR localized on DA fibers was expressed as a percentage of the volume of GFP-positive fibers. In SD rats (n = 3), the co-localization of a2A-AR in DA fibers was expressed as a percentage of the volume of simultaneous TH- and FR-fluorescent fibers. Finally, the number of positive (i.e. cells exhibiting 1 or >1 a2A-AR punctate immunoreactivity) and negative cells per ROI (x = 98 lm; y = 98 lm; z = 20 lm) was counted manually and expressed as the percentage of total DA cell bodies identified. The density of a2A-ARs (volume of a2A-AR IR) localized on DA cell bodies was expressed as a percentage of the volume of DA-positive-cells identified.

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Statistical analysis a2A-AR mRNA analysis was performed using a pool of DA neurons from VTA and SNc from three different rats. Analysis of a2A-AR and TH immunoreactivity was performed from three coronal sections from each animal out of four/six. All numerical data are expressed as mean ± SEM. As we used a relatively small number of cases in each group, statistical significance between different groups was calculated using the non-parametric Mann–Whitney test.

RESULTS Expression of a2A-AR mRNA in DA neurons Quantitative expression of mRNA for a2A-AR was analyzed in DA neurons in the VTA and SNCc of hTH-GFP rats, a transgenic line genetically encoded with a green fluorescent reporter protein specifically tagging TH in DA neurons, allowing the visualization of DA neurons without antibody staining (Iacovitti et al., 2014). Under epifluorescence illumination, 12 pools of 3 identified DA neurons each were collected from three rats via UV-laser microdissection from coronal cryosections of the medial-posterior VTA, where most DA neurons projecting to the mPFC are located (Bjo¨rklund and Dunnett, 2007). The mRNA expression

of a2A-AR was estimated by rtqPCR, comparing the Ct values of a2A-AR and GAPDH (-dCt) (Wittwer et al., 2001). As shown in Fig. 1A, amplification plots indicate that every neuronal pool contained significant amounts of mRNA for a2A-AR, the -dCt ranging between 3.83 and 7.04 (Fig. 1C). These results indicate that the a2A-AR abundances were only between 23.83 and 27.04 times lower than that of the highly abundant housekeeping gene GAPDH (Walker et al., 2004). To compare the relative expression of a2A-AR in DA neurons from VTA and SNc, 3 pools of 12 DA neurons from VTA and 3 pools from SNc, were collected and analyzed from three rats. As shown (Fig. 1D), DA neurons from VTA exhibited about 5-fold higher a2A-AR expression, compared to DA neurons from SNc. a2A-AR antibody specificity The specificity of the a2A-AR antibody was validated further using the most rigorous negative control, i.e. the a2A-AR-KO mice, in comparison with the positive WT C57BL6/J counterparts. Cell bodies of coronal sections of the LC obtained from a2A-AR KO (n = 3) and WT mice (n = 3) were doubly stained with the TH and a2A-AR antibodies. As shown in Fig. 2, a2A-AR immunoreactivity was detected in almost all the TH-immunoreactive cells of the LC in WT mice. TH immunoreactivity was diffuse green,

Fig. 1. qrt-PCR shows the presence of a2A-AR transcripts in Laser Microdissected DA-positive neurons from the VTA of hTH-GFP rats. Semilogarithmic qrt-PCR amplification plots, expressed as Delta reporter-normalized (Rn) vs. cycle number, for a2A-AR using cDNA from DA-positive neurons (12 samples in triplicate, each pool of three neurons) from the VTA of hTH-GFP rats. The threshold line for a2A-AR is shown (A). Semilogarithmic qrt-PCR amplification plots for a2A-AR and GADPH of a representative sample. Threshold lines for GADPH and a2A-AR are shown. Delta Rn: relative fluorescence, normalized to the internal fluorescence dye FAM (B). a2A-AR expression in DA-positive neurons from the VTA of hTH-GFP rats shown as dCT to the endogenous control GADPH. Data are expressed as mean ± SD of -dCt, which is the difference between the GADPH dCt and a2A-AR dCt in the number of cycles needed to pass the cycle threshold (Ct) (C). Relative quantification of a2A-AR mRNA showed a 5-fold increase in VTA compared to SN. Mann-Whitney test: a, U (3,3) = 0, *p = 0.02. Values were calculated using the ddCt strategy to compare the fold change in a2A-AR expression in VTA relative to SN, and were given as mean ± SEM of three rat pool samples (a pool of 12 DA neurons for each area). Relative expression was 5.97 ± 0.45 for VTA and 1.00 ± 0.21 for SN (D).

