Neuroscience Letters 583 (2014) 1–5
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Differences in acid-induced currents between oxytocin-mRFP1 and vasopressin-eGFP neurons isolated from the supraoptic and paraventricular nuclei of transgenic rats Jun-ichi Ohkubo a,b , Toyoaki Ohbuchi b , Mitsuhiro Yoshimura a , Takashi Maruyama a , Hirofumi Hashimoto a , Takanori Matsuura a , Hideaki Suzuki b , Yoichi Ueta a,∗ a
Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan b
h i g h l i g h t s • • • •
The differences between OXT-mRFP1 neurons and AVP-eGFP neurons in response to acid. Acid-induced currents were identified in isolated rat SON and PVN MNCs. The currents in the OXT-mRFP1 neurons were smaller than those in the AVP-eGFP neurons. The acid sensitivity of OXT neurons may be lower than that of AVP neurons.
a r t i c l e
i n f o
Article history: Received 27 June 2014 Received in revised form 28 August 2014 Accepted 1 September 2014 Available online 8 September 2014 Keywords: Acid-sensing ion channels Fluorescent protein SON PVN Oxytocin Vasopressin
a b s t r a c t The hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) consists of two types of magnocellular neurosecretory cells, oxytocin (OXT) and arginine vasopressin (AVP). We generated and characterized rats that express an OXT-monomeric red fluorescent protein 1 (mRFP1) and an AVP-enhanced green fluorescent protein (eGFP) fusion transgene. These transgenic rats enable the visualization of OXT or AVP neurons. Taking advantage of this, we examined the differences between OXT-mRFP1 neurons and AVP-eGFP neurons in response to acid. Acid-sensing ion channels (ASICs) are neuronal voltage-insensitive cationic channels that are activated by extracellular acidification. Although functional ASICs have been identified in AVP neurons, differences in acid-induced currents between OXT and AVP neurons in SON have not been reported. In the present study, we used the whole-cell patchclamp technique to investigate differences between OXT-mRFP1 neurons and AVP-eGFP neurons reaction to acid in SON and PVN. In voltage clamp mode, lowering extracellular pH evoked inward currents in both OXT-mRFP1 neurons and AVP-eGFP neurons. In our findings, the acid-induced currents in the OXT-mRFP1 neurons were significantly smaller than those in the AVP-eGFP neurons. These acid-induced currents were inhibited by amiloride, a known blocker of ASICs. Further, to compare the response to acid between OXT-mRFP1 and AVP-eGFP neurons in the same transgenic rat, we used a double transgenic rat by mating an OXT-mRFP1 transgenic rat with an AVP-eGFP transgenic rat. The acid-induced currents of OXT-mRFP1 neurons were significantly smaller than those of AVP-eGFP neurons from the double transgenic rats. These currents were almost completely inhibited by amiloride. The difference of acid-sensitivity between OXT and AVP neurons might contribute to maintaining systematic order in hypothalamic function. © 2014 Elsevier Ireland Ltd. All rights reserved.
∗ Corresponding author at: Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Tel.: +81 93 691 7420; fax: +81 93 692 1711. E-mail address: [email protected] (Y. Ueta). http://dx.doi.org/10.1016/j.neulet.2014.09.004 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Oxytocin (OXT) and arginine vasopressin (AVP) are synthesized and secreted from magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) and in the paraventricular nucleus (PVN) [1]. AVP plays a role in water homeostasis, cardiovascular regulation, controls osmolality, and blood pressure. In recent reports, the centrally released AVP has also been associated with regulation of circadian rhythm and/or social behavior [2,3]. The role
