Pre-synaptic histamine H3 receptors modulate glutamatergic transmission in rat globus pallidus

Neuroscience 176 (2011) 20 –31

PRE-SYNAPTIC HISTAMINE H3 RECEPTORS MODULATE GLUTAMATERGIC TRANSMISSION IN RAT GLOBUS PALLIDUS A. OSORIO-ESPINOZA,a A. ALATORRE,b J. RAMOS-JIMÉNEZ,a B. GARDUÑO-TORRES,a M. GARCÍA-RAMÍREZ,c E. QUEREJETAb AND J.-A. ARIAS-MONTAÑOa*

Histamine released from the nerve terminals of neurons located in the hypothalamus tuberomammillary nucleus regulates a variety of mammalian brain functions including sleep-wake rhythmicity, attention and learning as well as different aspects of body homeostasis (Haas et al., 2008). Histamine actions are mostly exerted through the activation of G protein-coupled receptors. Four such receptors (H1–H4) have been characterized to date, and three of them (H1, H2 and H3) are widely expressed in the brain (Haas and Panula, 2003; Haas et al., 2008). Histamine H3 receptors (H3Rs) are mainly expressed on nerve terminals and control the release and synthesis of histamine as well as the release of several other neuroactive substances namely acetylcholine, noradrenaline, dopamine, 5-hydroxytryptamine (5-HT), glutamate and ␥-aminobutyric acid (Leurs et al., 2005; Haas et al., 2008; Feuerstein, 2008). The globus pallidus (GP) belongs to the basal ganglia, a group of sub-cortical neuronal nuclei involved in the control of motor behavior (Bolam et al., 2000). Several lines of research have led to compelling evidence that the GP plays a unique role in basal ganglia physiology, and its dysfunction appears to constitute a central origin of motor disorders, including Parkinson’s disease, in which alterations in the pattern and synchrony of discharge of pallidal neurons have been reported (Chan et al., 2005). The GP is mainly made up by two populations of GABAergic neurons, with the predominant one projecting to the subthalamic nucleus while a second group sends projections to the striatum (Bolam et al., 2000; Chan et al., 2005). In turn, the main synaptic afferents to GP are striato-pallidal GABAergic axons (Bolam et al., 2000), glutamatergic fibers originated in the subthalamic nucleus and, to a lesser extent, in the cerebral cortex and thalamus (Naito and Kita, 1994; Bevan et al., 2002), and dopaminergic afferents from substantia nigra pars compacta (Anaya-Martinez et al., 2006). In spite of a modest histaminergic innervation (Panula et al., 1989) the rat GP expresses a high density of H3Rs, and the very low levels of the corresponding mRNA (Pillot et al., 2002a) indicate that the vast majority, if not all, of such receptors are located on the nerve terminals of neurons projecting to the nucleus. In line with the above, H3R mRNA is expressed by neurons intrinsic to those nuclei that provide the main synaptic afferents to GP (Pillot et al., 2002a,b). Very little is known in regard to the function of pallidal H3Rs, but these receptors could regulate GP synaptic flow at the pre-synaptic level, as shown for other brain regions such as the striatum (Doreulee et al., 2001) and thalamus (Garduno-Torres et al., 2007). Therefore, in this work we set out to study the effect of H3R activation on

a Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional 2508, Zacatenco, 07360 México, D.F., México b Departamento de Fisiología, Escuela Superior de Medicina (ESM), IPN; Plan de Ayala y Carpio s/n, 11340 México, D.F., México c

Departamento de Fisiología, Escuela Nacional de Ciencias Biológicas (ENCB), IPN; Plan de Ayala y Carpio s/n, 11340 México, D.F., México

Abstract—The globus pallidus, a neuronal nucleus involved in the control of motor behavior, expresses high levels of histamine H3 receptors (H3Rs) most likely located on the synaptic afferents to the nucleus. In this work we studied the effect of the activation of rat pallidal H3Rs on depolarizationevoked neurotransmitter release from slices, neuronal firing rate in vivo and turning behavior. Perfusion of globus pallidus slices with the selective H3R agonist immepip had no effect on the release of [3H]-GABA ([3H]-␥-aminobutyric acid) or [3H]-dopamine evoked by depolarization with high (20 mM) Kⴙ, but significantly reduced [3H]-D-aspartate release (ⴚ44.8ⴞ2.6% and ⴚ63.7ⴞ6.2% at 30 and 100 nM, respectively). The effect of 30 nM immepip was blocked by 10 ␮M of the selective H3R antagonist A-331440 (4=-[3-[(3(R)-dimethylamino-1-pyrrolidinyl]propoxy]-[1,1-biphenyl]-4=-carbonitrile). Intra-pallidal injection of immepip (0.1 ␮l, 100 ␮M) decreased spontaneous neuronal firing rate in anaesthetized rats (peak inhibition 68.8ⴞ10.3%), and this effect was reversed in a partial and transitory manner by A-331440 (0.1 ␮l, 1 mM). In free-moving rats the infusion of immepip (0.5 ␮l; 10, 50 and 100 ␮M) into the globus pallidus induced dose-related ipsilateral turning following systemic apomorphine (0.5 mg/kg, s.c.). Turning behavior induced by immepip (0.5 ␮l, 50 ␮M) and apomorphine was partially prevented by the local injection of A-331440 (0.5 ␮l, 1 mM) and was not additive to the turning evoked by the intra-pallidal injection of antagonists at ionotropic glutamate receptors (0.5 ␮l, 1 mM each of AP-5, DL-2-amino-5-phosphonovaleric acid, and CNQX, 6-nitro-7sulphamoylbenzo[f]quinoxaline-2,3-dione). These results indicate that pre-synaptic H3Rs modulate glutamatergic transmission in rat globus pallidus and thus participate in the control of movement by basal ganglia. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: histamine, H3 receptor, globus pallidus, basal ganglia, glutamate release. *Corresponding author. Tel: ⫹5255-5747-3964; fax: ⫹5255-5747-3754. E-mail address: [email protected] (J.-A. Arias-Montaño). Abbreviations: GABA, ␥-aminobutyric acid; GP, globus pallidus; H3R, histamine H3 receptor; [3H]-NMHA, N-␣-[methyl-3H]histamine.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.12.051

