Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens

Neuropharmacology xxx (2015) 1e11

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Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens
nchez a, Raúl Gonza
lez-Pantoja a, Guillermo Aquino-Miranda a, Juan Escamilla-Sa
-Antonio Arias-Montan ~ o a, * Antonio Bueno-Nava b, Jose a
n y de Estudios Avanzados (Cinvestav) del IPN, Av. IPN 2508, Zacatenco, Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigacio 07360 M
exico, D.F., Mexico b
n de Neurociencias, Instituto Nacional de Rehabilitacio
n, Secretaría de Salud, Calzada M
Divisio exico-Xochimilco 289, Arenal de Guadalupe, 14389 M
exico, D.F., Mexico

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 March 2015 Received in revised form 6 June 2015 Accepted 6 July 2015 Available online xxx

We studied the effect of activating histamine H3 receptors (H3Rs) on rat nucleus accumbens (rNAcc) dopaminergic transmission by analyzing [3H]-dopamine uptake by synaptosomes, and dopamine synthesis and depolarization-evoked [3H]-dopamine release in slices. The uptake of [3H]-dopamine by rNAcc synaptosomes was not affected by the H3R agonist RAMH (10
10e10
6 M). In rNAcc slices perfusion with RAMH (1 mM) had no significant effect on [3H]-dopamine release evoked by depolarization with 30 mM Kþ (91.4 ± 4.5% of controls). The blockade of dopamine D2 autoreceptors with sulpiride (1 mM) enhanced Kþ-evoked [3H]-dopamine release (168.8 ± 15.5% of controls), but under this condition RAMH (1 mM) also failed to affect [3H]-dopamine release. Dopamine synthesis was evaluated in rNAcc slices incubated with the L-dihydroxyphenylalanine (DOPA) decarboxylase inhibitor NSD-1015 (1 mM). Forskolin-induced DOPA accumulation (220.1 ± 10.4% of controls) was significantly reduced by RAMH (41.1 ± 6.5% and 43.5 ± 9.1% inhibition at 100 nM and 1 mM, respectively), and this effect was prevented by the H3R antagonist ciproxifan (10 mM). DOPA accumulation induced by preventing cAMP degradation with IBMX (iso-butyl-methylxantine, 1 mM) or by activating receptors for the vasoactive intestinal peptide (VIP)/ pituitary adenylate cyclase-activating peptide (PACAP) with PACAP-27 (1 mM) was reduced (IBMX) or prevented (PACAP-27) by RAMH (100 nM). In contrast, DOPA accumulation induced by 8-Bromo-cAMP (1 mM) was not affected by RAMH (100 nM). These results indicate that in rNAcc H3Rs do not modulate dopamine uptake or release, but regulate dopamine synthesis by inhibiting cAMP formation and thus PKA activation. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Histamine Dopamine H3 receptor Nucleus accumbens Dopamine synthesis Dopamine release

1. Introduction The nucleus accumbens (NAcc) plays a key role in the neural systems responsible for translating motivation derived from limbic regions to goal-directed behaviors (Sesack and Grace, 2010). At least 95% of NAcc neuronal cells are GABAergic medium spiny neurons (MSNs) with the rest of the neuronal population

Abbreviations: D2R, dopamine D2 receptor; DOPA, L-dihydroxyphenylalanine; GABA, g-aminobutyric acid; H3R, histamine H3 receptor; MSNs, medium spiny neurons; RAMH, (R)(-)-a-methylhistamine; rNAcc, rat nucleus accumbens; TH, tyrosine hydroxylase; VTA, ventral tegmental area. * Corresponding author. Departamento de Fisiología, Biofísica y Neurociencias,
xico, D.F., Mexico. Cinvestav-IPN, Apdo. postal 14-740, 07000, Me ~ o). E-mail address: jaarias@fisio.cinvestav.mx (J.-A. Arias-Montan

represented by GABAergic and cholinergic interneurons. NAcc MSNs expressing dopamine D1-like receptors innervate the ventral tegmental area (VTA) via the so-called direct pathway whereas in the indirect pathway MSNs expressing D2-like receptors (D2Rs) project to GABAergic neurons located in the ventral pallidum that in turn send projections to the VTA (Russo and Nestler, 2013). The NAcc also sends afferents to hypothalamus, brainstem, globus pallidus and substantia nigra pars reticulata (SNr). In turn the latter two nuclei innervate the mediodorsal and other thalamic divisions, thus completing cortico-NAcc-pallidal/SNr-thalamocortical loops (Sesack and Grace, 2010). The NAcc is innervated by excitatory glutamatergic afferents from cerebral cortex, amygdala, hippocampus and thalamus, and inhibitory GABAergic afferents from ventral pallidum (Humphries and Prescott, 2010; Sesack and Grace, 2010; Russo and Nestler,

http://dx.doi.org/10.1016/j.neuropharm.2015.07.006 0028-3908/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Aquino-Miranda, G., et al., Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.07.006

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G. Aquino-Miranda et al. / Neuropharmacology xxx (2015) 1e11

