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Local administration of l -DOPA in the chicken ventromedial hypothalamus increases dopamine release in a dose-dependent manner
Neuroscience Letters 529 (2012) 150–154
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Local administration of l-DOPA in the chicken ventromedial hypothalamus increases dopamine release in a dose-dependent manner Mohammad Rashedul Alam a,b , Fumiaki Yoshizawa b , Kunio Sugahara b,∗ a b
United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan Department of Bio-productive Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan
h i g h l i g h t s
In vivo microdialysis showed the effect of l-DOPA on extracellular DA levels in chick VMH. Extracellular DA levels increased significantly in the VMH after l-DOPA perfusion. l-DOPA has no effect on NE and 5-HT during microdialysis. The DA, NE and 5-HT within the dialysate were related to neural activity.
a r t i c l e
i n f o
Article history: Received 25 July 2012 Received in revised form 21 August 2012 Accepted 28 August 2012 Keywords: Chickens Dopamine l-DOPA Microdialysis Tetrodotoxin Ventromedial hypothalamus
a b s t r a c t l-DOPA induced extracellular dopamine (DA), norepinephrine (NE) and serotonin (5-HT) in the ventromedial hypothalamus (VMH) of chickens were measured by in vivo microdialysis. Several doses of 3,4-dihydroxy-l-phenylalanine (l-DOPA) were administered locally through the microdialysis probe into the VMH of chickens for 10 min. Local perfusion of l-DOPA increased the extracellular levels of DA. The increased DA was dose-related and was significantly higher compared to the baseline and control group. The maximal level of DA was 212% and 254%, respectively, of the baseline following administration of 1 and 2 g/ml l-DOPA. There were no changes in NE and 5-HT levels from baseline after l-DOPA perfusion. l-DOPA (1 g/ml) was mixed with Ca2+ -free Ringer, tetrodotoxin (TTX) (2 M) and high K+ and was perfused for 30 min into the chicken VMH. TTX and Ca2+ -free Ringer’s solution inhibited the effectiveness of l-DOPA in increasing DA release. The NE and 5-HT levels were significantly lower than the baseline. After administration of K+ a significant increase of DA, NE and 5-HT was observed. The microdialysis results are consistent with our objective that l-DOPA induced extracellular DA increases in the VMH in a dose-dependent manner and the released DA, NE and 5-HT within the dialysate were related to neuronal activity. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hypothalamic monoamines have been reported to be involved in regulation of food intake in rats and chickens using intracerebroventricular (ICV) injection of compounds [4]. However, there has been a little information presented on the involvement of endogenous monoamines in variations of food intake in chickens. Ichijo et al. [6] found that the decreased food intake due to feeding a lysine-free diet followed the decrease in the ventromedial hypothalamic (VMH) dopamine (DA) level in chickens using an in vivo brain microdialysis technique. This finding suggests that
∗ Corresponding author at: Department of Animal Science, Division of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya-shi, Tochigi 321-8505, Japan. Tel.: +81 286495441; fax: +81 286495401. E-mail address: [email protected] (K. Sugahara). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.08.054
if DA in the VMH can be pharmacologically or physiologically modified, then the variations of food intake of chickens can be alleviated. A previous study with rats showed that local perfusion of 3,4-dihydroxy-l-phenylalanine (l-DOPA) using a reverse brain microdialysis technique affects the DA level in the striatum [2]. However, the effect of local l-DOPA administration on extracellular DA levels in the chicken VMH remains unexplored. Therefore, this study was done to determine if local perfusion of l-DOPA using a reverse brain microdialysis technique affects the extracellular level of monoamines in the VMH of chickens similarly to that with rats. We also examined if changes of monoamine contents in the dialysate derived from neuronal activity in the VMH. 2. Materials and methods All procedures involving the uses of animals were performed in accordance with regulations for the care and use of animals
M.R. Alam et al. / Neuroscience Letters 529 (2012) 150–154
required by the Animal Experimentation Committee of Utsunomiya University. 2.1. Animals and their management Day old male egg-type chickens (Gallus gallus domesticus) were housed in a temperature-controlled (32 ◦ C) room with 12/12 light/dark cycle. They were given free access to water and a commercial starter diet (CP, 21%; ME, 2.95 Mcal/kg, Kumiai Feed Co. Ltd, Tokyo, Japan). The feed was manufactured according to the requirements of all essential amino acids recommended in Japanese Feeding Standard for Poultry [11]. 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 at a temperature of 28 ◦ C.
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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 l/min. After collection of the baseline value, l-DOPA was perfused via the dialysis probe into the VMH of a chicken for 10 min whereas Ringer’s solution without calcium or with 2 M TTX or a high concentration of K+ (100 mM) were perfused for 30 min and dialysates were collected. After this pharmacological treatment the perfusion medium was switched back to the normal Ringer’s solution and the dialysis continued. The collection time interval for each dialysis sample was 30 min and the dialysate was collected for 7 or 8 h. During microdialysis experiment, the chickens were given free access to water and the commercial diet.