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rat GP synaptic transmission by analyzing the depolarization-evoked release of labeled neurotransmitters ([3H]-␥aminobutyric acid, [3H]-GABA; [3H]-dopamine; [3H]-D-aspartate) from slices, changes in the firing frequency of GP neurons in vivo, and the induction of turning behavior following intra-pallidal injection of H3R ligands and the systemic administration of apomorphine.

EXPERIMENTAL PROCEDURES Animals Wistar male rats (bred in Cinvestav facilities) were maintained under thermo-regulated (22⫾2 °C) and light-controlled conditions (light on 6 –18 h), with food and water ad libitum. All procedures used in this study were reviewed and approved by the Animal Care Committees of Cinvestav, ENCB and ESM. All efforts were made to minimize animal suffering and to use only as many animals were required for proper statistical analysis.

Preparation of GP slices Animals were killed by decapitation, the brain was quickly removed from the skull, immersed in ice-cold Krebs-Henseleit (KH) solution and coronal slices (350 ␮m thick) were obtained with a vibratome (Campden Instruments, Loughborough, UK). GP discs (2-mm diameter) were punched out from the slices, avoiding the adjacent striatal tissue which contains high levels of H3Rs. The composition of the KH solution was (mM): NaCl, 116; KCl, 3; MgSO4, 1; KH2PO4, 1; NaHCO3, 25; CaCl2, 1.8; D-glucose, 11; pH 7.4 after saturation with O2/CO2 (95:5% v:(v).

Binding of N-␣-[methyl-3H]histamine ([3H]-NMHA) to GP membranes GP punches (circa 45 discs from six animals per experiment) were frozen on dry ice immediately after preparation as above. The slices were then placed in 6 ml 10 mM Tris–HCl buffer containing 1 mM EGTA (pH 7.4) and homogenized using 12 strokes of a hand-held homogenizer. The homogenate was brought up to 20 ml and centrifuged (20,000⫻g, 20 min at 4 °C). The pellet thus formed was re-suspended in 20 ml 50 mM Tris–HCl (pH 7.4) and centrifuged again. The resulting pellet (crude-membrane preparation) was re-suspended in incubation buffer (50 mM Tris–HCl, 5 mM MgCl2, pH 7.4). Saturation analysis was carried out in 0.25 ml buffer containing [3H]-NMHA (0.01–12 nM) and ⬃75 ␮g protein (as determined by the bicinchoninic acid method), whereas for inhibition experiments incubations contained [3H]-NMHA (⬃1.5 nM) and increasing concentrations (10⫺11⫺10⫺5 M) of H3R ligands. Equilibration was for 90 min at 30 °C and terminated by filtration through Whatman GF/B glass fiber paper, pre-soaked in 0.3% polyethylenimine. Non-specific binding was determined as that insensitive to 10 ␮M histamine and accounted for 25–30% of total binding. Filters were soaked in 5 ml scintillator and the tritium content was determined by scintillation counting. Saturation binding data were fitted to a hyperbola by nonlinear 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⫽IC50/1⫹{[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 saturation analysis (0.58 nM, see results).

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Depolarization-evoked release of labeled neurotransmitters from GP slices After preparation as described in section 2.2, GP punches (six to eight discs from each of five to six rats, 40 discs per experiment) were equilibrated for 30 min at 37 °C in 100 ml of KH solution gassed continuously with O2/CO2 (95:5, v/v), with medium changes every 10 min. Slices were then transferred to 2.5 ml KH solution containing one of the following transmitters: [3H]-GABA (40 nM), [3H]-dopamine (80 nM) or [3H]-D-aspartate (100 nM). To prevent degradation of the tritiated neurotransmitters, the labeling solutions were supplemented with 10 ␮M aminooxyacetic acid ([3H]-GABA) or 10 ␮M pargyline and 500 ␮M ascorbic acid ([3H]dopamine). For [3H]-D-aspartate uptake the labeling solution contained dihydrokainic acid (500 ␮M) which by blocking the glutamate transporter-1 (GLT-1) (Kd 23 ␮M) (Wang et al., 1998) prevents [3H]-D-aspartate incorporation into glial cells (Kawahara et al., 2002). After 30 min at 37 °C, slices were washed several times with KH solution and then apportioned randomly between the chambers of a superfusion apparatus (20 chambers in parallel; two slices per chamber) and perfused with KH medium (0.5 ml/ min) for 30 min. For [3H]-GABA release, the perfusing solution contained 10 ␮M aminooxyacetic acid and 10 ␮M nipecotic acid (inhibitor of GABA uptake). For [3H]-dopamine release, the perfusion solution was supplemented with 10 ␮M pargyline, 500 ␮M ascorbic acid and 10 ␮M nomifensine (inhibitor of dopamine uptake). Pallidal dopamine D2-like receptors control in an inhibitory manner GABA release from striato-pallidal afferents (Floran et al., 1997) and dopamine release from nigro-striatal axons (L’Hirondel et al., 1998) which provide collaterals to GP (Anaya-Martinez et al., 2006). To prevent the effect of endogenous dopamine released upon depolarization, for [3H]-GABA and [3H]-dopamine release experiments perfusion solutions used after the wash-out period contained the D2-like receptor antagonist sulpiride (10 ␮M). Basal neurotransmitter release was measured by collecting four fractions of the superfusate at 2-min intervals (i.e. 1-ml fractions) before release was stimulated by changing to a solution containing 20 mM K⫹ (KCl substituted for NaCl) and a further six fractions collected. Drugs under test were present 4 min before and throughout the perfusion with high K⫹-medium (fractions 3–10). For experiments where the Ca2⫹-dependence of neurotransmitter release was studied, labeled GP slices were perfused with KH solution with no CaCl2 added for 15 min before and throughout the collection of fractions (i.e. four basal fractions and six high-K⫹ fractions). The tritium content in the superfusate fractions was determined by scintillation counting. To determine the total amount of tritium remaining in the tissue, the contents of each chamber were collected and treated with 1 ml HCl (1 M) for 1 h before addition of scintillator. The release of labeled neurotransmitters was initially expressed as a fraction of release: (tritium in fraction n)/{(tritium in fraction n)⫹(tritium in all subsequent fractions)⫹(tritium remaining in the tissue)}. Basal release values were usually in the range 0.002– 0.008, that is, 0.2– 0.8% of total tritium content. To allow for variations between chambers, fractional values were then transformed to a percentage of the fraction collected immediately before the change to the medium containing 20 mM K⫹ (i.e. the release in that fraction was set to 100%). To test for statistical differences between treatments, the area under the release curve after the change to high K⫹ (basal release subtracted) was calculated for each individual chamber and the data then analyzed as described previously (Garcia et al., 1997). All calculations were based on the radioactivity content expressed as disintegrations per minute (dpm) after automatic correction of counts per min (cpm) values according to the counting efficiency for individual samples.

