Effects of nociceptive stimuli on brain histamine dynamics.

The effects of nociceptive stimuli on the metabolism of brain histamine (HA) were examined, because of a previous observation that the exposure of mice to electric foot shock increased the brain HA turnover. In mice, the exposure to tail pinch markedly increased the brain level of tele-methylhistamine (t-MH), a predominant metabolite of brain HA, while the level of HA was not changed. The HA turnover, measured either by the accumulation of t-MH after pargyline injection or by the HA depletion after the treatment with alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase, was enhanced by the exposure to tail pinch, like the enhancement produced by the exposure to foot shock. The exposure of rats to tail pinch increased the t-MH level in the telencephalon and the midbrain. Other types of noxious stimuli, such as placing mice on a hot plate or subjecting mice to acetic acid-induced writhing, also significantly elevated the level of t-MH but not that of HA in the mouse brain. These results suggest that nociceptive stimuli produce an increase in the brain HA turnover.

Although there have been many reports concerning the effects of various types of stress on the brain metabolism of histamine (HA), a putative neurotransmitter in mam malian brain (1,2), the results that have been obtained are extremely inconsistent. Res traint and/or exposure to cold temperatures have been reported to induce a decrease (3,4), an increase (5) or no change (6) in the HA level. Blowing air onto the backs of rats has been shown to produce an increase in the brain HA level (7). The HA turnover, as measured by the conversion of 3H-histidine to 3H-HA, increased in the rat hypothalamus with exposure to cold and restraint stress (3). However, it is lowered in the mouse brain after exposure to restraint stress (8). There fore, the response of the brain histaminergic (HAergic) systems to stressful situations may depend largely on the type of stress and on the animal species.
Brain HA is almost exclusively metabolized to to/e-methylhistamine (t-MH) (9), which is further deaminated to te/e-methylimidazole acetic acid by type B monoamine oxidase * To whom all correspondence should be addressed . (10). Following an injection of pargyline, the t-MH level increases linearly for up to 2-4 hr (11)(12)(13). Therefore, it is possible to estimate the HA turnover from the rate of t-MH ac cumulation after pargyline administration. The non-radioisotopic estimation of HA turnover is also possible by measuring the rate of HA depletion after the inhibition of histidine decarboxylase by a-fluoromethyl histidine (a-FMH) (14), a highly specific inhibitor of this enzyme (15).
Using the above methods, we reported that the exposure of mice to electrical foot shock produced an enhancement of brain HA turnover (16). To determine if nociceptive stimuli are involved in the activation of brain HAergic systems, we examined the effects of other types of nociceptive stimuli, such as tail pinch, on the brain HA dynamics and com pared them with that of the foot shock.

Materials and Methods
Animals and experimental procedure: Male ddY mice, weighing 25-30 g and male Wistar rats weighing 200-250 g (Seiwa Experimental Animals, Fukuoka, Japan), were used. They were housed in groups and given free access to food and water. The animals were subjected to the following noxious stimuli: 1) Foot shock: An electric shock (1 mA AC, 0.25 sec in duration) was delivered at 15 sec intervals over either 60 or 90 min periods to mice by placing them on the copper-wire grid floor of a plastic chamber.
2) Tail pinch: The tail was pinched with forceps 1 cm (in the case of mice) or 3 cm (in the case of rats) from its root for a 10 sec in duration. The pinch was given at 5 min in tervals over a 15-120 min period at the threshold pressure for pain (biting or at tacking the forceps). 3) Hot plate: Mice were placed on a plate heated to a temperature of 50°C for 2 min. This procedure was repeated 4 times every 15 min. 4) Writhing: Acetic acid (0.7%) was injected i.p. in a volume of 0.1 ml/10 g of body weight. a-FMH hy drochloride was kindly donated by Dr. J.

Kollonitsch of Merck, Sharp & Dohme Research
Laboratories (Rahway, NJ). Pargyline hydrochloride was obtained from Sigma (St. Louis, MO). All drugs were dis solved in 0.9% saline solution. a-FMH (50 mg/kg) or pargyline (65 mg/kg) was in jected i.p. immediately before the exposure to foot shock or tail pinch. The doses of drugs were expressed as the respective free base.
Determination of HA and t-MH: The animals were decapitated following the ex posure to various noxious stimuli, and the brains, excluding the cerebellum, were im mediately removed. Since the t-MH level in the brain shows a circadian variation (17), the decapitation was performed between 12:00-2:00 P.M. In some experiments, brains were placed on ice and divided into 4 regions including the telencephalon, midbrain, thalamus and hypothalamus. The tissues were homogenized in 2-5 ml of 0.4 N per chloric acid containing an appropriate amount of pros methyl histamine as an internal standard. The HA and t-MH contents were determined simultaneously by the method used by Tsuruta et al. (18), as modified by Oishi et al. (19), using high performance liquid chromatography with fluorescence detection. For multiple comparisons, the data were first analyzed by one-way analysis of variance. Then, the significances of the inter group difference were evaluated by Dunnett's test. The data, concerning the regional ef fects of the exposure to tail pinch on the brain amine contents, were analyzed by the two tailed Student's t-test. The data concerning the effects of the exposure to tail pinch or foot shock on the rate of t-MH accumulation after pargyline and on the rate of HA deple tion after a-FMH were analyzed by the two way analysis of variance to determine the interaction of two factors (drug treatment and exposure to noxious stimuli).

