- Email: [email protected]
GABAergic modulation of noradrenaline release in the median preoptic nucleus area in the rat
Neuroscience Letters 342 (2003) 77–80 www.elsevier.com/locate/neulet
GABAergic modulation of noradrenaline release in the median preoptic nucleus area in the rat Kazuhiro Sakamakia, Masahiko Nomuraa, Satoko Hatakenakab, Hiroko Miyakubob, Junichi Tanakab,* a
Department of Physiology, Saitama Medical School, Iruma-gun, Saitama 350-0495, Japan Department of Curriculum, Teaching and Memory, Naruto University of Education, Naruto, Tokushima 772-8502, Japan
b
Received 14 January 2003; received in revised form 6 February 2003; accepted 14 February 2003
Abstract Microdialysis was employed to investigate whether g-aminobutyric acid (GABA) receptor mechanisms are involved in the regulation of noradrenaline (NA) release in the median preoptic nucleus (MnPO) in awake, freely moving rats. Perfusion with the GABA receptor antagonists as well as agonists was performed in the region of the MnPO through a microdialysis probe and dialysate levels of NA were measured. Perfusion with either bicuculline (10 and 50 mM), a GABAA receptor antagonist, or phaclofen (10 and 50 mM), a GABAB receptor antagonist, enhanced the release of NA in the MnPO area. Higher-dose perfusion with the GABAA agonist muscimol (50 mM) or the GABAB agonist baclofen (250 mM) decreased dialysate NA in the MnPO area. An iso-osmotic reduction of fluid volume following subcutaneous treatment with polyethylene glycol (PEG, 30%, 5 ml) significantly increased the NA level in the MnPO area. The increased levels of NA caused by the PEG treatment were attenuated by perfusion with muscimol (10 mM), but not by baclofen (50 mM). These results show the participation of both GABAA and GABAB receptors in the modulation of the release of NA in the MnPO area, and imply that the GABAA receptor mechanism may play an important role in the noradrenergic regulatory system of body fluid balance. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Median preoptic nucleus; Noradrenaline; g-Aminobutyric acid; Muscimol; Baclofen; Fluid balance
The median preoptic nucleus (MnPO), a midline structure within the anteroventral third ventricle (AV3V) region, plays vital roles in the regulation of body fluid balance and cardiovascular function [2,3,6,12 – 14,16,18]. The MnPO receives noradrenergic afferent projections from the brainstem [4,11]. Localized catecholamine depletion [3] or inactivation of adrenoceptors [14] in the AV3V region attenuates the drinking response produced by angiotensin II (ANG II). Reduction in extracellular fluid volume increases release [16] and turnover [18] of noradrenaline (NA) in the region of the MnPO. Theses observations show that the noradrenergic system in the MnPO is implicated in the modulation of the body fluid homeostasis. The MnPO area contains g-aminobutyric acid (GABA) neurons and terminals [7]. With respect to the action of the GABAergic system, experimental findings have shown that GABA and its analogs influence the ANG II-induced * Corresponding author. Tel./fax: þ 81-88-687-6277. E-mail address: [email protected] (J. Tanaka).
drinking and pressor responses [1,17]. Additionally, an electrophysiological study has indicated that GABAergic projections from the MnPO alter the excitability of neurohypophyseal neurons in the supraoptic nucleus [8]. The findings offer the proposition that the GABAergic system in the MnPO may be also involved in the control of extracellular fluid volume. In an attempt to verify the proposition, we examined the effects of perfusion with GABA receptor antagonists as well as agonists on NA release in the MnPO area. We also investigated the effects of an iso-osmotic reduction of fluid volume following subcutaneous treatment with polyethylene glycol (PEG) on the release of NA in the MnPO area and the effects of perfusion with the GABA receptor agonists on the PEGinduced NA release. Adult male Wistar rats (n ¼ 85) were used for the experiments and weighed 250 – 320 g at the time of testing. All animals were housed in animal quarters maintained on a 12 h light/dark cycle with ad libitum access to food and water.