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In vivo measurement of the firing rate of GP neurons Rats (180 –220 g) were anesthetized with chloral hydrate (300 mg/kg, i.p.; supplemented as needed) and positioned in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). A heating pad and rectal thermometer system was employed to maintain the body temperature at 36 –38 °C. A 4 mm burr hole was drilled into the skull to allow the stereotactical guide of glass electrodes and of a stainless-steel double-cannula system (30gauge syringe needles) into the right GP. The tips of both cannulae were separated by 0.1 mm. The recording electrode was placed following the co-ordinates (Querejeta et al., 2005): 0.2 mm posterior to bregma, 3.0 –3.3 mm lateral to the midline and 5– 6 mm ventral to the brain surface according to a rat brain atlas (Paxinos and Watson, 1998). The injection cannulae were placed at an angle of 70° in the latero-medial direction to the following coordinates: 1.1 mm posterior to bregma, 5.7 mm lateral to the midline and 5.6 mm ventral to the brain surface. Standard extracellular recordings were performed using glass electrodes (2– 6 M⍀) filled with 2 M NaCl solution containing 2% Pontamine Sky Blue Dye. Extracellular signals were amplified 10,000-fold, band-pass filtered between 0.3 and 3 kHz (DAM-80 amplifier; WPI, Saratosa, FL, USA) and stored on an audiocassette device. Single-unit activity was isolated using a window discriminator (WPI-121), spike times were pre-processed on-line and further analyzed off-line using the INF-386 program for spike data analysis (Soto and Vega, 1987). Drugs were dissolved in sterile saline solution (pH 7.4) and infused into the GP using a 1-␮l syringe connected to a precision micrometer that allowed an infusion rate of 50 nl/15 s. The volume injected per individual drug infusion was 100 nl. Only neurons with 8 min of stable basal firing were selected for drug application (one neuron per animal). Basal firing was determined by the average frequency in the 60-s period before vehicle or drug injection. Drug or vehicle effects were quantified in the 30-s period following the injection. Our recordings showed basal firing rate to vary by 9.13⫾0.17% (mean⫾standard deviation, n⫽15) and therefore only cells in which drug injection resulted in a 20% change in firing rate (i.e. over twofold the standard deviation) were taken for further analysis, in line with work by Walters and colleagues in which changes of 15–20% over the mean basal firing were considered significant (Bergstrom et al., 1982; Bergstrom and Walters, 1984). A previous study by one of us showed locally applied drugs to diffuse within an area with estimated diameter 950⫾30 ␮m (Querejeta et al., 2005). Therefore, only neurons within a 470⫾30 ␮m radius from the tip of the injection cannula were considered adequately perfused and taken into account for further analysis. At the end of the experiment the position of the recorded unit was marked by dye ejection from the electrode by passing negative constant current (10 ␮A) for 20 min. A lethal overdose of pentobarbital was then administered and animals were intra-cardially perfused with 4% formaldehyde. Following overnight incubation of the brain in formaldehyde, coronal slices were prepared to verify the position of the recording electrode and the injection cannula.

Testing of turning behavior Rats (180 –210 g) were anaesthetized with ketamine/xylazine (80/8 mg/kg, i.p.) and positioned in a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA) with the incisor bar 8 mm above the horizontal plane. The skull was then exposed and a 10-mm length of a 23-gauge stainless steel tubing was implanted into the GP according to the following co-ordinates (in mm): 0 mm posterior to bregma, 3.0 mm lateral to the midline, and 4.5 mm ventral to the brain surface. These co-ordinates differ slightly from those reported above for in vivo recording the reason being the use of a different stereotaxic apparatus as the position of the injection cannula was also verified in brain slices after the administration of