2.5. Determination of monoamines via high performance liquid chromatography (HPLC) with electrochemical detection (ECD)
2.2. Surgical procedures for implantation of the guide cannula Seventeen- or 18-day-old chickens were fasted for at least 2 h before surgery. At 17 or 18 days of age, the chickens were stereotaxically implanted with a guide cannula aimed at one side of the hypothalamus, specifically the ventromedial hypothalamus (VMH). In preparation for cannulae implantation, chickens were anaesthetized with intraperitoneal administration of sodium pentobarbital (4 mg/100 g body weight). They were then placed in a stereotaxic frame (David Kopf Instruments, California, USA) and a small incision with a scalpel was made to expose the skull. A highspeed microdrill was then used to drill a hole in each chicken skull overlying the VMH (VMH, anterior to the interaural line by 7 mm; lateral to the midline by 0.2 mm and 7.2 mm below the skull surface [10]) and the guide cannula (0.5 × 8 mm, diameter and length, respectively, Eicom, Kyoto, Japan) was lowered below the skull surface. Two additional holes were drilled in the skull for anchoring of the stainless-steel support screws (2 × 6 mm, diameter × length, respectively). The guide cannula was fixed in place with dental cement (Shofu Inc., Tokyo, Japan). A dummy cannula was then inserted to prevent obstruction. The chickens were individually housed and allowed at least 4 days for post-surgical recovery before the start of the microdialysis experiment [8]. During this period, the chickens were given ad libitum feed and water. 2.3. Preparation of solutions
Monoamines in the dialysate were analyzed by a HPLC system with a reversed phase ion-exchange column (CAX, Eicom) according to the method described by Tachibana et al. [14]. The mobile phase contained 0.1 M acetic acid–citric acid buffer, 100 mg/ml sodium-octyl-sulphate, 5 mg/ml Na2 EDTA, and 15% methanol, at pH 3.5. This solution was pumped through the system at 0.25 ml/min using a pump (EP-300, Eicom). The monoamines (DA, NE and 5-HT) were detected using an electrochemical detector (ECD-300, Eicom) with an applied potential of +0.45 V. The concentration of monoamine was determined by calculating peak areas and comparing with standard solutions.
2.6. Histological verification of the position of the dialysis probe After microdialysis chickens were deeply anaesthetized by intraperitoneal injection of sodium pentobarbital (8 mg/100 g b.w.) and perfused via cardiac puncture with Ringer’s solution (0.9% NaCl) followed by Zamboni fixative solution (10% paraformaldehyde). Then the chickens were decapitated and their brains were fixed in same fixative. Serial coronal sections were cut, mounted and stained with cresyl violet and analyzed in the light microscope according to the chicken brain atlas of Kuenzel and Masson [10]. An example of this microdialysis probe placement in the VMH is shown in Fig. 1.
The composition of normal Ringer’s solution was 147 mM NaCl, 4 mM KCl, and 4 mM CaCl2 . Calcium-free Ringer solution was made by replacing CaCl2 with equimolar MgCl2 . High K+ Ringer solution contained 100 mM KCl and the Na+ concentration was lowered accordingly to maintain osmolarity. l-DOPA (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in Ringer’s solution. The l-DOPA solution was then filtered through a 0.20 m membrane filter (Dismic-25AS, Advantec, Kyoto, Japan) before perfusion into the chickens. TTX (Wako Chemical Co. Ltd., Osaka, Japan) was dissolved in Ringer’s solution and diluted to obtain 2 M TTX. 2.4. Brain microdialysis experiment The microdialysis experiment was carried out on 21 or 22 days of age. At about 9:30 am each day the microdialysis probes were inserted into the VMH via the implanted guide cannulae while gently restraining the fully conscious chickens. The animal was then placed individually into the microdialysis cages (cages 1 and 2). Microdialysis was carried out in accordance with a previously reported method [14]. Briefly, the 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 cannulae.
Fig. 1. Representative coronal section of the VMH of chicken that was inserted microdialysis probe. Asterisk (*) represents the trace of microdialysis probe. Dotted circle shows the region of VMH. Scale bar = 400 m. Abbreviations are VMH, ventromedial hypothalamus; III, third ventricle.
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2.7. Statistical methods
DA (% of baseline)
The monoamines in the dialysate were expressed as a percentage of the baseline in each individual chicken. The average monoamine levels in the 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. The t-test was used to compare the results between the control and treatment groups at given time. The level of significance was P < 0.05. Data were expressed as the mean ± SEM.