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00242-8
78
K. Sakamaki et al. / Neuroscience Letters 342 (2003) 77–80
The rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and implanted with a microdialysis probe guide cannula (G-12, Eicom Co., Kyoto, Japan). The anesthetized animals were placed in a stereotaxic instrument and the scalp was incised and retracted laterally. The skull was leveled between lambda and bregma, and a 2 mm hole, centered on bregma, was drilled. The microdialysis probe guide cannula was lowered to coordinates which were 1 mm dorsal to the MnPO 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 MnPO were 0.3 mm posterior to bregma, 0.0 mm lateral to the midline, and 6.6 mm ventral to the cortical surface. The guide cannula was secured to the skull with acrylic dental cement and small stainless steel screws, and sealed with a dummy cannula after implantation. A dialysis experiment was carried out 3 days after the implantation of the guide cannula. The dummy cannula was removed, and the dialysis probe (BDP-1-12-01; Eicom Co.), the tip of which had a 1.0 mm long semipermeable membrane (0.2 mm outside diameter, molecular weight cut-off 50 kDa, in vivo recovery at 2 ml/min of almost 25%), was inserted into the implanted guide cannula. The probe was continuously perfused at a rate of 2 ml/min using a perfusion pump (EP-60; Eicom Co.) and gas-tight syringe (Hamilton Co., Reno, NEV) with modified physiological Ringer’s solution (NaCl 147 mM, CaCl2 2.3 mM, KCl 4 mM, pH 6.5). Samples were collected at 20 min intervals. Muscimol, baclofen, phaclofen, and bicuculline methiodide (Sigma, St. Louis, MO) were dissolved in the modified physiological Ringer’s solution. Following the collection of four stable baseline dialysate samples (80 min), the drugs were applied for 40 min directly into the MnPO through the dialysis probe. The extracellular fluid volume was decreased iso-osmotically by subcutaneous PEG injections. Since previous studies have demonstrated that 5 ml injections of 30% PEG are effective for eliciting reduced fluid volume [16,18], this concentration was utilized in this study. To investigate the effects of the GABA agonists on the PEG treatment-induced NA release, the agonists were applied for 60 min at 80 min after the injection of PEG. Sample collections were continued 6 h after the injection of PEG. The dialysates were analyzed for concentrations of NA, 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 monoamine from a reverse phase column (3.0 £ 100 mm SC-3ODS column; Eicom Co.). The graphite working electrode was set at þ 750 mV versus an Ag/AgCl reference electrode and the flow rate was 0.5 ml/min. At the termination of each experiment, the 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% neutral buffered
formalin for fixation. The brain was removed and stored in the formalin saline before being cut on a freezing microtome at 50 mm in transverse sections. Sections were mounted on glass slides and stained with Neutral Red for microscope examination. Changes in the concentrations of NA in brain dialysate were expressed as a percentage of the basal level calculated from the last four samples before drug perfusion or subcutaneous treatment. Chemical data were expressed as mean ^ SEM. The data were analyzed by one-way analysis of variance and subsequent t-test. A probability of less than 0.05 was required for significance. In 81 out of 85 rats tested, the microdialysis probes were located within the MnPO. The data from the remaining four rats having the probe placement outside the MnPO were not included in the analysis. Basal levels of NA in 20 min dialysate samples from the MnPO area were 16.7 ^ 0.7 pg/40 ml (n ¼ 81). Fig. 1A illustrates dialysate NA concentrations before, during and after applications of the GABA receptor antagonists. Perfusion with bicuculline methiodide (10 mM (n ¼ 6) and 50 mM (n ¼ 5)), a GABAA receptor antagonist, elicited significant increases in NA levels. Perfusion with phaclofen (10 mM (n ¼ 6) and 50 mM (n ¼ 5)), a GABAB receptor antagonist, also resulted in a significant increase in dialysate NA concentrations. Vehicle perfusion did not cause a significant change in NA levels (n ¼ 5). These results suggest that basal NA levels in the MnPO area may be tonically inhibited by endogenous GABA acting on GABAA and GABAB receptors. The effects of GABA receptor agonists on the NA release in the MnPO area are shown in Fig. 1B. Lower-dose perfusion with neither muscimol (10 mM, n ¼ 7), a GABAA receptor agonist, nor baclofen (50 mM, n ¼ 7), a GABAB receptor agonist, through a microdialysis probe elicited a significant alteration in NA levels. Higher-dose perfusion with either muscimol (50 mM, n ¼ 6) or baclofen (250 mM, n ¼ 5), on the other hand, produced a significant decrease in the NA release. No significant alterations in the NA release in the MnPO area were observed in the vehicle control group (n ¼ 5). The observations from the application of the antagonists and agonists show the participation of GABAA and GABAB receptors in the modulation of NA release in the MnPO area. The effects of PEG treatment on NA concentrations in the MnPO area and the effects of perfusion of the GABA agonists on the PEG-induced response were examined. Subcutaneous PEG injections significantly increased the NA release in the MnPO area (Fig. 2, n ¼ 8), and are consistent with the previous findings [16]. The increase in the NA release caused by the PEG treatment was significantly attenuated by the perfusion of muscimol (10 mM, n ¼ 8), but not by baclofen (50 mM, n ¼ 8), as shown in Fig. 2. These results imply that GABAA receptor mechanisms in the MnPO area may participate in the noradrenergic regulatory system of body fluid homeostasis,
K. Sakamaki et al. / Neuroscience Letters 342 (2003) 77–80
79
Fig. 2. Changes in NA concentrations in the region of the MnPO in response to an iso-osmotic reduction of fluid volume following subcutaneous treatment with PEG and the effects of administration of GABA receptor agonists on the PEG treatment-induced NA release. The changes in NA levels are expressed as percentages ^ SEM of basal pretreatment levels (calculated from four samples before treatment). The data are the mean ^ SEM of eight animals in each group. The arrow indicates the time of PEG injection. The horizontal bars indicate the duration of drug perfusion. Injections of PEG (30%, 5 ml) caused a significant increase compared to the basal level. Perfusion with muscimol (10 mM), but not baclofen (50 mM), significantly suppressed the PEG treatment-induced increase in NA concentrations in the MnPO area. *P , 0:05, **P , 0:01, compared with the basal level in the PEG-vehicle group and the PEGbaclofen group. #P , 0:05, ##P , 0:01, compared with the corresponding value in the PEG-vehicle group and the PEG-baclofen group.