a lethal overdose of pentobarbital, intra-cardial perfusion with 4% formaldehyde and overnight incubation of the brain in formaldehyde. The guide cannula was anchored to the skull with a stainless steel screw and dental acrylic cement, and a mandrel made with a 30-gauge stainless steel needle was placed into the cannula to prevent plugging. Cannula implantation was equally performed in either the left or the right GP. After the surgery animals were caged individually and a minimum of 5 days was allowed before the experiment. All experiments were carried out between 16 and 18 h. Animals were clothed with a plastic bandage connected to an automatic 10-channel rotameter, which registered rightward and leftward full turns separately. Rats were placed in individual hemispherical plastic bowls and allowed to adapt for 15 min before drug administration. Drugs were injected with a Hamilton 10 ␮lmicrosyringe connected via polyethylene tubing to a 30-gauge stainless-steel needle protruding 1 mm beyond the top of the guide cannula into the GP. The length of the tubing was such that it allowed drug injection without any movement restriction. Drugs were infused in a 0.5-␮l volume over 1 min and the infusion was monitored by following the movement of an air bubble introduced into the plastic tubing, allowing a further 3 min before the needle was removed. Apomorphine (0.5 mg/kg) was administered s.c. For time and treatment factors statistical analysis was carried out by two-way repeated measures (RM) analysis of variance (ANOVA) followed by the appropriate post-hoc test.

Drugs A-331440 (4=-[3-[(3(R)-dimethylamino-1-pyrrolidinyl]propoxy]-[1,1biphenyl]-4=-carbonitrile dihydrochloride), aminooxyacetic acid hemihydrochloride, apomorphine, ascorbic acid, clobenpropit dihydrobromide, dihydrokainic acid, histamine dihydrochloride, immepip dihydrobromide, (⫾)-nipecotic acid, pargyline hydrochloride, (⫾)-sulpiride and thioperamide maleate were purchased from Sigma Aldrich (Mexico City, Mexico). CNQX (6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione) and AP-5 (DL-2-amino-5phosphonovaleric acid) were purchased from Tocris (Ballwin, MO, USA). [2,3-3H]-␥-Aminobutyric acid ([3H]-GABA, specific activity 82 Ci/mmol) and [3H]-D-aspartate (35 Ci/mmol) were from Amersham (Piscataway, NJ, USA). N-␣-[methyl-3H]-histamine (85 Ci/ mmol) and [3H]-dopamine (48 Ci/mmol) were from PerkinElmer (Boston, MA, USA).

RESULTS 3

[ H]-NMHA binding to rat GP membranes Specific [3H]-NMHA binding to GP membranes yielded maximum binding (Bmax.) 162⫾29 fmol/mg protein (mean⫾ standard error, SEM; four determinations, Fig. 1A) and equilibrium dissociation constant (Kd) 0.58⫾0.09 nM, the latter in good agreement with values reported for rodent brain (Korte et al., 1990; Kathmann et al., 1993; Nickel et al., 2001). The H3R agonist immepip and the antagonists clobenpropit, thioperamide and A-331440 inhibited in a concentration-dependent manner [3H]-NMHA binding (Fig. 1B). For none of the drugs tested the fit was significantly improved by fitting the data to a two-site model rather than to a one-site model. This is the expected output for antagonists, but for the agonist immepip the result may be due to the number of experimental points used to build up the curve, because in membrane preparations from other rat

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Effect of H3R activation on depolarization-evoked neurotransmitter release from GP slices [3H]-GABA release. Changing the K⫹ concentration in the superfusion medium from 4 to 20 mM resulted in a marked increase in the release of [3H]-GABA from rat GP slices (mean stimulation 615⫾101% of basal at the peak of release, n⫽4; Fig. 2A). Previous work has shown K⫹evoked [3H]-GABA release to depend strongly on the presence of Ca2⫹ ions in the extracellular medium (Floran et al., 2005). The presence of the H3R agonist immepip (100 nM) in the perfusion medium had no significant effect on

Fig. 1. Binding of N-␣-[methyl-3H]histamine ([3H]-NMHA) to membranes from rat globus pallidus. (A) Saturation binding. Membranes were prepared as described in Experimental procedures and incubated with the indicated concentrations of [3H]-NMHA. Specific receptor binding was determined by subtracting the binding in the presence of 10 ␮M histamine from total binding. Points are means from triplicate determinations from a single experiment, which was repeated a further three 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 H3R ligands. Membranes were incubated with ⬃1.5 nM [3H]-NMHA and the indicated concentrations of H3R ligands. Values are expressed as the percentage of control specific binding and are means from triplicate determinations from a representative experiment. 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.

brain regions (v.gr. thalamus; Garduno-Torres et al., 2007) our experiments showed the presence of high- and lowaffinity sites, with the former likely to represent the ternary complex agonist-receptor-G protein (Park et al., 2008). Estimates for the inhibition constant (⫺log10 Ki, pKi) were: immepip 9.49⫾0.13 (n⫽8), clobenpropit 8.83⫾ 0.07 (n⫽7), thioperamide 7.75⫾0.16 (n⫽5) and A331440 7.90⫾0.07 (n⫽4). These values are in good agreement with those reported for cloned H3Rs (Leurs et al., 2005). However, we have found differences in affinities between rat brain regions and these determinations allowed thus for a more accurate calculation of receptor occupancy by agonists and antagonists in functional experiments.

Fig. 2. Lack of effect of H3R activation on depolarization-evoked [3H]-GABA or [3H]-dopamine release from rat globus pallidus slices. (A) [3H]-GABA release. The concentration of K⫹ in the perfusion medium was raised from 4 to 20 mM for the period indicated by the horizontal black bar. Where required, immepip (100 nM) was present for the period indicated by the gray horizontal bar. (B) [3H]-dopamine release. Perfusion with 20 mM K⫹ and immepip (100 nM) was as indicated by the black or gray horizontal bars, respectively. In both panels values are expressed as a percentage of the fractional release in fraction 4 and represent the means⫾SEM of 4 – 6 replicates from individual experiments, repeated a further three ([3H]-GABA) or two ([3H]-dopamine) times with similar results.

