- Email: [email protected]
Involvement of NMDA receptor mechanisms in the modulation of serotonin release in the lateral parabrachial nucleus in the rat
Brain Research Bulletin 71 (2006) 311–315
Involvement of NMDA receptor mechanisms in the modulation of serotonin release in the lateral parabrachial nucleus in the rat Junichi Tanaka a,∗ , Hiroko Miyakubo a , Ayako Kawakami a , Yasushi Hayashi b , Masahiko Nomura c,d a
Department of Curriculum, Teaching and Memory, Naruto University of Education, Takashima, Naruto-cho, Naruto, Tokushima 772-8502, Japan Department of Foods and Human Nutrition, Faculty of Human Life Sciences, Notre Dame Seishin University, Ifuku, Okayama 700-8516, Japan c International Education and Training Center, Saitama Medical University, Iruma-gun, Saitama 350-0495, Japan d Department of Physiology, Saitama Medical University, Iruma-gun, Saitama 350-0495, Japan
b
Received 22 August 2006; received in revised form 24 September 2006; accepted 25 September 2006 Available online 17 October 2006
Abstract Microdialysis was employed to investigate whether N-methyl-d-asparatate (NMDA) glutamate receptor mechanisms are involved in the modulation of serotonin (5-hydoxytryptamine, 5-HT) release in the region of the lateral parabrachial nucleus (LPBN) in freely moving rats. Perfusion of NMDA (10 and 50 M) through the microdialysis probe significantly enhanced extracellular concentrations of 5-HT and its metabolite 5hydroxyindoleacetic acid (5-HIAA) in the LPBN area. Local perfusion of the NMDA antagonist dizocilpine (MK801, 10 and 50 M) did not change the basal 5-HT and 5-HIAA levels in the LPBN area. MK801 (10 M) administered together with NMDA antagonized the stimulant effect of NMDA (10 M). The intake of 0.3 M NaCl and water induced by subcutaneous injections of the diuretic furosemide (FURO, 10 mg/kg) and the angiotensin converting enzyme inhibitor captopril (CAP, 5 mg/kg) produced significant increases in the 5-HT and 5-HIAA concentrations in the LPBN area. The increased levels of 5-HT and 5-HIAA caused by the combined treatment with FURO and CAP were attenuated by perfusion of MK801 (10 M). These results indicate the participation of NMDA receptors in the control of 5-HT release in the LPBN area. © 2006 Elsevier Inc. All rights reserved. Keywords: Lateral parabrachial nucleus; 5-Hydoxytryptamine; Glutamate; NMDA receptor; Sodium appetite; Thirst
1. Introduction It is known that serotonin (5-hydroxytryptamine, 5-HT) receptor mechanisms in the lateral parabrachial nucleus (LPBN), a structure lying dorsolateral to the superior cerebellar peduncle in the pons, play important roles in the control of sodium appetite and thirst. Bilateral injections of methysergide, a 5HT antagonist, into the LPBN enhance the salt and water intake induced by several physiological and pharmacological stimuli [1,2,11–13]. Conversely, pretreatment with bilateral injections of 2,5-dimethoxy-4-indoamphetamine, a 5-HT2a/2c receptor agonist, into the LPBN attenuates hypertonic NaCl and water intake induced by subcutaneous administration of the diuretic furosemide (FURO) and the angiotensin converting enzyme inhibitor captopril (CAP) [10,13]. Additionally,
∗
Corresponding author. Tel.: +81 88 687 6277; fax: +81 88 687 6277. E-mail address: [email protected] (J. Tanaka).
0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2006.09.017
the intake of salt and water elicited by acute sodium depletion induced by the combined treatment with FURO and CAP increases the release of 5-HT in the LPBN area [17]. Previous investigations have demonstrated that N-methyl-dasparatate (NMDA) glutamate receptors in the rat brain participate in the maintenance of body fluid and electrolyte [5,19,20]. The existence of NMDA receptors in the LPBN has been shown [3,18]. Experimental observations in several brain regions have revealed the interaction between glutamate release and 5-HT release via NMDA receptors [7,9,15,16]. The present study was designed to investigate whether NMDA receptor mechanisms are involved in the control of the serotonergic regulatory system of sodium and water intake in the LPBN in freely moving rats. We used in vivo microdialysis methods to examine the effects of perfusion of NMDA and its antagonist dizocilpine (MK801) on extracellular concentrations of 5-HT and its metabolite 5hydroxyindoleacetic acid (5-HIAA) in the LPBN area. We also investigated the effects of perfusion of MK801 on the changes in the 5-HT and 5-HIAA concentrations in the LPBN area caused
312
J. Tanaka et al. / Brain Research Bulletin 71 (2006) 311–315
by subcutaneous injections of FURO and CAP under the condition that 0.3 M NaCl and water are available for drinking. 2. Materials and methods The experiment was performed according to the guiding principles of the Physiological Society of Japan.
2.1. Animals Experiments were performed on adult male Wistar rats (n = 49) weighing 260–340 g. The animals were housed in individual stainless steel cages with free access to normal sodium diet (sodium content 0.5%, Oriental Yeast Co. Ltd.,), water and 0.3 M NaCl solution. Lights were on in the animal rooms for 12 h per day (light at 7:00–19:00 h), and temperature was maintained at 23–25 ◦ C.
2.2. Surgery The animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and placed in a stereotaxic frame. The microdialysis probe guide cannula (AG12, Eicom Co.) was lowered to a coordinate which was 1 mm dorsal to the LPBN since the probe assembly protrudes 1 mm below the ventral tip of the guide cannula when inserted. The stereotaxic coordinates of the guide cannula for the LPBN were 0.2 mm posterior from the interaural line, 1.8 mm lateral to the midline, and 5.4 mm ventral to the cortical surface. The guide cannula was then fixed to the skull with acrylic dental cement and small stainless steel screws, and sealed with a dummy cannula after implantation.
isotonic saline. CAP was injected subcutaneously at 5 mg/kg of body weight (0.13–0.17 ml). The doses of the drugs utilized in this study were same that previously demonstrated effects on NaCl and water intake [10–13,17]. Water and 0.3 M NaCl were available to the animals immediately after the treatment with FURO and CAP. Volumes of each liquid was measured every 20 min for 3 h.
