inverse agonist ABT-239 enhances acetylcholine and histamine release and increases c-Fos expression

Neuropharmacology 70 (2013) 131e140

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Selective brain region activation by histamine H3 receptor antagonist/inverse agonist ABT-239 enhances acetylcholine and histamine release and increases c-Fos expression L. Munari 1, 2, G. Provensi 1, M.B. Passani, P. Blandina* Dipartimento di Farmacologia Preclinica e Clinica, Universitá degli Studi di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 October 2012 Received in revised form 17 January 2013 Accepted 20 January 2013

Histamine axons originate solely from the tuberomamillary nucleus (TMN) to innervate almost all brain regions. This feature is consistent with a function for histamine over a host of physiological processes, including regulation of appetite, body temperature, cognitive processes, pain perception and sleepewake cycle. An important question is whether these diverse physiological roles are served by different histamine neuronal subpopulations. Here we report that systemic administration of the non-imidazole histamine H3 receptor antagonist 4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile (ABT-239, 3 mg/kg) increased c-Fos expression dose-dependently in rat cortex and nucleus basalis magnocellularis (NBM) but not in the nucleus accumbens (NAcc) nor striatum, and augmented acetylcholine and histamine release from rat prefrontal cortex. To further understand functional histaminergic pathways in the brain, dual-probe microdialysis was used to pharmacologically block H3 receptors in the TMN. Perfusion of the TMN with ABT-239 (10 mM) increased histamine release from the TMN, NBM, and cortex, but not from the striatum or NAcc. When administered locally, ABT-239 increased histamine release from the NBM, but not from the NAcc. Systemic as well as intra-TMN administration of ABT-239 increased c-Fos expression in the NBM, and cortex, but not in the striatum or NAcc. Thus, as defined by their sensitivity to ABT-239, histaminergic neurons establish distinct pathways according to their terminal projections, and can differentially modulate neurotransmitter release in a brain regionspecific manner. This implies independent functions of subsets of histamine neurons according to their terminal projections, with relevant consequences for the development of specific compounds that affect only subsets of histamine neurones, thus increasing target specificity. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Acetylcholine c-Fos Histamine Histamine H3 receptor Microdialysis

1. Introduction The histamine H3 receptor (H3R) is largely confined to the nervous system (Leurs et al., 2005), where it acts as a presynaptic autoreceptor that restricts histamine release and synthesis (Arrang et al., 1987, 1983). The H3R is located also on histaminergic somata where it provides a tonic inhibition of firing (Haas and Panula,

Abbreviations: H3R, histamine H3 receptor; ACh, acetylcholine; ADHD, attention-deficit hyperactivity disorder; i.p., intraperitoneally; PBS, phosphate-buffer saline; TMN, tuberomamillary nucleus; NBM, nucleus basalis magnocellularis; NAcc, Nucleus Accumbens. * Corresponding author. Tel.: þ39 (0)554271239. E-mail address: patrizio.blandina@unifi.it (P. Blandina). 1 L. Munari and G. Provensi contributed equally. 2 Present address: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA. 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.01.021

2003). However, its presence is not restricted to histaminergic neurons, and H3 heteroreceptors modulate the release of several neurotransmitters, including acetylcholine (ACh), glutamate, noradrenaline and serotonin, from different brain regions (Blandina et al., 2010). Pharmacological blockade of H3Rs exert procognitive effects, increase wakefulness and reduce bodyweight in animal models (Passani and Blandina, 2011; Passani et al., 2007). As a result, an increasing number of H3R antagonists/inverse agonists progressed to clinical trials for the treatment of a variety of conditions, including narcolepsy as well as cognitive and wakefulness disorders associated with Alzheimer’s disease, Parkinson’s disease, schizophrenia and attention-deficit hyperactivity disorder (ADHD) (Benarroch, 2010; Passani and Blandina, 2011). Moreover, the use of H3R antagonists/inverse agonists that weaken traumatic memories may alleviate disorders such as post-traumatic stress syndrome, panic attacks, specific phobias and generalized anxiety (Blandina et al., 2010; Passani et al., 2001). Recent evidence suggests more

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areas of potential therapeutic interest, such as in the treatment of alcoholism (Nuutinen et al., 2011) and pain (Hsieh et al., 2010). ABT239 is a selective, non-imidazole H3R antagonist/inverse agonist with similar high potency in both human and rat (Cowart et al., 2004). In cognition studies, it improved acquisition of a five-trial, inhibitory avoidance test in rat pups, and social memory in adult and aged rats (Fox et al., 2005). In microdialysis studies, it enhanced ACh release from rat frontal cortex and hippocampus as well as dopamine release from the frontal cortex, but not from the striatum (Fox et al., 2005). The goal of the present study was to evaluate whether neurochemical effects induced by ABT-239, such as c-Fos activation, ACh and histamine release modulation, were restricted through region-specific regulation to distinct brain regions involved in cognitive processes. 2. Experimental procedure 2.1. Animals Male SpragueeDawley rats (225e275 g) or CD1 mice (25e30 g; both Harlan, Milano, Italy) were used for experiments, all of which complied with the European Communities Council Directive of 24 November 1986 (86/609/EEC) recommendations for the care and use of laboratory animals, and local ethical review. Animals were housed in groups of three/five in a temperature-controlled room (20e24  C),