2013). The Nacc also receives modulatory afferents from the brain stem, including dopaminergic projections from the VTA, serotonergic fibers from the dorsal raphe nucleus, a small noradrenergic projection from the locus coeruleus and the nucleus tractus solitarii, and sparse projections from the pedunculopontine tegmentum, the parabrachial nucleus and the periaqueductal gray (Beckstead et al., 1979; Sesack and Grace, 2010). Dopaminergic axons innervating the NAcc release their neurotransmitter in response to reward-related stimuli, and alterations in accumbal dopaminergic synaptic transmission have been implicated in the acquisition, maintenance and relapse of addiction (Koob and Volkow, 2010; Sesack and Grace, 2010). In the mammalian brain histamine acts as a neuromodulator through the activation of three (H1, H2 and H3) of the four G proteincoupled receptors cloned so far (Panula and Nuutinen, 2013). The rat NAcc (rNAcc) receives histaminergic innervation (Inagaki et al., 1988) and possesses a high density of both histamine H3 receptors (H3Rs) and the corresponding mRNA (Pillot et al., 2002). H3Rs are primarily expressed on nerve terminals, where they regulate the synthesis and release of histamine as well as the release of other neuroactive substances (for reviews see Feuerstein, 2008; OsorioEspinoza et al., 2010; Panula and Nuutinen, 2013). Dopaminergic neurons located in sustantia nigra pars compacta (SNc) and that innervate the striatum (Ikemoto, 2007) express H3R mRNA (Pillot et al., 2002), and in mouse striatal slices H3R activation inhibited depolarization-evoked [3H]-dopamine release (Schlicker et al., 1993). However, the virtual absence of H3R mRNA in the rat VTA reported by Pillot et al. (2002) did not support a similar action in the rNAcc. The rNAcc derives its dopaminergic innervation not only from the VTA but also from the dorsal tier of the SNc and the A8 (retrorubral) cell group (Hasue and Shammah-Lagnado, 2002; Bjor
lezklund and Dunnett, 2007). Moreover, recent data by Gonza Sepúlveda et al., (2013) using an enhanced probe showed that VTA dopaminergic neurons co-express mRNAs coding for the H3R and tyrosine hydroxylase (TH), the limiting enzyme in dopamine synthesis, as well as immunoreactivity for the corresponding proteins. This evidence led us to examine the effect of H3R activation on rNAcc dopaminergic transmission by analyzing [3H]-dopamine uptake by synaptosomes and [3H]-dopamine release and dopamine synthesis in slices. A preliminary account of this work was presented in the abstract form to the European Histamine Research Society (Aquino-Miranda et al., 2014). 2. Materials and methods Rats (males, Wistar strain, 250e300 g, bred in the Cinvestav facilities) were used throughout the experiments. All procedures were in accordance to the guidelines for the care and use of laboratory animals issued by the National Institutes of Health (NIH Publications No. 8023, revised 1978) and the Mexican Council for Animal Care, as well as approved by the Cinvestav Animal Care Committee. All efforts were made to minimize animal suffering and to use only as many animals were required for proper statistical analysis. 2.1. Preparation of rNAcc slices Animals were decapitated, the brain was quickly removed from the skull, immersed in ice-cold Krebs-Henseleit (KH) solution and coronal slices (300 mm thick) were obtained with a vibratome (World Precision Instruments, Sarasota, FL, USA). The NAcc was carefully dissected from the slices, with special care to avoid the adjacent striatum which contains high levels of H3Rs. The

composition of the KH solution was (mM): NaCl, 116; KCl 3, MgSO4, 1; KH2PO4, 1; NaHCO3, 25; D-glucose, 11; pH 7.4 after saturation with O2/CO2 (95:5% v:v). In order to reduce excitoxicity, CaCl2 was not added to this solution. 2.2. Synaptosome preparation Synaptosomes were prepared using a modification of the method of Gray and Whittaker (1962). Briefly, NAcc slices from 5 rats were placed in 5 ml 0.32 M sucrose/5 mM Hepes (pH 7.4 with NaOH) and homogenized using 10 strokes of a hand-held homogenizer. The homogenate was brought up to 15 ml with 0.32 M sucrose/5 mM Hepes and centrifuged at 1,000  g (10 min, 4  C). The supernatant was collected, adjusted to 20 ml with 0.32 M sucrose/ 5 mM Hepes and centrifuged at 20,000  g (20 min, 4  C). The pellet was resuspended in 7 ml 0.32 M sucrose/5 mM Hepes, layered onto 20 ml 0.8 M sucrose/5 mM Hepes and centrifuged at 20,000  g for 20 min (4  C). The pellet (synaptosomes) was used for binding or uptake experiments as described below. 2.3. Binding of N-a-[methyl-3H]-histamine ([3H]-NMHA) to synaptosomal membranes 2.3.1. Obtention of membranes The synaptosomal pellet was resuspended in 20 ml of lysis solution (10 mM TriseHCl, 1 mM EGTA, pH 7.4) and incubated for 20 min at 4  C. The suspension was then centrifuged (32,000  g, 20 min, 4  C) and the resulting pellet (synaptosomal membranes) was resuspended in incubation buffer (50 mM TriseHCl, 5 mM MgCl2, pH 7.4). 2.3.2. [3H]-NMHA binding assay For saturation analysis membranes (~20 mg protein aliquots; BCA assay) were incubated in 100 ml incubation buffer containing [3H]-NMHA (0.01e10 nM), whereas for inhibition experiments incubations contained [3H]-NMHA (3 nM) and increasing concentrations (10
11e10
6 M) of H3R ligands. Equilibration was for 60 min at 30  C and terminated by filtration through Whatman GF/ B glass fiber paper, pre-soaked in 0.3% polyethylenimine. Nonspecific binding was determined as that insensitive to 10 mM histamine and accounted for ~30% of total binding. Filters were soaked in 3 ml scintillator and the tritium content was determined by scintillation counting. Saturation binding data were fitted to a hyperbola by non-linear regression with GraphPad Prism 5 (Graph Pad Software, San Diego, CA, USA). Inhibition curves were fitted to a logistic (Hill) equation and values for inhibition constants (Ki) were calculated according to the equation (Cheng and Prusoff, 1973): Ki ¼ IC50/1 þ {[D]/Kd}, where [D] is the concentration of [3H]-NMHA present in the assay and Kd the mean value for the equilibrium dissociation constant estimated from the saturation analysis (1.32 nM, see Results). 2.4. Uptake of [3H]-dopamine by rNAcc synaptosomes Synaptosomes were resuspended in Krebs-Ringer-Hepes (KRH) buffer and aliquoted (140 ml) into plastic tubes. Drugs under test were added in a 10-ml volume and equilibrated for 10 min at 37  C before the addition of [3H]-dopamine in a 50-ml volume to yield 50 nM as the final concentration. After 30 min at 37  C, incubations were filtered through Whatman GF/B glass fiber paper, pre-soaked for 2 h in 0.3% polyethylenimine. Filters were washed 3 times with ice-cold KRH buffer, soaked in 3 ml scintillator and the tritium content was determined by scintillation counting. Nonspecific uptake was determined in samples incubated at 4  C or in the presence of 1 mM GBR-12909. The composition of the KRH buffer was

Please cite this article in press as: Aquino-Miranda, G., et al., Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.07.006

