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Central l -proline attenuates stress-induced dopamine and serotonin metabolism in the chick forebrain
Neuroscience Letters 460 (2009) 78–81
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Central l-proline attenuates stress-induced dopamine and serotonin metabolism in the chick forebrain Kousuke Hamasu a , Kazutaka Shigemi a , Yusuke Kabuki a , Shozo Tomonaga a , D. Michael Denbow b , Mitsuhiro Furuse a,∗ a
Laboratory of Advanced Animal and Marine Bioresources, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0306, USA
b
a r t i c l e
i n f o
Article history: Received 17 April 2009 Received in revised form 12 May 2009 Accepted 12 May 2009 Keywords: Stress l-Proline Chick Microdialysis NMDA
a b s t r a c t Using microdialysis, we investigated the effect of l-proline on monoamine release in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) of freely moving and restricted chicks. A 30 min handlingstress resulted in a significant increase in extracellular homovallinic acid (HVA), a dopamine metabolite, and 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite, in the MNH. l-Proline, perfused through the microdialysis probe into the MNH during the stressed condition, significantly attenuated the average dialysate concentration of HVA produced by handling-stress. Handling-stress resulted in a significant increase in 5-HIAA levels in the control group, which were attenuated by profusion with l-proline. lProline did not significantly modify basal concentrations of HVA or 5-HIAA in the MNH during control conditions. These results show that perfusion of l-proline modified the turnover/metabolism of dopamine and serotonin in the MNH caused by handling-stress. © 2009 Elsevier Ireland Ltd. All rights reserved.
While l-glutamate is well established as a neurotransmitter in the central nervous system (CNS), little is known about the possible role of l-proline as a neurotransmitter. We have previously demonstrated that the amount of l-proline in the brain was reduced under stressful conditions, and intracerebroventricular (i.c.v.) injection of l-proline had sedative and hypnotic effects under an acute stressful condition in neonatal chicks [11]. Therefore, it appears that l-proline might function through several neurotransmitter systems in the CNS. Past studies showed that l-proline can activate the strychnine-sensitive glycine receptor and the N-methyl-daspartate (NMDA) glutamate receptor [2,3,13,20]. The NMDA, but not glycine, receptor appears involved in the action of l-proline to induce sedative and hypnotic effects [12]. Activation of dopaminergic and serotonergic pathways is related to behavioral and emotional changes [1,9]. Some experiments suggested a modulatory interaction between the glutamatergic and monoaminergic pathway, which is mediated via NMDA receptor activation [10,15]. From these findings, we assumed that l-proline activates the NMDA receptor and alters activation of dopaminergic and serotonergic pathways. Indeed, i.c.v. injection of l-proline suppressed the increase in serotonin turnover rate and slightly suppressed the increase in dopamine turnover rate induced by isolation
∗ Corresponding author. Tel.: +81 92 642 2953; fax: +81 92 642 2953. E-mail address: [email protected] (M. Furuse). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.05.036
stress in the telencephalon (Hamasu et al., unpublished). However, the effect of l-proline on the release of stress-induced monoamines is still obscure. In the present study, we investigated the interaction of l-proline and monoamine release in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) during stress. The MNH, a presumed analogue of the mammalian prefrontal cortex [17,18], was identified to be involved in the stress response [9,19]. One-day-old male layer chicks (Julia) purchased from a local hatchery (Murata Hatchery, Fukuoka, Japan) were maintained in a windowless room at a constant temperature of 30 ± 1 ◦ C. Lighting was provided continuously for 24 h. Chicks were given free access to a commercial starter diet (Toyohashi Feed and Mills Co. Ltd., Aichi, Japan) and water. Experimental procedures followed the guidance for Animal Experiments in the Faculty of Agriculture and in the Graduate Course of Kyushu University and the Law (No. 105) and Notification (No. 6) of the Japanese Government. Under sodium pentobarbital anesthesia (3 mg/100 g body weight), a guide-cannula was stereotaxically implanted in the brain to accommodate microdialysis probes in the MNH [17] according to the chick brain atlas [16,30] at the following stereotaxic coordinates: anterior 6.0 mm, lateral 1.0 mm, and 2.5 mm depth (Fig. 1). The microdialysis probe, to be inserted later, extended 1 mm beyond the guide-cannula. The guide-cannula assembly was then fixed to the skull by surrounding the cannula and two anchorage screws with dental cement. After implantation of the guide-cannula, the chicks were housed individually and allowed to
K. Hamasu et al. / Neuroscience Letters 460 (2009) 78–81
79
Fig. 1. The image of the medio-rostral neostriatum/hyperstriatum ventrale (MNH).