allowed free access to food and water, and kept on a 12-h light/dark cycle (light starts at 7:00 AM). Alternatives to in vivo techniques were not available, but all efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2. Microdialysis in freely moving rats Rats, anesthetized with chloral hydrate (400 mg/kg i.p.) and positioned in a stereotaxic frame (Stellar, Stoelting Co., Wood Dale, IL), were implanted with one or two guide cannulae (Metalant, Sweden) according to the following coordinates from bregma (Paxinos and Watson, 1998): TMN, AP ¼ 4.3, L ¼ 1.1, DV ¼ þ7.2; nucleus basalis magnocellularis (NBM), AP ¼ 0.8; L ¼ 2.8; DV ¼ þ6.5; Dorsal Striatum, AP ¼ 0, L ¼ 4, DV ¼ þ4; Nucleus Accumbens (NAcc), AP ¼ þ1,7, L ¼ 1.4, DV ¼ þ6.3; Prefrontal Cortex, AP ¼ þ3.2; L ¼ 1.0; DV ¼ þ2.8. In the experiments aimed at measuring the release of both histamine and ACh from the prefrontal cortex, rats were implanted with two guide cannulae in the prefrontal cortex according to the following coordinates: AP ¼ þ3.2; L ¼ þ0.8; DV ¼ þ1.3 at 12 angle for the ACh probe, and AP ¼ þ3.2; L ¼ 0.8; DV ¼ þ2.3 at 12 angle for the histamine probe. A surgical screw served as an anchor and the cannulae were fixed to the skull with acrylic dental cement (Fig. 1). The microdialysis experiments were performed 48 h after surgery during which rats, housed one per cage, recovered from surgery. The stylet was removed from the guide cannulae, and the microdialysis probes were inserted; the probe for ACh (molecular weight cut-off ¼ 20,000 Da, CMA USA) protruded 3 mm from the cannula tip, whereas that for HA (molecular weight cut-off ¼ 6000 Da, Microbiotech, Sweden) protruded 2 mm. Probes were perfused with Ringer’s solution (in mM: NaCl, 147; CaCl2, 1.2 and KCl, 4.0 at pH 7.0) at a flow rate of 2 ml/min using a microperfusion

Fig. 1. Schematic diagram showing the position of the microdialysis probes. Rats were implanted with two contralateral probes in the prefrontal cortex to measure the release of ACh and histamine (A). In another set of experiments, rats were implanted with one probe in the TMN to deliver drugs locally and measure neurotransmitter release, and a second probe in the prefrontal cortex (B), the NAcc (C), the dorsal striatum (D) or the NBM (E) to measure histamine release.

L. Munari et al. / Neuropharmacology 70 (2013) 131e140 pump (Carnegie Medicine, Sweden; Mod CMA/100). Fractions were collected at 15 min intervals. Spontaneous release was defined as the average value of the four/ five 15-min fractions collected during 60/75 min of perfusion with Ringer’s solution prior to drug treatment. All subsequent fractions were expressed as percentage of this value. In the dual-probe experiments, both probes were perfused with control medium in the first four 15-min fractions to measure histamine spontaneous release; ABT-239 was then added only to the TMN-perfusing medium, dissolved into the Ringer’s solution. Drug addition did not modify the pH of the medium. In the single-probe experiments the brain region of interest was perfused with control medium in the first four fractions, and ABT-239 was then added to the perfusing medium. In another set of experiments, ABT-239 was administered systemically. In the experiments aimed at measuring ACh release from the prefrontal cortex, neostigmine bromide (0.1 mM), a cholinesterase inhibitor, was added to the medium perfusing the prefrontal cortex to recover detectable ACh concentrations in the dialysate. To prevent degradation of histamine, 1.5 ml of 5 mM HCl was added to each sample. The dialysates were kept at 80  C until analysis. 2.3. Histology The placement of microdialysis membranes was verified post-mortem. Rats were overdosed with chloral hydrate, the brains removed and stored in 10% formalin for 10 days. Forty mm sections were then sliced on a cryostat, mounted on gelatincoated slides and then stained with cresyl violet for light microscopic observation. Data from rats in which the membranes were not correctly positioned were discarded (less than 5%). 2.4. Determination of histamine Histamine contents in the dialysates were determined by HPLC-fluorimetry (Cenni et al., 2006). Briefly, the column (Hypersil ODS, 3 mm, 2.1  100 mm; Thermo Electron Corporation, Bellefonte, PA, USA) was eluted with 0.25 M potassium dihydrogen phosphate containing 5% octanesulfonic acid (Sigma) at a flow rate of 0.4 ml/min. The eluate from the column was mixed first with 0.1% o-phthalaldehyde solution at a flow rate of 0.1 ml/min and then to a solution containing 4 M sodium hydroxide and 0.2 M boric acid (flow rate 0.137 ml/min) to adjust the reaction mixture to pH 12.5. The reaction took place at 45  C. Then 17% orthophosphoric acid was added to the solution (flow rate 0.137 ml/min) to reach a final reaction mixture at pH 3. The fluorescent intensity was measured with a spectrofluorometer (Agilent series 1100, Waldbronn, Germany) at 450 nm with excitation at 360 nm. The sensitivity limit was 10 fmol and the signal/noise ratio was higher than 3. Histamine levels in the dialysate samples were calculated as fmol/15 min, and were not corrected for probe recovery (about 40%). 2.5. Determination of ACh To prevent degradation of ACh, 5 ml of 0.5 mM HCl was added to each sample. ACh was assayed in the dialysate by HPLC with electrochemical detection using an ACh/choline assay chromatographic kit (Bio Analytical System Inc, West Lafayette, Indiana, USA) consisting of an ACh analytical column (BAS MF-6150) and an ACh/ choline Immobilized Enzyme Reactor (IMER, BAS MF-6151). The mobile phase was 50 mM Tris/NaClO4 containing 0.05% ProClin (BAS CF-2150, Bio Analytical Systems Inc, West Lafayette, Indiana, USA), a broad spectrum antimicrobial suited as a preservative for enzymes, pH 8.5, at 1 ml/min flow rate. ACh, separated in the analytical column, was hydrolized in the IMER by acetylcholinesterase to acetate and choline that was oxidized by choline oxidase to produce betaine and hydrogen peroxide. Hydrogen peroxide was electrochemically detected by a platinum-working electrode at þ500 mV with an Ag/AgCl reference electrode. The sensitivity limit was 125 fmol, and the signal/noise ratio was higher than 3. To evaluate the amounts of ACh in the samples, a linear regression curve was made with ACh standards, and the peak areas of this compound in the samples were compared with those of the standards by means of an integrator (P.E. Nelson model 1020; The Perkin Elmer Corporation, Norwalk, CT, USA). ACh levels in the dialysate samples were calculated as fmol/15 min, and were not corrected for probe recovery (about 60%). 2.6. Immunohistochemistry-c-Fos activation c-Fos immunohistochemistry was used as an indicator of in vivo neuronal activation using the methodology previously described (Bacciottini et al., 2002). Briefly, the tuberomamillary nucleus (TMN) of rats was perfused for 60 min through a microdialysis probe with Ringer’s solution alone or containing 10 mM ABT-239 at a flow rate of 2 ml/min using a microperfusion pump (Carnegie Medicine, Sweden; Mod CMA/100). Ninety min after the end of perfusion, rats were killed and perfused transcardially with ice-cold 4% paraformaldehyde. In another set of experiments, ABT-239 was administered i.p. (1 or 3 mg/kg), and rats were killed and perfused transcardially with ice-cold 4% paraformaldehyde 120 min after dosing. Controls received an injection of vehicle (saline). The brains were removed and post-fixed in 4% paraformaldehyde overnight and then immersed in 20% sucrose for 72 h. Coronal sections were cut at 40 mm on a freezing cryostat, and were collected in phosphatebuffer saline (PBS) until processed for immunohistochemistry. Free-floating sections

