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Increased brain histamine in an alcohol-preferring rat line and modulation of ethanol consumption by H₃ receptor mechanisms ¹

MINNAMAIJA LINTUNEN^*, PETRI HYYTIÄ, TINA SALLMEN^*, KAJ KARLSTEDT^*, LEENA TUOMISTO, ROB LEURS, KALERVO KIIANMAA, ESA R. KORPI and PERTTI PANULA^*,#2

^* Department of Biology, Åbo Akademi University, Turku, Finland;
Department of Mental Health and Alcohol Research, National Public Health Institute, Helsinki, Finland;
Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland;
Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Vrije Universiteit, Amsterdam, the Netherlands; Department of Pharmacology and Clinical Pharmacology, University of Turku, Turku, Finland; and
^# Department of Anatomy, Institute of Biomedical Sciences, University of Helsinki, Helsinki, Finland

²Correspondence: Department of Biology, Åbo Akademi University, BioCity, Tykistökatu 6A, 20520 Turku, Finland. E-mail: pertti.panula{at}abo.fi

SPECIFIC AIM

It has been suggested that the histaminergic system regulates brain reward mechanisms, the molecular basis of which is poorly understood. By applying molecular and behavioral methods to alcohol-preferring (AA) and alcohol-avoiding (ANA) rats, the animal models displaying aberrant reward-related behavior, we studied the possible role that the histaminergic system plays in regulating ethanol drinking.

PRINCIPAL FINDINGS

1. High brain concentration of histamine neurotransmitter and its metabolite tele-methylhistamine in alcohol-preferring AA rats
HPLC analysis revealed 120% and 170% higher histamine concentrations in the hypothalamus and septum in AA rats relative to ANA rats. Significantly higher histamine concentration was also found in the frontal cortex (60%) and hippocampus (90%) of AA rats. Histamine levels in Sprague-Dawley rats, which were used as nonselected animals, were slightly higher than in ANA rats. This indicates clearly elevated histamine levels in AA rats. In the same areas, the concentrations of tele-methylhistamine, the first metabolite in the catabolic pathway of histamine, were also significantly higher (i.e., frontal cortex, 40%; hypothalamus, 60%; and hippocampus, 70%) in AA rats compared with ANA rats. Because of the known extraneuronal methylation of histamine, these results suggest that the high histamine levels of AA rats are associated with elevated brain histamine release and turnover.

2. High density of histaminergic nerve fibers, but no change in histamine synthesizing tuberomammillary (TM) neurons in alcohol-preferring AA rats
Although the histamine concentrations in AA rats were higher, no difference was found in the mRNA expression levels of L-histidine decarboxylase (HDC), the enzyme catalyzing histamine synthesis, in the hypothalamic TM area between AA and ANA rats. Neither the intensity of cellular histamine staining nor the number of immunoreactive TM neurons revealed any differences. However, histamine-immunoreactive nerve fibers displayed a higher density in the motor cortex, septal region, nucleus accumbens, preoptic area, and hippocampus of AA rats

3. Lower histamine H₁ and H₃ receptor levels in alcohol-preferring AA rats
To find out whether the concentration changes in the histaminergic system were associated with altered histamine receptor expression or binding, we performed in situ hybridization and autoradiographic binding studies. The H₁ receptor mRNA expression was 12%–22% lower in the primary motor cortex, septal and preoptic regions, hippocampus and amygdaloid complex of AA rats when compared with ANA rats. These differences were only partly associated with the corresponding differences in H₁ receptor binding. Measurements of [³H]mepyramine binding to brain sections revealed 15% lower levels of H₁ receptor binding in the primary motor cortex and hippocampus of AA rats (Fig. 1a ). In situ hybridization studies with H₂ receptor probe revealed no differences between AA or ANA rats. The autoinhibitory H₃ receptor that controls histamine synthesis and release and acts as a heteroreceptor is potentially important in addictive behavior. Measurements of [³H]NAMH binding revealed 13%–35% lower levels of H₃ receptor binding in the primary motor and insular cortex, accumbal region and hippocampus of AA rats (Fig. 1b) , which also displayed higher histamine and tele-methylhistamine levels.

View larger version (89K): [in this window] [in a new window]   Figure 1. Binding of H1 and H3 receptor ligands to AA and ANA rat brains. The upper panels reveal autoradiographic pseudocolor film images from ligand binding studies to a) H1 and b) H3 receptors. Arrowheads indicate the areas with differences in binding between AA and ANA rats. Bregma 0.20 mm for the septal level and –3.30 mm for the hippocampal level. The lower panels reveal quantification of binding signals of H1 (left) and H3 (right) receptors in AA and ANA rat brains measured from autoradiographic films by computer-based image analysis. The values from the dentate gyrus (DG) of ANA rats were set as 100% for both receptors separately. The bar charts show the mean ± SE. Py, Rad, and LMol CA1 or CA3, pyramidal, radial, and lacunosum molecular layers of field CA1 or CA3 of the hippocampus, respectively; Gr and Mol DG, granular and molecular layers of dentate gyrus of the hippocampus, respectively. Scale bars, 1 mm. *P < 0.05; **P < 0.01; ***P < 0.001.

