to the work of Claude Bernard (Bernard, 1849). The involvement of the ..... Bitar M., Koulu M., Rapoport S. I., and Linnoila M. (1986) Diabe- tes-induced alteration ...
Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry
Microdialysis Study of Modification of Hypothalamic Neurotransmitters in Streptozotocin-Diabetic Rats Nobuyo Ohtani, Mitsuaki Ohta, and Tsukasa Sugano Department of Veterinary Physiology, Osaka Prefecture Universily, Osaka, Japan
Abstract: Neurochemical changes in the ventromedial hypothalamus (VMH) after a single intravenous injection of streptozotocin were examined, using in vivo brain microdialysis under free-moving conditions. Although streptozotocin-induced diabetes produced significant decreases in extracellular concentrations of noradrenaline (NA), serotonin (5-HT), and their metabolites in the VMH, the ratios of 3-methoxy-4-hydroxyphenylglycol/ NA and 5-hydroxyindoleacetic acid (5-HIAA)/5-HT were increased. Experimental diabetes led to a pronounced increase in extracellular GABA, which correlated strongly with the decrease in dialysate levels of NA, and to a smaller extent with that of 5-HT. A modification of dopamine (DA) metabolism was induced in the VMH of diabetic rats, whereas there was no change in dialysate DA levels. Daily injections of insulin were able to restore their levels to normal in the areas tested in the microdialysis study. The equal increases in dialysate 5-HT and 5-HIAA and the better restoration of the 5-HIAA/5-HT ratio after insulin therapy indicate that serotonergic activity may depend on the levels of circulating insulin more than on noradrenergic activity. Circulating NA was reduced in streptozotocin-diabetic rats, suggesting that the diabetes-induced reduction in sympathetic activity is accompanied by decreases in NA, or 5-HT, or both, in the VMH. Key Words: Ventromedial hypothalamus— In vivo microdialysis— Noradrenaline—Serotonin GABA— Streptozotocin-induced diabetes. J. Neurochem. 69, 1622—1628 (1997). —
The concept that the hypothalamic region of the brain is important in glucose homeostasis dates back to the work of Claude Bernard (Bernard, 1849). The involvement of the ventromedial hypothalamus (VMH), which is a rostral center of the sympathetic nervous system (Kurotsu, 1947), in a neural glucoregulatory mechanism (Woods and Porte, 1974; Niijima, 1989) has been established by a variety of techniques (Frohman and Bernardis, 1971; Steffens, 1981; Steffens et al., 1988) as well as by measurement of alterations in neuronal activity in response to changes in systemic blood glucose levels (Niijima et al., 1988). In this study, we investigated how the release of neurotransmitters in the VMH is modified under streptozotocin-
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induced diabetes, using in vivo brain microdialysis under free-moving conditions. Streptozotocin lesions the insulin-secreting B cells of pancreatic islets and induces a remarkable rise in blood glucose, resulting in a state of insulin-dependent diabetes mellitus (Arison et al., 1967; Hohenegger and Rudas, 1971). Streptozotocin-induced diabetes is accompanied by changes in not only a turnover rate, but also a steady-state level of brain monoamines (Crandall and Fernstrom, 1983; Lackovi~et al., 1990). The precursors and the rate-limiting enzymes of monoamines are also affected by experimental diabetes (Trulson and MacKenzie, 1980; Crandall et al., 1981; Mayanil et al., 1982; Bitar et al., 1986; Gupta et al., 1992). Although there are many lines of evidence that brain monoamines and their metabolites rise and/or fall in diabetes, neuronal modification of hypothalamic activity in homeostatic failure remains to be investigated. In the present study, we estimated the hypothalamic activities of monoaminergic neurons including noradrenaline (NA), serotonin (5-HT), or dopamine (DA) in streptozotocin-treated rats, measuring the extracellular concentrations of monoamines and their major metabolites in the VMH. In addition, recent evidence suggests that GABA, a primary inhibitory neurotransmitter in the CNS, is involved in the brain/blood glucose control system (Beverly et al., 1995). Our preliminary observation of the remarkable decreases in the dialysate levels of NA and its metabolite, 3methoxy-4-hydroxyphenylglycol (MHPG), in diabetic rats led us to also measure GABA in the extracellular fluid in the VMH.
Received August 12, 1996; revised manuscript received May 21, 1997; accepted June 10, 1997. Address correspondence and reprint requests to Dr. N. Ohtani at Department of Veterinary Physiology, College ofAgriculture, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 593, Japan. Abbreviations used: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonm; Ins, insulin; MHPG, 3-methoxy-4-hydroxyphenylglycol; NA, noradrenaline; OPA, o-phthaldialdehyde; STZ, streptozotocin; VMH, ventromedial hypothalamus; wks, weeks.
HYPOTHALAMIC NEUROTRANSMITTERS IN DIABETIC RATS MATERIALS AND METHODS
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position. Rats were allowed at least 3 days for recovery after brain surgery.