recover from surgery for approximately 24 h. During this period, the chicks had free access to food and water. The microdialysis probe was inserted and experiments were performed on free moving chicks. During this period, the chicks had free access to food and water. Probes were I-shaped (A-I-4-01, EICOM, Kyoto, Japan) ending in a dialysis membrane (1 mm long, 0.22 mm wide) with a molecular weight cut-off of 50,000 Da. Using a syringe pump (EP-64, EICOM, Kyoto, Japan) with a 2.5 ml gastight syringe, the probe was perfused with a Ringer-type solution (147 mM NaCl, 4 mM KCl, 3 mM CaCl2 ) at a flow rate of 2.0 l/min. During all experiments the microdialysates were collected every 30 min. After establishment of a baseline, different treatments were given to the chicks and samples were taken. Samples were stored at −80 ◦ C until they were analyzed. Dialysate concentrations of homovallinic acids (HVAs), a dopamine (DA) metabolite, and 5-hydroxyindoleacetic acid (5HIAA), a serotonin (5-HT) metabolite were determined. To test the effects of l-proline on basal dialysate concentrations of HVA and 5-HIAA, l-proline (100 mM) (gift from Kyowa Hakko Kogyo, Tokyo, Japan) was dissolved in Ringer-type solution and perfused through the microdialysis probe for 60 min (the time of two microdialysis samples) in Experiment 1. To test the effects of l-proline on stress-stimulated dialysate concentrations of HVA and 5-HIAA, l-proline (100 mM) was dissolved in Ringer-type solution and perfused through the microdialysis probe for 60 min (the time of two microdialysis samples) in Experiment 2. Samples were collected to determine if there was an increase in HVA and 5-HIAA caused by a 30 min handling-stress by gently restricting their movement. Chicks were sacrificed with an overdose of sodium pentobarbital. After Cresyl violet was perfused through the guide-cannulae, the brain was removed and fixed in 2% buffered paraformaldehyde. Serial coronal sections were cut to determine the location of the dialysis probe. The levels of monoamines and their metabolites were determined using a high performance liquid chromatography (HPLC) with electrochemical detection. DA, 5-HT, HVA and 5-HIAA were determined. Dialysate aliquots were collected every 30 min (60 l) into the fraction collector (Eicom, EFC-82, Kyoto, Japan). The tubes were moved to an autosampler (Model-231XL, Gilson, Middleton, WI, USA) and the dialysate samples (30 l) were automatically injected into HPLC system (Eicom, Kyoto, Japan) with a
Fig. 2. Effects of l-proline perfused through the microdialysis probe during 60 min (black bar) on dialysate concentrations of HVA (upper panel) and 5-HIAA (lower panel) in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) of awake 8or 9-day-old layer chicks. Results are expressed as means ± S.E.M. The number of chicks was 5.