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Fig. 2. Representative 3D confocal reconstruction of TH-immunoreactive neurons in the locus coeruleus of wild type C57BL (A) and a2A-AR KO (B) mice. a2A-AR-immunoreactive products (yellow puncta) are colocalized with TH immunoreactivity (diffuse green) in cells of WT but not of a2A-AR KO. Bregma: AP  5.3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1. a2A-AR immunoreactivity in DA cells in the VTA and SNc of hTH-GFP and Sprague–Dawley rats hTH-GFP rats

1

DA cells per ROI DA cells a2A-AR positive2 a2A-ARIR/THIR volume/volume3 1 2 3 * ****

Sprague–Dawley rats

VTA

SNc

VTA

SNc

5.7 ± 0.7 93.5 ± 2.6 4.5 ± 0.9*,a

5.7 ± 0.3 91.8 ± 3.6 1.1 ± 0.1

5.8 ± 0.4 93.8 ± 3.3 5.3 ± 0.4****,b

5.5 ± 0.2 91.4 ± 2.9 1.8 ± 0.2

Each value is mean ± SEM of cells counted in each ROI (n = 40; x = 90 lm; y = 90 lm; z = 10 lm) from three animals per group. Each value represents the percentage (mean ± SEM) of DA cells showing a2A-AR co-localization. Each value represents volume (means ± SEM) of a2A-AR IR expressed as percentage of volume of TH-immunoreactivity (IR). Mann-Whitney test: aU (32,40) = 439. p < 0.05 compared to SNc of hTH-immunoreactivity (IR); bU (30,40) = 89. p < 0.0001 compared to SNc of Sprague–Dawley rats.

whereas that of a2A-AR was visualized as intense puncta (yellow, <1 lm diameter). By contrast, a2A-AR immunoreactivity was not detected in any of the TH-positive cells in the LC of a2A-AR KO mice, confirming the specificity of the antibody for a2A-AR. Co-localization of a2A-AR and TH immunoreactivity in the VTA and SNc DA cell bodies in hTH-GFP rats were identified by their specific GFP fluorescence, whereas in SD rats they were detected with the TH antibody. Both in hTH-GFP rats and in their SD WT counterparts, DA cells displaying a2A-AR immunoreactivity were counted manually in the VTA and in the SNc. As listed in Table 1, in SD and hTH-GFP rats, the vast majority of DA cells in the VTA and SNc were doubly labeled for a2A-AR and TH. However, since the density of a2A-AR staining per individual DA neuron was stronger in the VTA than in the SNc (Fig. 3), the degree of a2A-AR immunoreactivity colocalized in individual DA cells in both regions was quantified as described in materials and methods. The fluorescence volume of a2A-AR immunoreactivity colocalized with TH immunoreactivity in cells in the VTA was 3–4 times higher than the corresponding fluorescence volume of a2A-AR

colocalized with TH immunoreactivity in the cells of the SNc (Table 1). Colocalization of a2A-AR and TH immunoreactivity in DA axons in the mPFC and caudate nucleus In hTh-GFP rats, DA fibers were identified by their specific fluorescence, while in SD rats, DA projections to the mPFC from the VTA were identified by the colocalization of the anterogradely transported FR and TH immunoreactivity. As shown in Fig. 4, in hTH-GFP rats, the colocalization of a2A-AR immunoreactivity with GFP positivity is observed in a higher number of DA axons in the mPFC than in the dorsal caudate. Consistently, the density of a2A-AR immunoreactivity in DA fibers was seven times higher in the mPFC than in the caudate nucleus (Table 2). Fig. 5 shows that a2A-AR and TH immunoreactivity were colocalized on the anterogradely labeled mPFC axons of SD rats.