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were incubated in anti c-Fos polyclonal antibodies 1:5000 in PBS containing 0.3% Triton X-100 (Sigma) and 5 mg/ml bovine serum albumin (BSA), overnight. The immunoreactive product was detected with the avidin-biotin peroxidase system (Vectastain kit; Vector Laboratories, Burlingame CA USA). After washing with PBS, sections were mounted on gelatin-coated slides, dehydrated in increasing concentrations of EtOH and coverslipped. Sections were observed using an Olympus BX40 microscope equipped with a Nikon DS-F1 camera. Atlas coordinates (relative to the bregma) for the sections analyzed were from þ3.2 to þ2.7 for the cortex, from þ1.7 to þ1.2 for the NAcc, from þ0.5 to þ0.0 for the striatum and from 0.4 to 0,6 for the NBM (Paxinos and Watson, 1998). The number of c-Fos immunopositive nuclei were counted bilaterally using the Image J software (NIH, USA) on three to six sections per region for each rat. Statistics were calculated on the average values from three to six sections of individual animals. Thus, sample size and statistics were based on number of rats. The number of positive nuclei were normalized to an area of 1 mm2.

2.7. Statistical analysis All values are expressed as means  SEM, and the number of animals used in each experiment is also indicated. The presence of significant treatment effects was first determined by a one-way ANOVA followed by Bonferroni/Dunn test or NewmaneKeuls test. Comparisons using paired or unpaired Student’s t test were used to assess differences between groups in the object recognition test. For all statistical tests, P < 0.05 was considered significant. For clarity, we reported in figures and figure legends only the significant differences vs. the last sample before drug treatment. However, differences were significant vs. all baseline samples. Statistical analysis was performed using StatView (Abacus Concepts, Inc. Berkley, CA, USA), GraphPad Prism (GraphPad Software, Inc. La Jolla, CA, USA) or JMP (SAS Institute, Cary, North Carolina, USA).

2.8. Materials ABT-239 was synthesized at Abbott Laboratories (Abbott Park, IL 60064, USA). All other reagents and solvents were of HPLC grade or the highest grade available (Sigma, UK).