Results
Effect of the exposure to tail pinch on the levels of HA and t-MH in the mouse brain: As shown in Fig. 1, the steady-state level of t MH in the mouse brain increased significantly with the exposure to tail pinch. A significant increase in the t-MH level was already observed 15 min after starting the tail pinch test. As the period of exposure to tail pinch increased, the increase in the t-MH level became more marked. On the other hand, the HA level remained unchanged despite the exposure to tail pinch.
Effects of the exposure to tail pinch or foot shock on the HA turnover in the mouse brain: To determine if the elevation of the t-MH level induced by tail pinch resulted from an enhancement of the HA turnover, we ex amined the effect of the exposure of mice to tail pinch for 60 min on the HA turnover , which was estimated either by pargyline induced t-MH accumulation or by a-FMH induced HA depletion, and compared the result with that of foot shock. The t-MH level in the control group increased by 104.8 ng/g during the 90 min following the pargyline ad ministration (Fig. 2A). The exposure to foot shock or tail pinch significantly increased the t-MH level in the pargyline treated mice, thereby suggesting an enhancement of HA turnover. However, since the steady-state t MH level was also significantly increased by the exposure to foot shock or tail pinch, the pargyline-induced t-MH accumulation was only 132.0 and 128.0 ng/g in the foot shock and the pinch-subjected group, respectively. These values may represent underestimation and no significant interactions were shown between the pargyline or saline treatment and the exposure to noxious stimuli. When the

Fig. 2. Effects of the exposure to foot shock or tail pinch on A) the pargyline-induced t-M H accumulation and B) the a-FMH-induced
HA depletion in the mouse brain. Pargyline (65 mg/kg, i.p.) or a-FMH (50 mg/kg, i.p.) was administered immediately before the exposure to foot shock or tail pinch. Mice were decapitated 90 min and 60 min after the injection of pargyline and a-FMH, respectively.
animals were treated with a-FMH, the HA level in the control group decreased by 41.6% during the 60 min period following the ad ministration (Fig. 2B). The HA level in foot shock and tail pinch-subjected groups de creased by 54.8 and 62.6%, respectively, during the same period of time after the ad ministration of a-FMH. There were significant interactions (P<0.05) between the treatment with saline or a-FMH and the exposure to foot shock or tail pinch, indicating a facili tation of HA turnover by these treatments.
Regional effects of the exposure to tail pinch on the levels of HA and t-MH in the rat brain: Since the increase in the steady state t-MH level may have resulted from an enhancement of HA turnover, we examined the effect of the exposure to tail pinch on the steady-state levels of HA and t-MH in dif ferent regions of the rat brain. When the rats were exposed to tail pinch for 60 min, a significant rise of the t-M H level was observed in the telencephalon and the midbrain, but not in either the thalamus or the hypothalamus (Fig. 3).
Effects of various noxious stimuli on the levels of HA and t-MH in the mouse brain: To further confirm that noxious stimulation causes an activation of brain HAergic systems, we examined the effects of other types of noxious stimuli, such as placing mice on a hot plate or subjecting mice to acetic acid induced writhing, on the levels of HA and t MH in the mouse brain. All the noxious stimuli tested significantly elevated the level of t-MH but not that of HA (Fig. 4). Fig. 4.

Discussion
Effects of various noxious stimuli on the levels of HA and t-MH in the mouse brain. The HA and t-MH contents were determined 60 min after the exposure of the mice to the respective stimuli. Each column represents the mean±S.E.M. of 6-10 animals. *P<0.01, compared to the control group. ment of the HA turnover in the brain, which was accompanied by a marked increase in the t-MH level (16). In the present study, the exposure to tail pinch increased the brain t MH level. Both the pargyline-induced t-MH accumulation and a-FMH-induced HA de pletion were enhanced by the exposure to tail pinch to extents similarto those produced by the exposure to foot shock. Therefore, the in crease in the t-MH level induced by the ex posure to tail pinch seems to be due to an enhancement of brain HA turnover. Like foot shock or tail pinch, other types of nociceptive stimuli, such as heat stress on a hot plate and ii p. injection of acetic acid, increased the t MH level in the mouse brain, thereby sug gesting that nociceptive stimuli faciliate brain HA turnover. On the other hand, the HA level was not changed by any type of nociceptive stimuli tested in the mouse brain. This is con sistent with our previous results, in which phencyclidine (20) and opioids (21), strong enhancers of brain HA turnover, had no in fluence on the steady-state level of HA. Regulatory mechanisms may be set in motion rapidly to maintain the HA level within a relatively narrow range. Significant increases in the t-MH level were observed in the telencephalon and the midbrain of rats exposed to tail pinch. How ever, the levels in the thalamus and the hypo thalamus were not altered. This is consistent with the effect of the exposure to foot shock. These results suggest that activation of the HAergic systems by nociceptive information occurs predominantly in the midbrain and the telencephalic regions.
The nociceptive information seems to enhance the activity of HAergic nerve terminals in some restricted brain areas outside the hypothala mus.
The increase in HA turnover in the mouse brain induced by electric foot shock is at tenuated by systemic administration of nalox one (25). Therefore, it is likely that the ex posure to foot shock causes an activation of endogenous opioid systems, which in turn increases the HAergic outflow. Indeed, opioids enhance the HA turnover by acting on the opioid a receptors and increasing the release of HA from nerve endings (21). Various noxious stimuli reportedly cause an activation of endogenous opioid systems by facilitating the release of met-enkephalin (26) and 3-endorphin (27). Taken together, these results suggest that nociceptive stimuli in crease the HA turnover partly by releasing endogenous opioid peptides.
HA injected intracerebroventricularly (28) or into the midbrain raphe region (29) pro duces analgesia. In addition, foot shock induced analgesia is attenuated by HA H2 antagonists, such as cimetidine and ranitidine (30). Therefore, the activation of the HAergic systems, as suggested in the present study by various nociceptive stimuli, may be involved in antinociception, although the precise physiological roles of endogenous HA in the brain still remain to be determined.