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depolarization-evoked [3H]-GABA release (93.0⫾4.1% of controls, P⬎0.05, student t test, n⫽4). [3H]-dopamine release. Depolarization with 20 mM K caused a significant increase in the release of [3H]dopamine from GP slices (198⫾8% of basal at the peak of release, n⫽18; Fig. 2B). K⫹-evoked [3H]-dopamine release from rat pallidal slices has been shown to be Ca2⫹dependent (Dewar et al., 1987). Perfusion with the H3R agonist immepip (100 nM) also failed to modify K⫹-evoked [3H]-dopamine release (93.9⫾6.4% of controls, P⬎0.05, student t test, n⫽3). ⫹

[3H]-D-aspartarte release. Fig. 3 shows that depolarization with 20 mM K⫹ caused a modest but significant increase in [3H]-D-aspartate release from GP slices (143.6⫾5.0% of basal at the peak of release, n⫽18). Perfusion with KH solution with no CaCl2 added markedly reduced K⫹-evoked [3H]-D-aspartate release (⫺70⫾8%, three determinations; Fig. 3A) as compared with slices

perfused with normal medium (1.8 mM CaCl2), 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 (data not shown). Fig. 3A also shows that the H3R agonist immepip (100 nM) significantly inhibited K⫹-evoked [3H]-D-aspartate release (⫺63.7⫾6.2%, n⫽3, P⬍0.05, student t test). In a different set of experiments (Fig. 3B) the inhibitory effect of 30 nM immepip (55.2⫾2.6% of control values, n⫽6; P⬍0.05, ANOVA and Student-Newman-Keuls post-hoc test) was blocked by the non-imidazole H3R antagonist A-331440 (10 ␮M, 89.1⫾5.3% of control values, P⬎0.05 when compared with control values; Fig. 3C, D). The extent of K⫹-evoked [3H]-D-aspartate release varied among determinations but both the immepip inhibition and the blockade by A-331440 were observed in each of six experiments as illustrated in Fig. 3C. Further, in two determinations the H3R antagonist A-331440 had no effect of its

Fig. 3. Effect of H3R activation on depolarization-evoked [3H]-D-aspartate release from globus pallidus slices. (A) Ca2⫹-dependence and inhibition by immepip. Labeled slices were perfused with KH medium (1.8 mM Ca2⫹ or no added CaCl2) before raising the K⫹ concentration in the perfusion medium from 4 to 20 mM for the period indicated by the horizontal black bar. Where required, immepip (100 nM) was present for the period indicated by the gray bar. Values are expressed as a percentage of the fractional release of [3H]-D-aspartate in fraction 4 and represent the means⫾SEM of 4 – 6 replicates from a representative experiment, repeated a further twice with similar results. (B) Blockade by the H3R antagonist A-331440. Perfusion with 20 mM K⫹ and drugs (30 nM immepip in the absence or presence of 10 ␮M A-331440) was as indicated by the black or gray horizontal bars, respectively Values are means⫾SEM of 4 – 6 replicates from an individual experiment, repeated a further five times with similar results. (C) Individual results from six experiments as in (B). The effect of drugs on K⫹-evoked [3H]-D-aspartate release was analyzed by comparing the areas under the appropriate release curves (arbitrary units, a.u.), after subtracting the basal release. (D) Statistical analysis of the data presented in (C). Values are means⫾SEM from the six experiments presented in (C). a P⬍0.05, ns, non-significantly different when compared with control release (no drugs added); b P⬍0.05 when compared with immepip alone; ANOVA and Student-Newman–Keuls post-hoc test.

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Fig. 4. Effect of the local administration of the H3R agonist immepip on the spontaneous firing rate of globus pallidus neurons. (A) Frequency histogram showing the effect of the intra-pallidal injection of the H3R agonist immepip (10 pmol/100 nl, 100 ␮M). A similar result was obtained in 12 further neurons recorded in different animals. (B) Expanded traces and interspike-interval histograms for basal firing and immepip inhibition from the neuron shown in panel (A). Basal firing and immepip effect were analyzed in 30-s periods before and after drug administration. Similar results were obtained in a further three neurons analyzed.

own on depolarization-evoked [3H]-D-aspartate release (data not shown). In order to block the inhibitory effect of immepip we first tested the classical H3R antagonist clobenpropit. However, at 10 ␮M (the concentration calculated to fully block the action of 30 nM immepip) clobenpropit markedly reduced by itself the release of [3H]-D-aspartate evoked by K⫹, an effect shared by thioperamide (data not shown), another imidazole-containing antagonist, and therefore likely to be related to the inhibition by micromolar concentrations of imidazole-containing compounds of P- and N-type voltageactivated calcium channels (Milhaud et al., 2002) involved in glutamate exocytosis (Vazquez and Sanchez-Prieto, 1997; Martin et al., 2007). The non-imidazole antagonist A-331440 was used instead throughout this work.

In four neurons the local injection of the H3R antagonist A-331440 (100 pmol/100 nl, 1 mM solution) once the inhibitory effect of immepip (100 ␮M; 39.7⫾2.7% of controls for this series of determinations) was established, brought the firing rate to values (76.9⫾5.8% of basal rate) significantly different from immepip alone (P⬍0.01, student t test), although the antagonist effect was transient (Fig. 5). Neither the H3R antagonist A-331440 (100 nl, 1 mM solution) nor an equal volume of saline solution had significant effect on spontaneous firing (102.1⫾1.2% and 101.9⫾2.3% of basal, respectively, mean⫾SEM) in five neurons recorded for each condition (data not illustrated).