3. Results 3.1. Microdialysis experiments ACh and histamine were released at a stable rate after 120 min of equilibration following the insertion of the dialysis membranes. Electrophysiological recordings of histamine neurons in freelymoving animals indicate that their activity is high during waking and attention, and low or absent during sleep (Takahashi et al., 2006). In the present study, since the experiments were performed during the light phase, histamine cells activity was likely low. Consistently, about 90% of the rats were sleeping before ABT239 administration that produced clear signs of awakening (eating, drinking, grooming, moving in the cage), but not of agitation or irritability, as described also in Giannoni et al. (2009). Rats were one/cage and experiments were performed in a quiet room dedicated to perform microdialysis experiments. c-Fos experiments were performed under the same conditions. Histological analysis confirmed that the probes were located in the correct areas, with no signs of unusual tissue damage or bleeding. 3.2. Systemic administration of ABT-239 increased the release of both histamine and ACh from the prefrontal cortex After collection of five, 15-min baseline samples, ABT-239 was administered i.p. at the dose of 3 mg/kg. ABT-239 significantly increased both ACh and histamine release immediately after the administration, up to peak values of 177  21% (ANOVA, F(15,75) ¼ 4.966, P < 0.0001), and 132  36% (ANOVA, F(15,75) ¼ 7.626, P < 0.0001), respectively. ACh and histamine release remained significantly elevated for about 60 min, and then returned to basal levels (Fig. 2). Mean spontaneous releases were 550  61 fmol/15 min for ACh, and 47  5 fmol/15 min for histamine (N ¼ 6).

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-25 0 Fig. 2. Time course of ACh and histamine release from the prefrontal cortex of freely moving rats following systemic administration of ABT-239. Neurotransmitters release was measured in fractions collected every 15 min. Neostigmine bromide (0.1 mM), a cholinesterase inhibitor, was added to the medium perfusing the ACh probe. Control values of spontaneous neurotransmitter release were calculated for each experiment by averaging the mean of five initially collected 15-min samples, and averaged 550  61 fmol/15 min for ACh, and 47  5 fmol/15 min for histamine. Neurotransmitter releases were expressed as a percentage of spontaneous release. The arrow indicates the time of drug administration. Represented are means  SEM of 6 rats. ***P < 0.001 vs. last sample before drug treatment (ANOVA and Bonferroni/Dunn test).

3.3. Effects of TMN perfusion with ABT-239 on histamine release from the TMN and the prefrontal cortex Using a double-probe microdialysis protocol, histamine release was monitored from both the TMN and the prefrontal cortex. After collection of five, 15-min baseline samples, ABT-239 (10 mM) was infused for 60 min locally into the TMN, where all histaminergic neuronal cell bodies are localized (Inagaki et al., 1988), while the prefrontal cortex was always perfused with control medium. Infusion of ABT-239 elicited a significant increase of histamine release from both the TMN (ANOVA, F(12,58) ¼ 7.743, P < 0.0001) and the prefrontal cortex (ANOVA, F(12,60) ¼ 8.351, P < 0.0001). Histamine release was restored to control levels during subsequent TMN perfusion with control medium (Fig. 3). In the TMN the mean spontaneous release of histamine was 52  4 fmol/15 min, and the maximal increase produced by ABT-239 was 86  10% (n ¼ 6). In the posterior hypothalamus histamine was likely released by short projections, as histaminergic neurons display an extensive axonal arborizations within this brain region (Inagaki et al., 1988). In the prefrontal cortex, where histamine was released from TMN projections, the mean spontaneous release was 47  7 fmol/15 min, and the maximal increase 95  33% (N ¼ 6).

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Fig. 3. Influence of ABT-239 administration into the TMN on histamine release from the TMN (lower panel) and the prefrontal cortex (upper panel) of freely moving rats. Histamine was measured and calculated as described in Fig. 4. The mean spontaneous release of histamine was 52  4 fmol/15 min in the TMN, and 47  7 fmol/15 min in the prefrontal cortex. ABT-239, at a concentration of 10 mM, was infused into the TMN and histamine release was measured from the TMN and the prefrontal cortex. Bar indicates the period of drug application. Shown are means  SEM of 6 rats. ***P < 0.001 vs. last sample before drug treatment (ANOVA and Bonferroni/Dunn test).

3.4. Effects of TMN perfusion with ABT-239 on histamine release from the TMN and the dorsal striatum Perfusion of the TMN with 10 mM ABT-239 increased significantly histamine release from the TMN (ANOVA, F(12,25) ¼ 4.328, P < 0.001) but not from the striatum. The mean of histamine spontaneous release from the TMN was 51  1 fmol/15 min (n ¼ 3), and ABT-239 elicited a maximal increase of 124  25% (Fig. 6). Histamine levels returned to basal values during washout of the compound. Conversely, no significant change was observed in the release of histamine from the dorsal striatum, the changes being within variability range (approx. 20%) observed between individual 15-min collection periods during perfusion with control medium (Fig. 4). Histamine spontaneous release from the dorsal striatum was 56  2 fmol/15 min (N ¼ 3). 3.5. Effects of TMN perfusion with ABT-239 on histamine release from the TMN and NBM Histamine release was monitored from both the TMN and NBM. ABT-239 induced a significant increase of histamine release from

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TIME (min) Fig. 4. Influence of ABT-239 administration into the TMN on histamine release from the TMN (lower panel) and the dorsal striatum (upper panel) of freely moving rats. Histamine was measured and calculated as described in Fig. 4. The mean of histamine spontaneous release from the TMN was 56  2 fmol/15 min and 51  1 fmol/15 min from the dorsal striatum. TMN was perfused with 10 mM ABT-239, and histamine release was measured from the TMN and the dorsal striatum. Bar indicates the period of ABT-239 application. Shown are means  SEM of 3 rats. **P < 0.01 vs. last sample before drug treatment (ANOVA and Bonferroni/Dunn test).

