European Journal of Neuroscience, Vol.12, pp.3823±3832, 2000 ã Federation of European Neuroscience Societies Modulation of histamine H3 receptors in the brain of 6-hydroxydopamine-lesioned rats Oleg V. Anichtchik,1,2 Marko Huotari,3,4 Nina Peitsaro,1 John W. Haycock,5 Pekka T. MaÈnnistoÈ 3 and Pertti Panula1,6 1Department of Biology, AÊ bo Akademi University, BioCity, Artillerigatan 6, 20520, Turku, Finland 2Graduate School of Biomedical Sciences (TuBS), Turku, Finland 3Department of Pharmacology and Toxicology, University of Kuopio, Finland 4Graduate School of Pharmacy, University of Kuopio, Finland 5Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, USA 6Institute of Biomedicine, Department of Anatomy, University of Helsinki, Finland 35 Keywords: GTP-g-[ S] autoradiography, H3 receptor binding, immunohistochemistry, Parkinson's disease Abstract Parkinson's disease is a major neurological disorder that primarily affects the nigral dopaminergic cells. Nigral histamine innervation is altered in human postmortem Parkinson's disease brains. However, it is not known if the altered innervation is a consequence of dopamine de®ciency. The aim of the present study was to investigate possible changes in the H3 receptor system in a well- characterized model of Parkinson's disease ± the 6-hydroxydopamine (6-OHDA) lesioned rats. Histamine immunohistochemistry showed a minor increase of the ®bre density index but we did not ®nd any robust increase of histaminergic innervation in the ipsilateral substantia nigra on the lesioned side. In situ hybridization showed equal histidine decarboxylase mRNA expression on both 3 sides in the posterior hypothalamus. H3 receptors were labelled with N-alpha-[3H]-methyl histamine dihydrochloride ([ H] NAMH). Upregulation of binding to H3 receptors was found in the substantia nigra and ventral aspects of striatum on the ipsilateral side. An 35 increase of GTP-g-[ S] binding after H3 agonist activation was found in the striatum and substantia nigra on the lesioned side. In situ hybridization of H3 receptor mRNA demonstrated region-speci®c mRNA expression and an increase of H3 receptor mRNA in ipsilateral striatum. Thus, the histaminergic system is involved in the pathological process after 6-OHDA lesion of the rat brain at least through H3 receptor. On the later stages of the neurotoxic damage, less H3 receptors became functionally active. Increased H3 receptor mRNA expression and binding may, for example, modulate GABAergic neuronal activity in dopamine-depleted striatum. Introduction Animal models of Parkinson's disease are needed for better et al., 1989). Histamine-containing cell bodies are located exclusively understanding the pathogenesis and for drug screening. in the tuberomammillary nucleus of the posterior hypothalamus, and Neurotoxins, such as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine their projections extend to all regions of the brain. The involvement (MPTP) or 6-hydroxydopamine (6-OHDA), produce parkinsonism in of the histaminergic system in control of movement has been several species, including ®sh, rodent, primates and humans (Burns suggested, since intracerebroventricular administration of histamine et al., 1983; Langston et al., 1983; Perese et al., 1989; Pollard et al., produces biphasic changes in the locomotor activity (Nistico et al., 1992) and provide useful experimental models. 6-OHDA cannot pass 1980; Tuomisto & Eriksson, 1980), and the histamine-induced through the brain blood barrier, so that intraventicular or intracerebral hypoactivity is inhibited by administration of the histamine H3 injections are needed to produce lesions. 6-OHDA is taken up and receptor antagonist thioperamide (Chiavegatto et al., 1998). accumulates in catecholaminergic nerve terminals, hence the damage Moreover, the central histaminergic system has been suggested to occurs quite speci®cally in these neurotransmitter pathways. The have an inhibitory role in methamphetamine-induced stereotyped cytotoxic effect of 6-OHDA is considered to be related to the behaviour (Ito et al., 1997). formation of oxygen-based free radicals and oxidation products Parkinson's disease is one of the major brain-degenerative (Reader & Dewar, 1999) in catecholaminergic terminals. However, disorders, characterized by bradykinesia, rigidity and resting tremor. during the development of experimental parkinsonism, different other The pathophysiological core of Parkinson's disease is a speci®c neurotransmitter pathways are also modulated, e.g. the serotoniner- degeneration of dopamine-containing nerve cells in the substantia gic, GABAergic and cholinergic ones (Lloyd & Hornykiewicz, 1973; nigra and ventral tegmental area with their projections to striatum, Scatton et al., 1983; Reader & Dewar, 1999). hypothalamus and thalamus. A growing body of evidence indicates The histaminergic cell bodies and ®bres in the rat brain have been possible involvement of the histaminergic system in the pathogenesis mapped with antibodies against histamine (Panula et al., 1984; Panula of Parkinson's disease or animal models of this disease. H3 receptor binding is upregulated in the substantia nigra and striatum of 6-OHDA lesioned rats (Ryu et al., 1994, 1996), but is not changed in the striatum Correspondence: Professor Pertti Panula, as above. of the Parkinson's disease patients (Goodchild et al., 1999). Central E-mail: ppanula@abo.