150 mm × 2.1 mm octadecyl silane (ODS) column (SC-5ODS, Eicom) and electrochemical detector (ECD-300, Eicom) at an applied potential of +700 mV versus an Ag/AgCl reference analytical electrode as described previously [22]. The changes in electric current (nA) were recorded in a computer using an interface system (Power Chrom ver 2.3.2.J; AD Instruments, Tokyo, Japan). The mobile phase was composed of aceto-citric acid buffer (pH 3.5, 0.1 mol/L), methanol, sodium-1-octane sulfonate (0.46 mol/L) and disodium ethylenediaminetetraacetic acid (0.015 mmol/L) (830:170:1.9:1) at a flow rate of 0.2 ml/min. Results from Experiment 1 were analyzed by one-way repeated measure analysis of variance (ANOVA) with respect to time. In Experiment 2 data were analyzed by one-way and two-way repeated measure ANOVA with respect to time-treatment. When the ANOVA showed significant effects, Dunnett’s test was used to compare samples collected 30 min before and after treatment as a post hoc test. Statistical analyses were conducted using a commercially available package StatView (version 5, SAS Institute, Cary, NC, USA 1998). Fig. 2 shows the effects of l-proline (100 mM) on basal dialysate concentrations of HVA (upper panel) and 5-HIAA (lower panel). At the 100 mM dose, l-proline did not significantly modify basal dialysate HVA or 5-HIAA concentrations. Fig. 3 shows the effects of l-proline (100 mM) on stressstimulated dialysate concentrations of HVA (upper panel) and 5-HIAA (lower panel). There was a significant interaction (F(5, 60) = 2.463, P < 0.05) between the control and l-proline group on HVA, suggesting that l-proline significantly reduced the average increases of dialysate concentration of HVA produced by handlingstress. In contrast, there was no significant interaction in 5-HIAA (F(5, 60) = 1.819, P = 0.1227). However, the 30 min handling-stress resulted in a significant increase in 5-HIAA levels in the control group (F(5, 35) = 9.990, P < 0.001), while there was no increase in the l-proline group (F(5, 25) = 0.960, P = 0.4606).
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K. Hamasu et al. / Neuroscience Letters 460 (2009) 78–81
Furthermore, l-pipecolic acid (hexahydropicolinic acid) has a similar structure to l-proline, since it is an imino acid having a ring structure. According to Takagi et al. [25], l-pipecolic acid activated both GABA-A and GABA-B receptors. l-Proline may also stimulate GABAergic interneurons and may reduce local transmitter release, so the increases of dialysate concentration of HVA and 5-HIAA in the MNH caused by handling-stress may be attenuated. Several studies suggest the possibility that an interaction between glutamate and monoamine seems to exist in the prefrontal cortex [10,15]. In support of this interaction, microdialysis studies have suggested that DA release in the prefrontal cortex induced by stress was, in part, a consequence of an increased glutamatergic neurotransmission [5,14,26]. However, other studies did not support this claim, because it has been shown that stress did not increase extracellular concentrations of glutamate in the prefrontal cortex [4,28]. Thus, the release of monoamine during stress in the prefrontal cortex is still controversial. The present results indicated that l-proline might be involved in the release of monoamines during stress in the prefrontal cortex mediated by the NMDA receptor. Indeed, there is a long history of evidence suggesting that l-proline may be a neuronal modulator or transmitter in the central nervous system [7,8,21,24,29]. It is known that l-proline containing neurons and transporters is localized in several brain areas [7,21,27]. However, the effect of stress on extracellular l-proline is still obscure. Further study is necessary to verify the hypothesis. Acknowledgements
Fig. 3. Effects of l-proline (100 mM), perfused through the microdialysis probe for 60 min, on increases of HVA and 5-HIAA produced by a 30 min handling-stress in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) of awake 8- or 9-day-old layer chicks. Results are expressed as means ± S.E.M. The number of chicks was 8 for control and 6 for l-proline, respectively. *Significantly different from the data that were collected just before the beginning of stress.
In the present study, DA and 5-HT were not detectable, due to low concentrations, but their metabolites, HVA and 5-HIAA, were clearly detected. These metabolites may, at least in part, reflect release of their respective neurotransmitters [9,10]. The present results demonstrated that the increases of dialysate concentration of HVA and 5-HIAA in the MNH caused by handlingstress were attenuated by the simultaneous perfusion of l-proline (100 mM). It is suggested that these effects of l-proline were mediated by the NMDA receptor, given that a previous study indicated that l-proline modulated activation of the NMDA receptor [12]. The microdialysis study by Gruss et al. [10] which showed a modulatory interaction between glutamatergic and monoaminergic neurotransmission mediated by the NMDA receptor further supports this suggestion. They demonstrated that the dialysate concentration of HVA and 5-HIAA was decreased by local intracerebral infusion of NMDA, and these effects were antagonized by an NMDA antagonist. However, no decrease was confirmed by local intracerebral infusion of l-proline in the present study (Fig. 2). The cause of this discrepancy may be the concentration of l-proline. The dialysate concentration of HVA and 5-HIAA might be decreased by infusion of higher concentrations of l-proline. Del Arco and Mora [4] reported that NMDA (100 M) significantly reduced the average increases of dialysate concentration of DA without modifying basal dialysate in DA. Regarding the mechanisms of action, NMDA may stimulate GABAergic interneurons in the MNH region, which inhibit monoaminergic synaptic boutons and thereby reduce local neurotransmitter release. This suggestion is supported by studies in which endogenous glutamate increases GABA [5] and GABA agonists inhibit dopamine release in the prefrontal cortex [6,23].