both the TMN (ANOVA, F(12,26) ¼ 9.429, P < 0.0001) and the NBM (ANOVA, F(12,26) ¼ 7.458, P < 0.0001), which returned to baseline values after perfusion with ABT-239 ended. In the TMN the maximal increase was 105  10% (Fig. 5). When ABT-239 was infused into the TMN, histamine release from TMN projections into the NBM increased up to a maximum of 107  27% (Fig. 5). Histamine spontaneous release from the TMN averaged 40  4 fmol/15 min, and from the NBM averaged 44  5 fmol/15 min (N ¼ 3). 3.6. Effects of TMN perfusion with ABT-239 on histamine release from the TMN and the NAcc When ABT-239 (10 mM) was added to the TMN-perfusing medium for 60 min, the spontaneous release of histamine from the TMN increased significantly by a maximum of 114  18% (ANOVA, F(12,61) ¼ 13.163, P < 0.0001), but was not significantly changed in the NAcc, since the changes remained within variability range (approx. 20%) observed between individual 15-min collection periods during perfusion with control medium (Fig. 6). The mean spontaneous release of histamine was 41  4 fmol/15 min in the TMN, and 33  3 fmol/15 min in the NAcc (N ¼ 6). 3.7. Single-probe experiments: effects of local perfusion with ABT239 on histamine release from the NBM and nucleus accumbens To learn the effects of ABT-239 at axonal domains of histaminergic neurons, each rat was implanted with a single probe, in the

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TIME (min) Fig. 5. Influence of ABT-239 administration into the TMN on histamine release from the TMN (lower panel) and the NBM (upper panel) of freely moving rats. Histamine was measured and calculated as described in Fig. 4. Histamine spontaneous release averaged 44  5 fmol/15 min in the TMN, and 40  4 fmol/15 min in the NBM. TMN was perfused with 10 mM ABT-239, and histamine release was measured from the TMN and the NBM. Bar indicates the period of ABT-239 application. Shown are means  SEM of 3 rats. **P < 0.01 vs. last sample before drug treatment (ANOVA and Bonferroni/Dunn test).

NBM or the NAcc, to administer locally ABT-239 and monitor changes in histamine release. NBM perfusion with 10 mM ABT-239 for 45 min elicited a significant, transient increase of histamine release (ANOVA, F(11,48) ¼ 10.52, P < 0.0001) with a maximal value of 111  15% (Fig. 7A). Increased histamine levels persisted during perfusion with ABT-239, after which basal histamine levels were quickly attained. The mean spontaneous release of histamine from the NBM was 59  8 fmol/15 min (n ¼ 5). Perfusion of the NAcc with 10 mM ABT-239 for 45 min failed to produce any significant change in the spontaneous release of histamine (Fig. 7B). The mean spontaneous release of histamine from the nucleus accumbens was 37  5 fmol/15 min (N ¼ 6).

3.8. Systemic administration of ABT-239 increased c-Fos expression Compared with vehicle-treated animals the density of c-Fos staining was increased dose-dependently following i.p. administration of ABT-239 (1 and 3 mg/kg) in rat prefrontal cortex and NBM but not in NAcc nor striatum (Fig. 8A and B). Statistical analysis (ANOVA and NewmaneKeuls, F(2,10) ¼ 13.85) revealed that cFos immunoreactive nuclei/mm2 in cortical slices from rats treated with either 1 mg/kg ABT-239 (60.7  21.4; N ¼ 3; P < 0.05) or 3 mg/ kg ABT-239 (80.8  14.7; N ¼ 5; P < 0.01) were significantly more

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TIME (min) Fig. 6. Influence of ABT-239 administration into the TMN on histamine release from the TMN (lower panel) and the nucleus accumbens (NAcc, upper panel) of freely moving rats. Histamine was measured and calculated as described in Fig. 4. The mean spontaneous release of histamine was 41  4 fmol/15 min in the TMN, and 32  3 fmol/ 15 min in the nucleus accumbens. TMN was perfused with 10 mM ABT-239, and histamine release was measured from the TMN and the NAcc. Bar indicates the period of ABT-239 application. Shown are means  SEM of 6 experiments. ***P < 0.001 vs. last sample before drug treatment (ANOVA and Bonferroni/Dunn test).

numerous than those from rats injected with saline (18.7  11.5; N ¼ 3). Similarly, c-Fos immunoreactive nuclei/mm2 in NBM slices from rats treated with either 1 mg/kg ABT-239 (36.1  10.6; N ¼ 3; P < 0.05) or 3 mg/kg ABT-239 (61.6  11.8; N ¼ 5; P < 0.001) were significantly more numerous than those from rats injected with saline (15.3  5.46; N ¼ 3; NewmaneKeuls, F(2,10) ¼ 19.75). No significant changes were observed in NAcc and striatum. 3.9. Intra-TMN administration of ABT-239 increased c-Fos expression in selected brain regions Compared with vehicle-treated animals the density of c-Fos staining was increased in rat brain following the perfusion of TMN through a microdialysis probe for 60 min with a solution containing 10 mM ABT-239 (Fig. 9A). Quantification of this effect showed that ABT-239 induced statistically significant increases in c-Fos expression in the NBM (unpaired Student’s t test, P < 0.05) and the prefrontal cortex (unpaired Student’s t test, P < 0.05) but not in the striatum nor the NAcc (Fig. 9B). 4. Discussion ABT-239 displays good bioavailability, excellent blood brain barrier penetration (Esbenshade et al., 2005), and has previously been shown to improve cognitive performance in a number of animal models, including acquisition of a five-trial, inhibitory avoidance test, social memory, and performances in a spatial