DISCUSSION The present study shows that DA neurons in the VTA and SNc express mRNA encoding a2A-AR and exhibit a2A-AR immunoreactivity. Single-cell UV laser microdissection and qrt-PCR show high and low levels

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Fig. 3. Representative 3D confocal reconstruction of DA cells (green) and co-localized a2A-AR immunoreactivity (yellow dots) in the VTA (A) and substantia nigra compacta (SNc) (B) of hTH-GFP rats. The density of a2A-AR immunoreactivity is higher in the VTA than in SNc cells. Bregma: AP 6.0 to 6.6. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of a2A-AR mRNA in all DA neurons in the VTA and SNc, respectively, of hTH-GFP rats, a transgenic rat model in which DA neurons can be identified by their specific fluorescence. Prior studies using radioactive in situ hybridization indicated that a2A-AR mRNA has a widespread distribution in almost all supraspinal noradrenergic areas in the rat brain, being most abundant in the LC, but also present in noradrenergic projection areas (Nicholas et al., 1993, 1996; Wang et al., 1996). This suggests that a2A-ARs mediate presynaptic autoreceptor function in noradrenergic neurons as well as postsynaptic functions (Scheinin et al., 1994). However, while some of these studies determined that a2A-AR mRNA are localized in neuronal cells, they did not define, except for noradrenergic neurons (Aoki et al., 1994), the cell-type localization, which is needed to correlate mRNA expression with physiological significance. Our results are the first demonstration of a2A-AR mRNA expression in DA neurons of VTA and SN. Moreover, they demonstrate, both in hTH-GFP and their wild-type SD counterparts, that a2A-AR immunoreactivity is localized in virtually all DA cells in the VTA and SNc and in a high percentage of DA axons in the mPFC but, to a lesser extent, in the caudate nucleus. In contrast to our results, an earlier study by Rosin et al. (1993), using double labeling with antisera to TH or to phenylethanolamine-N-methyl transferase, found that a2A-AR immunoreactivity was present in most, perhaps all, noradrenergic and adrenergic cells of the brainstem, but only in an insignificant percentage of DA cells in the SN and VTA. Rosin et al. (1993) cautioned against the possibility that some double-labeled cells might have been obscured by overlying cells in the tissue section or by TH immunoreactivity. Indeed, their rather negative results might be explained by the use of the immunoenzymatic peroxidase-antiperoxidase technique and optical microscopy (instead of fluorescence and confocal microscopy), which does not allow a clear separate

identification of targets at very different concentrations (White et al., 1987). The most important outcome of our results is that both mRNA and immunoreactivity of a2A-AR were much higher in the VTA than in the SN DA neurons, as well as in the DA axons in the mPFC than in those in the dorsal caudate. Further experiments in the transport, trafficking, stability, and surface expression of the receptor might contribute to clarify the large differences in the level of a2A-AR immunoreactivity between meso-prefrontal and nigrostriatal neurons. Indeed, previous studies indicate that trafficking, expression, and the functionality of a receptor in heterologous cell types may be enhanced, or permitted, by the co-expression of an appropriate G proteincoupled receptor partner (Uberti et al., 2003; Xu et al., 2003; Hague et al., 2004, 2005). For example, trafficking to the plasma membrane, functionality and stability of a2C-ARs are controlled by the co-expression of b2-ARs (Prinster et al., 2006). Future research should also clarify whether the higher mRNA expression and density of a2AARs in the meso-prefrontal than in nigro-striatal DA neurons are correlated with the paucity or absence of D2 autoreceptors, and whether this difference might account for the high effectiveness of a2A-AR ligands for modifying dopamine release in the mPFC, while they are ineffective in the caudate nucleus (Gresch et al., 1995; Tanda et al., 1996; Devoto et al., 2001, 2004). In contrast to their earlier observation of an insignificant presence of a2A-AR immunoreactivity in DA neurons in the VTA and SN, Rosin et al. (1996) (see also Lee et al., 1998) detected high levels of a2C-AR subtype immunoreactivity in most TH-positive neurons, including those present in the VTA and SN. They suggested that a2C-ARs act as autoreceptors for central noradrenergic neurons and are also responsible for the suppressant effect of a2A-AR agonists on dopamine release. This conclusion is in apparent contrast with the findings of

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Fig. 4. Representative 3D confocal reconstruction of a2A-AR immunoreactivity colocalized in DA terminals in the mPFC (A) and dorsal caudate (B) of hTH-GFP rats. a2A-AR immunoreactivity (yellow dots) is higher in DA axons (green) in the mPFC (a, Bregma: AP + 3.7) than in the dorsal caudate (B, Bregma: AP + 1.0). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2. a2A-AR immunoreactivity (IR) in DA fibers in the mPFC and dorsal caudate of hTH-GFP rats