This work was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (No. 18208023) and the SKYLARK Food Science Institute. References [1] E.D. Abercrombie, K.A. Keefe, D.S. Difrischia, M.J. Zigmond, Differential effect of stress on in vivo dopamine release in striatum, nucleus accumbens, and medial frontal cortex, J. Neurochem. 52 (1989) 1655–1658. [2] S.M. Cohen, J.V. Nadler, Proline-induced potentiation of glutamate transmission, Brain Res. 761 (1997) 271–282. [3] M. David, A. Brian, N. Victor, NMDA receptor-mediated depolarizing action of proline on CA1 pyramidal cells, Eur. J. Pharmacol. 219 (1992) 59–66. [4] A. Del Arco, F. Mora, Dopamine release in the prefrontal cortex during stress is reduced by the local activation of glutamate receptors, Brain Res. Bull. 56 (2001) 125–130. [5] A. Del Arco, J.L. Gonzalez-Mora, V.R. Armas, F. Mora, Amphetamine increases extracellular concentrations of glutamate in striatum of the awake rat, Neuropharmacology 38 (1999) 943–954. [6] M.D. Doherty, A. Gratton, Effects of medial prefrontal cortical injections of GABA receptor agonists and antagonists on the local and nucleus accumbens dopamine responses to stress, Synapse 32 (1999) 288–300. [7] R.T. Fremeau Jr., M.G. Caron, R.D. Blakely, Molecular cloning and expression of a high affinity l-proline transporter expressed in putative glutamatergic pathways of rat brain, Neuron 8 (1992) 915–926. [8] J.A. Gogos, M. Sautha, Z. Takacs, K.D. Beck, V. Luine, L.R. Lucas, J.V. Nadler, M. Karayiorgou, The gene encoding proline dehydrogenase modulates sensorimotor gating in mice, Nat. Genet. 21 (1999) 434–439. [9] M. Gruss, K. Braun, Distinct activation of monoaminergic pathway in chick brain in relation to auditory imprinting and stressful situations: a microdialysis study, Neuroscience 76 (1997) 891–899. [10] M. Gruss, M. Bredenkotter, K. Braun, N-methyl-d-aspartate receptor-mediated modulation of monoaminergic metabolites and amino acids in the chick forebrain: an in vivo microdialysis and electrophysiology study, J. Neurobiol. 40 (1999) 116–135. [11] K. Hamasu, T. Haraguchi, Y. Kabuki, N. Adachi, S. Tomonaga, H. Sato, D.M. Denbow, M. Furuse, l-Proline is a sedative regulator of acute stress in the brain of neonatal chicks, Amino Acids, in press. [12] K. Hamasu, K. Shigemi, Y. Tsuneyoshi, H. Yamane, H. Sato, D.M. Denbow, M. Furuse, Intracerebroventricular injection of l-proline and d-proline induces sedative and hypnotic effects by different mechanisms under an acute stressful condition in chicks, Amino Acids, in press. [13] V. Henzi, D.B. Reichling, S.W. Helm, A.B. Macdermott, l-Proline activates glutamate and glycine receptors in cultured rat dorsal horn neurons, Mol. Pharmacol. 41 (1992) 793–801. [14] H.P. Jedema, B. Moghaddam, Glutamatergic control of dopamine release during stress in the rat prefrontal cortex, J. Neurochem. 63 (1993) 785–788.