Fig. 1. Effects of GABA receptor antagonists (A) and agonists (B) on extracellular NA concentrations in the region of the MnPO. Changes in NA levels are expressed as percentages ^ SEM of basal pretreatment levels (calculated from four samples before treatment). Each data point represents the mean ^ SEM for five to seven experiments. The horizontal bars indicate the duration of drug perfusion. (A) Perfusion with either bicuculline (10 and 50 mM) or phaclofen (10 and 50 mM) dose-dependently enhanced NA levels in the MnPO area. *P , 0:05, **P , 0:01, comparison between the vehicle group and the bicuculline-perfused group. #P , 0:05, ##P , 0:01, comparison between the vehicle group and the phaclofen-perfused group. (B) Perfusion with either muscimol (10 mM) or baclofen (50 mM) did not cause significant changes in NA levels. Highdose perfusion with muscimol (50 mM) or baclofen (250 mM), on the other hand, elicited a significant change in NA concentrations in the MnPO area. *P , 0:05, **P , 0:01, comparison between the vehicle group and the muscimol-perfused group. #P , 0:05, ##P , 0:01, comparison between the vehicle group and the baclofen-perfused group.
and play important roles in the inhibitory modulation of NA release in the MnPO. The present study demonstrated that the lower-dose perfusion with muscimol, but not phaclofen, reduces the PEG treatment-induced enhancement of the NA release in the MnPO area, whereas the higher-dose perfusion with
either muscimol or baclofen alone suppresses basal NA levels in the MnPO area. Although it is impossible to explain the precise mechanism underlying these results, it is tempting to speculate that GABA receptors are preferentially implicated in treatment-evoked NA release rather than basal NA releases in the MnPO area. Additionally, it might be expected that the GABA receptor subtypes may play distinctly different roles in the modulation of NA release. It has been shown that the noradrenergic system in the MnPO plays a critical role in the elicitation of drinking and pressor responses [2,3,12,14]. Previous studies have demonstrated that GABA and its analogs influence the ANG IIinduced drinking and pressor responses through the lamina terminalis along the anterior wall of the third ventricle [1, 17]. These observations and the present results suggest that the GABAergic system in the MnPO may serve to inhibit the responses. Although the precise mechanisms underlying the inhibition of NA release cannot be decided from our data, it is possible that in the MnPO area, GABA released from GABAergic nerve terminals acts on NA terminals which result in reduced NA release. However, there are no anatomical data showing that GABAergic fibers terminate on NA terminals in the region of the MnPO. Additionally, despite the fact that the presence of GABAergic neurons and terminals in the MnPO area has been demonstrated [7], the precise location of the GABA interneurons acting in the
80
K. Sakamaki et al. / Neuroscience Letters 342 (2003) 77–80
inhibitory pathway is still unknown. Because neural inputs to the MnPO come from the organum vasculosum lamina terminalis, subfornical organ (SFO), medial hypothalamus, and several brainstem regions [4,5,10,11], it is possible to speculate that one of the projections from these sites is the GABAergic inhibitory pathway. Experimental observations have indicated that the interaction between the angiotensinergic and catecholaminergic systems in the MnPO is important for drinking behavior [2,3,9,12,14]. The MnPO is innervated by angiotensinergic nerve terminals derived from the SFO [5]. Activation of the SFO causes an increase in the excitability of MnPO neurons via ANG II receptors [13] and the NA release in the MnPO area [15]. These findings and our data suggest that the noradrenergic inputs to the MnPO that are implicated in the maintenance of extracellular fluid balance may be modulated by both the angiotensinergic projections from the SFO and the GABAergic system. It is suggested that the GABAergic system inhibits the release of ANG II from the angiotensinergic nerve terminals of the SFO projections, which resulted in the reduced NA release in the MnPO area. To clarify more precisely the action of the GABAergic system on the release of NA and ANG II in the MnPO area may help to resolve the neural mechanism for eliciting dipsogenic responses. Further studies are in progress.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