Control †*
250 200
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100
0 -1
When Ca2+ was omitted from the perfusion fluid the NE decreased by 19 ± 8% and 15 ± 13% from baseline at 1 and 1.5 h, respectively (Fig. 3). When l-DOPA was mixed (1 g/ml) with Ca2+ free Ringer’s solution, the extracellular level of DA remained close to the baseline. The 5-HT level significantly decreased (P < 0.05) at 1 and 1.5 h. The monoamines returned to the baseline when the perfusion fluid was switched back to normal Ringer’s solution. 3.3. Effect of TTX plus l-DOPA perfusion on extracellular NE, DA and 5-HT in the VMH TTX was perfused via the dialysis membrane into the VMH. At 2 M TTX the NE decreased by26 ± 12%, 20 ± 25% and 21 ± 8% from the baseline at 1, 1.5 and 2 h, respectively (Fig. 3). TTX inhibited
*†
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Time-course of experiment (h)
3.1. Effect of l-DOPA perfusion on extracellular DA, NE and 5-HT level in the VMH
3.2. Effect of Ca2+ free Ringer’s solution plus l-DOPA perfusion on extracellular NE, DA and 5-HT in the VMH
2µg/ml
50
3. Results
Control
NE (% of baseline)
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Time -course of experiment (h) Control
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5-HT (% of baseline)
Several pilot experiments were done to study the dose response effect of l-DOPA in chicken VMH. Subsequently, a small response of DA level to l-DOPA was observed at 0.5 g/ml. The extracellular DA level was gradually increased to 1 g/ml. Finally, a two- to two and half-fold increase in extracellular DA levels from the baseline was observed using 1 or 2 g/ml l-DOPA solution that was perfused into the chicken VMH at a rate of 1 l/min for 10 min. The changes in the extracellular level of DA (upper panel), NE (middle panel) and 5-HT (lower panel) from the baseline over time are shown in Fig. 2. Local administration of l-DOPA with perfusate significantly increased the DA levels compared with the baseline. At 30 min following administration there was no increase in DA. Both 1 and 2 g/ml l-DOPA caused a measurable increase of DA compared to the baseline by 60 min, and this increase was significantly different from the control group and baseline. The DA reached its peak value of 212 ± 17% and 254 ± 27%, respectively, of the baseline, at 1 h following administration of l-DOPA at 1 and 2 g/ml. The level of DA at 1.5 and 2 h was 155 ± 11% and 125 ± 12%, respectively, for 1 g/ml, whereas it was 173 ± 33% and 141 ± 20%, respectively, for the same time periods but at 2 g/ml. The DA level returned to the baseline at 2.5 h after l-DOPA perfusion at both doses. The DA in the control group remained fairly stable over time during the experiment. An increase of DA levels at 144% of the baseline at 60 min following l-DOPA administration was observed using 0.5 g/ml, but this increment was not significantly different from the baseline (data are not shown). The NE and 5-HT levels at both doses of l-DOPA did not show significant changes compared with the basal level and the control group during microdialysis. They remained almost stable throughout the experiment. However, there were some variations from the baseline in 5-HT at 2 to 3 h in the 1 g/ml l-DOPA treated chickens. The reason for this variation cannot be explained.
1µg/ml
300
1µg/ml
2µg/ml
200 150 100 50 0 -1
0
1
2
3
4
5
Time-course of experiment (h) Fig. 2. Effects of l-DOPA treatment on the extracellular dopamine (DA, upper panel), norepinephrine (NE, middle panel) and serotonin (5-HT, lower panel) concentrations in the VMH of chicken. The levels of DA, NE and 5-HT are expressed as a percentage of each compound’s baseline value. l-DOPA was administered using a reverse microdialysis after the last baseline sample measurement. The arrow indicates the beginning of l-DOPA perfusion for 10 min. Data are expressed as means ± SEM (n = 5 or 6 per group). *Significantly different from the control group (P < 0.05). †Significantly different from the basal level (P < 0.05).
the ability of l-DOPA to increase extracellular DA and DA remained close to the baseline. In the presence of TTX, l-DOPA did not significantly augment the extracellular level of DA. The 5-HT significantly decreased (P < 0.05) at 1, 1.5 and 2 h when compared with the baseline. 3.4. Effect of 100 mM K+ plus l-DOPA perfusion on extracellular NE, DA and 5-HT levels in the VMH Addition of KCl plus l-DOPA to the perfusion fluid produced a significant increase (P < 0.05) in the VMH extracellular NE, DA and 5-HT to 663 ± 223, 550 ± 184 and 695 ± 131% of the baseline, respectively, at 1 h. At 30 min following administration there was no increase in NE, DA and 5-HT level. The level of NE, DA and 5-HT at 1.5 h were 455 ± 117%, 486 ± 129% and 346 ± 40% of the baseline,
M.R. Alam et al. / Neuroscience Letters 529 (2012) 150–154
L-DOPA + Ca2+ -free Ringer
200 180
NE
DA
5-HT
% of baseline
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*
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200 180 160 140 120 100 80 60 40 20 0
NE
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5-HT
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Time-course of experiment (h)
% of baseline
L-DOPA + high K 1000 900 800 700 600 500 400 300 200 100 0
NE
DA
5-HT
* * *
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Time-course of experiment (h) Fig. 3. Effects of Ca2+ -free Ringer’s plus l-DOPA (upper panel), TTX plus l-DOPA (middle panel) and 100 mM K+ plus l-DOPA (lower panel) on the extracellular levels of NE, DA and 5-HT in the chicken VMH. The duration of perfusion was 30 min. The arrow indicates the beginning of perfusion. The results are expressed as a percentage of the basal levels, which are set at 100% and are given as means ± SEM (n = 6 in each group). Asterisks (*P < 0.05) represent values significantly different from the mean basal value.
