L-DOPA induced extracellular dopamine increases in the ventromedial hypothalamus affects food intake by chickens on a lysine-free diet

Previous work from our laboratory suggests that ventromedial hypothalamic (VMH) dopamine levels were associated with decreased in food intake by chicken on a lysine-free diet. Dopamine in the VMH started to decrease from its baseline after presenting a lysine-free diet and subsequently food intake decreased. In the present study, the dopamine levels were manipulated by perfusing L-3-4-dihydroxyphenylalanine (L-DOPA) into the VMH of chicken using the in vivo microdialysis technique and food intake was concomitantly measured when chickens received an experimental lysine-free diet. A microdialysis probe was implanted into the VMH. L-DOPA was then administered locally at 2 μg/ml through the dialysis probe into the VMH of free moving chicken for 15 min and the extracellular levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT) were measured. Hourly food intake was also measured simultaneously both for control and experimental groups. Microdialysates collected from the VMH were analyzed using high performance liquid chromatography with electrochemical detection. Local administration of L-DOPA in chicken VMH increased extracellular levels of DA, which was observed at 1–2.5 h. There were no differences of NE and 5-HT levels from baseline in either group. Food intake was higher in L-DOPA treated chickens than controls at 2 h. Chickens received the lysine-free diet ate as much of their diet as the controls in the subsequent 2 h when the DA level was kept higher than the baseline. The findings suggest that L-DOPA induced extracellular DA increased in the VMH which was temporarily followed by the restoration of food intake in the lysine-free group. Keywords L-DOPA Dopamine VMH Lysine-free diet Microdialysis Monoaminergic neurotransmitters such as dopamine (DA), norepinephine (NE) and serotonin (5-HT) in the hypothalamus are involved in the central regulation of food intake. A different pattern of release for each monoamine has been observed, with either an increase or a decrease in concentration associated with food intake in rats [10,16] . In relation to the monoamines, the studies of lesions have shown that both the lateral hypothalamus (LH) and ventromedial hypothalamus (VMH) are commonly associated with food intake regulation in chicken [15] . Essential amino acids are an important component of the chicken's diet, with lysine often being the first limiting amino acid. A disproportional amount of a single essential amino acid decreases the food intake in chicken [20] . The hypothalamic circuit in rats triggers an anorectic response to an essential amino acid deficient diet [2,18,23] . Recognition of and subsequent anorectic responses to a lysine-free diet also had a hypothalamic origin, which was reported as being an integration centre for food intake [9,22] . The release of DA, NE and 5-HT in the LH or VMH is connected with either inhibition or stimulation of food intake [17,21] . In recent years the accumulated evidence indicates that hypothalamic DA plays a role in the regulation of food intake. It regulates hunger and satiety, and is central to the reward system. It is reported that DA in the LH is associated with inhibition of food intake, while that in the VMH is associated with stimulation of food intake [8] . Recently Ichijo et al. [11] found that DA levels in the VMH were associated with decreased food intake by chickens on a lysine-free diet. In this report, DA levels in VMH started to decrease (about 40% from baseline) at 1 h after presenting the lysine-free diet and remained lower than that of baseline and the control group until the end of the feeding trial. Food intake of the lysine-free group was significantly less than that of the control at 4 and 5 h. These findings lead to the hypothesis that keeping the DA level in VMH at or beyond the basal level restores the food intake of chickens on the lysine-free diet to close to the control group. Therefore, in the present study, the DA levels were manipulated by perfusing L-DOPA into the VMH of chicken using the in vivo microdialysis technique and food intake was concomitantly measured when chickens received an experimental lysine-free diet. It was necessary to establish the dose of L-DOPA in chicken VMH to increase the DA level in VMH by two- or three-fold higher