K. Hamasu et al. / Neuroscience Letters 460 (2009) 78–81 [15] H.P. Jedema, B. Moghaddam, Characterization of excitatory amino acid modulation of dopamine release in the prefrontal cortex of conscious rats, J. Neurochem. 66 (1996) 1448–1453. [16] W.J. Kuenzel, M. Masson, A Stereotaxic Atlas of the Brain of Chick (Gallus domesticus), Johns Hopkins University Press, Baltimore, 1988, p. 166. [17] M. Metzger, S. Jiang, K. Braun, A quantitative immuno-electron microscopic study of dopamine terminals in forebrain regions of the domestic chick involved in filial imprinting, Neuroscience 111 (2002) 611–623. [18] M. Metzger, S. Jiang, J. Wang, K. Braun, Organisation of the dopaminergic innervation of forebrain areas relevant to learning: a combined immunohistochemical/retrograde tracing study in the domestic chick, J. Comp. Neurol. 376 (1996) 1–27. [19] S.C. Muller, H. Scheich, Social stress increases [14 C]2-deoxyglucose incorporation in three rostral forebrain areas of the young chick, Behav. Brain Res. 19 (1986) 93–98. [20] J.G. Ortiz, M.L. Cordero, A. Rosado, Proline-glutamate interactions in the CNS, Prog. Neuro-Psychopharmacol. 21 (1996) 141–152. [21] S.E. Renick, D.T. Kleven, J. Chan, K. Stenius, T.A. Milner, V.M. Pickel, R.T. Fremeau Jr., The mammalian brain high-affinity l-proline transporter is enriched preferentially in synaptic vesicles in a subpopulation of excitatory nerve terminals in rat forebrain, J. Neurosci. 19 (1999) 21–33. [22] S. Saito, T. Takagi, T. Koutoku, E.S. Saito, H. Hirakawa, S. Tomonaga, T. Tachibana, D.M. Denbow, M. Furuse, Differences in catecholamine metabolism and behaviour in neonatal broiler and layer chicks, Br. Poult. Sci. 45 (2004) 158–162.
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[23] M. Santiago, A. Machado, J. Cano, Regulation of the prefrontal cortical dopamine release by GABAa and GABAb receptor agonists and antagonists, Brain Res. 630 (1993) 28–31. [24] S.H. Snyder, A.B. Young, J.P. Bennett, A.H. Mulder, Synaptic biochemistry of amino acids, Fed. Proc. 32 (1973) 2039–2047. [25] T. Takagi, T. Bungo, T. Tachibana, E.S. Saito, S. Saito, I. Yamasaki, S. Tomonaga, D.M. Denbow, M. Furuse, Intracerebroventricular administration of GABA-A and GABA-B receptor antagonists attenuate feeding and sleep-like behavior induced by l-pipecolic acid in the neonatal chick, J. Neurosci. Res. 73 (2003) 270–275. [26] R. Takahata, B. Moghaddam, Glutamatergic regulation of basal and stimulusactivated dopamine release in the prefrontal cortex, J. Neurochem. 71 (1998) 1443–1449. [27] Y. Takemoto, R. Semba, Immunohistochemical evidence for the localization of neurons containing the putative transmitter l-proline in rat brain, Brain Res. 1073–1074 (2006) 311–315. [28] W. Timmerman, G. Cisci, A. Nap, J.B. De Vries, B.H. Westerink, Effects of handling on extracellular levels of glutamate and other amino acids in various areas of the brain measured by microdialysis, Brain Res. 833 (1999) 150–160. [29] S. Yoneda, E. Roberts, A new synaptosomal biosynthetic pathway of proline from ornithine and its negative feedback inhibition by proline, Brain Res. 239 (1982) 479–488. [30] O.M. Youngren, R.E. Phillips, A stereotaxic atlas of the brain of the three-day-old domestic chick, J. Comp. Neurol. 181 (1978) 567–600.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Central l-proline attenuates stress-induced dopamine and serotonin metabolism in the chick forebrain Kousuke Hamasu a , Kazutaka Shigemi a , Yusuke Kabuki a , Shozo Tomonaga a , D. Michael Denbow b , Mitsuhiro Furuse a,∗ a
Laboratory of Advanced Animal and Marine Bioresources, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0306, USA
b
a r t i c l e
i n f o
Article history: Received 17 April 2009 Received in revised form 12 May 2009 Accepted 12 May 2009 Keywords: Stress l-Proline Chick Microdialysis NMDA
a b s t r a c t Using microdialysis, we investigated the effect of l-proline on monoamine release in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) of freely moving and restricted chicks. A 30 min handlingstress resulted in a significant increase in extracellular homovallinic acid (HVA), a dopamine metabolite, and 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite, in the MNH. l-Proline, perfused through the microdialysis probe into the MNH during the stressed condition, significantly attenuated the average dialysate concentration of HVA produced by handling-stress. Handling-stress resulted in a significant increase in 5-HIAA levels in the control group, which were attenuated by profusion with l-proline. lProline did not significantly modify basal concentrations of HVA or 5-HIAA in the MNH during control conditions. These results show that perfusion of l-proline modified the turnover/metabolism of dopamine and serotonin in the MNH caused by handling-stress. © 2009 Elsevier Ireland Ltd. All rights reserved.