respectively, which were significantly different from the baseline (P < 0.05). Thereafter the NE, DA and 5-HT gradually dropped and returned to the baseline when normal Ringer’s solution was reintroduced. 4. Discussion This study was done to elucidate the effect of l-DOPA perfusion on the extracellular DA levels with parallel measurements of NE and 5-HT in the VMH of freely moving chicken using an in vivo brain microdialysis technique. The results showed that local
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l-DOPA perfusion clearly elevates the in vivo dialysate level of DA for short durations. This study also investigated whether lDOPA-induced DA, NE and 5-HT in the dialysate was related to neuronal activity by applying TTX, Ca2+ -free Ringer and high K+ to the perfusion medium. The DA, NE and 5-HT obtained in the dialysate showed TTX sensitivity, Ca2+ dependency and high K+ reactivity. In the present study, l-DOPA at the rate of 1 and 2 g/ml was perfused locally into the chicken VMH for 10 min and the extracellular level of DA increased in a dose-dependent manner. Maximum DA levels in the dialysates increased twofold and two and half-fold from the baseline, respectively, at 60 min following administration. After discontinuation of the l-DOPA perfusion, the DA levels returned to the baseline in 2.5 h. The effects of l-DOPA on the extracellular DA levels in different areas of the rat brain regions including the hypothalamus, striatum, and brain stem have been previously reported. Adachi et al. [2] examined the effects of l-DOPA in the rat brain striatum using an in vivo microdialysis technique at the rate of 2 and 5 g/ml of l-DOPA in the perfusate. The peak level of DA was 140% and 270% of the basal level, respectively, at 60 min. Effect of intraperitoneal injection of l-DOPA on extracellular DA levels was observed to be dose-dependent in the rat striatum. The DA levels in the dialysates increased at 120 min at the level of 3-, 5and 7-fold of the baseline following the administration of 100, 200 and 400 mg/kg of l-DOPA, respectively [9]. Abercrombie et al. [1] injected several concentrations of l-DOPA plus DOPA decarboxylase inhibitor intraperitoneally and found that the response of DA at 60 min was 1.5–2 fold of the baseline and lasted for at least 150 min. On the other hand, Kabuki et al. [7] observed that the DA in the extraction of the whole brain increased to 4 and 6-fold of the baseline, respectively, in hamsters 35 min after intraperitoneal injection of l-DOPA at 50 and 250 mg/kg plus DOPA decarboxylase inhibitor. Considering the methodological aspects and the results of other investigations, it is likely that local perfusion of l-DOPA into the chicken VMH at the rate of 1 and 2 g/ml for 10 min is suitable for stimulating DA within less than 60 min. On the other hand, the NE and 5-HT levels were almost stable throughout the microdialysis experiments, which indicated that perfusion of l-DOPA had no effect on them. This is consistent with other reports that showed that l-DOPA increased the DA content of a variety of tissues in rats, while it has little or no effect on NE and 5-HT levels [5,13]. Kabuki et al. [7] demonstrated that intraperitoneal injection of l-DOPA caused an increase in NE and a decrease in 5-HT in the whole brain extraction of hamsters in a dose-dependent manner. Some other reports indicated that lDOPA increased DA metabolites, i.e., dihydroxyphenyl acetic acid (DOPAC) and homovalinic acid (HVA) in the rat striatum [3,12]. The differences in the response of NE and 5HT or metabolites of DA to l-DOPA administration likely depend on the dose of l-DOPA and the presence of some inhibitors (DOPA decarboxylase or tyrosine hydroxylase). From the time-course variations of level of monoamine in the dialysate from the VMH during perfusion of l-DOPA with TTX, Ca2+ free or high K+ solution, it is confirmed that the monoamines in the dialysate are related to the neuronal activity. In conclusion, l-DOPA perfusion increased extracellular DA levels in the chicken VMH. There were no changes from baseline of the NE and 5-HT levels after l-DOPA perfusion. The results suggested that l-DOPA stimulated extracellular DA levels in the VMH in a dose-related manner without impact on NE and 5HT levels using a reverse microdialysis technique. The released DA, NE and 5-HT within the dialysate resulted from neuronal activity.