Effect of intra-pallidal injection of the H3R agonist immepip on neuronal firing

The local administration of the H3R agonist immepip (5, 25 or 50 pmol) in 0.5 ␮l saline solution (i.e. 10, 50 or 100 ␮M, respectively) followed by systemic apomorphine (0.5 mg/ kg, s.c.) resulted in turning behavior, ipsilateral to the cannulated side, dose-dependent and significantly different from controls (Fig. 6A, F(3,18)⫽9.58, P⬍0.01 for differences among treatments; F(6,36)⫽33.58, P⬍0.01 for time course; F(18,18)⫽3.41, P⬍0.01 for treatment and time interactions; P⬍0.05 for 10, 50 and 100 ␮M immepip versus controls; two-way RM ANOVA and post hoc Student-Newman–Keuls test). Turning behavior reached a peak between 10 and 20 min after apomorphine administration, depending on the immepip dose, to decline after-

The mean spontaneous firing rate of the recorded GP neurons was 29.7⫾4.1 spikes/s (n⫽21; range 7– 68 spikes/s). Local infusion of immepip (10 pmol/100 nl, i.e. 100 ␮M solution) resulted in inhibition of the spontaneous neuronal firing in each of 13 neurons tested (Fig. 4A), with a peak inhibition of 68.8⫾10.3%. The inhibitory effect began shortly after the drug infusion with partial recovery in most cases (nine out of 13 neurons; 56.3⫾13.1% recovery). Panel B of Fig. 4 shows recordings for basal firing and immepip inhibition of the neuron illustrated in panel A and the corresponding inter-spike interval histograms.

Induction of turning behavior by the intra-pallidal injection of the H3R agonist immepip

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Fig. 5. Partial reversal by the H3R antagonist A-331440 of the inhibitory effect of immepip on the spontaneous firing rate of globus pallidus neurons. (A) Once the action of locally injected immepip (10 pmol/100 nl, 100 ␮M) was established, the administration of the H3R antagonist A-331440 (100 pmol/100 nl, 1 mM; as indicated by the bars) reversed in a partial and transient manner the agonist effect. Similar results were obtained in a further three neurons from different animals. (B) Statistical analysis. The effect of immepip in the presence or absence of A-331440 is expressed as a percentage of basal firing. Values are means⫾SEM from four neurons from different animals and were analyzed with student’s t-test. For A-331440 effect, values from the three applications showed in panel (A) were averaged and the resulting figures from the four neurons tested were then pooled.

wards to values not significantly different from controls after 50 – 60 min. In a different set of determinations, the intra-pallidal injection of the H3R antagonist A-331440 (0.5 ␮l, 1 mM) after immepip (0.5 ␮l, 50 ␮M) and apomorphine resulted in a reduction in turning behavior as compared with animals injected with immepip alone (Fig. 6B). The effect was statistically significant in the first two 10-min periods, that is, for 20 min after apomorphine administration (F⫽32.07, P⬍0.01 for time; P⫽0.04 at 10 min and P⫽0.03 at 20 min after apomorphine, two-way RM ANOVA and StudentNewman–Keuls post-hoc test). The intra-pallidal injection of the glutamate ionotropic receptor antagonists AP-5 and CNQX has been shown to produce ipsilateral turning behavior (Chen et al., 2002). In accord with this observation, in a different set of experiments the local injection (0.5 ␮l) of a mixture containing 1 mM of each antagonist resulted in ipsilateral turning behavior, significantly different from controls, following apomorphine administration (Fig. 6C, F(1,18)⫽91.12, P⬍0.01 for differences among treatments; F(8,5)⫽8.12, P⬍0.01 for time course; and F(1,5)⫽8.12, P⬍0.01 for treatment and time interactions, two-way RM ANOVA and StudentNewman–Keuls test). The co-administration of AP-5/ CNQX and the H3R agonist immepip (100 ␮M) in a 0.5 ␮l-volume (Fig. 6C) did not result in an additive effect (F⫽0.36, DF⫽5, P⫽0.66 for treatment and F⫽0.70 for treatment and time interaction, two-way RM ANOVA).

DISCUSSION There is increasing evidence for a pivotal role of GP in basal ganglia physiology and pathophysiology (Chan et al., 2005). Out of the three types of GP neurons characterized on the basis of their electrophysiological and morphological properties, type B and a subpopulation of type A neurons fire spontaneous action potentials (Cooper and Stanford, 2000), and this activity is modulated by glutamatergic afferents (Plenz and Kital, 1999; Magill et al., 2000, 2001). For instance, in anaesthetized rats the intra-pallidal infusion of the AMPA ((RS)-␣-amino-3-hydroxy-5-methyl-4isoxazole propionic acid)/kainate antagonist 2,3-dihydroxy-

6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) markedly reduced neuronal firing (Soltis et al., 1994) and in awake monkeys local infusion of AMPA/kainate or N-methyl-Daspartate (NMDA) antagonists, or muscimol blockade of the glutamatergic subthalamic nucleus-GP input decrease the firing rate of external GP neurons (Kita et al., 2004). The experimental data reported herein indicate that in turn GP glutamatergic transmission is modulated by histamine through the activation of pre-synaptic H3Rs. Effect of histamine H3R activation on depolarizationevoked neurotransmitter release from GP slices GABAergic striato-pallidal neurons, the main source of GP afferents, express high levels of H3R mRNA (Ryu et al., 1994; Pillot et al., 2002b) making GABA release a likely target for the action of pallidal H3Rs, as shown for striatonigral neurons (Garcia et al., 1997; Arias-Montano et al., 2001, 2007). However, we failed to observe any significant effect of H3R activation on [3H]-GABA release from GP slices. On the other hand, H3Rs appear to modulate the activity of striato-pallidal neurons at the striatal level because their activation decreases dopamine D2-like receptor-induced locomotor activity in reserpinized mice (Ferrada et al., 2008), an effect that may rely on the antagonistic H3/D2 receptor intra-membrane interaction shown to occur in sheep striatal homogenates and in transfected cells in the same study. The activity of pallidal neurons is modulated by D2-like receptors by reducing GABAergic inhibition pre- and postsynaptically (Cooper and Stanford, 2001; Shin et al., 2003). Because H3R activation decreases dopamine release from terminals (Schlicker et al., 1993) and dendrites (Garcia et al., 1997) of nigro-striatal neurons, which provide collaterals to GP (Anaya-Martinez et al., 2006), it was conceivable that pre-synaptic H3Rs modulated the activity of intrinsic pallidal neurons by inhibiting dopamine release. However, our experiments also showed a lack of effect of H3R activation on K⫹-evoked [3H]-dopamine release from GP slices. In contrast to [3H]-GABA and [3H]-dopamine release and in spite of the modest extent of release, our data show