References [14] [1] M. Abe, T. Tokunaga, K. Yamada, T. Furukawa, Gamma-aminobutyric acid and taurine antagonize the central effects of angiotensin II and renin on the intake of water and salt, and on blood pressure in rats, Neuropharmacology 27 (1988) 309 –318. [2] S.I. Bellin, R.K. Bhatnagar, A.K. Johnson, Periventricular noradrenergic systems are critical for angiotensin-induced drinking and blood pressure responses, Brain Res. 403 (1987) 105–112. [3] S.I. Bellin, S.K. Landas, A.K. Johnson, Localized injections of 6hydroxydopamine into lamina terminalis-associated structures: effects on experimentally induced drinking and pressor responses, Brain Res. 416 (1987) 75 –83. [4] H. Kawano, S. Masuko, Synaptic inputs of neuropeptide Yimmunoreactive noradrenergic nerve terminals to neurons in the nucleus preopticus medianus which project to the paraventricular nucleus of the hypothalamus of the rat: a combined immunohisto-
[15]
[16]
[17]
[18]
chemical and retrograde tracing method, Brain Res. 600 (1993) 74–80. R.W. Lind, L.W. Swanson, D. Ganten, Angiotensin II immunoreactivity in neural afferents and efferents of the subfornical organ of the rat, Brain Res. 321 (1984) 209 –215. M.L. Mangiapane, T.N. Thrasher, L.C. Keil, J.B. Simpson, W.F. Ganong, Deficits in drinking and vasopressin secretion after lesions of the nucleus medianus, Neuroendocrinology 37 (1983) 73–77. E. Mugnaini, W.H. Oertel, An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunocytochemistry, in: A. Bjorklund, T. Hokfelt (Eds.), Handbook of Chemical Neuroanatomy, 4, Elsevier, New York, 1985, pp. 436 –608. R. Nissen, L.P. Renaud, GABA receptor mediation of median preoptic nucleus-evoked inhibition of supraoptic neurosecretory neurons in rat, J. Physiol. 479 (1994) 207–216. E.M. Rechards, K. Hermann, C. Summers, M.K. Raisada, M.I. Phillips, Release of immunoreactive angiotensin II from neuronal cultures: adrenergic influences, Am. J. Physiol. 257 (1989) C588–C595. C.B. Saper, D. Levisohn, Afferent connections of the median preoptic nucleus in the rat: anatomical evidence for a cardiovascular integrative mechanism in the anteroventral third ventricular (AV3V) region, Brain Res. 288 (1983) 21– 31. C.B. Saper, D.J. Reis, T. Joh, Medullary catecholamine inputs to the anteroventral third ventricular cardiovascular regulatory region in the rat, Neurosci. Lett. 42 (1983) 285–291. R.K.P. Silva, J.V. Menani, W.A. Saad, A. Renzi, J.E.N. Silveira, A.C. Luiz, L.L.A. Camargo, Role of the a1-, and a2- and b-adrenoceptors of the median preoptic area on the water intake, renal excretion, and arterial pressure induced by ANG II, Brain Res. 717 (1996) 38–43. J. Tanaka, Involvement of the median preoptic nucleus in the regulation of paraventricular vasopressin neurons by the subfornical organ in the rat, Exp. Brain Res. 76 (1989) 47–54. J. Tanaka, a-Adrenergic control of ANG II-induced drinking in the rat median preoptic nucleus, Physiol. Behav. 77 (2002) 155–160. J. Tanaka, Y. Hayashi, S. Shimamune, K. Hori, M. Nomura, Subfornical organ efferents enhance extracellular noradrenaline concentrations in the median preoptic area in rats, Neurosci. Lett. 230 (1997) 171 –174. J. Tanaka, K. Hori, K. Koito, M. Nomura, Enhanced monoamine release in the median preoptic area following reduced extracellular fluid volume in rats, Neurosci. Lett. 147 (1992) 110–113. T. Unger, F. Bles, D. Ganten, R.E. Lang, R. Rettig, N.A. Schwab, GABAergic stimulation inhibits central actions of angiotensin II: pressor responses, drinking and release of vasopressin, Eur. J. Pharmacol. 90 (1983) 1–9. L.D. Wilkin, K.P. Patel, P.G. Schmid, A.K. Johnson, Increased norepinephrine turnover in the median preoptic nucleus following reduced extracellular fluid volume, Brain Res. 423 (1987) 369– 372.