2.6. Measurement of 5-HT and 5-HIAA Immediately after collection, the dialysates were analyzed for concentrations of 5-HT and 5-HIAA, using HPLC (EP-10, Eicom Co.) with electrochemical detection (ECD-100, Eicom Co.). A mobile phase consisting of 0.1 M sodium acetate, 0.1 M citric acid, 0.75 mM sodium 1-octanesulfonate, 0.3 mM EDTA and 21% methanol (pH 3.9) was used to elute the monoamines from a reverse phase column (3.0 mm × 100 mm SC-3ODS column, Eicom Co.). The graphite working electrode was set at +750 mV versus a Ag/AgCl reference electrode and the flow rate was 0.5 ml/min.
2.7. Histology At the termination of each experiment, the microdialysis probe was perfused with Ringer’s solution containing 2% Pontamine sky blue dye to confirm more precisely the dialysis site by staining the structure. Each animal was then sacrificed with an overdose of sodium pentobarbital and perfused through the heart with isotonic saline to clear blood, which was followed by 10% formalin for fixation. The brain was removed and stored in the formalin saline for 24 h. Transverse sections of 50 m were cut on a freezing microtome. Sections were mounted on glass slides and stained with Neutral red for microscopic examination.
2.3. Microdialysis 2.8. Statistics A dialysis experiment was carried out 3 days after the implantation of the guide cannula. The dummy cannula was removed, and the dialysis probe (AI-112-1, Eicom Co.) whose tip had 1.0-mm long semipermeable membrane (0.2 mm outside diameter, molecular weight cut-off 50 kDa, in vivo recovery at 2 l/min is almost 25%) was inserted into the implanted guide cannula. Each rat was then placed in the metabolism cage. The probe was continuously perfused at a rate of 2 l/min using a perfusion pump with Ringer’s solution (NaCl 147 mM, CaCl2 2.3 mM, KCl 4 mM; pH 6.5) for more than 12 h. On the next day, the dialysis experiments were performed. Water and 0.3 M NaCl were removed 2 h before the NMDA or MK801 application or the treatment with FURO and CAP. Dialysis samples were collected at 20 min intervals.
2.4. Treatment with NMDA and MK801 NMDA (Sigma Chemical Co., St. Louis, MO) and its antagonist MK801 (Sigma Chemical Co.) were dissolved in pure dimethylsulfoxide (DMSO). Immediately before use, these drug stocks were diluted with the Ringer’s solution. Subsequently, the drugs were applied for 20 min directly into the LPBN area through the dialysis probe. NMDA and MK801 application was achieved in doses of 10 and 50 M. Vehicle perfusion refers to perfusion solution plus 0.1% DMSO, corresponding to the highest concentration of DMSO in animals treated with the solution containing the two drugs. To investigate the effects of local application of the NMDA antagonist on the FURO and CAP treatment-induced changes in the 5-HT release in the LPBN area, the antagonist was applied for 20 min at 40 min after the FURO and CAP treatment. Sample collections were continued 3 h after the treatment.
2.5. Administration of FURO and CAP The treatment with FURO and CAP was performed under the condition that 0.3 M NaCl and water are available for drinking. Two metal drinking spouts were presented, one with tap water, and one with 0.3 M NaCl solution, both at room temperature. Each liquid was provided from a burette with 0.1-ml divisions that was fitted with the drinking spout. FURO (Sigma Chemical Co.) was dissolved in distilled water. FURO was administered subcutaneously at 10 mg/kg of body weight (0.26–0.34 ml). CAP (Sigma Chemical Co.) was dissolved in
All values reported are mean ± S.E.M. Data were analyzed by means of one-way or two-way repeated measures analysis of variance (ANOVA) and subsequent t-test. The criterion for significance was P < 0.05 in all cases.
3. Results 3.1. The dialysis probe placement and basal concentrations of 5-HA and 5-HIAA Histological analysis of the rat brains showed that the microdialysis probes of 44 out of 49 rats tested were located within the LPBN. The remaining five animals had the probe placement outside the LPBN; four and one animals received perfusion of NMDA (10 M) and vehicle, respectively. Basal levels of 5-HT and 5-HIAA in 20 min dialysate samples from the LPBN area were 10.5 ± 1.1 pg/40 l (n = 44) and 698.4 ± 15.6 pg/40 l (n = 44), respectively. Basal levels of 5-HT and 5-HIAA in 20 min dialysate samples from the area outside the LPBN were 4.2 ± 1.9 pg/40 l (n = 5) and 312.0 ± 51.8 pg/40 l (n = 5), respectively. 3.2. Effects of perfusion of NMDA and MK801 on extracellular concentrations of 5-HT and 5-HIAA in the LBPN and the area outside the LBPN Perfusion of NMDA (10 M, n = 5 and 50 M, n = 5) through the microdialysis probe produced significant increases in both the 5-HT (Fig. 1A) and 5-HIAA (Fig. 1B) concentrations in the LPBN area. Neither lower (10 M, n = 5) nor higher (50 M, n = 6) perfusion of MK801 elicited significant changes in the
J. Tanaka et al. / Brain Research Bulletin 71 (2006) 311–315
313
Fig. 1. Effects of perfusion of N-methyl-d-asparatate (NMDA: 10 M, n = 5 and 50 M, n = 5) or the NMDA antagonist dizocilpine (MK801: 10 M, n = 5 and 50 M, n = 6) through the microdialysis probe on extracellular concentrations of serotonin (5-hydoxytryptamine, 5-HT (A) and (C)) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA (B) and (D)) in the region of the lateral parabrachial nucleus (LPBN), and the effects of MK801 (10 M) administered together with the NMDA (10 M) on the NMDA-induced changes in the 5-HT (A) and 5-HIAA (B) levels (n = 6). Values are expressed as percentage of the sample taken immediately before the perfusion. Results are shown as mean ± S.E.M. The horizontal bars show the duration of drug application. The data obtained from vehicle perfusion (vehicle, n = 5) are represented in (A)–(D). Perfusion of NMDA (10 and 50 M) significantly increased the 5-HT (A) and 5-HIAA (B) in the LPBN area. Perfusion of MK801 (10 and 50 M) did not cause significant changes in the 5-HT (C) and 5-HIAA (D) levels in the LPBN area. MK801 (10 M) administered together with NMDA antagonized the stimulant effect of NMDA (10 M) (A and B). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the basal control level (0 min). # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the corresponding value in NMDA (10 M).