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(mM): NaCl 113, NaHCO3 25, KCl 3, MgCl2 1, KH2PO4 1, CaCl2 1.8, Dglucose 11, Hepes 20; pH 7.4 with NaOH. 2.5. Depolarization-evoked [3H]-dopamine release from rNAcc slices Because of the large number of animals required if neurotransmitter release was to be assayed with synaptosomes, these experiments were performed in rNacc slices (300 mm thick) prepared as described above. Slices from 5 animals were equilibrated at 37  C in normal KH solution (1.8 mM CaCl2), with changes of solution every 10 min. After 30 min the slices were transferred to 2 ml KH solution containing 50 nM [3H]-dopamine in the presence of 10 mM pargyline and 200 mM ascorbic acid (to prevent degradation of the tritiated neurotransmitter). Desipramine and fluoxetine (1 mM each) were added to the solution to prevent the possible uptake by noradrenergic or serotonergic terminals, respectively (see Results). After the labeling period, the slices were thoroughly washed with KH solution and then apportioned randomly between the chambers of a superfusion apparatus (15 chambers in parallel; 2 slices per chamber) and superfused (1 ml/min) with KH medium supplemented with 10 mM pargyline and 200 mM ascorbic acid. Slices were perfused for 20 min before the collection of 15 fractions of 1 ml (1 min) each. Neurotransmitter release was stimulated by changing to a solution containing 30 mM Kþ (KCl substituted for NaCl) for fractions 2 and 11, returning to normal KH solution between these fractions and after the second Kþ-stimulus. Drugs under test were present 5 min before and throughout the second Kþ-stimulus (i.e. fractions 6e12). The perfusion solution was supplemented with 1 mM GBR-12909 to prevent [3H]-dopamine re-uptake. For experiments where the Ca2þ-dependence of [3H]-dopamine release was tested, neurotransmitter release was evoked by a single depolarizing stimulus, and labeled slices were perfused for 15 min before and throughout the collection of fractions with KH solution (normal or high Kþ as required) with no CaCl2 added. The tritium content in the superfusate fractions was determined by scintillation counting. The contents of each chamber were collected and treated with 0.5 ml HCl (1 M) for 3 h before addition of scintillator in order to determine the amount of tritium remaining in the tissue. Tritium efflux into the superfusate was calculated as a fraction of tritium present in the tissues at the onset of the respective collection period. To allow for variations between chambers, fractional values were transformed to a percentage of the fraction collected immediately before the first change to the high Kþ medium (i.e. the release in fraction 1 was set to 100%). To test for statistical differences between treatments, after subtraction of basal release the area under the release curve for 5 fractions after the change to high Kþ (i.e. fractions 2e6 and 11e15) was determined for each individual chamber and the ratio of the second over the first Kþ stimulus (S2/S1) was calculated. Statistical comparisons were performed with Student's t test or one-way ANOVA and post hoc Dunnett's or Tukey's tests (Graph Pad Prism 5.0) as appropriate. 2.6. Determination of DOPA (L-dihydroxyphenylalanine) accumulation in rNAcc slices Slices were equilibrated in normal KH solution for 30 min at 37  C, with changes of solution every 10 min. At the end of this period, slices (2 per tube; ~200 mg protein) were placed in 250 ml of KH solution containing 200 mM L-tyrosine and 1 mM NSD-1015 (inhibitor of DOPA decarboxylase) and incubated at 37  C. For experiments with forskolin or IBMX, at the end of the pre-

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incubation with NSD-1015 (15 min), 250 ml of KH solution (controls) or KH solution containing 20 mM forskolin or 2 mM IBMX were added and incubations continued for 15 min. For experiments with PACAP-27 slices were pre-incubated in 250 ml of KH solution for 5 min before the addition of PACAP-27 in a 10 ml-volume, and incubations were continued for 30 min. For 8-Bromo-cAMP (8-BrcAMP) slices were pre-incubated (15 min) in 250 ml of KH solution before the addition of 8-Br-cAMP in a 10 ml-volume (25 mM) and incubations were continued for 30 min. Where required the H3R agonist RAMH or the D2R agonist quinpirole were added in a 10 mlvolume 5 min before the stimulating agent (forskolin, IBMX, PACAP-27 or 8-Br-cAMP). The antagonist/inverse agonist ciproxifan was added (10 ml) 5 min before RAMH. All solutions were supplemented with L-tyrosine (200 mM) and NSD-1015 (1 mM). At the end of the incubation 1 ml of ice-cold KH solution was added and the medium was aspirated before the addition of 250 ml of an ice-cold HClO4 solution (0.4 M) containing EGTA (5 mM) and Na2S2O5 (2.5 mM). Tubes were left to stand on ice and after 60 min adrenaline (50 pmol in 50 ml ice-cold HCLO4/Na2S2O5 solution) was added to each sample as an internal standard. The slices were then homogenized and the homogenates centrifuged (15,000  g, 10 min, 4  C). The supernatants were recovered and passed through 0.22 mm filters (Millex-GV) and the filtrates analyzed by HPLC-EC. Pellets were treated for 24 h with 500 ml NaOH (0.1 M) and proteins levels were assayed. Determination of DOPA, dopamine and adrenaline by HPLC-EC was performed essentially as described previously (AlfaroRodriguez et al., 2013). Briefly, 50-ml samples were injected into a HPLC system (Alltech HPLC pump, model 626, Grace Discovery Sciences, Deerfield, IL, USA) coupled to an electrochemical detector (ESA, Coulochem III, Thermo Scientific, Sunnyvale, CA, USA). The detection settings were: guard cell þ350 mV (ESA 5020) and analytical cell (ESA 5011A) potentials E1 þ200 mV and E2
200 mV. The peak signals were automatically delivered to a computer using the program EZCrom SI (version 3.2.1). DOPA, dopamine and adrenaline levels were estimated by interpolating a curve derived from five samples with known concentrations (5e80 nM). A 100  2 mm analytical column with a particle size of 3 mm (Microbore, BASi) was used and the mobile phase consisted of phosphate buffer solution (29 mM, pH 3.0) containing 3.5 mM sodium octyl sulfate, 0.43 mM EDTA, 0.6 mM tetrahydrofuran and methanol (16.5% v/v). The flow rate was 0.3 ml/min. 2.7. Drugs The following drugs were purchased from Sigma Aldrich (Mexico City, Mexico): 8-Br-cAMP (8-Bromoadenosine 30 ,50 -cyclic monophosphate), ciproxifan hydrochloride, clobenpropit dihydrobromide, desipramine hydrochloride, fluoxetine hydrochloride, GBR-12909 (1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3phenylpropyl)piperazine dihydrochloride), histamine dihydrochloride, IBMX (3-isobutyl-1-methylxanthine), NSD-1015 (3hydroxybenzylhydrazine dihydrochloride), PACAP-27 (Pituitary Adenylate Cyclase Activating Polypeptide-27), pargyline hydrochloride, (-)-quinpirole dihydrochloride and (R)(-)-a-methylhistamine dihydrochloride. N-a-[methyl-3H]-histamine (84.1 Ci/mmol) and [3H]-dopamine (48 Ci/mmol) were from Perkin Elmer (Boston, MA, USA). 3. Results 3.1. [3H]-NMHA binding to rNAcc membranes Saturation determinations of [3H]-NMHA binding to membranes obtained from rNAcc synaptosomes (Fig. 1A) yielded values

Please cite this article in press as: Aquino-Miranda, G., et al., Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.07.006

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Fig. 1. Binding of N-a-[methyl-3H]-histamine ([3H]-NMHA) to membranes from rNAcc. A) Saturation binding. Membranes were incubated with the indicated concentrations of [3H]NMHA in the absence and presence of 10 mM histamine, and specific receptor binding was determined by subtracting the binding in the presence of histamine from total binding. Points are means ± s.e.m. from triplicate determinations from a single experiment, which was repeated a further 3 times. The line drawn is the best fit to a hyperbola. Best-fit values for the equilibrium dissociation constant (Kd) and maximum binding (Bmax) are given in the text. B) Inhibition by the H3R agonist RAMH and the antagonists/inverse agonists clobenpropit and ciproxifan. Membranes were incubated with 3 nM [3H]-NMHA and the indicated concentrations of H3R ligands. Values are expressed as the percentage of control specific binding and are means ± range from duplicate determinations from representative determinations repeated 2 or 3 times with similar results. The line drawn is the best fit to a logistic equation for a one-site model. pKi values calculated from the best-fit IC50 estimates are given in the text.