GABAergic modulation of noradrenaline release in the median preoptic nucleus area in the rat Kazuhiro Sakamakia, Masahiko Nomuraa, Satoko Hatakenakab, Hiroko Miyakubob, Junichi Tanakab,* a
Department of Physiology, Saitama Medical School, Iruma-gun, Saitama 350-0495, Japan Department of Curriculum, Teaching and Memory, Naruto University of Education, Naruto, Tokushima 772-8502, Japan
b
Received 14 January 2003; received in revised form 6 February 2003; accepted 14 February 2003
Abstract Microdialysis was employed to investigate whether g-aminobutyric acid (GABA) receptor mechanisms are involved in the regulation of noradrenaline (NA) release in the median preoptic nucleus (MnPO) in awake, freely moving rats. Perfusion with the GABA receptor antagonists as well as agonists was performed in the region of the MnPO through a microdialysis probe and dialysate levels of NA were measured. Perfusion with either bicuculline (10 and 50 mM), a GABAA receptor antagonist, or phaclofen (10 and 50 mM), a GABAB receptor antagonist, enhanced the release of NA in the MnPO area. Higher-dose perfusion with the GABAA agonist muscimol (50 mM) or the GABAB agonist baclofen (250 mM) decreased dialysate NA in the MnPO area. An iso-osmotic reduction of fluid volume following subcutaneous treatment with polyethylene glycol (PEG, 30%, 5 ml) significantly increased the NA level in the MnPO area. The increased levels of NA caused by the PEG treatment were attenuated by perfusion with muscimol (10 mM), but not by baclofen (50 mM). These results show the participation of both GABAA and GABAB receptors in the modulation of the release of NA in the MnPO area, and imply that the GABAA receptor mechanism may play an important role in the noradrenergic regulatory system of body fluid balance. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Median preoptic nucleus; Noradrenaline; g-Aminobutyric acid; Muscimol; Baclofen; Fluid balance
The median preoptic nucleus (MnPO), a midline structure within the anteroventral third ventricle (AV3V) region, plays vital roles in the regulation of body fluid balance and cardiovascular function [2,3,6,12 – 14,16,18]. The MnPO receives noradrenergic afferent projections from the brainstem [4,11]. Localized catecholamine depletion [3] or inactivation of adrenoceptors [14] in the AV3V region attenuates the drinking response produced by angiotensin II (ANG II). Reduction in extracellular fluid volume increases release [16] and turnover [18] of noradrenaline (NA) in the region of the MnPO. Theses observations show that the noradrenergic system in the MnPO is implicated in the modulation of the body fluid homeostasis. The MnPO area contains g-aminobutyric acid (GABA) neurons and terminals [7]. With respect to the action of the GABAergic system, experimental findings have shown that GABA and its analogs influence the ANG II-induced * Corresponding author. Tel./fax: þ 81-88-687-6277. E-mail address: [email protected] (J. Tanaka).
drinking and pressor responses [1,17]. Additionally, an electrophysiological study has indicated that GABAergic projections from the MnPO alter the excitability of neurohypophyseal neurons in the supraoptic nucleus [8]. The findings offer the proposition that the GABAergic system in the MnPO may be also involved in the control of extracellular fluid volume. In an attempt to verify the proposition, we examined the effects of perfusion with GABA receptor antagonists as well as agonists on NA release in the MnPO area. We also investigated the effects of an iso-osmotic reduction of fluid volume following subcutaneous treatment with polyethylene glycol (PEG) on the release of NA in the MnPO area and the effects of perfusion with the GABA receptor agonists on the PEGinduced NA release. Adult male Wistar rats (n ¼ 85) were used for the experiments and weighed 250 – 320 g at the time of testing. All animals were housed in animal quarters maintained on a 12 h light/dark cycle with ad libitum access to food and water.