5-HT (Fig. 1C) and 5-HIAA (Fig. 1D) levels. MK801 (10 M) administered together with NMDA antagonized the stimulant effect of NMDA (10 M) (n = 6; Fig. 1A and B). Perfusion of vehicle did not cause any significant changes in the 5-HT (Fig. 1A and C) and 5-HIAA (Fig. 1B and D) concentrations in the LBPN area (n = 5). Perfusion of NMDA (10 M, n = 4) slightly enhanced the 5-HT (the maximum was 107.2 ± 4.8% of the control level at the administration period) and 5-HIAA (the maximum was 122.6 ± 15.9% of the control level at the 20 min after the drug application) concentrations in the area outside the LBPN (data not shown). 3.3. Changes in 5-HT and 5-HIAA concentrations in the LBPN and 0.3 M NaCl and water intake in to subcutaneous injections of FURO and CAP, and the effects of perfusion of MK801 on the changes in 5-HT and 5-HIAA levels Perfusion of vehicle combined with subcutaneous injections of FURO and CAP significantly enhanced the 5-HT (Fig. 2A) and 5-HIAA (Fig. 2B) concentrations in the LPBN area, and produced robust intake of 0.3 M NaCl (Fig. 2C) and water (Fig. 2D) (n = 6). Perfusion of MK801 significantly attenuated the enhancement of 5-HT (Fig. 2A) and 5-HIAA (Fig. 2B) in the LPBN area induced by the combined treatment with FURO and CAP, while the perfusion did not elicit significant differences
in the amount of the 0.3 M NaCl (Fig. 2C) and water (Fig. 2D) intake (n = 6). 4. Discussion The present study shows that NMDA glutamate receptors are involved in the modulation of 5-HT release in the LPBN area. It is known that almost all neurons in the central nervous system carry the NMDA subtype of ionotropic glutamate receptors, which can mediate postsynaptic Ca2+ influx [4]. Although it is impossible to explain the precise mechanisms underlying the enhancement of 5-HT release in the LPBN area, it is tempting to speculate that glutamate released from glutamatergic nerve terminals acts directly on 5-HT terminals through NMDA receptors, which result in increased 5-HT release induced by the Ca2+ influx. Since the regulation of adenosine release by glutamate receptors and calcium/calmodulin-dependent protein kinase II have important consequences in the presynaptic control of glutamate release [16], it is possible that the modulation of 5-HT release may be mediated by the presynaptic control mechanisms of glutamate release. However, there is no evidence showing that NMDA receptors in the 5-HT terminals do exist and glutamatergic fibers terminate on 5-HT terminals in the LPBN area. The findings in which the enhanced release of 5-HT in the LPBN area accompanied with robust intake of salt and water in response to the FURO and CAP treatment was diminished
314
J. Tanaka et al. / Brain Research Bulletin 71 (2006) 311–315
Fig. 2. Effects of perfusion of MK801 (10 M) through the microdialysis probe on the changes in the 5-HT (A) and 5-HIAA (B) concentrations in the LPBN area and the intake of 0.3 M NaCl (C) and water (D) induced by subcutaneous treatment with furosemide (FURO) and captopril (CAP). Values are expressed as percentage of the sample taken immediately before the combined treatment with FURO and CAP. Results are shown as mean ± S.E.M. The arrows show the time of the combined treatment with FURO and CAP. The horizontal bars indicate the duration of drug application. (A and B) The treatment with FRO and CAP produced significant increases in the 5-HT (A) and 5-HIAA (B) concentrations in the LPBN area (unfilled circles, n = 6). Perfusion of MK801 significantly reduced the treatment-induced increases in the 5-HT (A) and 5-HIAA (B) levels (filled circles, n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the basal control level (0 min). # P < 0.05, ### P < 0.001 compared with the corresponding value in FURO + CAP-vehicle. (C and D) The treatment with FURO and CAP elicited robust 0.3 M NaCl (C) and water (D) intake. Perfusion of MK801 did not cause significant changes in the 0.3 M NaCl and water intake.
by perfusion of MK801 imply that the NMDA receptor mechanisms in the LPBN may play an important role in the control of sodium appetite and thirst. Previous investigations have shown that the 5-HT receptor antagonist methysergide injected bilaterally into the LPBN markedly increase [1,2,11–13], while the 5-HT receptor agonist 2,5-dimethoxy-4-indoamphetamine reduces [10,13], salt and water intake induced by central administration of angiotensin II or dipsogenic/natriorexigenic treatments, suggesting that the 5-HT receptor mechanisms in the LPBN have an inhibitory action on sodium and water consumption. In this study, the MK801 application-induced reduction of 5-HT release in the LPBN area did not cause significant changes in the intake of sodium and water elicited by the FURO and CAP treatment. It is hypothesized that the decrease in the 5-HT release in the local area of the unilateral LPBN could not produce a marked alteration in the salt and water intake. It is also suggested the possibility that the inhibitory system of sodium and water intake, which are activated by 5-HT, may be damaged by a penetration of the dialysis probe. In addition, it is possible to speculate that perfusion of MK801 may influence the release of neurotransmitters or nouromodulaters except for 5-HT and the activity of LPBN neurons involved in the modulation of sodium and water intake. Indeed, electrophysiological observations have indicated that glutamate depolarizes LPBN neurons through NMDA receptors [21]. The serotonergic nerve terminals within the LPBN are derived from the area postrema and the medial nucleus of
the solitary tract [8], sites known to receive ascending projections from arterial baroreceptors, cardiopulmonary receptors, and gustatory receptors that can influence water and salt intake [6,14]. It is possible that the 5-HT release in the LPBN may be modulated by peripheral information arising from these receptors. Thus, it might be expected that the glutamatergic inputs through NMDA receptors in the LPBN may modulate the peripheral information for controlling sodium appetite and thirst. The precise physiological role of the glutamatergic system in the LPBN and the involvement of non-NMDA receptor mechanisms in the regulation of 5-HT release remain to be explained. Further studies that examine changes in the glutamine extracellular concentration within the LPBN during the intake of NaCl and water induced by the combined treatment with FURO and CAP may help to explain the action of glutamatergic mechanisms on the control of the serotonergic regulatory system of sodium appetite and thirst in the LPBN.
