Neurosctence Letters, 125 (1991) 235-237 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 0304394091001995

NSL 07716

235

Feeding-induced increase in the extracellular concentration of histamine in rat hypothalamus as measured by in vivo microdialysis

Yosh ino r i I toh , R y o z o Olshi and K i y o m i Saeki

Department of Pharmacology, Okayama Umverstty Medical School, Okayama (Japan)

(Received 12 November 1990, Rewsed version recetved 16 January 1991, Accepted l February 1991)

Key words H~stamme, Mlcrodmlysls, Rat, Hypothalamus; Fasting, Feeding

The extracellular concentration of histamine (HA) m the hypothalamus of conscious and freely moving rats was measured by m vivo mlcrodialysls and the effects of fasting and feeding on the HA concentration were examined In non-fasted rats, the basal HA concentration was almost constant from 11 00 to 17 00 h on the day following implantation of the dmlysls probe, the mean value being l 1 1 pg/30 mm No s~gmficant change m the HA concentration was observed m rats deprwed of food for 24 h In 24-h fasted rats, feeding for 15 rain produced a transient and slgmficant increase m the HA concentration These results suggest that histammergac actwity m the rat hypothalamus increases dunng feeding.

Several hnes of evtdence have suggested that histamine (HA), one of the putative neurotransmltters in the mam- malian brain [13], is involved m the regulation of food retake: mtracerebral [1] or intrahypothalamlc injection [11] of HA receptor agomsts and antagomsts affects feedmg behavior We previously demonstrated that the tissue level of te/e-methylhlstamine (t-MH), a predomi- nant metabohte of HA m the brain [12], exhibits feeding- related circadmn varmtion both m the mouse and rat brain and that feeding produces a marked increase m the t-MH level in 24-h fasted animals [6]. However, it re- mains to be clarified whether or not the feeding-induced increase m brain t-MH level resulted from an enhance- ment of histammergtc actw~ty

Recent development of brain microdmlys~s technique has enabled the measurement of extracellular concentra- tions of a variety of neurotransmitters in behaving ani- mals. Using this technique, we have measured the extra- cellular concentratton of HA m the hypothalamus of urethane-anesthetized rats [3] The present experiment was designed to determine, using brain mlcrodialysis, whether the extracellular HA concentration increases durmg feeding in conscious and freely moving rats deprived of food for 24 h

Male Wlstar rats weighing 250-300 g (Selwa Experi- mental Ammals, Fukuoka, Japan) were housed m groups m a room controlled at 22 +__ 1 °C and maintained in an alternating 12-h light-dark cycle (hghts automati-

Correspondence K Saekl, Department of Pharmacology, Okayama University Medical School, 2-5-1 Shlkata-cho, Okayama 700, Japan

cally on at 07.00 h). Rats were anesthetized with sodium pentobarbital (50 mg/kg, t p.) and were placed on a ster- eotaxtc apparatus (Narishige Scientific Instrument Lab., Tokyo, Japan). A U-shaped dialysis probe with a loop of cellulose membrane tubing (4 mm long, 0.22 mm o.d., molecular weight cut off at 50,000; BDP-UI-12-02, Eicom, Kyoto, Japan) was implanted in the medial hypothalamus, which contains both the nucle~ dorsome- dialis and ventromedmhs. The stereotaxic coordinates were 2.4 mm posterior and 0.8 mm lateral to the bregma, and 10 3 mm below the surface of skull. The probe was fixed to the skull w~th an anchor screw and dental ce- ment. After ~mplantatlon of the dialysis probe, the non- fasted group was allowed free access to food and water and the 24-h fasted group was given only water ad libi- turn. Mlcrodlalysls was started at 10.00 h on the follow- mg day (18-22 h after implantation of the dialysis probe), when all rats showed complete recovery from anesthesia. Unless otherwise indicated, food and water were not gwen dunng the microdialysis experiment. Rmger's solution was perfused at a rate of 1 pl/min. This perfuslon medium was freshly prepared from sterilized distilled water ~mmediately before use, since bacterial contaminatton may cause an erroneously high amount of HA in the mlcrodialysates [3]. The mlcrodialysates were collected every 30 rain from 1 h after the start of perfuslon into mlcrotubes containing 30 /tl of 0.2 M perchloric acid. HA was determined by post column o- phthalaldehyde derivatizatlon according to the method of Yamatodam et al. [16] with modifications [2], using HPLC with fluorescence detection. A 50/11 ahquot of the

236

H Non-fasted

o o Fasted for 24 h

~" 15

o \

o

o | I l I I I

12:00 13,00 14.'00 15:00 16:00 1~.00

Time (h)

Fag 1 Tame course of the basal histamine concentration dunng con-

tlnuous perfuslon of the medml hypothalamus in non-fasted and 24-h

fasted rats Sterile Ringer's solutmn was perfused at a rate of I pl/mm

from 10.00 h and 30-ram mlcrodmlysate fractions were collected from

l I 00 to 17.00 h into rmcrotubes containing 30/~1 of 0 2 M perchlonc

acid Each point represents the mean + S E M of 6 ammals

mixture of dmlysate and perchlonc acid was directly rejected onto HPLC. At the end of the experiments rats were decapitated under pentobarbital anesthesm and the brains were removed and frozen m a refrigerator at a temperature of - 40°C . Coronal sections of 200 /zm thickness were cut m a cryostat at - 2 0 ° C and they were placed on shde glasses to verify the locatton of the dialy-

sis probe under the microscope. Data were first evaluated by one-way or two-way anal-

ysis of variance When signifcant differences were observed, the statistical comparison of 30-min post- treatment values w~th the average of basal (pretreat- ment) values was performed by either Dunnett 's test or