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00242-8
78
K. Sakamaki et al. / Neuroscience Letters 342 (2003) 77–80
The rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and implanted with a microdialysis probe guide cannula (G-12, Eicom Co., Kyoto, Japan). The anesthetized animals were placed in a stereotaxic instrument and the scalp was incised and retracted laterally. The skull was leveled between lambda and bregma, and a 2 mm hole, centered on bregma, was drilled. The microdialysis probe guide cannula was lowered to coordinates which were 1 mm dorsal to the MnPO 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 MnPO were 0.3 mm posterior to bregma, 0.0 mm lateral to the midline, and 6.6 mm ventral to the cortical surface. The guide cannula was secured to the skull with acrylic dental cement and small stainless steel screws, and sealed with a dummy cannula after implantation. A dialysis experiment was carried out 3 days after the implantation of the guide cannula. The dummy cannula was removed, and the dialysis probe (BDP-1-12-01; Eicom Co.), the tip of which had a 1.0 mm long semipermeable membrane (0.2 mm outside diameter, molecular weight cut-off 50 kDa, in vivo recovery at 2 ml/min of almost 25%), was inserted into the implanted guide cannula. The probe was continuously perfused at a rate of 2 ml/min using a perfusion pump (EP-60; Eicom Co.) and gas-tight syringe (Hamilton Co., Reno, NEV) with modified physiological Ringer’s solution (NaCl 147 mM, CaCl2 2.3 mM, KCl 4 mM, pH 6.5). Samples were collected at 20 min intervals. Muscimol, baclofen, phaclofen, and bicuculline methiodide (Sigma, St. Louis, MO) were dissolved in the modified physiological Ringer’s solution. Following the collection of four stable baseline dialysate samples (80 min), the drugs were applied for 40 min directly into the MnPO through the dialysis probe. The extracellular fluid volume was decreased iso-osmotically by subcutaneous PEG injections. Since previous studies have demonstrated that 5 ml injections of 30% PEG are effective for eliciting reduced fluid volume [16,18], this concentration was utilized in this study. To investigate the effects of the GABA agonists on the PEG treatment-induced NA release, the agonists were applied for 60 min at 80 min after the injection of PEG. Sample collections were continued 6 h after the injection of PEG. The dialysates were analyzed for concentrations of NA, 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 monoamine from a reverse phase column (3.0 £ 100 mm SC-3ODS column; Eicom Co.). The graphite working electrode was set at þ 750 mV versus an Ag/AgCl reference electrode and the flow rate was 0.5 ml/min. At the termination of each experiment, the 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% neutral buffered
formalin for fixation. The brain was removed and stored in the formalin saline before being cut on a freezing microtome at 50 mm in transverse sections. Sections were mounted on glass slides and stained with Neutral Red for microscope examination. Changes in the concentrations of NA in brain dialysate were expressed as a percentage of the basal level calculated from the last four samples before drug perfusion or subcutaneous treatment. Chemical data were expressed as mean ^ SEM. The data were analyzed by one-way analysis of variance and subsequent t-test. A probability of less than 0.05 was required for significance. In 81 out of 85 rats tested, the microdialysis probes were located within the MnPO. The data from the remaining four rats having the probe placement outside the MnPO were not included in the analysis. Basal levels of NA in 20 min dialysate samples from the MnPO area were 16.7 ^ 0.7 pg/40 ml (n ¼ 81). Fig. 1A illustrates dialysate NA concentrations before, during and after applications of the GABA receptor antagonists. Perfusion with bicuculline methiodide (10 mM (n ¼ 6) and 50 mM (n ¼ 5)), a GABAA receptor antagonist, elicited significant increases in NA levels. Perfusion with phaclofen (10 mM (n ¼ 6) and 50 mM (n ¼ 5)), a GABAB receptor antagonist, also resulted in a significant increase in dialysate NA concentrations. Vehicle perfusion did not cause a significant change in NA levels (n ¼ 5). These results suggest that basal NA levels in the MnPO area may be tonically inhibited by endogenous GABA acting on GABAA and GABAB receptors. The effects of GABA receptor agonists on the NA release in the MnPO area are shown in Fig. 1B. Lower-dose perfusion with neither muscimol (10 mM, n ¼ 7), a GABAA receptor agonist, nor baclofen (50 mM, n ¼ 7), a GABAB receptor agonist, through a microdialysis probe elicited a significant alteration in NA levels. Higher-dose perfusion with either muscimol (50 mM, n ¼ 6) or baclofen (250 mM, n ¼ 5), on the other hand, produced a significant decrease in the NA release. No significant alterations in the NA release in the MnPO area were observed in the vehicle control group (n ¼ 5). The observations from the application of the antagonists and agonists show the participation of GABAA and GABAB receptors in the modulation of NA release in the MnPO area. The effects of PEG treatment on NA concentrations in the MnPO area and the effects of perfusion of the GABA agonists on the PEG-induced response were examined. Subcutaneous PEG injections significantly increased the NA release in the MnPO area (Fig. 2, n ¼ 8), and are consistent with the previous findings [16]. The increase in the NA release caused by the PEG treatment was significantly attenuated by the perfusion of muscimol (10 mM, n ¼ 8), but not by baclofen (50 mM, n ¼ 8), as shown in Fig. 2. These results imply that GABAA receptor mechanisms in the MnPO area may participate in the noradrenergic regulatory system of body fluid homeostasis,
K. Sakamaki et al. / Neuroscience Letters 342 (2003) 77–80
79
Fig. 2. Changes in NA concentrations in the region of the MnPO in response to an iso-osmotic reduction of fluid volume following subcutaneous treatment with PEG and the effects of administration of GABA receptor agonists on the PEG treatment-induced NA release. The changes in NA levels are expressed as percentages ^ SEM of basal pretreatment levels (calculated from four samples before treatment). The data are the mean ^ SEM of eight animals in each group. The arrow indicates the time of PEG injection. The horizontal bars indicate the duration of drug perfusion. Injections of PEG (30%, 5 ml) caused a significant increase compared to the basal level. Perfusion with muscimol (10 mM), but not baclofen (50 mM), significantly suppressed the PEG treatment-induced increase in NA concentrations in the MnPO area. *P , 0:05, **P , 0:01, compared with the basal level in the PEG-vehicle group and the PEGbaclofen group. #P , 0:05, ##P , 0:01, compared with the corresponding value in the PEG-vehicle group and the PEG-baclofen group.