References [1] D.S.A. Colombari, J.V. Menani, A.K. Johnson, Forebrain angiotensin type 1 receptors and parabrachial serotonin in the control of NaCl and water intake, Am. J. Physiol. 271 (1996) R1470–R1476. [2] J.I.F. De Gobbi, L.A. De Luca Jr., J.V. Menani, Serotonergic mechanisms of the lateral parabrachial nucleus on DOCA-induced sodium intake, Brain Res. 880 (2000) 131–138.
J. Tanaka et al. / Brain Research Bulletin 71 (2006) 311–315 [3] A. Guthmann, H. Herbert, Expression of N-methyl-d-aspartate receptor subunits in the rat parabrachial and Kolliker–Fuse nuclei and in selected pontomedullary brainstem nuclei, J. Comp. Neurol. 415 (1999) 501– 517. [4] M.R. Hynd, H.L. Scott, P.R. Dodd, Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer’s disease, Neurochem. Int. 45 (2004) 583–595. [5] J.W. Jahng, T.A. Houpt, MK801 increases feeding and decreases drinking in nondeprived, freely feeding rats, Pharmacol. Biochem. Behav. 68 (2001) 181–186. [6] A.K. Johnson, R.L. Thunhorst, Sensory mechanisms in the behavioral control of body fluid balance: thirst and salt appetite, in: S.J. Fluharty, A.R. Morrisom, J.M. Sprague, E. Stellar (Eds.), Progress in Psychobiology and Physiological Psychology, vol. 16, Academic, New York, 1995, pp. 145–176. [7] H.S. Kim, H.S. Jang, K.H. Hahn, M.J. Kim, S.H. Yoon, Y.H. Jo, M.S. Kim, D.J. Rhie, Serotonin inhibits the induction of NMDA receptor-dependent lomg-term potentiation in the rat primary visual cortex, Brain Res. 1103 (2006) 49–55. [8] A.J. Lanca, D. van der Kooy, A serotonin-containing pathway from the area postrema to the parabrachial nucleus in the rat, Neuroscience 14 (1985) 1117–1126. [9] M. Marcoli, C. Cervetto, P. Paluzzi, S. Guarnieri, M. Raiteri, G. Maura, Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors, Neurochem. Int. 49 (2006) 12–19. [10] L.O. Margatho, S.P. Barbosa, L.A. De Luca Jr., J.V. Menani, Central serotonergic and adrenergic/imidazoline inhibitory mechanisms on sodium and water intake, Brain Res. 956 (2002) 103–109. [11] J.V. Menani, L.A. De Luca Jr., A.K. Johnson, Lateral parabrachial nucleus serotonergic mechanisms and salt appetite induced by sodium depletion, Am. J. Physiol. 274 (1998) R555–R560.
315
[12] J.V. Menani, A.K. Johnson, Lateral parabrachial nucleus serotonergic mechanisms: angiotensin-induced pressor and drinking responses, Am. J. Physiol. 269 (1995) R1044–R1049. [13] J.V. Menani, R.L. Thunhorst, A.K. Johnson, Lateral parabrachial nucleus and serotonergic mechanisms in the control of salt appetite in rats, Am. J. Physiol. 270 (1996) R162–R168. [14] R. Norgren, The central organization of the gustatory and visceral systems in the nucleus of the solitary tract, in: Y. Katsuki, R. Norgren, M. Sato (Eds.), Brain Mechanisms of Sensation, Wiley, New York, 1981, pp. 143–160. [15] J.C. Owen, P.S. Whitton, Vhronic treatment with antidepressant drugs reversibly alters NMDA mediated regulation of extracellular 5-HT in rat frontal cortex, Brain Res. Bull. 70 (2006) 62–67. [16] R. Paes-de-Carvalho, B.V. Dias, R.A. Martins, M.R. Pereira, C.C. Portugal, C. Lanfredi, Activation of glutamate receptors promotes a calciumdependent and transporter-mediated release of purines in cultured avian retinal cells: possible involvement of calcium/calmodulin-dependent protein kinase II, Neurochem. Int. 46 (2005) 441–451. [17] J. Tanaka, Y. Hayashi, K. Yamato, H. Miyakubo, M. Nomura, Involvement of serotonergic systems in the lateral parabrachial nucleus in sodium and water intake: a microdialysis study in the rat, Neurosci. Lett. 357 (2004) 41–44. [18] M. Watanabe, M. Mishina, Y. Inoue, Distinct distributions of five NMDA receptor channel subunit mRNAs in the brainstem, J. Comp. Neurol. 343 (1994) 520–531. [19] Z. Xu, A.K. Johnson, Non-NMDA receptor antagonist-induced drinking in rat, Brain Res. 808 (1998) 124–127. [20] Z. Xu, L.M. Lane, J. Herbert, Dizocilpine maleate, an N-methyl-d-aspartate antagonist, inhibits dipsogenic responses and c-fos expression induced by intracerebral infusion of angiotensin II, Neuroscience 78 (1997) 203–214. [21] J.A. Zidichouski, J.C. Easaw, J.H. Jhamandas, Glutamate receptor subtypes mediate excitatory synaptic responses of rat lateral parabrachial neurons, Am. J. Physiol. 270 (1996) H1557–H1567.