Table 1. Comparison of the binding characteristics of rat brain and cloned histamine H3 receptors.

I. [125I]-iodoproxyfan binding Cloned rat H3R445a,h,i Rat striatumb,j II. [3H]-NMHA binding Cloned rat H3R445c,k Nucleus accumbensd,l Rat cerebral cortexe,k Rat cerebral cortexe,m Globus pallidusd,n Olfactory bulbd,o Thalamusd,p

Bmax (fmol/mg protein)

Kd (nM)

pKi RAMH

Immepip

Clobenpropit

Ciproxifan

300 78 ± 7

0.07 0.07

8.44 8.95

ND ND

8.85 9.32

8.40 ND

NR 196 ± 19 NR NR 1327 ± 79 106 ± 19 141 ± 12

0.80 1.32 NR NR 0.74 0.68 0.78

8.72 8.71 8.82 NR ND 9.10 ND

8.80 ND 9.30 NR 9.55 9.96 10.35f 8.49g

9.40 8.35 9.30 9.45 8.07 9.24 8.89

ND 7.80 ND 9.20 ND ND ND

ND, not determined; NR, not reported. Where indicated, values are means ± s.e.m. [3H]-NMHA, [3H]-N-methyl-histamine; H3R445, histamine H3 receptor of 445 amino acids. Bold indicates that these data were obtained in this work, and then compared with reported data. a CHO cells. b Total striatum membranes. c SK-N-MC cells. d Synaptosomal membranes. e Total cerebral cortex membranes. f High-affinity site. g Low-affinity site. h Data were taken from Ligneau et al., 2000. i Data were taken from Morisset et al., 2001. j Data were taken from Ligneau et al., 1994. k Data were taken from Lovenberg et al., 2000. l Data were taken from this work. m Data were taken from Esbenshade et al., 2003. n Data were taken from Morales-Figueroa et al., 2014. o Data were taken from Aquino-Miranda et al., 2012. p ~ o-Torres et al., 2007. Data were taken from Gardun

of 196 ± 19 fmol/mg protein (mean ± standard error, s.e.m., 4 experiments) for maximum specific binding (Bmax) and 1.32 nM for the equilibrium dissociation constant (Kd; pKd 8.88 ± 0.08). Specific [3H]-NMHA binding was inhibited in a concentrationdependent manner by the H3R agonist RAMH and the antagonists/ inverse agonists clobenpropit and ciproxifan (Fig. 1B). Values for the elog10 of the inhibition constant (pKi) obtained from 3 to 4 determinations were: RAMH 8.71 ± 0.07 (Ki 1.95 nM), clobenpropit 8.35 ± 0.10 (Ki 4.46 nM), and ciproxifan 7.80 ± 0.07 (Ki 15.85 nM). This series of experiments confirmed the presence of H3Rs on rNAcc nerve terminals with affinities for H3R ligands in agreement

with those reported for the cloned H3R isoform of 445 amino acids, the more abundant in rat brain (Morisset et al., 2001) and for native rat H3Rs (Table 1). However, we have found some differences in receptor affinity amongst several rat brain regions (Table 1), and these determinations allowed thus for a more accurate calculation of receptor occupancy by agonists and antagonists in functional experiments. 3.2. [3H]-dopamine uptake by rNAcc synaptosomes Fig. 2A depicts that the specific uptake of [3H]-dopamine by rNAcc synaptosomes was fully and potently inhibited by GBR-

Please cite this article in press as: Aquino-Miranda, G., et al., Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.07.006

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Fig. 2. Effect of selective inhibitors and the H3R agonist RAMH on [3H]-dopamine uptake by rNAcc synaptosomes. Synaptosomes were incubated for 30 min with [3H]-dopamine (50 nM) in the absence and presence of the indicated concentrations of the drugs under test, added 10 min before. The uptake of [3H]-dopamine is expressed as percentage of control values after subtraction of non-specific uptake, determined in parallel samples incubated at 4  C (A) or 10 mM GBR-12909 (B). A) Effect of the selective inhibitors GBR-12909 (dopamine transporter, DAT), desipramine (noradrenaline transporter, NET) and fluoxetine (serotonin transporter, SERT). Values are means ± s.e.m. of 3 replicates from a representative experiment repeated a further twice with similar results. Curves drawn are the best-fit adjust to a logistic equation. IC50 values are given in the text. B) Effect of the H3R agonist RAMH. Values are means ± s.e.m. from the combined values of 3 experiments. None of the values in the presence of RAMH was significantly different from control uptake (ANOVA and Dunnett's test).

Fig. 3. Effect of H3R ligands on depolarization-evoked [3H]-dopamine release from rat or mouse NAcc slices. A) Representative experiment for the effect of RAMH (1 mM) on [3H]dopamine release from rNAcc slices. Labeled slices were perfused with KH solution (4 mM Kþ) and [3H]-dopamine release was evoked by raising the Kþ concentration in the perfusion medium to 30 mM for the periods indicated by the vertical gray bars. Where required, RAMH was present for the period indicated by the horizontal black bar. Values are expressed as a percentage of [3H]-dopamine release in fraction 1 and represent the means ± s.e.m. of 5 replicates. B) Statistical analysis of 4 experiments for RAMH or ciproxifan. [3H]-Dopamine release was analyzed by comparing the areas under the curve after each stimulus and S2/S1 ratios were then constructed. To allow for differences between experiments values (means ± s.e.m.) are expressed as percentage of control release (no drugs added). C) Effect of RAMH in rNAcc slices perfused with the D2R antagonist sulpiride. Slices were perfused with 1 mM RAMH in the absence or presence of 1 mM sulpiride. Values are means ± s.e.m. from 3 experiments. D) Effect of RAMH or ciproxifan on Kþ-evoked [3H]-dopamine release from mouse NAcc slices. Values are means ± s.e.m. from 3 experiments. For experiments illustrated in panels B and D the statistical analysis was performed with ANOVA and Dunnett's test; ns, non-significantly different from control values. For experiments illustrated in panel C the statistical analysis was performed with ANOVA and Tukey's test; ns, non-significantly different.