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of OXT in lactation and parturition has been well characterized. In addition, centrally released OXT is involved in maternal bonding, sexual behavior, and social affiliation [4–7]. Acid-sensing ion channels (ASICs) are widely expressed in the peripheral and central nervous system where they perform physiological functions [8]. The ASICs are voltage-insensitive cation channels that are activated by extracellular acidification. Our recent electrophysiological and biochemical studies showed that functional ASICs are expressed in rat SON AVP neurons identified by the fluorescence generated from an enhanced green fluorescent protein (eGFP) fusion gene in the AVP-producing neurons [9,10]. However, the acid sensitivity of SON OXT neurons has not been assessed until now. In the present study, we used a whole-cell patch-clamp technique to identify the acidmediated currents in isolated rat hypothalamic MNCs that express an OXT-monomeric red fluorescent protein 1 (mRFP1) transgene [11] and/or an AVP-eGFP transgene. Interestingly, acid-induced currents in the OXT-mRFP1 neurons were significantly smaller than those in the AVP-eGFP neurons. All preparations and electrophysiological experiments were performed as previously reported [12]. Experiments were performed on transgenic rats at 3–5 weeks of age (young adults, weighing 80–150 g). These transgenic rats express chimeric OXTmRFP1 [11] and/or AVP-eGFP gene products [9]. The animals were housed in standard plastic cages at 23–25 ◦ C on a 12-h light (07.00–19.00 h)–12-h dark cycle. All experiments in this study were carried out in accordance with the guidelines of the Physiological Society of Japan under the control of the Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health, Japan. A modified Krebs–Henseleit solution (KHB) was used in the experimental steps where SON slice preparations were manipulated; the KHB solution contained: 124 mm NaCl, 5 mm KCl, 1.3 mm MgSO4 , 1.24 mm KH2 PO4 , 2 mm CaCl2 , 25.9 mm NaHCO3 and 10 mm glucose. The solution was continuously oxygenated with a mixture of 95% O2 –5% CO2 . In the experiments with dissociated neurons, HEPES buffered solution (HBS) was used: 140 mm NaCl, 5 mm KCl, 1.2 mm KH2 PO4 , 2 mm CaCl2 , 1.2 mm MgCl2 , 10 mm glucose and 10 mm HEPES, adjusted to pH 7.4 with NaOH. This solution was continuously oxygenated with 100% O2 , and the osmolarity was adjusted to 300 ± 5 mosmol l−1 with mannitol. The pipette solution used in the recording electrodes contained 140 mm potassium gluconate, 1 mm MgCl2 , 1 mm CaCl2 , 10 mm EGTA, 10 mm HEPES, and 2 mm Mg-ATP, adjusted to pH 7.3 with Tris base. Acid solutions were buffered using a combination of 10 mM HEPES and 10 mM 2-(N-morpholino) ethanesulfonic acid (MES) (Sigma). Young adult male transgenic rats were euthanized by decapitation. The brains were quickly removed and cooled in KHB at 4 ◦ C for 1 min. A block containing the hypothalamus was cut from the brain and glued onto the stage of a vibrating blade tissue slicer (Linearslicer Pro 7, DSK, Kyoto, Japan). After removal of the meninges, 500 m coronal slices containing the SON and PVN were sectioned in KHB at 4 ◦ C. SON slices were excised from the coronal brain sections using a circular tissue puncher (inner diameter 1.8 mm) and then incubated in KHB at room temperature (22–24 ◦ C) for 1 h before enzyme digestion. Neurons were dissociated by an enzymatic digestion method [13]. Briefly, slices were incubated in 4 ml HBS containing DNase I (Sigma, 0.5 mg ml−1 ) and papain (20 U ml−1 ; Worthington Biochemical Corp., Lakewood, NJ, USA) in a 50 ml centrifuge tube containing 100% O2 for 50 min at 30 ◦ C. The slices were then transferred to normal HBS and washed for at least 1 h prior to mechanical dissociation by trituration with fire-polished glass pipettes. The cell suspension was plated onto coverslips individually placed into the wells of multiwell culture plates with 2 ml of culture medium and maintained in a humidified incubator at
37 ◦ C with 5% CO2 for 1 day. The culture medium was NeurobasalA medium (Invitrogen) supplemented with 0.5 mm l-glutamine, B27 (Invitrogen), 5 ng ml−1 fibroblast growth factor (Sigma), and penicillin–streptomycin (Invitrogen). The osmolarity of the culture medium was adjusted to 300 ± 5 mosmol l−1 with NaCl. The coverslip with the plated neurons was placed in a glassbottomed chamber and continuously perfused with HBS at a rate of 1.5 ml min−1 using an eight-head peristaltic pump (MP-8, Gilson nucleus, Villiers le Bel, France). The volume of the recording chamber was 1 ml. The solution level was kept constant by a lowpressure aspiration system. Isolated MNCs expressing OXT-mRFP1 and AVP-eGFP were