Fig. 7. Effects of local perfusion with ABT-239 on histamine release from the NBM (A), and the NAcc (B) of freely moving rats: single-probe experiments. Each rat was implanted in the NBM or NAcc with a probe used to simultaneously administer ABT239 locally and monitor changes in histamine release. Histamine was measured and calculated as described in Fig. 4. The spontaneous release of histamine averaged 59  8 fmol/15 min (N ¼ 5) from the NBM, and 37  5 fmol/15 min (N ¼ 6) B) from the NAcc. Both regions were perfused with 10 mM ABT-239 for 45 min. Bars indicate the period of ABT-239 application. Shown are means  SEM of 5 (A), and 6 (B) rats. ***P < 0.001 vs. last sample before drug treatment (ANOVA and Bonferroni/Dunn test).

navigation test in the water maze at doses ranging 0.1e3 mg/kg (Fox et al., 2005). Although the translational relevance of these animal models to human cognition is uncertain, ABT-239 showed broad efficacy across several models and displayed similar high potency in both human and rat (Esbenshade et al., 2005), increasing confidence that a beneficial effect may be seen in humans. Since ABT-239 significantly enhanced ACh and histamine release from the prefrontal cortex of freely moving rats, it is possible that this improvement reflected enhanced cortical ACh and/or histamine tone. Another H3R antagonist, GSK189254, increased cortical ACh and histamine releases and improved rat performances in object recognition (Giannoni et al., 2010; Medhurst et al., 2007). Conversely, imetit and R-alpha-methylhistamine, two H3R agonists, reduced cortical ACh release and impaired rat performance in a passive avoidance and object recognition test (Blandina et al., 1996). These findings strongly suggest that H3R-elicited cognitive improvements or deficits are associated with changes in ACh release depending on whether H3Rs are blocked or activated. Nevertheless, alternative non-cholinergic mechanisms could also account for H3R procognitive effects, as ABT-239 influenced also the release of cortical dopamine that is involved in cognitive processes (Fox et al., 2005). Test conditions in which cholinesterase inhibitors are present in the perfusion medium may be criticized because cholinesterase inhibition might alter the modulation of ACh spontaneous release. Indeed, neostigmine altered the ability of dopaminergic agents to modulate striatal ACh release (DeBoer and Abercrombie, 1996), but the much lower levels of cortical compared with striatal ACh release, measured by microdialysis, required the presence of cholinesterase inhibitors in the perfusion fluid to reduce the variability

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Fig. 8. Influence of systemic administration of ABT-239 on c-Fos expression in rat brain regions. A) Representative photomicrographs illustrating c-Fos immunostaining in the prefrontal cortex, the nucleus accumbens (NAcc), the striatum (STR) and the NBM following i.p. injections of either vehicle or ABT-239 (1 and 3 mg/kg). Scale bar, 100 mm. B) Effects of ABT-239 (gray and black bars) on the density (counts per square millimeter) of c-Fos immunopositive nuclei compared with vehicle-treated animals (white bars). Shown are means  SEM of 3 (saline and 1 mg/kg) and 5 (3 mg/kg) rats. *P < 0.05; **P < 0.01; ***P < 0.001 vs. correspondent vehicle-treated animals (ANOVA and Newman/Keuls test).

of ACh release. Therefore, microdialysis studies characterizing histaminergic (Blandina et al., 1996; Cecchi et al., 2001; Fox et al., 2005), serotonergic (Consolo et al., 1996), glutamatergic (Giovannini et al., 1994) and GABAergic (Giorgetti et al., 2000) modulation of cortical ACh release employed cholinesterase inhibitors. Relevant in this regard is that concentrations up to 0.5 mM neostigmine failed to alter qualitatively cortical ACh release patterns in response to behavioral activation (Himmelheber et al., 1998). Following oral dosing, GSK189254, another H3R antagonist, induced a significant increase in ACh release from the anterior cingulate cortex (Medhurst et al., 2007). These experiments were performed without cholinesterase inhibitors in the perfusing medium, but microdialysis samples were collected every 30 min instead of 5e15 min as in the case of the other studies. Therefore, it is unlikely that the presence of 0.1 mM neostigmine in the perfusion

medium affects qualitatively the effect of ABT-239 on cortical ACh release. To gather more insight into the mechanism of action of ABT-239, we examined c-Fos activation pattern in rat brain regions after acute treatment with ABT-239 (3 mg/kg, i.p.). Despite the widespread distribution of histaminergic terminals and H3Rs (Haas and Panula, 2003), ABT-239 activated neuronal cells in restricted rat brain regions such as the prefrontal cortex and the NBM but not the striatum nor the NAcc. The same c-Fos activation pattern was induced by other H3R antagonists, JNJ-10181457 (Bonaventure et al., 2007) and GSK189254 (Medhurst et al., 2007). Further experiments are required to identify the neuronal population(s) expressing c-Fos, but their activation is possibly related with the selective increases of cortical cholinergic and/or histaminergic tone produced by ABT-239 at the same dose. This dose also improved