While l-glutamate is well established as a neurotransmitter in the central nervous system (CNS), little is known about the possible role of l-proline as a neurotransmitter. We have previously demonstrated that the amount of l-proline in the brain was reduced under stressful conditions, and intracerebroventricular (i.c.v.) injection of l-proline had sedative and hypnotic effects under an acute stressful condition in neonatal chicks [11]. Therefore, it appears that l-proline might function through several neurotransmitter systems in the CNS. Past studies showed that l-proline can activate the strychnine-sensitive glycine receptor and the N-methyl-daspartate (NMDA) glutamate receptor [2,3,13,20]. The NMDA, but not glycine, receptor appears involved in the action of l-proline to induce sedative and hypnotic effects [12]. Activation of dopaminergic and serotonergic pathways is related to behavioral and emotional changes [1,9]. Some experiments suggested a modulatory interaction between the glutamatergic and monoaminergic pathway, which is mediated via NMDA receptor activation [10,15]. From these findings, we assumed that l-proline activates the NMDA receptor and alters activation of dopaminergic and serotonergic pathways. Indeed, i.c.v. injection of l-proline suppressed the increase in serotonin turnover rate and slightly suppressed the increase in dopamine turnover rate induced by isolation
∗ Corresponding author. Tel.: +81 92 642 2953; fax: +81 92 642 2953. E-mail address: [email protected] (M. Furuse). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.05.036
stress in the telencephalon (Hamasu et al., unpublished). However, the effect of l-proline on the release of stress-induced monoamines is still obscure. In the present study, we investigated the interaction of l-proline and monoamine release in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) during stress. The MNH, a presumed analogue of the mammalian prefrontal cortex [17,18], was identified to be involved in the stress response [9,19]. One-day-old male layer chicks (Julia) purchased from a local hatchery (Murata Hatchery, Fukuoka, Japan) were maintained in a windowless room at a constant temperature of 30 ± 1 ◦ C. Lighting was provided continuously for 24 h. Chicks were given free access to a commercial starter diet (Toyohashi Feed and Mills Co. Ltd., Aichi, Japan) and water. Experimental procedures followed the guidance for Animal Experiments in the Faculty of Agriculture and in the Graduate Course of Kyushu University and the Law (No. 105) and Notification (No. 6) of the Japanese Government. Under sodium pentobarbital anesthesia (3 mg/100 g body weight), a guide-cannula was stereotaxically implanted in the brain to accommodate microdialysis probes in the MNH [17] according to the chick brain atlas [16,30] at the following stereotaxic coordinates: anterior 6.0 mm, lateral 1.0 mm, and 2.5 mm depth (Fig. 1). The microdialysis probe, to be inserted later, extended 1 mm beyond the guide-cannula. The guide-cannula assembly was then fixed to the skull by surrounding the cannula and two anchorage screws with dental cement. After implantation of the guide-cannula, the chicks were housed individually and allowed to
K. Hamasu et al. / Neuroscience Letters 460 (2009) 78–81
79
Fig. 1. The image of the medio-rostral neostriatum/hyperstriatum ventrale (MNH).