Fig. 1. Effects of GABA receptor antagonists (A) and agonists (B) on extracellular NA concentrations in the region of the MnPO. Changes in NA levels are expressed as percentages ^ SEM of basal pretreatment levels (calculated from four samples before treatment). Each data point represents the mean ^ SEM for five to seven experiments. The horizontal bars indicate the duration of drug perfusion. (A) Perfusion with either bicuculline (10 and 50 mM) or phaclofen (10 and 50 mM) dose-dependently enhanced NA levels in the MnPO area. *P , 0:05, **P , 0:01, comparison between the vehicle group and the bicuculline-perfused group. #P , 0:05, ##P , 0:01, comparison between the vehicle group and the phaclofen-perfused group. (B) Perfusion with either muscimol (10 mM) or baclofen (50 mM) did not cause significant changes in NA levels. Highdose perfusion with muscimol (50 mM) or baclofen (250 mM), on the other hand, elicited a significant change in NA concentrations in the MnPO area. *P , 0:05, **P , 0:01, comparison between the vehicle group and the muscimol-perfused group. #P , 0:05, ##P , 0:01, comparison between the vehicle group and the baclofen-perfused group.
and play important roles in the inhibitory modulation of NA release in the MnPO. The present study demonstrated that the lower-dose perfusion with muscimol, but not phaclofen, reduces the PEG treatment-induced enhancement of the NA release in the MnPO area, whereas the higher-dose perfusion with
either muscimol or baclofen alone suppresses basal NA levels in the MnPO area. Although it is impossible to explain the precise mechanism underlying these results, it is tempting to speculate that GABA receptors are preferentially implicated in treatment-evoked NA release rather than basal NA releases in the MnPO area. Additionally, it might be expected that the GABA receptor subtypes may play distinctly different roles in the modulation of NA release. It has been shown that the noradrenergic system in the MnPO plays a critical role in the elicitation of drinking and pressor responses [2,3,12,14]. Previous studies have demonstrated that GABA and its analogs influence the ANG IIinduced drinking and pressor responses through the lamina terminalis along the anterior wall of the third ventricle [1, 17]. These observations and the present results suggest that the GABAergic system in the MnPO may serve to inhibit the responses. Although the precise mechanisms underlying the inhibition of NA release cannot be decided from our data, it is possible that in the MnPO area, GABA released from GABAergic nerve terminals acts on NA terminals which result in reduced NA release. However, there are no anatomical data showing that GABAergic fibers terminate on NA terminals in the region of the MnPO. Additionally, despite the fact that the presence of GABAergic neurons and terminals in the MnPO area has been demonstrated [7], the precise location of the GABA interneurons acting in the
80
K. Sakamaki et al. / Neuroscience Letters 342 (2003) 77–80
inhibitory pathway is still unknown. Because neural inputs to the MnPO come from the organum vasculosum lamina terminalis, subfornical organ (SFO), medial hypothalamus, and several brainstem regions [4,5,10,11], it is possible to speculate that one of the projections from these sites is the GABAergic inhibitory pathway. Experimental observations have indicated that the interaction between the angiotensinergic and catecholaminergic systems in the MnPO is important for drinking behavior [2,3,9,12,14]. The MnPO is innervated by angiotensinergic nerve terminals derived from the SFO [5]. Activation of the SFO causes an increase in the excitability of MnPO neurons via ANG II receptors [13] and the NA release in the MnPO area [15]. These findings and our data suggest that the noradrenergic inputs to the MnPO that are implicated in the maintenance of extracellular fluid balance may be modulated by both the angiotensinergic projections from the SFO and the GABAergic system. It is suggested that the GABAergic system inhibits the release of ANG II from the angiotensinergic nerve terminals of the SFO projections, which resulted in the reduced NA release in the MnPO area. To clarify more precisely the action of the GABAergic system on the release of NA and ANG II in the MnPO area may help to resolve the neural mechanism for eliciting dipsogenic responses. Further studies are in progress.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