® histamine was unaffected by MPTP treatment in the neocortex and Received 30 March 2000, revised 7 July 2000, accepted 3 August 2000 hypothalamus in mice (Cumming et al., 1989), whereas nigral and 3824 O. V. Anichtchik et al. striatal levels were not examined. Recently we observed increased In order to visualize the histaminergic system in the brain of rats histaminergic innervation in the substantia nigra of Parkinson's bearing unilateral 6-OHDA lesion, we used the immunohistochemical disease patients (Anichtchik et al., 2000). The aim of this study was protocol described previously (Anichtchik et al., 2000) with minor therefore to examine the potential plastic changes of the histaminergic modi®cations. Brie¯y, slides with sections were incubated in the system in rats after 6-OHDA lesion of the medial forebrain bundle antiserum against histamine raised in the rabbit over two nights at lesion with special emphasis on the H3 receptor system. +4 °C. The immunoreactivity was detected using biotin-streptavidin method (Vectastain Elite Kit, Vector Labs, CA, USA). This was Materials and methods ®nished by the coupled oxidation reaction in a solution containing 0.025% diaminobenzidine (Sigma), 0.01% H2O2 and 0.3% NiSO4 in 6-OHDA lesion experiments 0.05 M Tris-HCl buffer, pH 7.6 for 2±3min until a bluish-black Unilateral lesions of the medial forebrain bundle were made in Wistar product of the reaction appeared. Parallel sections were processed for male rats (National Animal Center, University of Kuopio, Finland) antityrosine hydroxylase immunohistochemistry with polyclonal weighing 180±230 g. Thirty minutes before the 6-OHDA infusion the antibodies against tyrosine hydroxylase (2 mg/mL) raised in sheep, rats were injected with desipramine, a noradrenaline uptake blocker af®nity puri®ed and characterized as described (Haycock, 1989). The (20 mg/kg i.p.) to restrict the damage to the dopaminergic system reaction was stopped in distilled water, then slides were dried and only. The animals were anaesthetized with 350 mg/kg chloral hydrate coverslipped with Permount (Fisher Chemical, NJ, USA). Control (i.p.), placed in a Kopf stereotactic frame (David Kopf, Tujunga, sections were incubated as previously described (Panula et al., 1989) USA) and the skull was exposed. A stainless steel guide cannula was with preimmune serum or antiserum HA19C preabsorbed with a lowered through a drilled burr hole 2.0 mm above the target. An histamine±protein conjugate. As an additional control, absence of the infusion needle was lowered 2.0 mm below the lower tip of the guide primary antibody in the ®rst incubation was also used. No cannula. The coordinates measured from bregma for the ®nal infusion immunoreaction was seen in these samples. site in the medial forebrain bundle near substantia nigra were AP 4.4, L 1.2, DV 8.3(Paxinos & Watson, 1986). The volume of freshly H3 binding experiments 3 made infused 6-OHDA (12 mgin4mL of NaCl containing 0.2% H3 receptors were labelled with N-alpha-[methyl- H]-methylhista- ascorbic acid) was 4 mL, given over 8 min using CMA syringes mine dihydrochloride ([3H] NAMH) (NEN Life Science Products, (1 mL) connected to PE 10 type plastic tubing and run by a motor- Inc., MA, USA) (81.500 Ci/mmol). Rats, used for binding experi- driven slow-motion syringe pump (CMA/102, CMA Microdialysis, ments, were decapitated, brains were rapidly removed and fresh Solna, Sweden). The needle was retained in its position for 4 min frozen in a liquid nitrogen. 15 mm thick coronal sections were cut in a after the infusion of 6-OHDA. Following recovery from anaesthesia cryostat at ±20 °C, thaw-mounted on poly (L)-lysine-coated slides and (about 1 week), the animals were housed singly and after the recovery stored at ±70 °C until used. Before binding procedures, slides with period in groups of three to ®ve rats per cage. Water and food were sections were left to thaw and dry at room temperature. The binding available ad libitum. Appropriate permits were obtained for all buffer contained 150 mM Na2/K phosphate buffer pH 7.4, 2 mM 3 studies. Experiments were in accordance with