12909, a selective inhibitor of the dopamine transporter (DAT), with IC50 2.40 nM (pIC50 8.62 ± 0.15). This value is in accord with the IC50 values reported for GBR-12909 to inhibit either [3H]GBR12935 binding to rat striatal membranes (2 nM; Kimura et al., 2003) or [3H]-dopamine uptake by rat striatal synaptosomes

(7.3 nM; Zhang et al., 2000). In contrast, both desipramine and fluoxetine, selective inhibitors of the transporters for noradrenaline (NET) or serotonin (SERT), respectively, were weak inhibitors of [3H]-dopamine uptake (39 ± 5% and 73 ± 6% inhibition, respectively, at 10 mM, the maximal concentration tested).

Please cite this article in press as: Aquino-Miranda, G., et al., Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.07.006

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3.3. Effect of H3R activation on [3H]-dopamine uptake by rNAcc synaptosomes A recent study showed a physical interaction between Gbg complexes and DAT that results in the reduction of [3H]-dopamine uptake by rat striatal synaptosomes (Garcia-Olivares et al., 2013). We therefore tested the effect of H3R activation on [3H]-dopamine uptake by rNAcc synaptosomes. Fig. 2B shows that the selective H3R agonist RAMH had no significant effect on [3H]-dopamine uptake on a wide range of concentrations (0.1 nM-1 mM). 3.4. Effect of H3R activation on depolarization-evoked [3H]dopamine release from rNAcc slices In experiments with a single depolarizing pulse, raising the Kþ concentration in the incubation medium (1.8 mM CaCl2) from 4 to 30 mM increased [3H]-dopamine release to 1268 ± 217% of basal at the peak of release (4 experiments; data not illustrated). In parallel determinations in slices perfused with KH solution with no CaCl2 added, Kþ-evoked [3H]-dopamine release was reduced by 89.4 ± 1.2% (P < 0.001 when compared with the release in the presence of 1.8 mM CaCl2, Student's t test), indicating that a significant component of release was due to exocytosis. No significant difference in basal release was observed between slices perfused with normal medium or medium with no CaCl2 added (82.3 ± 8.6% of control values, P ¼ 0.087, Student's t test). In 2-pulse experiments Kþ-evoked [3H]-dopamine was 885 ± 40% and 692 ± 34% of basal at the peak of release in the first and second depolarizing stimuli, respectively (Fig. 3A), with an overall S2/S1 ratio of 0.73 ± 0.03 (20 experiments). Fig. 3B shows that the selective H3R agonist RAMH (1 mM) reduced Kþ-evoked [3H]-dopamine release in a modest manner (91.4 ± 4.5% of controls, 8 experiments), but the effect did not yield statistical significance. Dopamine release is under the control of D2 autoreceptors and in a different series of experiments the D2 receptor (D2R) agonist quinpirole (10 mM) had no effect on basal [3H]dopamine release (106.3 ± 6.7% of controls, 3 experiments, P ¼ 0.400, Student's t test; data not illustrated), but significantly reduced Kþ-evoked [3H]-dopamine release (80.6 ± 3.5% of controls, P ¼ 0.006; data not illustrated). The relatively modest inhibition can be attributed to D2R activation by endogenous dopamine, as evidenced by experiments with the antagonist sulpiride (see below). H3Rs may have spontaneous or constitutive activity (Arrang et al., 2007) and the lack the effect of H3R activation on Kþ-

evoked [3H]-dopamine release could be due to the receptor being fully active in a constitutive manner, exerting thus a tonic inhibition on neurotransmitter release. Because of the high affinity of H3Rs for histamine (Ki 5.7 nM for the rat receptor, Drutel et al., 2001), an alternative explanation was that tonic H3R activation by endogenous histamine masked the effect of the exogenous agonist. To test for these possibilities, rNAcc slices were perfused with the antagonist/inverse agonist ciproxifan (1 mM). The analysis of 3 experiments showed that ciproxifan had no significant effect on Kþevoked [3H]-dopamine release (99.8 ± 4.2% of control values, Fig. 3B), nor on basal release (88.0 ± 11.5% of controls, P ¼ 0.356, Student's t test; data not illustrated). In slices from mouse striatum both histamine and the H3R agonist RAMH inhibited [3H]-dopamine release induced by electrical stimulation by 16e18%, and the blockade of D2 autoreceptors enhanced the inhibitory action of both agonists (Schlicker et al., 1993). Fig. 3C shows that in rNAcc slices perfusion with the D2R antagonist sulpiride (1 mM) significantly increased Kþ-evoked release to 168.8 ± 15.5% of control values, indicating that D2 autoreceptors were activated by endogenous dopamine released upon depolarization. However, in the presence of sulpiride the H3R agonist RAMH (1 mM) also failed to reduce depolarization-evoked [3H]-dopamine release (Fig. 3C). Sulpiride also increased, although to a modest extent, basal [3H]-dopamine release (108.5 ± 2.6% of controls, 3 experiments, P ¼ 0.039, Student's t test; data not illustrated). As mentioned above, the study by Schlicker et al. (1993) used mouse striatal slices, and to test for an inter-species difference we performed experiments with mouse NAcc slices. However, neither the H3R agonist RAMH (1 mM) nor the antagonist/inverse agonist ciproxifan (1 mM) modified Kþ-induced [3H]-dopamine release (Fig. 3D). 3.5. Effect of H3R activation on DOPA accumulation in rNAcc slices In dopaminergic nerve terminals neurotransmitter synthesis is stimulated by the cAMP/PKA pathway through PKA-mediated phosphorylation of the enzyme TH (Onali and Olianas, 1989; Daubner et al., 2011). In rNAcc slices, direct adenylyl cyclase (AC) activation with forskolin (10 mM) increased DOPA accumulation to 220.1 ± 10.4% of control values (Fig. 4A), a stimulation similar to that reported for rNAcc homogenates (~240% of basal TH activity; Onali and Olianas, 1989). Fig. 4B shows that whereas in the range 1e30 nM the H3R agonist RAMH had no effect, at 100 nM and 1 mM

Fig. 4. Effect of H3R ligands on forskolin-induced DOPA accumulation in NAcc slices. A) Forskolin-induced DOPA accumulation. Slices were incubated with NSD-1015 (1 mM) for 15 min before further incubation (30 min) in the presence and absence of forskolin (10 mM). Values are means ± s.e.m. of 5 replicates from a representative experiment repeated 7 times with similar results. ***, P < 0.001, Student's t test. B) Effect of the H3R agonist RAMH on forskolin-induced DOPA accumulation. Slices were incubated with forskolin (10 mM) in the presence and the absence of the indicated concentrations of RAMH, added 5 min before. Values are expressed as a percentage of the effect of forskolin, after basal subtraction, and are means ± s.e.m. from 3 to 5 determinations for each concentration. C) Blockade by the H3R antagonist/inverse agonist ciproxifan of the effect of RAMH on forskolin-induced DOPA accumulation. Slices were incubated with forskolin (10 mM) in the presence and the absence of RAMH (100 nM), added 5 min before. Where required ciproxifan (cipro, 10 mM) was added 5 min before RAMH. Values are means ± s.e.m. from 5 experiments. For experiments illustrated in panels B and C the statistical analysis was performed with ANOVA and Dunnett's test; *, P < 0.05, ns, non-significantly different from controls.