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Fig. 9. Influence of ABT-239 administration into the TMN on c-Fos expression in rat brain regions. A) Representative photomicrographs illustrating c-Fos immunostaining in the NBM, the prefrontal cortex, the striatum (STR) and the nucleus accumbens (NAcc) following TMN perfusion with either vehicle or ABT-239 (10 mM) for 60 min. Scale bar, 100 mm. B) Effects of ABT-239 (black bars) on the density (counts per square millimeter) of c-Fos immunopositive nuclei compared with vehicle-treated animals (white bars). Shown are means  SEM of vehicle- (N ¼ 3) vs. ABT-239-treated (N ¼ 4) rats. *P < 0.05; *P < 0.05 vs. correspondent vehicle-treated animals (ANOVA and Bonferroni/Dunn test).

object recognition, thus further supporting the identification of the cortex as a neural substrate underlying object recognition processing. Moreover, cortical ACh increase can be also a consequence of the augmentation of histamine release in the NBM, as activation of H1Rs increased cortical ACh release (Cecchi et al., 2001) through direct stimulation of NBM cholinergic neurons (Khateb et al., 1995), which provide all cholinergic innervation to the cortex (Mesulam et al., 1983). Histamine axons originate from a single source, the TMN in the posterior hypothalamus, to innervate almost all CNS regions (Haas and Panula, 2003), and the histamine system was generally regarded as one single functional unit that provided histamine throughout the brain (Wada et al., 1991). However, evidence is beginning to accumulate in favor of diverse physiological roles served by different histamine neuronal subpopulation (Blandina et al., 2012). Since microdialysis provides a powerful means for defining the dynamics regulating histamine release in

discrete brain regions, the present study, using the double-probe microdialysis technique in freely moving animals, addressed the question whether histaminergic neurons are organized into distinct functional circuits impinging on different brain regions. Rats were implanted with one probe in the TMN, to deliver ABT-239 and measure histamine release locally, and another probe to measure histamine release from histaminergic projection areas. ABT-239, applied locally into the TMN, significantly increased histamine release from the TMN, the prefrontal cortex and the NBM, but not from the striatum or the nucleus accumbens. The present findings complement earlier observations that thioperamide and GSK189254, an imidazole and a non-imidazole H3R antagonist, respectively, if applied to the rat TMN invariably augmented histamine release from the TMN, the prefrontal cortex and the NBM, but not from the NAcc, nor the striatum (Giannoni et al., 2010, 2009). These results taken together strongly support the hypothesis

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that H3R antagonists, as a class of agents, differentiate histaminergic neurons according to their projection areas. In the TMN, where histamine was released from short projections that formed extensive axonal arborizations, ABT-239 increased local histamine release likely as a consequence of both somatic and presynaptic H3autoreceptors blockade. Indeed, blockade of somatic and presynaptic H3-autoreceptors converged in augmenting histamine levels in the synaptic cleft by increasing histamine neuronal firing and release from terminals (Haas and Panula, 2003). Histamine increases in the prefrontal cortex and NBM during TMN perfusion with ABT-239 were likely consequent of discharge potentiation of histamine neurons sending efferents to these regions, whereas the lack of increase in the striatum and the NAcc, despite the fact that these brain areas receive histaminergic innervation (Panula et al., 1989), indicated that histaminergic neurons projecting to these regions were insensitive to H3R blockade. The lack of response in the NAcc and the striatum was not explained by spatial segregation due to probe localization, as retrograde tracing with dye injections into the striatum or prefrontal cortex showed that most histaminergic somata are within the medial part of the ventral TMN (Kohler et al., 1985). This proximity suggested that histaminergic somata projecting to the striatum and prefrontal cortex had the same exposure to H3R antagonists, but were not affected in the same way. Two histaminergic neuronal populations that differed significantly for H3R expression levels were described (Blandina et al., 2012). Since the magnitude of neuronal responses to extracellular signals may depend on membrane receptor density, histamine neurons displaying low levels of the H3R are possibly those innervating the nucleus accumbens or striatum. Alternatively, as several isoforms of the H3R displaying strong pharmacological differences have been described (Bongers et al., 2007), in vivo insensitivity to H3R antagonists may depend on high expression of particular isoforms. Interestingly, local perfusion of the NBM with ABT-239 increased significantly the release of histamine locally, thus suggesting the presence of H3-autoreceptors. Conversely, local perfusion of the NAcc with ABT-239 did not alter spontaneous histamine release, thus indicating that the entire somato-dendritic domain of histaminergic neurons projecting to this region was insensitive to H3R blockade. In keeping with these results, increased expression of c-Fos after perfusion of TMN with ABT-239 occurred in the prefrontal cortex and in the NBM, but neither in the NAcc nor in the striatum. These findings clearly strengthen the hypothesis that histaminergic neurons are not a homogenous neuronal population, and that functional heterogeneity of responses among histaminergic neurons relates to TMN neurons heterogeneity with respect to projection fields. Noteworthy, intra-hypothalamic perfusion of bicuculline, a GABAA-R antagonists, that acts directly onto histaminergic neurons to augment cell firing (Haas et al., 2008), increased histamine release from the TMN, the nucleus accumbens and the prefrontal cortex, but not from the striatum (Giannoni et al., 2009). Moreover, administration of methanandamide, a cannabinoid receptor 1 agonists, in the TMN facilitated histamine release from the TMN, from the NBM and striatum but not from the perirhinal cortex (Cenni et al., 2006; Passani et al., 2007). Histamine has a major role in maintenance of arousal and contributes to modulation of appetite, energy homeostasis, motor behavior, and cognition. The extent to which these diverse physiological roles are served by subpopulation of histamine neurones is yet unclear, however, there is much experimental evidence, including the present paper, suggesting that the histaminergic system is organized into distinct functional pathways modulated by selective mechanisms. Consequences could be relevant for the development of specific compounds that affect only subsets of histamine cells, thus increasing the target specificity.