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Acknowledgments This research was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, Sports and Technology of Japan (No. 22580304). We are grateful to Prof. Joe M. Regenstein for editing the English. References [1] E.D. Abercrombie, A.E. Bonatz, M.J. Zigmond, Effects of l-DOPA on extracellular dopamine in striatum of normal and 6-hydroxydopamine-treated rats, Brain Research 525 (1990) 36–44. [2] 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 Research Bulletin 69 (2006) 593–596. [3] N.T. Buu, Vesicular accumulation of dopamine following l-DOPA administration, Biochemistry and Pharmacology 38 (1989) 1787–1792. [4] D.M. Denbow, H.P. Van Krey, M.P. Lacy, T.J. Dietrick, Feeding, drinking and body temperature of leghorn chicks: effects of ICV injections of biogenic amine, Physiology and Behavior 31 (1983) 85–90. [5] P.S. Doshi, D.J. Edwards, Effects of l-DOPA on dopamine and norepinephrine concentrations in rat brain assessed by gas chromatography, Journal of Chromatography 210 (1981) 505–511.
[6] 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, Journal of Poultry Science 45 (2008) 281–286. [7] Y. Kabuki, K. Shigemi, K. Hamasu, M. Furuse, l-DOPA attenuates hyperactivity of Roborovskii hamsters, Behavioural Pharmacology 20 (2009) 260–264. [8] A.M. Khalil, A. Ichijo, F. Yoshizawa, K. Sugahara, Effect of surgery for cannula implantation into the hypothalamus on the behaviour and neural activity of broiler chicks, British Poultry Science 51 (2010) 146–151. [9] K. Koshimura, T. Ohue, Y. Akiyama, A. Itoh, S. Miwa, l-DOPA administration enhances exocytic dopamine release in vivo in the rat striatum, Life Sciences 51 (1992) 747–755. [10] W.J. Kuenzel, M. Masson, A Stereotaxic Atlas of the Brain of the Chick (Gallus domesticus), The Johns Hopkins University Press, Baltimore, 1988. [11] National Agriculture and Food Research Organization, Japanese Feeding Standard for Poultry, Japan Livestock Industry Association, Tokyo, 2004. [12] F. Ponzio, G. Achilli, C. Perego, G. Rinaldi, S. Algeri, Does acute l-DOPA increase active release of dopamine from dopaminergic neurons, Brain Research 273 (1983) 45–51. [13] G.L. Snyder, M.J. Zigmond, The effects of l-DOPA on in vitro dopamine release from striatum, Brain Research 508 (1990) 181–187. [14] T. Tachibana, M. Tazawa, K. Sugahara, Feeding increases 5-hydroxytryptamine and norepinephrine within the hypothalamus of chicks, Comparative Biochemistry and Physiology Part A 130 (2001) 715–722.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Local administration of l-DOPA in the chicken ventromedial hypothalamus increases dopamine release in a dose-dependent manner Mohammad Rashedul Alam a,b , Fumiaki Yoshizawa b , Kunio Sugahara b,∗ a b
United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan Department of Bio-productive Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan
h i g h l i g h t s
In vivo microdialysis showed the effect of l-DOPA on extracellular DA levels in chick VMH. Extracellular DA levels increased significantly in the VMH after l-DOPA perfusion. l-DOPA has no effect on NE and 5-HT during microdialysis. The DA, NE and 5-HT within the dialysate were related to neural activity.
a r t i c l e
i n f o
Article history: Received 25 July 2012 Received in revised form 21 August 2012 Accepted 28 August 2012 Keywords: Chickens Dopamine l-DOPA Microdialysis Tetrodotoxin Ventromedial hypothalamus
a b s t r a c t l-DOPA induced extracellular dopamine (DA), norepinephrine (NE) and serotonin (5-HT) in the ventromedial hypothalamus (VMH) of chickens were measured by in vivo microdialysis. Several doses of 3,4-dihydroxy-l-phenylalanine (l-DOPA) were administered locally through the microdialysis probe into the VMH of chickens for 10 min. Local perfusion of l-DOPA increased the extracellular levels of DA. The increased DA was dose-related and was significantly higher compared to the baseline and control group. The maximal level of DA was 212% and 254%, respectively, of the baseline following administration of 1 and 2 g/ml l-DOPA. There were no changes in NE and 5-HT levels from baseline after l-DOPA perfusion. l-DOPA (1 g/ml) was mixed with Ca2+ -free Ringer, tetrodotoxin (TTX) (2 M) and high K+ and was perfused for 30 min into the chicken VMH. TTX and Ca2+ -free Ringer’s solution inhibited the effectiveness of l-DOPA in increasing DA release. The NE and 5-HT levels were significantly lower than the baseline. After administration of K+ a significant increase of DA, NE and 5-HT was observed. The microdialysis results are consistent with our objective that l-DOPA induced extracellular DA increases in the VMH in a dose-dependent manner and the released DA, NE and 5-HT within the dialysate were related to neuronal activity. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hypothalamic monoamines have been reported to be involved in regulation of food intake in rats and chickens using intracerebroventricular (ICV) injection of compounds [4]. However, there has been a little information presented on the involvement of endogenous monoamines in variations of food intake in chickens. Ichijo et al. [6] found that the decreased food intake due to feeding a lysine-free diet followed the decrease in the ventromedial hypothalamic (VMH) dopamine (DA) level in chickens using an in vivo brain microdialysis technique. This finding suggests that