than that of the level before perfusion while maintaining stable conditions for NE and 5-HT, to prove the above hypothesis. For this purpose, several pilot experiments were carried out and the extracellular DA levels were increased by two- or three-fold from the baseline when L-DOPA solution of 2 μg/ml was perfused at a rate of 1 μl/min for 15 min. Day old male egg-type chickens ( Gallus gallus domesticus ) were housed in a temperature-controlled (32 °C) room with an automatic 12:12 h light/dark cycle (light on at 09.00 h). Chickens were given free access to water and a commercial starter diet (CP, 21%; ME, 2.95 Mcal/kg, Kumiai Feed Co. Ltd, Tokyo, Japan). At 7 days of age, chickens were weighed and selected so that the average body weight was as uniform as possible and they were housed in individual cages in an experimental room at a temperature of 28 °C. All procedures involving the uses of animals were performed according to the guidelines for the care and use of animals, as approved by the Animal Experimentation Committee of Utsunomiya University. All chickens were given free access to the commercial starter diet until 15 days of age. At 16 days, the commercial diet was changed to a control diet (CP, 14.5%; ME, 2.95 Mcal/kg). The control diet consisted of cornstarch, crystalline amino acid mixture, corn oil, mineral and vitamin mixtures, and contained 11.9 g/kg of lysine hydrochloride. The control diet was formulated according to Sugahara and Kubo [20] . The lysine-free diet had the same composition as the control diet, except for the absence of lysine hydrochloride. The two diets were kept isonitrogenous and isocaloric by replacing lysine hydrochloride with glutamic acid and cornstarch. Food intake was measured at every hour for 5 h of feeding trials for both the groups. At 17 or 18 days of age, the chickens were implanted with a guide cannula (0.5 mm × 8 mm, diameter and length, respectively, Eicom, Kyoto, Japan) aimed at one side of the ventromedial hypothalamus (anterior to the interaural line, 7 mm, lateral to the midline, 0.2 mm and below the skull surface, 7.2 mm) under sodium pentobarbital anaesthesia (4 mg/100 g body weight). The site was selected based on a study of a brain atlas [14] . The guide cannula was fixed to the skull with stainless-steel support screws (2 mm × 6 mm, diameter × length, respectively) and dental cement. The chickens were individually housed and allowed at least 4 days for post-surgical recovery. During this period, the chickens were given ad libitum food (control diet) and water. L-DOPA was obtained from Sigma (Sigma Chemical Co., St. Louis, MO, USA) and dissolved in Ringer's solution (147 mM NaCl, 4 mM KCl, and 4 mM CaCl 2 ). The L-DOPA solution was then filtered through a 0.20 μm membrane filter (Dismic-25AS, Advantec, Kyoto, Japan) before perfusion into the chickens. Freshly prepared L-DOPA solution was used in each experimental day. The L-DOPA solution was sonicated and taken by a Hamilton gas tight syringe and administered into the chicken VMH. Each experimental day, at about 9:30 am, the microdialysis probes (3 mm long, 0.22 mm wide, molecular weight cut-off of approximately 50,000 Da, Eicom) were inserted into the VMH via the implanted guide cannula. Using a syringe pump (EP 60, Eicom) with a 2.5 ml gas tight syringe, the probe was perfused with a Ringer's solution at a flow rate of 1 μl/min. No treatment was administered until the basal level of monoamines became stable and this did not occur until at least 3 h after probe insertion. After collecting the baseline samples, the L-DOPA was perfused via the dialysis probe into the VMH of a chicken for 15 min as well as changing the control diet to a lysine-free diet. After completing the L-DOPA treatment, the perfusion medium was switched back to normal Ringer's solution and the dialysis was continued. On the other hand, only Ringer's solution was perfused into the control chicken throughout the experiment and these chickens were always provided the control diet. The collection time for each dialysis sample was 30 min and the dialysate was collected for 8 h for each pair of chickens. The dialysate was collected into a fraction collector (EF-80, Eicom) containing 10 μl of phosphate buffer to prevent degradation of the monoamines. Monoamines in the dialysates were detected by reverse phase high performance liquid chromatography (HPLC) with an electrochemical