identified by their red and green fluorescence, respectively. The electrodes used in this study were triple-pulled from a glass capillary with a puller (P-87, Sutter Instrument Co., Novato, CA, USA). The pipettes had a final resistance of 5–8 M when filled. An Axopatch 200B amplifier (Axon Instruments) was used to record ionic currents in the tight-seal, whole-cell configuration. Acidic-solution and the non-specific ASIC blocker, amiloride (Sigma–Aldrich, St. Louis, MO), were pressure-applied via a Y-tube system [14], which is a rapid drug exchange technique that allows the external solution surrounding a neuron to be exchanged within 20 ms. The Y-tube tip was positioned 1.5 mm from the recording electrode in the cell body. Membrane voltages were controlled and recorded with a computer running pCLAMP10.3 software (Molecular Devices, Sunnyvale, CA, USA). Recording commenced at least 3 min after membrane rupture when the currents reached a steady state. To avoid current variances due to differences in cell size, current density (pA/picofarads; pF) was set as peak current amplitude (pA) divided by membrane capacitance (pF) from the same cell [15]. Recordings included in the data analysis were collected during periods of stable series resistance. All experiments were carried out at room temperature (22–24 ◦ C). Results are expressed as the mean ± S.E.M. Statistical comparisons were performed with Student’s t-test or Welch’s t-test. In all cases, p < 0.05 was considered significant. When neurons were held at −70 mV, fast reduction of the extracellular pH from 7.4 to 5.5 induced rapidly desensitizing inward currents in both SON OXT neurons and SON AVP neurons. Peak amplitude of current induced by pH 5.5 in the OXTmRFP1 neurons was 8.79 ± 2.2 pA/pF (n = 18, Fig. 1a and b). On the other hand, peak amplitude of current induced by pH 5.5 in the AVP-eGFP neurons was 73.1 ± 7.6 pA/pF (n = 10, Fig. 1c and d, p < 0.0001 vs. OXT neurons). Co-application of 10 M amiloride significantly reduced pH 5.5-induced current to 2.25 ± 0.62 pA/pF (p = 0.0029) and 14.1 ± 3.9 pA/pF (p < 0.0001) in OXT-mRFP1 neurons and AVP-eGFP neurons, respectively (Fig. 1). Similar results have been observed in double transgenic rats by mating an OXTmRFP1 transgenic rat with an AVP-eGFP transgenic rat (Fig. 2a and b). That is, the peak amplitude of current induced by pH 5.5 in the OXT-mRFP1 neurons and AVP-eGFP neurons were 9.72 ± 2.1 pA/pF (n = 20) and 53.6 ± 5.5 pA/pF (n = 21), respectively (p < 0.0001, Fig. 2c–e). Application of 10 M amiloride significantly reduced pH 5.5-induced current to 2.94 ± 0.56 pA/pF (p = 0.0008) and 8.59 ± 1.5 pA/pF (p < 0.0001) in OXT-mRFP1 and AVP-eGFP neurons, respectively (Fig. 2c–e). We confirmed the reaction to acidic condition in the magnocellular PVN neurons with the same experimental manner of the SON neurons. As a result, the peak amplitude of current induced by pH 5.5 in the OXT-mRFP1 neurons and AVPeGFP neurons were 16.2 ± 3.6 pA/pF (n = 4) and 64.8 ± 10 pA/pF (n = 4), respectively (p = 0.0045). Application of 10 M amiloride significantly reduced pH 5.5-induced current to 4.86 ± 1.5 pA/pF (p = 0.0272) and 8.56 ± 1.8 pA/pF (p = 0.0129) in OXT-mRFP1 and AVP-eGFP neurons, respectively (Fig. 2f). Furthermore, to examine whether the other acid evoked the similar inward currents in both SON OXT neurons and SON AVP neurons, we have been observed the reaction to the other acid
J.-i. Ohkubo et al. / Neuroscience Letters 583 (2014) 1–5
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Fig. 1. Effects of acid and the ASICs antagonists on inward currents in OXT-mRFP1 and AVP-eGFP neurons. Sample traces of the inward currents observed in response to (pH 5.5) and the ASICs antagonist amiloride (10 M) are shown in an OXT-mRFP1 neuron (a). Sample traces of the inward currents observed in response to acid (pH 5.5) and the ASICs antagonist amiloride (10 M) are shown in an AVP-eGFP neuron (c). The effect of acidification and amiloride was compared between OXT-mRFP1 and AVP-eGFP neurons (b, d). All data are expressed as the mean ± S.E.M. (OXT-mRFP1 neurons; n = 18, AVP-eGFP neurons; n = 10) **: pH 5.5 vs. pH 5.5 + amiloride, ††: OXT-mRFP1 neurons vs. AVP-eGFP neurons, p < 0.01.