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Disclosure statement P.B. has received a research grant from Abbott, which also provided ABT-239. Abbott agreed in the decision to submit the article for publication, but played no roles in the study design, the collection, analysis and interpretation of data, and the writing of the article. L.M., G.P. and M.B.P. declare no conflict of interest. Author contribution L. Munari and G. Provensi: performed the microdialysis experiments, analyzed the microdialysis samples and prepared the manuscript. G. Provensi and M.B. Passani: performed and analyzed the anatomical and immunohistochemical data. P. Blandina and M.B. Passani: provided the experimental design, analyzed the data and prepared the manuscript. Acknowledgments This research was supported by Universitá di Firenze funds (M.B.P.), PRIN 2009 (MIUR/Italy) (M.B.P.), and Compagnia di San Paolo-Torino (I) (P.B.). The authors are grateful to Dr. Jorge D. Brioni for reading the manuscript. We thank Abbott for gift of ABT-239. References Arrang, J.M., Garbarg, M., Lancelot, J.C., Lecont, J.M., Pollard, H., Robba, M., Schunack, W., Schwartz, J.C., 1987. Highly-potent and selective ligands for histamine-H3 receptors. Nature 327, 117e123. Arrang, J.M., Garbarg, M., Schwartz, J.C., 1983. Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine receptors. Nature 302, 832e 837. Bacciottini, L., Passani, M., Giovannelli, L., Cangioli, I., Mannaioni, P., Schunack, W., Blandina, P., 2002. Endogenous histamine in the medial septum-diagonal band complex increases the release of acetylcholine from the hippocampus: a dualprobe microdialysis study in the freely moving animal. Eur. J. Neurosci. 15, 1669e1680. Benarroch, E.E., 2010. Histamine in the CNS: multiple functions and potential neurologic implications. Neurology 75, 1472e1479. Blandina, P., Giorgetti, M., Bartolini, L., Cecchi, M., Timmerman, H., Leurs, R., Pepeu, G., Giovannini, M.G., 1996. Inhibition of cortical acetylcholine release and cognitive performance by histamine H3 receptor activation in rats. Br. J. Pharmacol. 119, 1656e1664. Blandina, P., Munari, L., Giannoni, P., Mariottini, C., Passani, M.B., 2010. Histamine neuronal system as therapeutic target for the treatment of cognitive disorders. Future Neurol. 5, 543e555. Blandina, P., Munari, L., Provensi, G., Passani, M.B., 2012. Histamine neurons in the tuberomamillary nucleus: a whole center or distinct subpopulations? Front. Syst. Neurosci. 6. 33e33. Bonaventure, P., Letavic, M., Dugovic, C., Wilson, S., Aluisio, L., Pudiak, C., Lord, B., Mazur, C., Kamme, F., Nishino, S., Carruthers, N., Lovenberg, T., 2007. Histamine H3 receptor antagonists: from target identification to drug leads. Biochem. Pharmacol. 73, 1084e1096. Bongers, G., Bakker, R.A., Leurs, R., 2007. Molecular aspects of the histamine H3 receptor. Biochem. Pharmacol. 73, 1195e1204. Cecchi, M., Passani, M.B., Bacciottini, L., Mannaioni, P.F., Blandina, P., 2001. Cortical acetylcholine release elicited by stimulation of histamine H1 receptors in the nucleus basalis magnocellularis: a dual probe microdialysis study in the freely moving rat. Eur. J. Neurosci. 13, 68e78. Cenni, G., Blandina, P., Mackie, K., Nosi, D., Formigli, L., Giannoni, P., Ballini, C., DellaCorte, L., Mannaioni, P.F., Passani, M.B., 2006. Differential effect of cannabinoid agonists and endocannabinoids on histamine release from distinct regions of the rat brain. Eur. J. Neurosci. 24, 1633e1644. Consolo, S., Arnaboldi, S., Ramponi, S., Nannini, L., Ladinsky, H., Baldi, G., 1996. Endogenous serotonin facilitates in vivo acetylcholine release in rat frontal cortex through 5-HT1B receptors. J. Pharmacol. Exp. Ther. 277, 823e830. Cowart, M., Pratt, J.K., Stewart, A.O., Bennani, Y.L., Esbenshade, T.A., Hancock, A.A., 2004. A new class of potent non-imidazole H(3) antagonists: 2-aminoethylbenzofurans. Bioorg. Med. Chem. Lett. 14, 689e693. DeBoer, P., Abercrombie, E.D., 1996. Physiological release of striatal acetylcholine in vivo: modulation by D1 and D2 receptor subtypes. J. Pharmacol. Exp. Ther. 277, 775e783. Esbenshade, T.A., Fox, G.B., Krueger, K.M., Miller, T.R., Kang, C.H., Denny, L.I., Witte, D.G., Yao, B.B., Pan, L., Wetter, J., Marsh, K., Bennani, Y.L., Cowart, M.D.,

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