detector (ECD-300, Eicom) using an ion-exchange column (CAX, Eicom) and an analytical cell (ATC-300, Eicom) with an applied potential of +0.45 V. The mobile phase contained 0.1 M acetic acid-citric acid buffer, 100 mg/ml sodium-octyl-sulphate, 5 mg/ml EDTA. 2Na, 15% methanol, at pH 3.5 and was pumped through the system at 0.25 ml/min using a pump (EP-300, Eicom). Chromatograms were integrated and compared with standards and analyzed using a computer-based data acquisition system (eDAQ Power Chrome 280, City, NSW, Australia). Freshly prepared standard solutions were used for the HPLC-ECD runs and included the following: norepinephrine, 3-hydroxytyramine hydrochloride and 5-hydroxytryptamine creatine sulphate complex (Sigma Chemical Co. Ltd). After completion of the dialysis, chickens were decapitated and brain was fixed in 10% paraformaldehyde. Serial coronal sections were cut, mounted and stained with cresyl violet and analyzed in the light microscope. The monoamines in the dialysate were expressed as a percentage of the baseline in each individual chicken. The average monoamine level in three samples immediately preceding the drug application was defined as the baseline (100%). Data were analyzed using a two-way ANOVA with the Dunnett's test at each time point. To compare the results of food intake, the t -test was performed. Values of P < 0.05 were considered to be significant. Data were expressed as mean ± SEM. The microscopic observations of brain slices after exsanguinations confirmed that eight chickens for each diet had the correct placement of the microdialysis probe in the VMH of the chickens being studied. A representative microscopic example is shown in Fig. 1 . The effect of L-DOPA administration on food intake in chickens fed ad libitum over a 5 h period is shown in Fig. 2 . The hourly changes of food intake were measured for 5 h in the control ( n = 8) and the lysine-free plus L-DOPA treated groups ( n = 8). The control chickens ate their diet at a similar rate (0.8–1.1 g/100 g body weight hour) during the experiments. This response of the control chickens is consistent with our previous findings [11] . At 2 h, food intake was significantly higher in lysine-free chickens compared to the controls. At 3 h, food intake also tended to be higher ( P = 0.25). The food intake in the lysine-free chickens was lower at 4 and 5 h compared with the controls and a significant difference was observed at 5 h. Fig. 3 shows the effects of local perfusion of L-DOPA solution on the extracellular levels of DA (upper panel), NE (middle panel) and 5-HT (lower panel) in the VMH. The basal concentrations of DA (pg/μl) were 1.01 ± 0.03 and 1.00 ± 0.04, for the control and the lysine-free plus L-DOPA treated chicken, respectively. Local perfusion of L-DOPA significantly increased the DA level compared with the baseline. At 30 min following administration there was no increase in DA. At 60 min following perfusion with L-DOPA, the extracellular DA reached its maximum level of 324 ± 32% of the baseline. The levels of DA at 1.5, 2, 2.5 and 3 h were 247 ± 29%, 167 ± 13%, 153 ± 14% and 140 ± 16%, respectively. The DA returned to the basal level at 3.5 h after L-DOPA perfusion. Dopamine in the control chickens remained close to the basal level throughout the experiments. Exogenously administered L-DOPA is known to increase the concentration of DA in many brain regions including the hypothalamus, striatum, and brain stem [4,12] . However, the effect of L-DOPA upon interstitial DA levels remains unexplored in chicken VMH. The results of the present investigation demonstrate that perfusion of L-DOPA solution (2 μg/ml) into VMH significantly increased the extracellular level of DA in the lysine-free chicken. The maximal response was 3-fold of the basal level at 1 h ( Fig. 3 ). L-DOPA is the precursor of DA, which is then transformed to DA in neurons after its administration and the extracellular level is increased [12,19] . The current results are consistent with Adachi et al. [1] , who demonstrated that local administration of L-DOPA increased DA levels in a rat striatum in a dose dependant manner. An increase in DA level (144% of baseline, data not shown) to L-DOPA administration was observed at 0.5 μg/ml. The extracellular DA level increased to 195% of baseline (data not shown) when the concentration of perfused solution was 1 μg/ml. Therefore, DA level in the VMH responded