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Fig. 5. Effect of H3R activation on IBMX-induced DOPA accumulation in rNAcc slices. A) Representative experiment. Slices were incubated with NSD-1015 (1 mM) for 15 min before further incubation (15 min) in the presence and absence of IBMX (1 mM). Where required the H3R agonist RAMH (100 nM) or the D2R agonist quinpirole (10 mM) were added 5 min before IBMX. Values are means ± s.e.m. from 4 replicates. *, P < 0.05, ***, P < 0.001, when compared with IBMX alone, ANOVA and Tukey's test. B) Analysis of 4 experiments. Values are expressed as a percentage of the effect of IBMX, after basal subtraction, and are means ± s.e.m. *, P < 0.05, ***, P < 0.001, when compared with IBMX alone, ANOVA and Dunnett's test.

Fig. 6. Effect of H3 or D2 receptor activation on DOPA accumulation induced by 8-bromo-cAMP (Br-cAMP) in rNAcc slices. Slices were incubated with NSD-1015 (1 mM) for 15 min before further incubation (30 min) in the presence and absence of 8-Br-cAMP (1 mM). Where required the H3R agonist RAMH (100 nM) or the D2R agonist quinpirole (10 mM) were added 5 min before 8-Br-cAMP. A) Representative experiment for RAMH. Values are means ± s.e.m. from 4 replicates. B) Analysis of 4 experiments with RAMH. Values are expressed as a percentage of basal DOPA accumulation and are means ± s.e.m. C) Representative experiment for quinpirole. Values are means ± s.e.m. from 5 replicates. D) Analysis of 3 experiments with quipirole. Values are expressed as a percentage of basal DOPA accumulation and are means ± s.e.m. For all panels, the statistical analysis was performed with ANOVA and Tukey's test; ns, non-significantly different, *, P < 0.05; **, P < 0.01.

significantly reduced forskolin-induced DOPA accumulation (
41.1 ± 6.5% and
43.5 ± 9.1%, respectively). The inhibitory effect of 100 nM RAMH was prevented by the H3R antagonist/inverse agonist ciproxifan (10 mM; Fig. 4C), indicating a receptor-mediated action. Likewise for release experiments, ciproxifan itself had no effect on forskolin-induced DOPA accumulation (Fig. 4C) or basal

accumulation (101.0 ± 11.3% of controls, 3 experiments, P ¼ 0.927, Student's t test; data not illustrated), supporting that H3Rs present on rNAcc dopaminergic terminals lack constitutive activity. In a different series of experiments, the D2R agonist quinpirole (10 mM) had no effect on basal DOPA accumulation (91.0 ± 6.2% of control values, P > 0.05, one-way ANOVA and Tukey's test, 4

Please cite this article in press as: Aquino-Miranda, G., et al., Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.07.006

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Fig. 7. Effect of H3R activation on DOPA accumulation induced by PACAP-27 in rNAcc slices. Slices were incubated with NSD-1015 (1 mM) for 5 min before further incubation (30 min) in the presence and absence of PACAP-27 (1 mM); where required the H3R agonist RAMH (100 nM) was added 5 min before PACAP-27. A) Representative experiment. Values are the means ± s.e.m. from 5 replicates for each condition. B) Analysis of 4 experiments. Values are expressed as a percentage of basal DOPA accumulation, and are means ± s.e.m. For both panels the statistical analysis was performed with ANOVA and Dunnett's test; **, P < 0.01, ns, non-significantly different from basal accumulation.

experiments) but significantly reduced the stimulatory action of forskolin by 52.7 ± 9.8% (P < 0.05). The concentration of quinpirole employed was selected for maximal receptor activation on the basis of the concentrationeresponse curves reported for inhibition of forskolin-stimulated TH activity in rNAcc homogenates (Onali and Olianas, 1989) or electrically-evoked dopamine release from rNAcc slices (Yamada et al., 1994). In 2 experiments, blockade of D2Rs with sulpiride (1 mM) enhanced forskolin-stimulated DOPA accumulation (175.0 ± 27.9% of forskolin alone after basal subtraction, mean ± range, data not illustrated), indicating D2 autoreceptor activation by endogenous dopamine. In the presence of sulpiride, the H3R agonist RAMH (1 mM) reduced the effect of forskolin by 49.3 ± 11.7% (mean ± range), a value similar to its effect in the absence of the D2R antagonist (43.5 ± 9.1%, see above). Intracellular levels of cAMP are determined by the rate of synthesis by ACs and the rate of degradation by the isoforms of cyclic nucleotide phosphodiesterases expressed by a particular type of cell (Sunahara and Taussig, 2002). Incubation with the general phosphodiesterase inhibitor IBMX (1 mM) significantly increased DOPA accumulation in rNAcc slices to 324 ± 53% of control values (Fig. 5A), suggesting that ACs present in rNAcc dopaminergic nerve terminals possess tonic activity. Activating H3Rs with RAMH (100 nM) inhibited in a modest but significant manner IBMXinduced DOPA accumulation (81.6 ± 2.2% of control values, Fig. 5B). In parallel determinations, the D2R agonist quinpirole (10 mM) markedly reduced the effect of IBMX on DOPA accumulation (23.7 ± 11.5% of control values; Fig. 5A, B). Incubation of rNAcc slices with the membrane-permeant cAMP analog 8-Br-cAMP (1 mM) increased DOPA accumulation to 223 ± 28% of control values (Fig. 6A), but this action was not affected by the H3R agonist RAMH (100 nM; Fig. 6B). In a different series of experiments, the effect of 8-Br-cAMP was significantly reduced by D2R activation with quinpirole (10 mM;
45.2 ± 10.4%; Fig. 6C, D). Receptors for the vasoactive intestinal (VIP) and pituitary ACactivating peptides (PACAPs) couple to Gas proteins and are activated by endogenous ligands that include VIP, PACAP-38 and PACAP-27 (Alexander et al., 2013). Significant amounts of PACAP-38 are found in the NAcc (Vaudry et al., 2000), and in rNAcc homogenates PACAP-27 increased DOPA accumulation with EC50 ~100 nM (Moser et al., 1999). Fig. 7 shows that in rNAcc slices PACAP-27 (1 mM) significantly increased DOPA accumulation to 148.9 ± 10.0% of basal values (Fig. 7A), and that H3R activation with