4. H₃ receptor but not H₁ receptor ligands modulate ethanol self-administration of alcohol-preferring rats
An H₁ receptor antagonist mepyramine did not significantly change ethanol self-administration in AA rats (Fig. 2a ). In contrast to H₁ receptor, H₃ receptor ligands modulated ethanol self-administration in a bidirectional manner in alcohol-preferring AA rats. Both H₃ receptor antagonists, thioperamide (Fig. 2b) and clobenpropit (Fig. 2c) dose-dependently and significantly suppressed ethanol consumption, whereas an H₃ receptor agonist, R--methylhistamine (Fig. 2d) , significantly increased ethanol intake. Contrary to ethanol drinking, water intakes during the sessions were not altered by any of the ligands used.

View larger version (60K): [in this window] [in a new window]   Figure 2. Effects of H1 and H3 receptor ligands on ethanol self-administration in AA rats. a) H1 receptor antagonist mepyramine did not have any significant effect on ethanol intake. H3 receptor antagonists b) thioperamide and c) clobenpropit significantly decreased ethanol intake. d) An H3 receptor agonist R--methylhistamine significantly increased ethanol intake. Data are expressed as the mean ± SE ethanol intake (g/kg) during 30-min sessions. Asterisks denote differences (*P<0.05) from the vehicle injections.

CONCLUSIONS AND SIGNIFICANCE

The present study demonstrates high brain histamine and its metabolite levels in AA rats. In addition, the increase in tele-methylhistamine concentration suggested an increase in histamine release (Fig. 3 ). Normally, activation of H₃ autoreceptors at the presynaptic nerve terminals of the histaminergic TM neurons by high histamine release will decrease the histamine synthesis and its release. The autoradiographic ligand binding to H₃ receptors revealed no differences in the reward-inhibiting TM neurons, the only known source of neuronal histamine in the brain, but the levels were significantly lower in the frontal cortex, nucleus accumbens, and hippocampus of AA rats (Fig. 3) . These differences in H₃ receptors (or a possible isoform of it) suggest an inability of H₃ autoreceptors to exercise, through normal signal transduction, the control on histamine synthesis and release (Fig. 3) . It has been shown that blocking of H₃ receptors will increase the activity of HDC. This finding suggests that, despite lack of differences in HDC mRNA levels, the HDC activity might be increased because of reduced autoreceptor function in AA rats (Fig. 3) .

View larger version (35K): [in this window] [in a new window]   Figure 3. Schematic diagram of the altered brain histaminergic system in alcohol-preferring AA rats. Double-framed boxes indicate putative regulatory sites of principal pathophysiology.

As for AA rats, an H₃ receptor agonist increased ethanol drinking at higher doses, and H₃ receptor antagonists decreased ethanol drinking in a dosedependent manner (Fig. 3) . This finding might suggest that the H₃ receptor-mediated histaminergic mechanisms are important in regulating ethanol drinking. The results also indicate that the H₃ receptor, as a heteroreceptor, is active in AA rats in regulating the drinking response, although it might be inactive, as an autoreceptor, in controlling histamine levels. The mechanism of this effect is not known. Dopamine and the mesocorticolombic pathway are known to induce reward-related processes, but a simple, direct mediation via altered dopamine release in the limbic areas is unlikely, because the behavioral effects should then be the opposite. However, the combined effect of the H₃ receptor blocking agents and ethanol on dopamine release may explain the results. Under normal conditions, histamine inhibits dopamine release via H₃ heteroreceptors in the striatum. The high histamine release and action through H₃ heteroreceptors may be responsible for the lower basal dopamine release in the caudate putamen and/or nucleus accumbens of AA rats. This low release may affect the high alcohol-preference of AA rats. As both H₃ receptor blocking and ethanol intake increase dopamine release, the maximum reward-inducing level may be reached at the highest H₃ receptor blocker doses used in our drinking experiments. It is possible that ethanol failed to induce any more dopamine release in the presence of H₃ receptor blocker; the AA rats reached satiety sooner for ethanol, and response was strongly reduced at later phases of the sessions. Although this finding may be a part of the pathophysiological mechanism underlying the abnormal behavior of AA rats, many other transmitters in addition to dopamine and histamine are obviously involved in the circuitry.

In addition to the action of histamine through H₃ receptors, H₁ receptors are involved in the reinforcing mechanism. The lower levels of H₁ receptor after in situ hybridization and ligand binding in alcohol-preferring AA rats agree with the earlier findings that H₁ receptor antagonism indicates a positively rewarding drug action. This receptor alteration may be secondary to the high histamine levels and release. However, the H₁ receptor ligand, mepyramine, failed to affect ethanol drinking in our study. This suggests that H₃ receptors are most important in the reinforcing actions of alcohol and may play a modulatory role in addictive behaviors. Further studies are needed to clarify the role of possible molecular forms of H₃ receptors in the regulation of histamine synthesis and release in AA rats, as well as possible differences in HDC enzyme, including mutations in the respective genes.

The main finding of the present study was the high brain histamine level of alcohol-preferring AA rats and the efficient bidirectional modulation of operant responding for ethanol solution by histamine H₃ receptor ligands in these rats. These data suggest a correlation between the altered histaminergic system and the genetic predisposition to high alcohol preference. Individual differences in alcohol preference may therefore be linked through H₃ receptor to differences in the functional activity of forebrain monoamine systems or their sensitivity to modification by ethanol.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0545fje ; to cite this article, use (February 20, 2001) FASEB J. 10.1096/fj.00-0545fje

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