Involvement of NMDA receptor mechanisms in the modulation of serotonin release in the lateral parabrachial nucleus in the rat Junichi Tanaka a,∗ , Hiroko Miyakubo a , Ayako Kawakami a , Yasushi Hayashi b , Masahiko Nomura c,d a
Department of Curriculum, Teaching and Memory, Naruto University of Education, Takashima, Naruto-cho, Naruto, Tokushima 772-8502, Japan Department of Foods and Human Nutrition, Faculty of Human Life Sciences, Notre Dame Seishin University, Ifuku, Okayama 700-8516, Japan c International Education and Training Center, Saitama Medical University, Iruma-gun, Saitama 350-0495, Japan d Department of Physiology, Saitama Medical University, Iruma-gun, Saitama 350-0495, Japan
b
Received 22 August 2006; received in revised form 24 September 2006; accepted 25 September 2006 Available online 17 October 2006
Abstract Microdialysis was employed to investigate whether N-methyl-d-asparatate (NMDA) glutamate receptor mechanisms are involved in the modulation of serotonin (5-hydoxytryptamine, 5-HT) release in the region of the lateral parabrachial nucleus (LPBN) in freely moving rats. Perfusion of NMDA (10 and 50 M) through the microdialysis probe significantly enhanced extracellular concentrations of 5-HT and its metabolite 5hydroxyindoleacetic acid (5-HIAA) in the LPBN area. Local perfusion of the NMDA antagonist dizocilpine (MK801, 10 and 50 M) did not change the basal 5-HT and 5-HIAA levels in the LPBN area. MK801 (10 M) administered together with NMDA antagonized the stimulant effect of NMDA (10 M). The intake of 0.3 M NaCl and water induced by subcutaneous injections of the diuretic furosemide (FURO, 10 mg/kg) and the angiotensin converting enzyme inhibitor captopril (CAP, 5 mg/kg) produced significant increases in the 5-HT and 5-HIAA concentrations in the LPBN area. The increased levels of 5-HT and 5-HIAA caused by the combined treatment with FURO and CAP were attenuated by perfusion of MK801 (10 M). These results indicate the participation of NMDA receptors in the control of 5-HT release in the LPBN area. © 2006 Elsevier Inc. All rights reserved. Keywords: Lateral parabrachial nucleus; 5-Hydoxytryptamine; Glutamate; NMDA receptor; Sodium appetite; Thirst
1. Introduction It is known that serotonin (5-hydroxytryptamine, 5-HT) receptor mechanisms in the lateral parabrachial nucleus (LPBN), a structure lying dorsolateral to the superior cerebellar peduncle in the pons, play important roles in the control of sodium appetite and thirst. Bilateral injections of methysergide, a 5HT antagonist, into the LPBN enhance the salt and water intake induced by several physiological and pharmacological stimuli [1,2,11–13]. Conversely, pretreatment with bilateral injections of 2,5-dimethoxy-4-indoamphetamine, a 5-HT2a/2c receptor agonist, into the LPBN attenuates hypertonic NaCl and water intake induced by subcutaneous administration of the diuretic furosemide (FURO) and the angiotensin converting enzyme inhibitor captopril (CAP) [10,13]. Additionally,
∗
Corresponding author. Tel.: +81 88 687 6277; fax: +81 88 687 6277. E-mail address: [email protected] (J. Tanaka).
0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2006.09.017
the intake of salt and water elicited by acute sodium depletion induced by the combined treatment with FURO and CAP increases the release of 5-HT in the LPBN area [17]. Previous investigations have demonstrated that N-methyl-dasparatate (NMDA) glutamate receptors in the rat brain participate in the maintenance of body fluid and electrolyte [5,19,20]. The existence of NMDA receptors in the LPBN has been shown [3,18]. Experimental observations in several brain regions have revealed the interaction between glutamate release and 5-HT release via NMDA receptors [7,9,15,16]. The present study was designed to investigate whether NMDA receptor mechanisms are involved in the control of the serotonergic regulatory system of sodium and water intake in the LPBN in freely moving rats. We used in vivo microdialysis methods to examine the effects of perfusion of NMDA and its antagonist dizocilpine (MK801) on extracellular concentrations of 5-HT and its metabolite 5hydroxyindoleacetic acid (5-HIAA) in the LPBN area. We also investigated the effects of perfusion of MK801 on the changes in the 5-HT and 5-HIAA concentrations in the LPBN area caused
312
J. Tanaka et al. / Brain Research Bulletin 71 (2006) 311–315
by subcutaneous injections of FURO and CAP under the condition that 0.3 M NaCl and water are available for drinking. 2. Materials and methods The experiment was performed according to the guiding principles of the Physiological Society of Japan.
2.1. Animals Experiments were performed on adult male Wistar rats (n = 49) weighing 260–340 g. The animals were housed in individual stainless steel cages with free access to normal sodium diet (sodium content 0.5%, Oriental Yeast Co. Ltd.,), water and 0.3 M NaCl solution. Lights were on in the animal rooms for 12 h per day (light at 7:00–19:00 h), and temperature was maintained at 23–25 ◦ C.
2.2. Surgery The animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and placed in a stereotaxic frame. The microdialysis probe guide cannula (AG12, Eicom Co.) was lowered to a coordinate which was 1 mm dorsal to the LPBN since the probe assembly protrudes 1 mm below the ventral tip of the guide cannula when inserted. The stereotaxic coordinates of the guide cannula for the LPBN were 0.2 mm posterior from the interaural line, 1.8 mm lateral to the midline, and 5.4 mm ventral to the cortical surface. The guide cannula was then fixed to the skull with acrylic dental cement and small stainless steel screws, and sealed with a dummy cannula after implantation.
isotonic saline. CAP was injected subcutaneously at 5 mg/kg of body weight (0.13–0.17 ml). The doses of the drugs utilized in this study were same that previously demonstrated effects on NaCl and water intake [10–13,17]. Water and 0.3 M NaCl were available to the animals immediately after the treatment with FURO and CAP. Volumes of each liquid was measured every 20 min for 3 h.