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that H3R activation inhibits K⫹-evoked [3H]-D-aspartate efflux, used as an indicator of synaptic glutamate release (Davies and Johnston, 1976; Gundersen et al., 1998; Martire et al., 2000; Raiteri et al., 2007; Wang and Nadler, 2007). H3R-mediated inhibition of glutamate release has been previously reported for rat hippocampus, striatum and thalamus by using electrophysiological and biochemical approaches (Doreulee et al., 2001; Molina-Hernandez et al., 2001; Garduno-Torres et al., 2007). This action appears to involve the action of G␤␥ complexes, released upon G␣i/o protein activation, at the pore-forming ␣1-subunit of N- and P/Q-type voltage-operated calcium channels (De Waard et al., 2005) because H3R agonists reduce depolarization-induced calcium entry in dissociated hypothalamic histaminergic neurons and striatal synaptosomes (Takeshita et al., 1998; Molina-Hernandez et al., 2001). Effect of H3R activation on neuronal firing Synaptically released glutamate controls the activity of intrinsic pallidal neurons (Plenz and Kital, 1999; Magill et al., 2000, 2001). The inhibition of glutamate release would thus decrease the activity of GP neuronal cells, and our results showed that H3R activation by a locally infused agonist results in diminished neuronal firing. This effect was reversed by the H3R antagonist A-331440, although only for a brief period following drug infusion, an action probably explained by the much higher affinity of pallidal H3Rs for immepip over A-331440, alongside probable differences in drug clearance. The large immepip doses injected could also imply non-specific actions not blocked by the antagonist. However, immepip is highly selective for H3Rs (Ki 0.5 nM) over H1 (Ki 16 ␮M) and H2 (Ki⬎365 ␮M) receptors (Vollinga et al., 1994) and H2 receptor activation stimulates GP neuronal activity, an effect opposite to that observed with immepip in this study, with no effect of H1 receptor agonists (Chen et al., 2005, see below). Further, whereas in the cardiovascular system the H3R agonists R-␣-methyl-histamine and imetit have been shown to act at ␣2-adrenoceptors and 5-HT3 receptors, respectively, immepip was devoid of these effects (Coruzzi et al., 1995).

Fig. 6. Effect of drugs acting at H3Rs or ionotropic glutamate receptors on turning behavior. (A) Ipsilateral turning induced by the intrapallidal injection of the H3R agonist immepip followed by systemic apomorphine. Vertical arrows indicate drug administration as follows:

immepip (0.5 ␮l) infused into the left or right globus pallidus; apomorphine (0.5 mg/kg, s.c.). Control animals were administered sterile saline (0.5 ␮l) into the globus pallidus. Values are means⫾SEM from the number of animals as follows: control, four; 10 ␮M, nine; 50 ␮M, seven; 100 ␮M, nine. (B) Partial blockade of the immepip action by the H3R antagonist A-331440. Immepip (0.5 ␮l, 50 ␮M) was injected into the globus pallidus followed by apomorphine (0.5 mg/kg, s.c.) as indicated. Three minutes after apomorphine, vehicle or the H3R antagonist A-331440 (0.5 ␮l, 1 mM) was infused through the same cannula. Vertical arrows indicate drug administration. Values are means⫾SEM from eight animals for each condition. *, Significant difference between treatments (P⬍0.05, two-way RM ANOVA and Student-Newman–Keuls post-hoc test). (C) Ipsilateral turning induced by the intra-pallidal injection of a mixture (0.5 ␮l) of glutamate ionotropic receptor antagonists (AP-5 and CNQX, 1 mM each) or AP-5/ CNQX plus immepip (100 ␮M) in a total volume of 0.5 ␮l, followed by apomorphine (0.5 mg/kg, s.c.) as indicated. Points are means⫾SEM from the values obtained from five (AP-5/CNQX) or eight (AP-5/CNQX plus immepip) animals.