Animals Male Sprague—Dawley rats (Kiwa Laboratories, Wakayama, Japan), with an initial weight of 240—260 g and a normal plasma glucose level of 130—150 mg/dl, were used in all studies. They were kept in individual stainless cages in a temperature-controlled room (23 ~ 2°C)and fed a commercial diet with water ad libitum. Illumination in the room was from 0900 to 2100 h. Animal care and handling throughout the experimental procedures were performed in complete accordance with The Guidelines fcr Animal Experiinentation of the Japanese Association for Laboratory Animal Science (Experimental Animals 36, 285—288, 1987). The experimental protocols were approved by the Animal Care and Use Committee of the Osaka Prefecture University.
Single intravenous injection of streptozotocin Streptozotocin was dissolved in 0.1 M citrate buffer, adjusted to pH 4.5, and injected into the jugular vein at 60 mg/kg body weight under light ether anesthesia. The blood glucose level was measured weekly and only animals showing >250 mg/dl plasma glucose 1 week after streptozotocin injection were used. Animals were divided into the following groups: (1) controls; (2) rats 2 weeks after streptozotocin injection (STZ2wks); (3) rats 5 weeks after streptozotocin injection (STZ5wks); (4) rats treated daily with 2 lU of porcine insulin for 7 days, 1 week after streptozotocin injection (STZ2wks + Ins); (5) rats treated with 2 lU of porcine insulin daily for 7 days, 4 weeks after streptozotocin injection (STZ5wks + Ins).
In vivo brain microdialysis In vivo brain microdialysis was performed under freemoving conditions. The microdialysis membrane was 1.0 mm long with a diameter of 0.22 mm (Fig. 1). According to in vitro calibration tests in this study, the recoveries were 15—20% each. The tip of microdialysis probe was within the VMH; it was perfused with artificial CSF (NaC1 147.0 mM, CaCl
2
2.3 mM, KC1 4.0 mM, pH 5.9—6.0) at 1.0 ~il/min,using a microinfusion pump (CMA/ 100, Carnegie Medicin, Stockholm, Sweden). The concentrations of monoamines and GABA became almost constant after 60 min of perfusion, and hence, samples were collected from 90 min after the start of perfusion. Samples were collected every 60 mm, in Eppendorf tubes kept on ice, and were immediately measured by a HPLC system. Placement of the microdialysis probe was verifiedby brain
Blood sampling Arterial blood samples for measurements of glucose and catecholamines were taken from precannulated conscious animals. Rats were anesthetized with pentobarbital sodium (35 mg/kg i.p.) and monitored throughout the surgical procedure. The right external jugular vein was isolated and a 4-cm segment of Silastic tubing (0.3 mm i.d. X 0.64 mm od., Kaneka Medix, Osaka, Japan) was inserted toward the heart. The catheter was secured with acotton suture, tunneled under the skin, and exteriorized through the incision on top of the head before the skin in the neck was closed with wound staples. A piece of 27-gauge stainless steel tubing was inserted into the end of the catheter, and the catheter was filled with a 20% polyvinylpyrrolidone (PVP-l0, SigmaAldrich Japan, Tokyo, Japan) solution containing 50 lU/mi heparin and 5mg/mi ampiciliin (Sigma-Aldrich Japan). The catheter was capped with a stainless steel pin to maintain patency, and the rat was then placed into a stereotaxic instrument (Narishige, Tokyo, Japan) for brain surgery. Aliquots of 1.0—1.5 ml were withdrawn from the carotid artery and chilled immediately on ice. Plasma catecholamines were isolated and measured according to the method of Davis et al. (1981), within 4 h of sampling.
Brain surgery The skull was exposed, and a small hole was drilled to implant a guide cannula (AG-12, E1COM, Kyoto, Japan) for a dialysis probe (A-I-12-01, EiCOM). The coordinate of the guide tip was A: 5.0; L: 0.7; and H: 1.5, according to the brain atlas of König and Kuppel (1963). Stainless steel screws were anchored into the skull, and dental cement was used to fIx the guide cannula and venous catheter in
FIG. 1. Schematic diagram (A) and representative photograph (B) of asagittal section showing a dialysis probe. The microdialysis membrane of the probe was 1 .0 mm long with a diameter of 0.22 mm (A), and hence, the area dialyzed by the membrane was in and near the VMH. Arrowheads (B), the tip of a dialysis probe (A: 5.0; L: 0.7; H: 1.5, according to the stereotaxiccoordinates). Bar, 1.0 mm. AH, anterior hypothalamus; CA, anterior commissure; CO, optic chiasma; P0, preoptic area; VMH, yentromedial hypothalamus.
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dissection or histological study, and in each case it was verified that the tip of the microdialysis probe was within the ventrolateral part of the VMH.
Brain dissection At the end ofthe experiment, the animals were killed and their brains were quickly removed and placed on ice. The VMH was microdissected stereomicroscopically on the cooling plate at — 10°Cand weighed immediately. The isolated tissue in 0.5 ml of 0.1 M perchloric acid containing 1 ng/ 10 ~tl dihydroxybenzylamine as an internal standard was sonicated at 0°C.The homogenate was centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through 0.2/.tm filters (Kurabou, Osaka, Japan) and was applied to a HPLC system.