to L-DOPA perfusion in a dose-dependent manner in chickens as in rats. However, at both concentrations, after discontinuation of the L-DOPA perfusion, the DA level returned to the baseline in 1 h. Neither food intake nor DA levels in the VMH of the control chicken changed throughout the experiment. If L-DOPA were to be perfused in chickens fed a control diet, that would increase the DA level in the VMH and would have some affect on the food intake. Recently, Bungo et al. [3] reported that when broiler chickens were fed a commercial diet and were intracerebroventricularly administered 50 μg of L-DOPA, there was no effect on food intake. Therefore, it is unlikely that L-DOPA perfusion affect food intake in the control chickens even though DA level increased in the VMH. At 1 h post-perfusion of L-DOPA, the DA level in the VMH reached a maximum level and food intake of chickens on the lysine free diet at 1–2 h was higher than that of the control. Thereafter, DA levels remained higher than the baseline up to 2.5 h post-perfusion and food intake during this period was not different between the two groups. Significantly lower food intake of the lysine free diet plus L-DOPA perfusion group was observed at 0.5–1 h after the DA level in the VMH returned to the baseline. The disappearance of additional food intake at 4–5 h could be due to the short term effect of L-DOPA on food intake which was dependent on the time-course changes in interstitial DA level. This response of food intake (at 4–5 h) was different from our previous study [11] in which the lower food intake of chickens receiving a lysine free diet without L-DOPA perfusion was observed 3 h after presenting the diet and thereafter. Therefore, the lower food intake in the lysine-free group was delayed by an hour due to the perfusion of L-DOPA in the VMH. This suggests that increase in DA in the VMH were associated with food intake variations in chickens on a lysine-free diet. Microinjections of catecholamine into different brain structures have been studied by different investigators over the years. The exogenous administration of catecholamine and serotonin into cerebral ventricles affected the food intake in meat- and egg-type chicken [13] . Intracerebroventricular injection of DA into the lateral ventricle did not alter food intake in chicken [5] . Microinjection of DA into the nucleus accumbens produced a dose-dependent stimulation of eating in rat while in the ventricular system in food deprived rat the same treatment resulted in a suppression of food intake [24] . Therefore, taken together the reported findings including the current results, it is likely that the effect of central DA on food intake varies with the brain regions. As shown in Fig. 3 , NE levels in the VMH of both groups remained stable throughout the microdialysis, although there was a tendency for NE to decrease from 1.5 to 4 h in the lysine-free plus L-DOPA treated chickens from the baseline. A significant difference was observed at 3.5 and 4 h when compared with the controls. The level of 5-HT also remained stable throughout the microdialysis experiment in both groups except for at 1.5 and 4.5 h in the lysine-free and control groups, respectively. A significant difference was shown at these two time points when compared between two groups. These results are similar to our previous study in which chickens were not perfused with L-DOPA [11] . Reports from other investigators indicate that although L-DOPA has been shown to increase the DA content of a variety of tissues in rats, it has little or no effect on NE level [3,6,19] . In addition to this, Everett and Borcherding [7] pointed out that the formation of NE from DA after injection of L-DOPA is minimal in mice. Therefore, it is likely that activity of at least NE neurons in the VMH is independent of the activity of DA neuron in chickens. In summary, the extracellular DA level significantly increased in the VMH after L-DOPA perfusion and this occurred at 1–2.5 h. The NE and 5-HT remained stable during the microdialysis in both groups. Food intake of chickens on lysine-free diet with DA level higher than the baseline was similar to that of the control chickens. These results suggest that L-DOPA perfusion increased extracellular DA in the VMH which in turn was temporarily followed by the restoration of food intake in chickens fed a lysine-free diet. Acknowledgements This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, Sports and Technology of Japan (No. 22580304 ). We thank Prof. Joe M. Regenstein, USA for editorial assistance. References [1] U. Adachi S. Yamada M. Satomoto K. Watanabe H. Higuchi T. Kazama M. Doi S. Sato Pentobarbital inhibits L-DOPA induced dopamine increases in the rat striatum: An in vivo microdialysis study Brain Res. Bull. 69 2006 593 596 [2] J.E. Blevins P.S. Teh C.X. Wang D.W. Gietzen Effects of amino acid deficiency on monoamines in the lateral hypothalamus (LH) in rats Nutr. Neurosci. 