RAMH (100 nM) prevented the PACAP-27 effect (Fig. 7A, B). None of the drugs that stimulated DOPA accumulation altered dopamine levels measured in the same samples, indicating full inhibition by the decarboxylase inhibitor NSD-1015 (1 mM) of the conversion of DOPA to dopamine (control values 508.5 ± 45.3 pmol/ mg protein, 38 experiments; forskolin, 103.9 ± 5.9%, P ¼ 0.513, n ¼ 20; IBMX, 109.6 ± 3.7%, P ¼ 0.062, n ¼ 3; 8-Br-cAMP, 103.6 ± 4.9% of control values, P ¼ 0.488, n ¼ 4; PACAP-27, 106.5 ± 9.3%, P ¼ 0.500, n ¼ 7; Student's t test). 4. Discussion The NAcc synaptic circuitry is modulated by dopamine released from axons of VTA neurons (Sesack and Grace, 2010). The NAcc is also innervated by histaminergic fibers (Inagaki et al., 1988) and we show herein that in rNAcc H3R activation inhibits dopamine synthesis but not its uptake or depolarization-evoked release. This action supports a role for histamine in the modulation of brain limbic functions. 4.1. Lack of effect of H3R activation on [3H]-dopamine uptake by rNAcc synaptosomes The potent inhibition by GBR-12909 and the weak inhibitory effect of both desipramine and fluoxetine indicate that [3H]-dopamine uptake by synaptosomes, and therefore [3H]-dopamine release from slices, take place in NAcc dopaminergic nerve terminals, and exclude that noradrenergic or serotonergic terminals were responsible, at least to some extent, for [3H]-dopamine uptake and release. In mouse striatum Gbg complexes interact physically with the carboxyl terminus of the dopamine transporter DAT and in HEK293 cells overexpression of Gb1g2, the most common Gbg dimer expressed in the brain, reduced the activity of the transfected human DAT. Further, direct activation of Gbg subunits by a cellpermeant myristoylated peptide (mSIRK) reduced both [3H]dopamine uptake by rat striatal synaptosomes and dopamine clearance in mouse striatum in vivo (Garcia-Olivares et al., 2013). It was therefore conceivable that upon H3R activation, Gbg complexes released from Gai/o proteins could inhibit [3H]-dopamine uptake by rNAcc synaptosomes. However, the lack of significant effect of the agonist RAMH on [3H]-dopamine uptake indicates that H3R activation does not modulate dopamine transport by the terminals of VTA neurons.

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4.2. Lack of effect of H3R activation on depolarization-evoked [3H]dopamine release Our data showed that H3R activation had no significant effect on Kþ-evoked [3H]-dopamine release from rNAcc slices. H3R-mediated inhibition of dopamine release has been observed in slices from mouse striatum (Schlicker et al., 1993) and rat SNr (Garcia et al., 1997). For mouse striatal slices both histamine and the agonist RAMH inhibited the electrically-evoked release of [3H]-dopamine (
18 ± 3% at 10 mM and
16 ± 6% at 1 mM, respectively), and the effect of histamine was significantly increased to 38 ± 4% by blocking D2 autoreceptors with haloperidol (Schlicker et al., 1993). In our experiments the D2R antagonist sulpiride significantly increased Kþ-evoked [3H]-dopamine release from rNAcc slices (168.8 ± 15.5% of control values) indicating that D2R activation by endogenous dopamine could have occluded the H3R action because both receptors signal through Gai/o proteins, but this possibility can be discarded by the lack of effect of H3R activation in the presence of sulpiride. The absence of effect of the H3R agonist RAMH on Kþ-evoked 3 [ H]-dopamine release could also be explained by endogenous histamine occupying H3Rs present on dopaminergic terminals or by receptor constitutive activity (Arrang et al., 2007) exerting a tonic inhibition on neurotransmitter release. However, the lack of effect of the antagonist/inverse agonist ciproxifan on depolarizationinduced [3H]-dopamine release argues against these two possibilities. 4.3. Inhibition by H3R activation of DOPA accumulation TH is the limiting enzyme in dopamine synthesis, and phosphorylation at Serine40 by PKA increases its catalytic activity (Dunkley et al., 2004). In rNAcc synaptosomes, forskolin increased TH phosphorylation at Serine40 and stimulated dopamine synthesis (Olianas et al., 2008), and in rNAcc homogenates both forskolin and dibutyryl-cAMP stimulated TH activity, with D2R activation inhibiting the effect of forskolin but not that of the cAMP analog. Further, rolipram, a phosphodiesterase inhibitor, stimulated TH activity and this effect was also inhibited by D2R activation (Onali and Olianas, 1989). Altogether, these data indicate that in NAcc dopaminergic terminals neurotransmitter synthesis is regulated by the cAMP/PKA pathway, which in turn is under the control of D2 autoreceptors. Our data show that H3R activation reduced by ~40% forskolininduced DOPA accumulation in rNAcc slices. For comparison, D2R activation reduced the effect of 10 mM forskolin on TH activity in NAcc homogenates and DOPA accumulation in NAcc slices by ~60% and 52.7%, respectively (Onali and Olianas, 1989, and this work). AC isoforms differ in their regulation by Gai/o subunits and the partial inhibition of TH activity by H3R activation could therefore be explained by the expression by dopaminergic neurons of AC isoforms sensitive (AC1,3,5,6,8,9) and insensitive (AC2,4,7) to the action of Gai/o proteins (Willoughby and Cooper, 2007) to which the H3R is coupled. In line with the study by Onali and Olianas (1989), in rNAcc slices inhibition of cAMP degradation by IBMX stimulated DOPA accumulation and this effect was inhibited by activating D2Rs and, to a lesser extent, by H3R activation. Taken together these data indicate that in rNAcc dopaminergic terminals H3 heteroreceptors can modulate dopamine synthesis by inhibiting cAMP formation and thus PKA activation. This conclusion is supported by the lack of effect of H3R activation on DOPA accumulation induced by the cAMP analog 8-Br-cAMP, which by binding to the PKA regulatory subunits circumvents the requirement of AC activation for PKA stimulation (Christensen et al., 2003). Unexpectedy, in rNAcc slices D2R activation partially inhibited DOPA accumulation stimulated by 8-Br-cAMP. TH phosphorylation