TH-IR % of ROI1 a2A-AR/TH volume/volume2

mPFC

Dorsal Caudate

3.17 ± 0.42****,a 14.17 ± 0.83****,b

33.74 ± 1.21 2.24 ± 0.14

1 Each value (mean ± SEM) represents the percentage of fluorescent volume of TH-IR per ROI (n = 40, x = 90 lm; y = 90 lm; z = 10 lm) from three animals. 2 Each value represents volume of a2A-AR IR (means ± SEM) expressed as percentage of TH-IR fluorescent volume calculated from 40 ROI in 3 animals. Mann-Whitney test: aU (40,40) = 0, ****p < 0.0001 compared to Dorsal caudate; b U (40,40) = 0, ****p < 0.0001 compared to Dorsal caudate.

Bu¨cheler et al. (2002), who used autoradiography and radioligand binding to show that deletion of the gene encoding the a2A-AR subtype eliminates approximately 90% of the total a2-AR density, while the a2C-AR gene deletion eliminates only 10% of the total a2-AR density in the mouse brain. However, these results do not preclude the possibility that high levels of a2C-ARs might be localized in DA neurons and even co-localize with

a2A-ARs, playing a complementary role in regulating DA release. Indeed, numerous studies of a2A-AR physiology in gene-targeted mice indicate that many biological functions of a2-ARs are mediated by more than one receptor subtype (Philipp et al., 2002). Future research should clarify whether the density of a2C-AR immunoreactivity in mesoprefrontal and nigrostriatal DA neurons is different from that observed for a2A-AR. In conclusion, the finding that a2-ARs are highly expressed in mesoprefrontal DA neurons supports the hypothesis that a2A-ARs function as heteroreceptors in mesoprefrontal DA neurons, acting as the target of noradrenaline released by LC noradrenergic terminals to modulate DA release (Gresch et al., 1995). Although an axo-axonic relationship between dopaminergic and noradrenergic terminals remains to be established, extracellular noradrenaline may diffuse trans-synaptically to access, by volume transmission, a2A-ARs on DA terminals (Fuxe et al., 2015). An exciting, but not necessarily alternative, possibility to the above mechanism may be that a2A-ARs function both as hetero and autoreceptors, being the target not

Fig. 5. Representative 3D confocal reconstruction of the colocalization of a2A-AR immunoreactivity with TH and Fluoro Ruby (FR) in the mPFC of Sprague-Dawley (SD) rats. Total TH immunoreactivity (magenta) of noradrenergic and DA fibers in the mPFC (A); TH-positive FR-traced fibers (red) in the mPFC (B); FR-stained TH-positive fibers (red) with colocalized a2A-AR immunoreactivity (yellow dots) (C). Bregma: AP + 3.7. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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only of noradrenaline, but also of DA released from the same neuron where they reside. Finally, it is also plausible that a2A-ARs on DA terminals are stimulated by dopamine putatively co-released with noradrenaline from noradrenergic terminals (Devoto et al., 2004). These hypotheses are consistent with a host of studies indicating interplay among catecholamine systems (Guiard et al., 2008). Importantly our study highlights the usefulness of the hTH-GFP rat as a valuable model for the study of the expression and localization of receptor proteins in DA neurons.

CONCLUSION The high a2A-AR expression in mesoprefrontal DA neurons raises the hypothesis that these receptors might function as autoreceptors replacing the lack of D2 receptors an hypothesis directly testable in electrophysiological recordings of identified mesoprefrontal in comparison with nigrostriatal dopaminergic neurons. Our results indicate a potential target for treatments of psychiatric disorders where altered prefrontal dopaminergic signaling is a key feature of pathophysiology.

CONFLICTS OF INTEREST The authors declare that they have no conflict of interests.

FUNDING This research was supported by the ‘‘Guy Everett Laboratory” Foundation. Acknowledgments—Sardegna Ricerche Scientific Park (Pula, CA, Italy) is acknowledged for free access to facilities of the Nanobiotechnology Laboratory.

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(Accepted 22 June 2016) (Available online 27 June 2016)