2.6. Measurement of 5-HT and 5-HIAA Immediately after collection, the dialysates were analyzed for concentrations of 5-HT and 5-HIAA, using HPLC (EP-10, Eicom Co.) with electrochemical detection (ECD-100, Eicom Co.). A mobile phase consisting of 0.1 M sodium acetate, 0.1 M citric acid, 0.75 mM sodium 1-octanesulfonate, 0.3 mM EDTA and 21% methanol (pH 3.9) was used to elute the monoamines from a reverse phase column (3.0 mm × 100 mm SC-3ODS column, Eicom Co.). The graphite working electrode was set at +750 mV versus a Ag/AgCl reference electrode and the flow rate was 0.5 ml/min.
2.7. Histology At the termination of each experiment, the microdialysis probe was perfused with Ringer’s solution containing 2% Pontamine sky blue dye to confirm more precisely the dialysis site by staining the structure. Each animal was then sacrificed with an overdose of sodium pentobarbital and perfused through the heart with isotonic saline to clear blood, which was followed by 10% formalin for fixation. The brain was removed and stored in the formalin saline for 24 h. Transverse sections of 50 m were cut on a freezing microtome. Sections were mounted on glass slides and stained with Neutral red for microscopic examination.
2.3. Microdialysis 2.8. Statistics A dialysis experiment was carried out 3 days after the implantation of the guide cannula. The dummy cannula was removed, and the dialysis probe (AI-112-1, Eicom Co.) whose tip had 1.0-mm long semipermeable membrane (0.2 mm outside diameter, molecular weight cut-off 50 kDa, in vivo recovery at 2 l/min is almost 25%) was inserted into the implanted guide cannula. Each rat was then placed in the metabolism cage. The probe was continuously perfused at a rate of 2 l/min using a perfusion pump with Ringer’s solution (NaCl 147 mM, CaCl2 2.3 mM, KCl 4 mM; pH 6.5) for more than 12 h. On the next day, the dialysis experiments were performed. Water and 0.3 M NaCl were removed 2 h before the NMDA or MK801 application or the treatment with FURO and CAP. Dialysis samples were collected at 20 min intervals.
2.4. Treatment with NMDA and MK801 NMDA (Sigma Chemical Co., St. Louis, MO) and its antagonist MK801 (Sigma Chemical Co.) were dissolved in pure dimethylsulfoxide (DMSO). Immediately before use, these drug stocks were diluted with the Ringer’s solution. Subsequently, the drugs were applied for 20 min directly into the LPBN area through the dialysis probe. NMDA and MK801 application was achieved in doses of 10 and 50 M. Vehicle perfusion refers to perfusion solution plus 0.1% DMSO, corresponding to the highest concentration of DMSO in animals treated with the solution containing the two drugs. To investigate the effects of local application of the NMDA antagonist on the FURO and CAP treatment-induced changes in the 5-HT release in the LPBN area, the antagonist was applied for 20 min at 40 min after the FURO and CAP treatment. Sample collections were continued 3 h after the treatment.
2.5. Administration of FURO and CAP The treatment with FURO and CAP was performed under the condition that 0.3 M NaCl and water are available for drinking. Two metal drinking spouts were presented, one with tap water, and one with 0.3 M NaCl solution, both at room temperature. Each liquid was provided from a burette with 0.1-ml divisions that was fitted with the drinking spout. FURO (Sigma Chemical Co.) was dissolved in distilled water. FURO was administered subcutaneously at 10 mg/kg of body weight (0.26–0.34 ml). CAP (Sigma Chemical Co.) was dissolved in
All values reported are mean ± S.E.M. Data were analyzed by means of one-way or two-way repeated measures analysis of variance (ANOVA) and subsequent t-test. The criterion for significance was P < 0.05 in all cases.
3. Results 3.1. The dialysis probe placement and basal concentrations of 5-HA and 5-HIAA Histological analysis of the rat brains showed that the microdialysis probes of 44 out of 49 rats tested were located within the LPBN. The remaining five animals had the probe placement outside the LPBN; four and one animals received perfusion of NMDA (10 M) and vehicle, respectively. Basal levels of 5-HT and 5-HIAA in 20 min dialysate samples from the LPBN area were 10.5 ± 1.1 pg/40 l (n = 44) and 698.4 ± 15.6 pg/40 l (n = 44), respectively. Basal levels of 5-HT and 5-HIAA in 20 min dialysate samples from the area outside the LPBN were 4.2 ± 1.9 pg/40 l (n = 5) and 312.0 ± 51.8 pg/40 l (n = 5), respectively. 3.2. Effects of perfusion of NMDA and MK801 on extracellular concentrations of 5-HT and 5-HIAA in the LBPN and the area outside the LBPN Perfusion of NMDA (10 M, n = 5 and 50 M, n = 5) through the microdialysis probe produced significant increases in both the 5-HT (Fig. 1A) and 5-HIAA (Fig. 1B) concentrations in the LPBN area. Neither lower (10 M, n = 5) nor higher (50 M, n = 6) perfusion of MK801 elicited significant changes in the
J. Tanaka et al. / Brain Research Bulletin 71 (2006) 311–315
313
Fig. 1. Effects of perfusion of N-methyl-d-asparatate (NMDA: 10 M, n = 5 and 50 M, n = 5) or the NMDA antagonist dizocilpine (MK801: 10 M, n = 5 and 50 M, n = 6) through the microdialysis probe on extracellular concentrations of serotonin (5-hydoxytryptamine, 5-HT (A) and (C)) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA (B) and (D)) in the region of the lateral parabrachial nucleus (LPBN), and the effects of MK801 (10 M) administered together with the NMDA (10 M) on the NMDA-induced changes in the 5-HT (A) and 5-HIAA (B) levels (n = 6). Values are expressed as percentage of the sample taken immediately before the perfusion. Results are shown as mean ± S.E.M. The horizontal bars show the duration of drug application. The data obtained from vehicle perfusion (vehicle, n = 5) are represented in (A)–(D). Perfusion of NMDA (10 and 50 M) significantly increased the 5-HT (A) and 5-HIAA (B) in the LPBN area. Perfusion of MK801 (10 and 50 M) did not cause significant changes in the 5-HT (C) and 5-HIAA (D) levels in the LPBN area. MK801 (10 M) administered together with NMDA antagonized the stimulant effect of NMDA (10 M) (A and B). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the basal control level (0 min). # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the corresponding value in NMDA (10 M).