∗ Corresponding author at: Department of Animal Science, Division of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya-shi, Tochigi 321-8505, Japan. Tel.: +81 286495441; fax: +81 286495401. E-mail address: [email protected] (K. Sugahara). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.08.054
if DA in the VMH can be pharmacologically or physiologically modified, then the variations of food intake of chickens can be alleviated. A previous study with rats showed that local perfusion of 3,4-dihydroxy-l-phenylalanine (l-DOPA) using a reverse brain microdialysis technique affects the DA level in the striatum [2]. However, the effect of local l-DOPA administration on extracellular DA levels in the chicken VMH remains unexplored. Therefore, this study was done to determine if local perfusion of l-DOPA using a reverse brain microdialysis technique affects the extracellular level of monoamines in the VMH of chickens similarly to that with rats. We also examined if changes of monoamine contents in the dialysate derived from neuronal activity in the VMH. 2. Materials and methods All procedures involving the uses of animals were performed in accordance with regulations for the care and use of animals
M.R. Alam et al. / Neuroscience Letters 529 (2012) 150–154
required by the Animal Experimentation Committee of Utsunomiya University. 2.1. Animals and their management Day old male egg-type chickens (Gallus gallus domesticus) were housed in a temperature-controlled (32 ◦ C) room with 12/12 light/dark cycle. They were given free access to water and a commercial starter diet (CP, 21%; ME, 2.95 Mcal/kg, Kumiai Feed Co. Ltd, Tokyo, Japan). The feed was manufactured according to the requirements of all essential amino acids recommended in Japanese Feeding Standard for Poultry [11]. 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 at a temperature of 28 ◦ C.
151
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 l/min. After collection of the baseline value, l-DOPA was perfused via the dialysis probe into the VMH of a chicken for 10 min whereas Ringer’s solution without calcium or with 2 M TTX or a high concentration of K+ (100 mM) were perfused for 30 min and dialysates were collected. After this pharmacological treatment the perfusion medium was switched back to the normal Ringer’s solution and the dialysis continued. The collection time interval for each dialysis sample was 30 min and the dialysate was collected for 7 or 8 h. During microdialysis experiment, the chickens were given free access to water and the commercial diet.
2.5. Determination of monoamines via high performance liquid chromatography (HPLC) with electrochemical detection (ECD)
2.2. Surgical procedures for implantation of the guide cannula Seventeen- or 18-day-old chickens were fasted for at least 2 h before surgery. At 17 or 18 days of age, the chickens were stereotaxically implanted with a guide cannula aimed at one side of the hypothalamus, specifically the ventromedial hypothalamus (VMH). In preparation for cannulae implantation, chickens were anaesthetized with intraperitoneal administration of sodium pentobarbital (4 mg/100 g body weight). They were then placed in a stereotaxic frame (David Kopf Instruments, California, USA) and a small incision with a scalpel was made to expose the skull. A highspeed microdrill was then used to drill a hole in each chicken skull overlying the VMH (VMH, anterior to the interaural line by 7 mm; lateral to the midline by 0.2 mm and 7.2 mm below the skull surface [10]) and the guide cannula (0.5 × 8 mm, diameter and length, respectively, Eicom, Kyoto, Japan) was lowered below the skull surface. Two additional holes were drilled in the skull for anchoring of the stainless-steel support screws (2 × 6 mm, diameter × length, respectively). The guide cannula was fixed in place with dental cement (Shofu Inc., Tokyo, Japan). A dummy cannula was then inserted to prevent obstruction. The chickens were individually housed and allowed at least 4 days for post-surgical recovery before the start of the microdialysis experiment [8]. During this period, the chickens were given ad libitum feed and water. 2.3. Preparation of solutions
Monoamines in the dialysate were analyzed by a HPLC system with a reversed phase ion-exchange column (CAX, Eicom) according to the method described by Tachibana et al. [14]. The mobile phase contained 0.1 M acetic acid–citric acid buffer, 100 mg/ml sodium-octyl-sulphate, 5 mg/ml Na2 EDTA, and 15% methanol, at pH 3.5. This solution was pumped through the system at 0.25 ml/min using a pump (EP-300, Eicom). The monoamines (DA, NE and 5-HT) were detected using an electrochemical detector (ECD-300, Eicom) with an applied potential of +0.45 V. The concentration of monoamine was determined by calculating peak areas and comparing with standard solutions.
2.6. Histological verification of the position of the dialysis probe After microdialysis chickens were deeply anaesthetized by intraperitoneal injection of sodium pentobarbital (8 mg/100 g b.w.) and perfused via cardiac puncture with Ringer’s solution (0.9% NaCl) followed by Zamboni fixative solution (10% paraformaldehyde). Then the chickens were decapitated and their brains were fixed in same fixative. Serial coronal sections were cut, mounted and stained with cresyl violet and analyzed in the light microscope according to the chicken brain atlas of Kuenzel and Masson [10]. An example of this microdialysis probe placement in the VMH is shown in Fig. 1.