References [14] [1] M. Abe, T. Tokunaga, K. Yamada, T. Furukawa, Gamma-aminobutyric acid and taurine antagonize the central effects of angiotensin II and renin on the intake of water and salt, and on blood pressure in rats, Neuropharmacology 27 (1988) 309 –318. [2] S.I. Bellin, R.K. Bhatnagar, A.K. Johnson, Periventricular noradrenergic systems are critical for angiotensin-induced drinking and blood pressure responses, Brain Res. 403 (1987) 105–112. [3] S.I. Bellin, S.K. Landas, A.K. Johnson, Localized injections of 6hydroxydopamine into lamina terminalis-associated structures: effects on experimentally induced drinking and pressor responses, Brain Res. 416 (1987) 75 –83. [4] H. Kawano, S. Masuko, Synaptic inputs of neuropeptide Yimmunoreactive noradrenergic nerve terminals to neurons in the nucleus preopticus medianus which project to the paraventricular nucleus of the hypothalamus of the rat: a combined immunohisto-
[15]
[16]
[17]
[18]
chemical and retrograde tracing method, Brain Res. 600 (1993) 74–80. R.W. Lind, L.W. Swanson, D. Ganten, Angiotensin II immunoreactivity in neural afferents and efferents of the subfornical organ of the rat, Brain Res. 321 (1984) 209 –215. M.L. Mangiapane, T.N. Thrasher, L.C. Keil, J.B. Simpson, W.F. Ganong, Deficits in drinking and vasopressin secretion after lesions of the nucleus medianus, Neuroendocrinology 37 (1983) 73–77. E. Mugnaini, W.H. Oertel, An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunocytochemistry, in: A. Bjorklund, T. Hokfelt (Eds.), Handbook of Chemical Neuroanatomy, 4, Elsevier, New York, 1985, pp. 436 –608. R. Nissen, L.P. Renaud, GABA receptor mediation of median preoptic nucleus-evoked inhibition of supraoptic neurosecretory neurons in rat, J. Physiol. 479 (1994) 207–216. E.M. Rechards, K. Hermann, C. Summers, M.K. Raisada, M.I. Phillips, Release of immunoreactive angiotensin II from neuronal cultures: adrenergic influences, Am. J. Physiol. 257 (1989) C588–C595. C.B. Saper, D. Levisohn, Afferent connections of the median preoptic nucleus in the rat: anatomical evidence for a cardiovascular integrative mechanism in the anteroventral third ventricular (AV3V) region, Brain Res. 288 (1983) 21– 31. C.B. Saper, D.J. Reis, T. Joh, Medullary catecholamine inputs to the anteroventral third ventricular cardiovascular regulatory region in the rat, Neurosci. Lett. 42 (1983) 285–291. R.K.P. Silva, J.V. Menani, W.A. Saad, A. Renzi, J.E.N. Silveira, A.C. Luiz, L.L.A. Camargo, Role of the a1-, and a2- and b-adrenoceptors of the median preoptic area on the water intake, renal excretion, and arterial pressure induced by ANG II, Brain Res. 717 (1996) 38–43. J. Tanaka, Involvement of the median preoptic nucleus in the regulation of paraventricular vasopressin neurons by the subfornical organ in the rat, Exp. Brain Res. 76 (1989) 47–54. J. Tanaka, a-Adrenergic control of ANG II-induced drinking in the rat median preoptic nucleus, Physiol. Behav. 77 (2002) 155–160. J. Tanaka, Y. Hayashi, S. Shimamune, K. Hori, M. Nomura, Subfornical organ efferents enhance extracellular noradrenaline concentrations in the median preoptic area in rats, Neurosci. Lett. 230 (1997) 171 –174. J. Tanaka, K. Hori, K. Koito, M. Nomura, Enhanced monoamine release in the median preoptic area following reduced extracellular fluid volume in rats, Neurosci. Lett. 147 (1992) 110–113. T. Unger, F. Bles, D. Ganten, R.E. Lang, R. Rettig, N.A. Schwab, GABAergic stimulation inhibits central actions of angiotensin II: pressor responses, drinking and release of vasopressin, Eur. J. Pharmacol. 90 (1983) 1–9. L.D. Wilkin, K.P. Patel, P.G. Schmid, A.K. Johnson, Increased norepinephrine turnover in the median preoptic nucleus following reduced extracellular fluid volume, Brain Res. 423 (1987) 369– 372.