5-HT (Fig. 1C) and 5-HIAA (Fig. 1D) levels. MK801 (10 M) administered together with NMDA antagonized the stimulant effect of NMDA (10 M) (n = 6; Fig. 1A and B). Perfusion of vehicle did not cause any significant changes in the 5-HT (Fig. 1A and C) and 5-HIAA (Fig. 1B and D) concentrations in the LBPN area (n = 5). Perfusion of NMDA (10 M, n = 4) slightly enhanced the 5-HT (the maximum was 107.2 ± 4.8% of the control level at the administration period) and 5-HIAA (the maximum was 122.6 ± 15.9% of the control level at the 20 min after the drug application) concentrations in the area outside the LBPN (data not shown). 3.3. Changes in 5-HT and 5-HIAA concentrations in the LBPN and 0.3 M NaCl and water intake in to subcutaneous injections of FURO and CAP, and the effects of perfusion of MK801 on the changes in 5-HT and 5-HIAA levels Perfusion of vehicle combined with subcutaneous injections of FURO and CAP significantly enhanced the 5-HT (Fig. 2A) and 5-HIAA (Fig. 2B) concentrations in the LPBN area, and produced robust intake of 0.3 M NaCl (Fig. 2C) and water (Fig. 2D) (n = 6). Perfusion of MK801 significantly attenuated the enhancement of 5-HT (Fig. 2A) and 5-HIAA (Fig. 2B) in the LPBN area induced by the combined treatment with FURO and CAP, while the perfusion did not elicit significant differences
in the amount of the 0.3 M NaCl (Fig. 2C) and water (Fig. 2D) intake (n = 6). 4. Discussion The present study shows that NMDA glutamate receptors are involved in the modulation of 5-HT release in the LPBN area. It is known that almost all neurons in the central nervous system carry the NMDA subtype of ionotropic glutamate receptors, which can mediate postsynaptic Ca2+ influx [4]. Although it is impossible to explain the precise mechanisms underlying the enhancement of 5-HT release in the LPBN area, it is tempting to speculate that glutamate released from glutamatergic nerve terminals acts directly on 5-HT terminals through NMDA receptors, which result in increased 5-HT release induced by the Ca2+ influx. Since the regulation of adenosine release by glutamate receptors and calcium/calmodulin-dependent protein kinase II have important consequences in the presynaptic control of glutamate release [16], it is possible that the modulation of 5-HT release may be mediated by the presynaptic control mechanisms of glutamate release. However, there is no evidence showing that NMDA receptors in the 5-HT terminals do exist and glutamatergic fibers terminate on 5-HT terminals in the LPBN area. The findings in which the enhanced release of 5-HT in the LPBN area accompanied with robust intake of salt and water in response to the FURO and CAP treatment was diminished
314
J. Tanaka et al. / Brain Research Bulletin 71 (2006) 311–315
Fig. 2. Effects of perfusion of MK801 (10 M) through the microdialysis probe on the changes in the 5-HT (A) and 5-HIAA (B) concentrations in the LPBN area and the intake of 0.3 M NaCl (C) and water (D) induced by subcutaneous treatment with furosemide (FURO) and captopril (CAP). Values are expressed as percentage of the sample taken immediately before the combined treatment with FURO and CAP. Results are shown as mean ± S.E.M. The arrows show the time of the combined treatment with FURO and CAP. The horizontal bars indicate the duration of drug application. (A and B) The treatment with FRO and CAP produced significant increases in the 5-HT (A) and 5-HIAA (B) concentrations in the LPBN area (unfilled circles, n = 6). Perfusion of MK801 significantly reduced the treatment-induced increases in the 5-HT (A) and 5-HIAA (B) levels (filled circles, n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the basal control level (0 min). # P < 0.05, ### P < 0.001 compared with the corresponding value in FURO + CAP-vehicle. (C and D) The treatment with FURO and CAP elicited robust 0.3 M NaCl (C) and water (D) intake. Perfusion of MK801 did not cause significant changes in the 0.3 M NaCl and water intake.
by perfusion of MK801 imply that the NMDA receptor mechanisms in the LPBN may play an important role in the control of sodium appetite and thirst. Previous investigations have shown that the 5-HT receptor antagonist methysergide injected bilaterally into the LPBN markedly increase [1,2,11–13], while the 5-HT receptor agonist 2,5-dimethoxy-4-indoamphetamine reduces [10,13], salt and water intake induced by central administration of angiotensin II or dipsogenic/natriorexigenic treatments, suggesting that the 5-HT receptor mechanisms in the LPBN have an inhibitory action on sodium and water consumption. In this study, the MK801 application-induced reduction of 5-HT release in the LPBN area did not cause significant changes in the intake of sodium and water elicited by the FURO and CAP treatment. It is hypothesized that the decrease in the 5-HT release in the local area of the unilateral LPBN could not produce a marked alteration in the salt and water intake. It is also suggested the possibility that the inhibitory system of sodium and water intake, which are activated by 5-HT, may be damaged by a penetration of the dialysis probe. In addition, it is possible to speculate that perfusion of MK801 may influence the release of neurotransmitters or nouromodulaters except for 5-HT and the activity of LPBN neurons involved in the modulation of sodium and water intake. Indeed, electrophysiological observations have indicated that glutamate depolarizes LPBN neurons through NMDA receptors [21]. The serotonergic nerve terminals within the LPBN are derived from the area postrema and the medial nucleus of
the solitary tract [8], sites known to receive ascending projections from arterial baroreceptors, cardiopulmonary receptors, and gustatory receptors that can influence water and salt intake [6,14]. It is possible that the 5-HT release in the LPBN may be modulated by peripheral information arising from these receptors. Thus, it might be expected that the glutamatergic inputs through NMDA receptors in the LPBN may modulate the peripheral information for controlling sodium appetite and thirst. The precise physiological role of the glutamatergic system in the LPBN and the involvement of non-NMDA receptor mechanisms in the regulation of 5-HT release remain to be explained. Further studies that examine changes in the glutamine extracellular concentration within the LPBN during the intake of NaCl and water induced by the combined treatment with FURO and CAP may help to explain the action of glutamatergic mechanisms on the control of the serotonergic regulatory system of sodium appetite and thirst in the LPBN.