The composition of normal Ringer’s solution was 147 mM NaCl, 4 mM KCl, and 4 mM CaCl2 . Calcium-free Ringer solution was made by replacing CaCl2 with equimolar MgCl2 . High K+ Ringer solution contained 100 mM KCl and the Na+ concentration was lowered accordingly to maintain osmolarity. l-DOPA (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in Ringer’s solution. The l-DOPA solution was then filtered through a 0.20 m membrane filter (Dismic-25AS, Advantec, Kyoto, Japan) before perfusion into the chickens. TTX (Wako Chemical Co. Ltd., Osaka, Japan) was dissolved in Ringer’s solution and diluted to obtain 2 M TTX. 2.4. Brain microdialysis experiment The microdialysis experiment was carried out on 21 or 22 days of age. At about 9:30 am each day the microdialysis probes were inserted into the VMH via the implanted guide cannulae while gently restraining the fully conscious chickens. The animal was then placed individually into the microdialysis cages (cages 1 and 2). Microdialysis was carried out in accordance with a previously reported method [14]. Briefly, the 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 cannulae.
Fig. 1. Representative coronal section of the VMH of chicken that was inserted microdialysis probe. Asterisk (*) represents the trace of microdialysis probe. Dotted circle shows the region of VMH. Scale bar = 400 m. Abbreviations are VMH, ventromedial hypothalamus; III, third ventricle.
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2.7. Statistical methods
DA (% of baseline)
The monoamines in the dialysate were expressed as a percentage of the baseline in each individual chicken. The average monoamine levels in the 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. The t-test was used to compare the results between the control and treatment groups at given time. The level of significance was P < 0.05. Data were expressed as the mean ± SEM.
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Several pilot experiments were done to study the dose response effect of l-DOPA in chicken VMH. Subsequently, a small response of DA level to l-DOPA was observed at 0.5 g/ml. The extracellular DA level was gradually increased to 1 g/ml. Finally, a two- to two and half-fold increase in extracellular DA levels from the baseline was observed using 1 or 2 g/ml l-DOPA solution that was perfused into the chicken VMH at a rate of 1 l/min for 10 min. The changes in the extracellular level of DA (upper panel), NE (middle panel) and 5-HT (lower panel) from the baseline over time are shown in Fig. 2. Local administration of l-DOPA with perfusate significantly increased the DA levels compared with the baseline. At 30 min following administration there was no increase in DA. Both 1 and 2 g/ml l-DOPA caused a measurable increase of DA compared to the baseline by 60 min, and this increase was significantly different from the control group and baseline. The DA reached its peak value of 212 ± 17% and 254 ± 27%, respectively, of the baseline, at 1 h following administration of l-DOPA at 1 and 2 g/ml. The level of DA at 1.5 and 2 h was 155 ± 11% and 125 ± 12%, respectively, for 1 g/ml, whereas it was 173 ± 33% and 141 ± 20%, respectively, for the same time periods but at 2 g/ml. The DA level returned to the baseline at 2.5 h after l-DOPA perfusion at both doses. The DA in the control group remained fairly stable over time during the experiment. An increase of DA levels at 144% of the baseline at 60 min following l-DOPA administration was observed using 0.5 g/ml, but this increment was not significantly different from the baseline (data are not shown). The NE and 5-HT levels at both doses of l-DOPA did not show significant changes compared with the basal level and the control group during microdialysis. They remained almost stable throughout the experiment. However, there were some variations from the baseline in 5-HT at 2 to 3 h in the 1 g/ml l-DOPA treated chickens. The reason for this variation cannot be explained.
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Time-course of experiment (h) Fig. 2. Effects of l-DOPA treatment on the extracellular dopamine (DA, upper panel), norepinephrine (NE, middle panel) and serotonin (5-HT, lower panel) concentrations in the VMH of chicken. The levels of DA, NE and 5-HT are expressed as a percentage of each compound’s baseline value. l-DOPA was administered using a reverse microdialysis after the last baseline sample measurement. The arrow indicates the beginning of l-DOPA perfusion for 10 min. Data are expressed as means ± SEM (n = 5 or 6 per group). *Significantly different from the control group (P < 0.05). †Significantly different from the basal level (P < 0.05).