recover from surgery for approximately 24 h. During this period, the chicks had free access to food and water. The microdialysis probe was inserted and experiments were performed on free moving chicks. During this period, the chicks had free access to food and water. Probes were I-shaped (A-I-4-01, EICOM, Kyoto, Japan) ending in a dialysis membrane (1 mm long, 0.22 mm wide) with a molecular weight cut-off of 50,000 Da. Using a syringe pump (EP-64, EICOM, Kyoto, Japan) with a 2.5 ml gastight syringe, the probe was perfused with a Ringer-type solution (147 mM NaCl, 4 mM KCl, 3 mM CaCl2 ) at a flow rate of 2.0 l/min. During all experiments the microdialysates were collected every 30 min. After establishment of a baseline, different treatments were given to the chicks and samples were taken. Samples were stored at −80 ◦ C until they were analyzed. Dialysate concentrations of homovallinic acids (HVAs), a dopamine (DA) metabolite, and 5-hydroxyindoleacetic acid (5HIAA), a serotonin (5-HT) metabolite were determined. To test the effects of l-proline on basal dialysate concentrations of HVA and 5-HIAA, l-proline (100 mM) (gift from Kyowa Hakko Kogyo, Tokyo, Japan) was dissolved in Ringer-type solution and perfused through the microdialysis probe for 60 min (the time of two microdialysis samples) in Experiment 1. To test the effects of l-proline on stress-stimulated dialysate concentrations of HVA and 5-HIAA, l-proline (100 mM) was dissolved in Ringer-type solution and perfused through the microdialysis probe for 60 min (the time of two microdialysis samples) in Experiment 2. Samples were collected to determine if there was an increase in HVA and 5-HIAA caused by a 30 min handling-stress by gently restricting their movement. Chicks were sacrificed with an overdose of sodium pentobarbital. After Cresyl violet was perfused through the guide-cannulae, the brain was removed and fixed in 2% buffered paraformaldehyde. Serial coronal sections were cut to determine the location of the dialysis probe. The levels of monoamines and their metabolites were determined using a high performance liquid chromatography (HPLC) with electrochemical detection. DA, 5-HT, HVA and 5-HIAA were determined. Dialysate aliquots were collected every 30 min (60 l) into the fraction collector (Eicom, EFC-82, Kyoto, Japan). The tubes were moved to an autosampler (Model-231XL, Gilson, Middleton, WI, USA) and the dialysate samples (30 l) were automatically injected into HPLC system (Eicom, Kyoto, Japan) with a
Fig. 2. Effects of l-proline perfused through the microdialysis probe during 60 min (black bar) on dialysate concentrations of HVA (upper panel) and 5-HIAA (lower panel) in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) of awake 8or 9-day-old layer chicks. Results are expressed as means ± S.E.M. The number of chicks was 5.
150 mm × 2.1 mm octadecyl silane (ODS) column (SC-5ODS, Eicom) and electrochemical detector (ECD-300, Eicom) at an applied potential of +700 mV versus an Ag/AgCl reference analytical electrode as described previously [22]. The changes in electric current (nA) were recorded in a computer using an interface system (Power Chrom ver 2.3.2.J; AD Instruments, Tokyo, Japan). The mobile phase was composed of aceto-citric acid buffer (pH 3.5, 0.1 mol/L), methanol, sodium-1-octane sulfonate (0.46 mol/L) and disodium ethylenediaminetetraacetic acid (0.015 mmol/L) (830:170:1.9:1) at a flow rate of 0.2 ml/min. Results from Experiment 1 were analyzed by one-way repeated measure analysis of variance (ANOVA) with respect to time. In Experiment 2 data were analyzed by one-way and two-way repeated measure ANOVA with respect to time-treatment. When the ANOVA showed significant effects, Dunnett’s test was used to compare samples collected 30 min before and after treatment as a post hoc test. Statistical analyses were conducted using a commercially available package StatView (version 5, SAS Institute, Cary, NC, USA 1998). Fig. 2 shows the effects of l-proline (100 mM) on basal dialysate concentrations of HVA (upper panel) and 5-HIAA (lower panel). At the 100 mM dose, l-proline did not significantly modify basal dialysate HVA or 5-HIAA concentrations. Fig. 3 shows the effects of l-proline (100 mM) on stressstimulated dialysate concentrations of HVA (upper panel) and 5-HIAA (lower panel). There was a significant interaction (F(5, 60) = 2.463, P < 0.05) between the control and l-proline group on HVA, suggesting that l-proline significantly reduced the average increases of dialysate concentration of HVA produced by handlingstress. In contrast, there was no significant interaction in 5-HIAA (F(5, 60) = 1.819, P = 0.1227). However, the 30 min handling-stress resulted in a significant increase in 5-HIAA levels in the control group (F(5, 35) = 9.990, P < 0.001), while there was no increase in the l-proline group (F(5, 25) = 0.960, P = 0.4606).