Assays A Bioanalytical Systems HPLC (West Lafayette, IN, U.S.A.) was used to measure monoamines and their metabolites by modified isocratic procedures (Davis et al., 1981; Mayer and Shoup, 1983). In brief, plasma NA and adrenaline, and brain NA, MHPG, 5-hydroxyindoleacetic acid (5HIAA), DA, and 3,4-dihydroxyphenylacetic acid (DOPAC), were analyzed by a reversed-phase HPLC system with an electrochemical detector (LC-4B, Bioanalytical Systems), whose carbon electrode was held at 0.75 V vs. the reference electrode. The mobile phase consisted of 0.1 M tartaric acid—sodium acetate buffer containing 0.5 mM EDTA-2Na, 200 mg/L sodium l-octanesulfonate, and 5% methanol at pH 4.0. For the determination of 5-HT, the carbon electrode was held at 0.45 V, and the mobile phase consisted of 0.1 M phosphate buffer containing 0.36 mM EDTA-2Na, 500 mg/L sodium 1-octanesulfonate, and 20% methanol at pH 6.0. The minimum detectable amount of monoamines was —~20pg. A Shimadzu HPLC system (Kyoto, Japan) was used to analyze GABA in dialysate and microdissected VMH, using a modification of an o-phthaldialdehyde (OPA) precolumn derivatization method (Roth, 1971; Griesmann et al., 1982). Fifty microliters of sample was injected onto a 250 X 4.6mm reverse-phase column [Shim-pack CLC-ODS (M), Shimadzu] and 10 X 4-mm guard column [Shim-pack G-ODS (4), Shimadzu]. Mobile phase was 20 mM sodium phosphate buffer (pH 2.4) containing 10 mM sodium hexanesulfonate delivered at a flow rate of 1.0 ml/min. Quantitation was by a fluorescence detector (346 nm excitation and 450 nm emission, LC-1OA, Shimadzu) after postcolunm derivatization with OPA reagent (OPA/mercaptoethanol in pH 10 borate buffer pumped at 0.3 ml/min). The minimum detectable amount of GABA was ~-~10pg. Plasma glucose was measured by glucose assay kit (Boehringer-Mannheim, Mannheim, Germany).
Histological study The brain tissues of killed animals, except those for the measurement of hypothalamic contents of monoamines and GABA, were fixed in 10% formol—saline. Serial frozen sections, 10 ~tmthick, were stained with 1% cresyl violet (Nissl stain). Tissue sections were examined under the light microscope for sites of microdialysis as well as for histological changes in the hypothalamus.
Statistical analysis Statistical analysis was performed by multifactor ANOVA
and Duncan‘ s multiple range test. Significance was set at p =
0.05. Results are expressed as mean ±SE values.
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RESULTS Mild diabetes was obtained 2 weeks after single intravenous injection of streptozotocin (STZ2wks; Fig. 2). Severer diabetes was produced 5 weeks after streptozotocin injection (STZ5wks, p < 0.01 vs. STZ2wks; Fig. 2). Although daily injection of 2 lU of porcine insulin for 7 days reduced plasma glucose levels of hyperglycemic rats to .—~250mg/dl, a difference was maintained in blood glucose levels between STZ2wks + Ins and STZ5wks + Ins. Plasma levels of NA were lowered significantly in both groups receiving a single injection of streptozotocin, and there was no significant difference between the two (Fig. 3). No changes in plasma levels of adrenaline were observed after treatments. The dialysate concentrations of NA and its metabolite, MHPG, were remarkably decreased even 2 weeks after a single injection of streptozotocin (Table 1). Although daily injection of insulin after 1 week of streptozotocin injection was able to restore the lowest levels of dialysate NA toward control, the dialysate NA in the group of STZ2wks + Ins was somewhat lower than that of STZ5wks + Ins (12.9 ±1.5 vs. 18.9 ±3.1 pg/~.tl).Although the calculated ratio of MHPG/NA in rats 5 weeks after streptozotocin injection was increased, the ratio may be difficult to interpret because both NA and MHPG were markedly reduced.
FIG. 2. Plasma glucose levels in rats that received a single intravenous injection of streptozotocin (STZ, 60 mg/kg body weight) and daily subcutaneous injections of insulin for 7 days (2 IU/ day). There were five experimental groups: control; rats 2 weeks after streptozotocin injection (STZ2wks); rats 5 weeks after streptozotocin injection (STZ5wks); rats treated daily with 2 lU of porcine insulin for 7 days after 1 week of streptozotocin injection
(STZ2wks + Ins); and rats treated with 2 lU of porcine insulin daily for 7 days after 4 weeks of streptozotocin injection (STZ5wks + Ins). Blood samples were taken from conscious rats through a cannula inserted into the jugular vein. Results were expressed as mean ± SE values of seven to 1p 10 animals. < 0.001 *p 0.001 vs. control. #p < 0.001 vs. STZ2wks. vs.