References [1] D.S.A. Colombari, J.V. Menani, A.K. Johnson, Forebrain angiotensin type 1 receptors and parabrachial serotonin in the control of NaCl and water intake, Am. J. Physiol. 271 (1996) R1470–R1476. [2] J.I.F. De Gobbi, L.A. De Luca Jr., J.V. Menani, Serotonergic mechanisms of the lateral parabrachial nucleus on DOCA-induced sodium intake, Brain Res. 880 (2000) 131–138.
J. Tanaka et al. / Brain Research Bulletin 71 (2006) 311–315 [3] A. Guthmann, H. Herbert, Expression of N-methyl-d-aspartate receptor subunits in the rat parabrachial and Kolliker–Fuse nuclei and in selected pontomedullary brainstem nuclei, J. Comp. Neurol. 415 (1999) 501– 517. [4] M.R. Hynd, H.L. Scott, P.R. Dodd, Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer’s disease, Neurochem. Int. 45 (2004) 583–595. [5] J.W. Jahng, T.A. Houpt, MK801 increases feeding and decreases drinking in nondeprived, freely feeding rats, Pharmacol. Biochem. Behav. 68 (2001) 181–186. [6] A.K. Johnson, R.L. Thunhorst, Sensory mechanisms in the behavioral control of body fluid balance: thirst and salt appetite, in: S.J. Fluharty, A.R. Morrisom, J.M. Sprague, E. Stellar (Eds.), Progress in Psychobiology and Physiological Psychology, vol. 16, Academic, New York, 1995, pp. 145–176. [7] H.S. Kim, H.S. Jang, K.H. Hahn, M.J. Kim, S.H. Yoon, Y.H. Jo, M.S. Kim, D.J. Rhie, Serotonin inhibits the induction of NMDA receptor-dependent lomg-term potentiation in the rat primary visual cortex, Brain Res. 1103 (2006) 49–55. [8] A.J. Lanca, D. van der Kooy, A serotonin-containing pathway from the area postrema to the parabrachial nucleus in the rat, Neuroscience 14 (1985) 1117–1126. [9] M. Marcoli, C. Cervetto, P. Paluzzi, S. Guarnieri, M. Raiteri, G. Maura, Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors, Neurochem. Int. 49 (2006) 12–19. [10] L.O. Margatho, S.P. Barbosa, L.A. De Luca Jr., J.V. Menani, Central serotonergic and adrenergic/imidazoline inhibitory mechanisms on sodium and water intake, Brain Res. 956 (2002) 103–109. [11] J.V. Menani, L.A. De Luca Jr., A.K. Johnson, Lateral parabrachial nucleus serotonergic mechanisms and salt appetite induced by sodium depletion, Am. J. Physiol. 274 (1998) R555–R560.
315
[12] J.V. Menani, A.K. Johnson, Lateral parabrachial nucleus serotonergic mechanisms: angiotensin-induced pressor and drinking responses, Am. J. Physiol. 269 (1995) R1044–R1049. [13] J.V. Menani, R.L. Thunhorst, A.K. Johnson, Lateral parabrachial nucleus and serotonergic mechanisms in the control of salt appetite in rats, Am. J. Physiol. 270 (1996) R162–R168. [14] R. Norgren, The central organization of the gustatory and visceral systems in the nucleus of the solitary tract, in: Y. Katsuki, R. Norgren, M. Sato (Eds.), Brain Mechanisms of Sensation, Wiley, New York, 1981, pp. 143–160. [15] J.C. Owen, P.S. Whitton, Vhronic treatment with antidepressant drugs reversibly alters NMDA mediated regulation of extracellular 5-HT in rat frontal cortex, Brain Res. Bull. 70 (2006) 62–67. [16] R. Paes-de-Carvalho, B.V. Dias, R.A. Martins, M.R. Pereira, C.C. Portugal, C. Lanfredi, Activation of glutamate receptors promotes a calciumdependent and transporter-mediated release of purines in cultured avian retinal cells: possible involvement of calcium/calmodulin-dependent protein kinase II, Neurochem. Int. 46 (2005) 441–451. [17] J. Tanaka, Y. Hayashi, K. Yamato, H. Miyakubo, M. Nomura, Involvement of serotonergic systems in the lateral parabrachial nucleus in sodium and water intake: a microdialysis study in the rat, Neurosci. Lett. 357 (2004) 41–44. [18] M. Watanabe, M. Mishina, Y. Inoue, Distinct distributions of five NMDA receptor channel subunit mRNAs in the brainstem, J. Comp. Neurol. 343 (1994) 520–531. [19] Z. Xu, A.K. Johnson, Non-NMDA receptor antagonist-induced drinking in rat, Brain Res. 808 (1998) 124–127. [20] Z. Xu, L.M. Lane, J. Herbert, Dizocilpine maleate, an N-methyl-d-aspartate antagonist, inhibits dipsogenic responses and c-fos expression induced by intracerebral infusion of angiotensin II, Neuroscience 78 (1997) 203–214. [21] J.A. Zidichouski, J.C. Easaw, J.H. Jhamandas, Glutamate receptor subtypes mediate excitatory synaptic responses of rat lateral parabrachial neurons, Am. J. Physiol. 270 (1996) H1557–H1567.