Student's t-test. As shown in Ftg, 1, m non-fasted rats, there were no

significant variations in the basal HA concentrations m 30-mln fractions from 11.00 to 17.00 h, and the average during this 6-h period was 11.1 + 0 9 pg/30 min (mean + S.E.M. of 6 animals). The basal HA concentrations

were also constant from 11.00 to 17 00 h m 24-h fasted rats, and the average during this period was 10.5 + 1.3 pg/30 mm, a value which was not significantly different from that in non-fasted rats. When 24-h fasted rats were given normal food for 15 mln, a s~gnlficant and transient increase (by 143%) in the HA concentratmn was observed (Fig. 2). In the other experiment, 24-h fasted rats were placed m a situation where they were able to see and smell the food but unable to eat it. Th~s situa- tion was achieved by placing the food covered with a 20- ml plexlglass beaker in the cage for 15 mm. In this exper- iment, the average of three consecutive pre-treatment

values was 11.6 _ 1 3 pg/30 mm and that of the experi- mental values was 18.9 + 2 4 pg/30 mm (mcluding 15- rain of the food-presentation period) (mean + S E M of 6 ammals) This mcrease (63%) m the HA concentration was significant (P < 0 05) but smaller than that produced by feedmg

Because of multiple compartmentalization of brain HA (neurons, mast cells and endothehal cells), tt seems to be difficult to d~scrtmmate neuronal HA from that derived from non-neuronal tissues [5]. However, the syn- thesis rate or turnover rate of HA in neurons ts much more rapid than that in non-neuronal tissues [5]. In our previous study [3], the extracellular HA concentration In the rat hypothalamus decreased by approximately 70% 4-5 h after treatment wtth ~-fluoromethylhtstLdine, an irreversible Inhibitor of histidine decarboxylase [4] Thts suggests that most of the extraceUular HA in th~s brain regmn is derived from neurons

In the present study, the extracellular concentratton of HA, corrected from the in vitro recovery of HA through the dialysis membrane (27 4%), m the medial hypothala- mus of conscious and freely mowng rats was 11 5-12.1 nM This value was higher than that (7.8 nM) reported prewously in the same brain region of urethane-anesthe- tlzed rats [3] or that (8 5 nM) reported by other mvestl-

O 03

Q.

O

t-

¢0 .-_= - r

25

20

15

10

5

0 ' 1tO0

Feeding IB

I | J I I I

12:.00 13:00 14:00 15:00 16:00

Time (h)

Fig 2 Effect of feeding on the extracellular concentrauon of hista-

mine in the medial hypothalamus of 24-h fasted rats Rats (n = 7) were

fed with normal food for 15 ram, as indicated by a sohd bar Each point represents the mean __+ S.E M * P < 0 01 as compared with the

average of the 5 basal (pretreatment) values

gators in the postenor basal hypothalamus of chloral hy- drate-anesthetized rats [10]. This may be due to the inhi- bitory action of the anesthetic agents used on HA turn- over in the brain [3, 8]. Considenng that the HA turn- over rate m the rat brain is reduced by 47% under urethane anesthesia [3], the value obtained m the present experiment in conscious rats seems to be reasonable.

Several investigators have observed that the HA con- centration m the rat brain showed circadian variation, although the results were rather inconsistent. Orr and Quay [7] reported that the concentratton of HA in the rat hypothalamus was highest early tn the hght period, whde Tuomisto and Tuomisto [14] obtained the highest value in the same region at the end of the light period. However, changes m tissue level of HA offer httle mfor- matron about the functional activtty of hlstaminerg~c systems. We also have observed that the concentration of t-MH but not of HA m the rat brain showed a clrca- dtan variation w~th the highest level m the middle of the dark period [6]. This diurnal change in the t-MH level disappeared m animals deprived of food for 24 h and the increase m the t-MH level was observed m these animals after feeding m the hght period, thereby suggesting that food intake is mvolved in the occurrence of the circadian variation of the t-MH level [6] However, it has been dif- ficult to clearly know the mechanisms underlying this feeding-related variation of the t-MH level. In the pres- ent study, feeding produced a marked increase in the extracellular HA concentratton. Therefore, it is sug- gested that the release of endogenous (probably neuro- nal) HA is enhanced during feeding. However, we cannot exclude the possibihty that other factors, such as behav- ioral excitation induced by the presentation of food, are revolved m the enhancement of hlstammerg~c actiwty m the food-deprived rats, since in the present experiment, a small but signtficant increase in the HA concentration was observed by showing the food inaccessible to the animals.

Immunohistochemical stu&es have demonstrated that cell bodies of HA containing neurons are present in the posterior part of the hypothalamus and their nerve ter- minals are found at the highest density m the hypothala- mus [15], where HA may play important roles in the regulation of some neural functtons, including food in- take (see review by Roberts and Calcutt [9]). Sakata et al. [11] have demonstrated in rats that bilateral microm- ject~on of H~ antagomsts into the ventromedlal hypotha- lamus elicited food intake, indicating that HA in this hypothalam~c nucleus mhibtts food retake. Therefore, the enhancement of hlstaminergic activity observed m the present experiment during feeding may be mvolved

237

tn the induction of the state of satiety. Further studies are needed to exactly correlate the hypothalamlc HA re- lease to feeding behavior.

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