the ability of l-DOPA to increase extracellular DA and DA remained close to the baseline. In the presence of TTX, l-DOPA did not significantly augment the extracellular level of DA. The 5-HT significantly decreased (P < 0.05) at 1, 1.5 and 2 h when compared with the baseline. 3.4. Effect of 100 mM K+ plus l-DOPA perfusion on extracellular NE, DA and 5-HT levels in the VMH Addition of KCl plus l-DOPA to the perfusion fluid produced a significant increase (P < 0.05) in the VMH extracellular NE, DA and 5-HT to 663 ± 223, 550 ± 184 and 695 ± 131% of the baseline, respectively, at 1 h. At 30 min following administration there was no increase in NE, DA and 5-HT level. The level of NE, DA and 5-HT at 1.5 h were 455 ± 117%, 486 ± 129% and 346 ± 40% of the baseline,
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Time-course of experiment (h) Fig. 3. Effects of Ca2+ -free Ringer’s plus l-DOPA (upper panel), TTX plus l-DOPA (middle panel) and 100 mM K+ plus l-DOPA (lower panel) on the extracellular levels of NE, DA and 5-HT in the chicken VMH. The duration of perfusion was 30 min. The arrow indicates the beginning of perfusion. The results are expressed as a percentage of the basal levels, which are set at 100% and are given as means ± SEM (n = 6 in each group). Asterisks (*P < 0.05) represent values significantly different from the mean basal value.
respectively, which were significantly different from the baseline (P < 0.05). Thereafter the NE, DA and 5-HT gradually dropped and returned to the baseline when normal Ringer’s solution was reintroduced. 4. Discussion This study was done to elucidate the effect of l-DOPA perfusion on the extracellular DA levels with parallel measurements of NE and 5-HT in the VMH of freely moving chicken using an in vivo brain microdialysis technique. The results showed that local
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l-DOPA perfusion clearly elevates the in vivo dialysate level of DA for short durations. This study also investigated whether lDOPA-induced DA, NE and 5-HT in the dialysate was related to neuronal activity by applying TTX, Ca2+ -free Ringer and high K+ to the perfusion medium. The DA, NE and 5-HT obtained in the dialysate showed TTX sensitivity, Ca2+ dependency and high K+ reactivity. In the present study, l-DOPA at the rate of 1 and 2 g/ml was perfused locally into the chicken VMH for 10 min and the extracellular level of DA increased in a dose-dependent manner. Maximum DA levels in the dialysates increased twofold and two and half-fold from the baseline, respectively, at 60 min following administration. After discontinuation of the l-DOPA perfusion, the DA levels returned to the baseline in 2.5 h. The effects of l-DOPA on the extracellular DA levels in different areas of the rat brain regions including the hypothalamus, striatum, and brain stem have been previously reported. Adachi et al. [2] examined the effects of l-DOPA in the rat brain striatum using an in vivo microdialysis technique at the rate of 2 and 5 g/ml of l-DOPA in the perfusate. The peak level of DA was 140% and 270% of the basal level, respectively, at 60 min. Effect of intraperitoneal injection of l-DOPA on extracellular DA levels was observed to be dose-dependent in the rat striatum. The DA levels in the dialysates increased at 120 min at the level of 3-, 5and 7-fold of the baseline following the administration of 100, 200 and 400 mg/kg of l-DOPA, respectively [9]. Abercrombie et al. [1] injected several concentrations of l-DOPA plus DOPA decarboxylase inhibitor intraperitoneally and found that the response of DA at 60 min was 1.5–2 fold of the baseline and lasted for at least 150 min. On the other hand, Kabuki et al. [7] observed that the DA in the extraction of the whole brain increased to 4 and 6-fold of the baseline, respectively, in hamsters 35 min after intraperitoneal injection of l-DOPA at 50 and 250 mg/kg plus DOPA decarboxylase inhibitor. Considering the methodological aspects and the results of other investigations, it is likely that local perfusion of l-DOPA into the chicken VMH at the rate of 1 and 2 g/ml for 10 min is suitable for stimulating DA within less than 60 min. On the other hand, the NE and 5-HT levels were almost stable throughout the microdialysis experiments, which indicated that perfusion of l-DOPA had no effect on them. This is consistent with other reports that showed that l-DOPA increased the DA content of a variety of tissues in rats, while it has little or no effect on NE and 5-HT levels [5,13]. Kabuki et al. [7] demonstrated that intraperitoneal injection of l-DOPA caused an increase in NE and a decrease in 5-HT in the whole brain extraction of hamsters in a dose-dependent manner. Some other reports indicated that lDOPA increased DA metabolites, i.e., dihydroxyphenyl acetic acid (DOPAC) and homovalinic acid (HVA) in the rat striatum [3,12]. The differences in the response of NE and 5HT or metabolites of DA to l-DOPA administration likely depend on the dose of l-DOPA and the presence of some inhibitors (DOPA decarboxylase or tyrosine hydroxylase). From the time-course variations of level of monoamine in the dialysate from the VMH during perfusion of l-DOPA with TTX, Ca2+ free or high K+ solution, it is confirmed that the monoamines in the dialysate are related to the neuronal activity. In conclusion, l-DOPA perfusion increased extracellular DA levels in the chicken VMH. There were no changes from baseline of the NE and 5-HT levels after l-DOPA perfusion. The results suggested that l-DOPA stimulated extracellular DA levels in the VMH in a dose-related manner without impact on NE and 5HT levels using a reverse microdialysis technique. The released DA, NE and 5-HT within the dialysate resulted from neuronal activity.
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