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Taken together, these pieces of information make unlikely a non-H3R action of immepip in our experiments. H3Rs may have spontaneous or constitutive activity, which for cloned receptors depends on the expression level and for native receptors has been reported mainly for H3 auto-receptors (Arrang et al., 2007; and references therein). The lack of effect of the H3R antagonist A-331440 on both K-evoked [3H]-D-aspartate release and spontaneous neuronal firing reported herein suggests that pallidal H3Rs are not significantly occupied by endogenous histamine nor do they have intrinsic activity. A previous electrophysiological study in the slice preparation (Chen et al., 2005) showed histamine (3–100 ␮M, EC50⬃10 ␮M) to increase the firing rate of GP neurons through a post-synaptic, H2 receptor-mediated action involving the cAMP/protein kinase A pathway. Together with our data, these results point out to a dual modulation by histamine of GP neuronal activity. Given the higher affinity of H3Rs for histamine compared to that of H2 receptors (Ki estimates for human receptors 2.5 nM and 2 ␮M, respectively; Leurs et al., 1994; Bongers et al., 2007), the H3Rmediated modulation of glutamate release and the subsequent decrease in neuronal firing may predominantly take place during the sleep states when the activity of histaminergic neurons is low, whereas the H2 receptor-mediated increase in firing would predominate in the arousal states, associated to high activity of histaminergic neurons and enhanced transmitter release in the target nuclei (Haas and Panula, 2003). Interestingly, brain histamine content as well as H3R-mRNA level and H3R density increase in striato-pallidal neurons of the ground squirrel monkey during hibernation, a state characterized by deep depression of central nervous system activity (Sallmen et al., 1999, 2003). According to the dis-inhibition model of basal ganglia function (Bolam et al., 2000), the H2 receptor-mediated increase in neuronal firing would lead to diminished activity of subthalamo-nigral and nigro-thalamic neurons and thus to dis-inhibition of thalamo-cortical pathways and facilitation of motor behavior. In turn, H3R-mediated inhibition of glutamate release and neuronal firing would lead to the opposite action (see below). Turning behavior after intra-pallidal immepip and systemic apomorphine Asymmetries in the basal ganglia synaptic outflow result in turning behavior, ipsilateral to the brain hemisphere where the firing of the basal ganglia output nuclei, substantia nigra pars reticulata and entopeduncular nucleus in rodents, is reduced leading thus to increased activity in the thalamic nuclei innervated by the output nuclei (Schwarting and Huston, 1996a,b). Turning behavior requires the activation of striatal D1-and D2-like receptors (Schwarting and Huston, 1996b), and this requirement is fulfilled by the general agonist apomorphine which stimulates the activity of the striato-nigral pathway while reducing the synaptic flow of the striato-pallidal pathway through D1- and D2-like receptor activation respectively. However, for apomorphine alone these actions would be symmetrical for both

brain hemispheres and no turning behavior is therefore elicited. Thus, ipsilateral turning induced by the intra-pallidal injection of immepip can be attributed to an imbalance in basal ganglia synaptic outflow between brain hemispheres most likely due to augmented inhibition of the indirect pathway in the treated GP with H3R-mediated inhibition of glutamate release functionally counteracting the apomorphine-induced increase in activity observed in most pallidal neurons (Napier et al., 1991; Nakao et al., 2000). The effect of H3R activation on turning behavior was similar and, more importantly, not additive to that induced by blocking ionotropic glutamate receptors, supporting an action of H3Rs on pallidal glutamatergic transmission. Limitations of this study We are aware that given the complexity of the complex basal ganglia synaptic circuitry our experimental approach bears intrinsic limitations that include, but are not restricted, to those discussed below. Neuronal firing in anaesthetized animals is different from that observed in awake subjects, although chloral hydrate anesthesia preserves some of the characteristics of neuronal activity recorded in free-moving rats, for instance the burst firing of midbrain dopamine neurons (Hyland et al., 2002). In our experiments H3R activation may also have resulted in trans-synaptic effects contributing, alongside the inhibition of glutamatergic transmission proposed herein, to the experimental output and which were not analyzed such as the pre-synaptic modulation of the release of 5-hydroxytryptamine (Threlfell et al., 2004), shown to increase the firing of GP neurons (Zhang et al., 2010). On the same line, in electrophysiological recordings the local application of H3R ligands should have affected not only the neuron under recording but also the surrounding neurons. Because GP projection neurons have local axon collaterals (Bolam et al., 2000), the synaptic actions of nearby neurons on the recorded neuron may also influence its firing. The injection of drugs, albeit at relatively small volumes, induces physical disturbances which alter the function of the recorded neuron or cell groups participating in specific behaviors, such as the turning behavior. Because of the larger volumes injected into the GP in our behavioral experiments, there exists the possibility of drugs diffusion to the adjacent striatum where H3R activation has been shown to modulate GABA and glutamate release (AriasMontano et al., 2001; Molina-Hernandez et al., 2001). However, the 0.5-␮l-volume injected in our experiments has been shown to diffuse to a ⬃1-mm diameter sphere (Myers, 1966), suggesting that the injected drugs are retained within the GP boundaries. Further, preliminary data (M. García, unpublished observations) indicate that although the intra-striatal injection of the H3R agonist immepip results in ipsilateral turning, the effect is significantly lesser than that observed when a similar drug concentration and volume is injected into the GP, supporting that the effects reported herein are related to actions in the GP and not to drug diffusion to the striatal tissue.

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CONCLUSION We have herein confirmed that H3Rs are present at high density in rat GP and provided evidence that their activation regulates the synaptic output of the basal ganglia in apomorphine-treated rats, as shown by the appearance of ipsilateral turning behavior. This effect appears to rely on the pre-synaptic inhibition of glutamate release leading to diminished activity of GP neurons, as evidenced by [3H]D-aspartate release experiments in slices and electrophysiological recordings in anaesthetized rats, respectively. The GP and the subthalamic nucleus have emerged as key players in the regulation of the control of the basal ganglia motor output through its reciprocal connections and afferents to other basal ganglia nuclei (Obeso et al., 2008; Hikosaka and Isoda, 2010). The modulation of rat GP glutamatergic transmission by pre-synaptic H3Rs described herein could therefore contribute to regulate the activity of GP neurons and thus basal ganglia function. Further studies are clearly required for a better understanding of the role of histamine and H3Rs in the regulation of GP neuron activity. Acknowledgments—This research was supported by Cinvestav, Conacyt (grant 47351M to J.-A.A.-M.) and IPN (grant IPN-SIP 20100899 to E.Q.). A.O.-E. holds a Conacyt pre-doctoral scholarship. 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.

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(Accepted 24 December 2010) (Available online 31 December 2010)