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Furthermore, l-pipecolic acid (hexahydropicolinic acid) has a similar structure to l-proline, since it is an imino acid having a ring structure. According to Takagi et al. [25], l-pipecolic acid activated both GABA-A and GABA-B receptors. l-Proline may also stimulate GABAergic interneurons and may reduce local transmitter release, so the increases of dialysate concentration of HVA and 5-HIAA in the MNH caused by handling-stress may be attenuated. Several studies suggest the possibility that an interaction between glutamate and monoamine seems to exist in the prefrontal cortex [10,15]. In support of this interaction, microdialysis studies have suggested that DA release in the prefrontal cortex induced by stress was, in part, a consequence of an increased glutamatergic neurotransmission [5,14,26]. However, other studies did not support this claim, because it has been shown that stress did not increase extracellular concentrations of glutamate in the prefrontal cortex [4,28]. Thus, the release of monoamine during stress in the prefrontal cortex is still controversial. The present results indicated that l-proline might be involved in the release of monoamines during stress in the prefrontal cortex mediated by the NMDA receptor. Indeed, there is a long history of evidence suggesting that l-proline may be a neuronal modulator or transmitter in the central nervous system [7,8,21,24,29]. It is known that l-proline containing neurons and transporters is localized in several brain areas [7,21,27]. However, the effect of stress on extracellular l-proline is still obscure. Further study is necessary to verify the hypothesis. Acknowledgements
Fig. 3. Effects of l-proline (100 mM), perfused through the microdialysis probe for 60 min, on increases of HVA and 5-HIAA produced by a 30 min handling-stress in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) of awake 8- or 9-day-old layer chicks. Results are expressed as means ± S.E.M. The number of chicks was 8 for control and 6 for l-proline, respectively. *Significantly different from the data that were collected just before the beginning of stress.
In the present study, DA and 5-HT were not detectable, due to low concentrations, but their metabolites, HVA and 5-HIAA, were clearly detected. These metabolites may, at least in part, reflect release of their respective neurotransmitters [9,10]. The present results demonstrated that the increases of dialysate concentration of HVA and 5-HIAA in the MNH caused by handlingstress were attenuated by the simultaneous perfusion of l-proline (100 mM). It is suggested that these effects of l-proline were mediated by the NMDA receptor, given that a previous study indicated that l-proline modulated activation of the NMDA receptor [12]. The microdialysis study by Gruss et al. [10] which showed a modulatory interaction between glutamatergic and monoaminergic neurotransmission mediated by the NMDA receptor further supports this suggestion. They demonstrated that the dialysate concentration of HVA and 5-HIAA was decreased by local intracerebral infusion of NMDA, and these effects were antagonized by an NMDA antagonist. However, no decrease was confirmed by local intracerebral infusion of l-proline in the present study (Fig. 2). The cause of this discrepancy may be the concentration of l-proline. The dialysate concentration of HVA and 5-HIAA might be decreased by infusion of higher concentrations of l-proline. Del Arco and Mora [4] reported that NMDA (100 M) significantly reduced the average increases of dialysate concentration of DA without modifying basal dialysate in DA. Regarding the mechanisms of action, NMDA may stimulate GABAergic interneurons in the MNH region, which inhibit monoaminergic synaptic boutons and thereby reduce local neurotransmitter release. This suggestion is supported by studies in which endogenous glutamate increases GABA [5] and GABA agonists inhibit dopamine release in the prefrontal cortex [6,23].
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