6 2003 291 299 [3] T. Bungo K. Yanagita J.I. Shirashi Feed intake after infusion of noradrenaline, dopamine or its precursor into the lateral ventricles in neonatal chicks J. Anim. Vet. Adv. 9 2010 760 763 [4] N.T. Buu Vesicular accumulation of dopamine following L-DOPA administration Biochem. Pharmacol. 38 1989 1787 1792 [5] D.M. Denbow J.A. Cherry P.B. Siegel H.P. Van Krey Eating, drinking and temperature response of chicks to brain catecholamine injections Physiol. Behav. 27 1981 265 269 [6] P.S. Doshi D.J. Edwards Effects of L-DOPA on dopamine and norepinephrine concentrations in rat brain assessed by gas chromatography J. Chromatogr. 210 1981 505 511 [7] G.M. Everett J.W. Borcherding L-DOPA: effect on concentrations of dopamine, norepinephrine and serotonin in brains of mice Science 168 1970 849 850 [8] S.O Fetissov M.M. Meguid C. Chen G. Miyata Synchronized release of dopamine and serotonin in the medial and lateral hypothalamus of rats Neuroscience 101 2000 657 663 [9] R.L. Hawkins M. Inoue M. Mori K. Torii Effects of inhibin follistation, or activin infusion into the lateral hypothalamus on operant behaviour of rats fed lysine deficient diet Brain Res. 704 1995 1 9 [10] B.G. Hoebel L. Hernandez D.H. Schwartz G.P. Mark G.A. Hunter Microdialysis studies of brain norepinephrine, serotonin and dopamine release during ingestive behaviour, theoretical and clinical implications Ann. N. Y. Acad. Sci. 575 1989 171 191 [11] A. Ichijo N. Hayashi C. Fukuoka J.J. Hu F. Yoshizawa K. Sugahara Dopamine release in the ventromedial hypothalamus of growing chickens decreases when they are fed a lysine devoid diet J. Poult. Sci. 45 2008 281 286 [12] K. Koshimura T. Ohue Y. Akiyama A. Itoh S. Miwa L-DOPA administration enhances exocytic dopamine release in vivo in the rat striatum Life Sci. 51 1992 747 755 [13] W.J. Kuenzel Central neuroanatomical systems involved in the regulation of food intake in birds and mammals J. Nutr. 124 1994 1355S 1370S [14] W.J. Kuenzel M. Masson A Stereotaxic Atlas of the Brain of the Chick ( Gallus domesticus ) 1988 The Johns Hopkins University Press Baltimore [15] S. Lepkovsky M. Yasuda Hypothalamic lesions, growth and body composition of male chickens Poult. Sci. 45 1966 582 588 [16] M.M. Meguid Z.J. Yang A. Laviano Meal size and number relationship to dopamine levels in the ventromedial hypothalamic nucleus Am. J. Physiol. 272 1997 1925 1930 [17] E.J.B. Ramos M.M. Meguid A.C.L. Campos J.C.U. Coelho Neuropeptide Y, α-melanocyte-stimulating hormone, and monoamines in food intake regulation Nutrition 21 2005 69 279 [18] M. Smriga M. Mori K. Torii Circadian release of hypothalamic norepinephrine in rats in vivo is depressed during early L-lysine deficiency J. Nutr. 130 2000 1641 1643 [19] G.L. Snyder M.J. Zigmond The effects of L-DOPA on in vitro dopamine release from striatum Brain Res. 508 1990 181 187 [20] K. Sugahara T. Kubo Involvement of food intake in the decreased energy retention associated with single deficiencies of lysine and sulphur-containing amino acids in growing chicks Br. Poult. Sci. 33 1992 805 814 [21] T. Tachibana D. Utimura H. Kato T. Kubo K. Sugahara Extracellular norepinephrine in the medial hypothalamus increases during feeding in chicks: a microdialysis study Comp. Biochem. Physiol. A 127 2000 331 338 [22] K. Torii T. Yokawa E. Tabuchi R.L. Hawkins M. Mori T. Kondoh T. Ono Recognition of deficient nutrient intake in the brain of rats with the L-lysine deficiency monitored by functional magnetic resonance imaging electrophysiologically and behaviourally Amino Acids 10 1996 73 81 [23] C.X. Wang H. Yang J.C. Perrot D.W. Gietzen Inhibition of norepinehrine release in the rat ventromedial hypothalamic nucleus in essential amino acid deficiency Neurosci. Lett. 259 1999 53 55 [24] P.J. Wellman Modulation of eating by central catecholamine systems Curr. Drug Targets 6 2005 191 199