9

at Serine40 is reduced by the activity of protein phosphatases (PP) 2A and 2C (Dunkley et al., 2004), and in mouse striatal slices D2R activation reduces the phosphorylation of spinophilin at Serine94 and PP-2A inhibition prevents this effect (Uematsu et al., 2005). D2R-mediated stimulation of PP-2A or PP-2C through a mechanism to be established could thus explain the effect on DOPA accumulation induced by 8-Br-cAMP. In rat striatal neurons D2R receptor stimulation also activates the Ca2þ-dependent phosphatase calcineurin or PP-2B (Hernandez-Lopez et al., 2000), but this phosphatase appears inactive at TH Serine40 (Dunkley et al., 2004). In contrast to our data, in rNAcc homogenates D2R activation had no effect on TH activity stimulated by dibutyryl-cAMP (Onali and Olianas, 1989). This discrepancy could be due to intracellular signaling mechanisms preserved in rNAcc slices but not in the homogenate preparation. In our experiments DOPA accumulation in rNAcc slices was stimulated by inducing cAMP formation, preventing cAMP hydrolysis or by a cAMP analog, maneuvers that are not physiological. In rNAcc homogenates PACAP-27, an agonist at VIP/PACAP receptors, coupled to Gas proteins, increased DOPA accumulation in an ATPdependent manner and this effect was potentiated by phosphodiesterase inhibition and reduced by protein kinase inhibition (Moser et al., 1999), indicating a cAMP/PKA-mediated action. In rNAcc slices H3R activation also prevented DOPA accumulation induced by PACAP-27, supporting a physiological role for histamine in the modulation of dopamine synthesis. In this regard, bilateral electrolytic lesion to the rat tuberomammillary nucleus decreased histamine levels in NAcc and increased the DOPAC/dopamine ratio, an effect reversed by the systemic administration of L-histidine, indicating that endogenous histamine reduces NAcc dopaminergic activity (Gong et al., 2007). As mentioned above, H3R-mediated inhibition of [3H]-dopamine release has been observed in slices from mouse striatum (Schlicker et al., 1993) and rat SNr (Garcia et al., 1997), but not in rat striatal slices (Smits and Mulder, 1991; and unpublished results of our own) and rNAcc slices (this work). Noteworthy, in the article by Schlicker et al. (1993) the authors mention that the effect of H3R activation on [3H]-dopamine release reported for mouse striatum was not reproduced in rat striatal slices. On the other hand, H3R activation does inhibit dopamine synthesis in slices of rat striatum
ndez et al., 2000; Gonza
lez-Sepúlveda et al., 2013) (Molina-Herna and rNAcc (this study). The activation of H3Rs results in two main intracellular actions, reduction of cAMP formation through Gai/o subunits (Bongers et al., 2007) and inhibition of voltage-operated calcium channels (Takeshita et al., 1998; Molina-Hernandez et al., 2001) most likely mediated by Gbg dimers (De Waard et al., 2005). Because in rNAcc slices the effect of H3R activation on dopamine synthesis appears to rely on the inhibition of the cAMP/PKA pathway, the discrepancy between the effects on dopamine synthesis and release may be explained by pre-synaptic H3 heteroreceptors being located away from the calcium channels involved in neurotransmitter release, but close to the ACs responsible for cAMP formation. In several areas of the rat brain such as cerebral cortex, hippocampus, cerebellum and olfactory bulb, presynaptic H3Rs regulate noradrenaline release (Feuerstein, 2008; Osorio-Espinoza et al., 2010; Panula and Nuutinen, 2013; Aquino-Miranda et al., 2012). The NAcc receives noradrenergic projections (Sesack and Grace, 2010), and the inhibition of DOPA accumulation reported herein could have occurred thus in noradrenergic nerve terminals endowed with H3Rs. However, the extent of the noradrenergic innervation is markedly lower than that by dopaminergic terminals, as judged by the at least 40-fold difference in the content of noradrenaline and
mez-Milane
s et al., 2012). dopamine found for the mouse NAcc (Go Therefore our results appear to reflect primarily an action of H3R

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activation on neurotransmitter synthesis by dopaminergic nerve terminals. Yanovsky et al. (2011) reported that a subpopulation of histaminergic neurons can take up L-DOPA and subsequently synthesize and release dopamine. It would then be possible that H3 autoreceptors actually modulate dopamine synthesis taking place in the histaminergic terminals that innervate the NAcc. However, these histaminergic neurons lack TH and therefore only synthesize dopamine when provided with L-DOPA, most likely released from catecholaminergic nerve terminals (Yanovsky et al., 2011). Because our evaluation of dopamine synthesis was based on the determination of DOPA accumulation after inhibition of DOPA decarboxylase, this information supports that the effect reported herein occurs mainly if not exclusively in the dopaminergic terminals originated in the VTA. Further, the expression of H3Rs on the NAcc histaminergic terminals appears to be very low and their activation does not modulate histamine release (Giannoni et al., 2010; Munari et al., 2013). 4.4. Conclusion We have herein provided evidence that in rNAcc H3R activation regulates dopamine synthesis, but not release or uptake. Dopamine plays a key role in the processing of synaptic information in the NAcc, and our results indicate that through its action at H3Rs histamine acts as a metamodulator of dopaminergic transmission by reducing the amount of neurotransmitter available for release. Author contributions G.A.-M. and J.-A.A.-M. designed the study; G.A.-M., A.B.-N., J.E.-S. and R.G.-P. conducted experiments; G.A.-M., A.B.-N. and J.-A.A.-M. performed data analysis. G.A.-M., A.B.-N., and J.-A.A.-M. wrote the manuscript. All authors revised and approved the manuscript. Acknowledgments Supported by Cinvestav and Conacyt (grant 220448 to J.-A.A.M.). G.A.-M. held a Conacyt M.Sc. scholarship. We are grateful to
noma de BarDr. Jordi Ortiz and Dr. Santi Rosell (Universidad Auto
Ayala celona) for valuable ideas and criticisms. We thank Biol. Jose
vila for technical advice. The authors declare that the research Da was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funding sources were not involved at all in the study design, collection, analysis and interpretation of data, writing of the manuscript or the decision to submit this report. References Alexander, S.P.H., et al., 2013. The concise guide to pharmacology 2013/14. Br. J. Pharmacol. 170, 1449e1867. Alfaro-Rodriguez, A., Alonso-Spilsbury, M., Arch-Tirado, E., Gonzalez-Pina, R., Arias~ o, J.A., Bueno-Nava, A., 2013. Histamine H3 receptor activation prevents Montan dopamine D1 receptor-mediated inhibition of dopamine release in the rat striatum: a microdialysis study. Neurosci. Lett. 552, 5e9.
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Please cite this article in press as: Aquino-Miranda, G., et al., Histamine H3 receptor activation inhibits dopamine synthesis but not release or uptake in rat nucleus accumbens, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.07.006