solution (HEPES buffered solution adjusted to pH 5.5 with HCl) in the double transgenic rats SON OXT neurons and AVP neurons. Similar results have been observed. That is, the peak amplitude of current induced by pH 5.5 in the OXT-mRFP1 neurons and AVPeGFP neurons were 6.51 ± 0.52 pA/pF (n = 5) and 64.8 ± 6.2 pA/pF (n = 5), respectively (p = 0.0007). Application of 10 M amiloride significantly reduced pH 5.5-induced current to 2.44 ± 0.72 pA/pF (p = 0.0018) and 8.71 ± 1.8 pA/pF (p = 0.0003) in OXT-mRFP1 and AVP-eGFP neurons, respectively. In the present study, we revealed that the acid sensitivity was significantly lower in the OXT neurons than in the AVP neurons. A pH of 6.3 did not evoke any changes in SON OXT-mRFP1 neurons (data not shown), whereas SON AVP-eGFP neurons were potentiated by pH 6.3, a putative physiological/pathophysiological pH-range as determined by a previous study [10]. These results indicate that there is negligible acid sensitivity in OXT neurons, with respect to physiological/pathophysiological pH conditions, and pH 5.5-induced currents appear to play only a minor role in potentiating OXT neurons. Our previous study demonstrated that the activity of ASICs expressed in SON AVP neurons is modulated by lactate, which is increased by SON hypoxia that results from
the stimulation of perivascular V1a -receptors by AVP that is locally released from dendrites in the rat SON and causes the constriction of local arterioles [10]. The difference in the magnitude of acid-induced currents in the OXT and AVP neurons may attribute to differences in ASICs density, unitary current, and/or affinity to acidic condition due to composition of the ASIC subunits. Previous findings suggest that OXT dose-dependently decreased the amplitude of ASIC currents in DRG neurons and altered acidosisevoked membrane excitability of DRG neurons and significantly decreased the amplitude of the depolarization and number of action potentials induced by acid stimuli [16]. This report may support our conclusion in our present study and may be associated with the difference in the magnitude of acid-induced currents in the OXT and AVP neurons. Our current findings convincingly validate that AVP is closely related to the local SON hypoxia that follows vasoconstriction, but not OXT, which has only a minor effect on vasoconstriction. From a technical point of view, the present study proved the utility of fluorescence tagging OXT with mRFP1 and AVP with eGFP. The double transgenic rats were also helpful for identifying living OXT and AVP neurons confirming the usefulness of these techniques.
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J.-i. Ohkubo et al. / Neuroscience Letters 583 (2014) 1–5
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Fig. 2. Effects of acid and the ASICs antagonist on inward currents in OXT-mRFP1 and AVP-eGFP neurons obtained from the SON and magnocellular PVN of a double transgenic rat. The OXT-mRFP1-expressing magnocellular neurosecretory cells (MNCs) were identified by red fluorescence, and the AVP-eGFP-expressing MNCs were identified by green fluorescence in the SON. The merged view of fluorescence of mRFP1 and eGFP (a). MNCs isolated from the SON (b). Scale bar indicates 50 m. MNCs isolated from SON of the double transgenic rats were examined in response to acid and the ASICs antagonist amiloride (c, d, e). MNCs isolated from the magnocellular PVN of the double transgenic rats were examined in response to acid and the ASICs antagonist amiloride (f). All data are expressed as the mean ± S.E.M. (SON: OXT-mRFP1 neurons; n = 20, AVP-eGFP neurons; n = 21, PVN: OXT-mRFP1 neurons; n = 4, AVP-eGFP neurons; n = 4) **: pH 5.5 vs. pH 5.5 + amiloride, ††: OXT-mRFP1 neurons vs. AVP-eGFP neurons, p < 0.01, *: pH 5.5 vs. pH 5.5 + amiloride, p < 0.05.
Certain issues remain unresolved in this study. We cannot definitively affirm that acid-induced currents in OXT-mRFP1 and AVP-eGFP neurons are modulated by ASICs since amiloride does not necessarily exhibit strict pharmacological specificity for ASICs. In addition, we cannot identify the ASIC subtypes in OXT-mRFP1 neurons. Further biochemical evidence is needed to confirm the molecular composition of these channels. Despite these limitations, our results strongly suggest that the hypothalamic MNCs are divided into the acid-insensitive OXT neurons and the acidsensitive AVP neurons, which might contribute to maintaining systematic order in hypothalamus functions. Acknowledgements We thank Ms. Kanako Shoguchi (University of Occupational and Environmental Health, Kitakyushu, Japan) for her technical assistance. This study was supported by a UOEH Grant for Advanced Research from the University of Occupational and Environmental Health for Y.U., by a Grant-in-Aid for Scientific Research (B), No 22390044 to Y.U. from the Japan Society for the Promotion of Science (JSPS), by a Grant-in-Aid for Scientific Research on Innovative Area, No 23113518 to Y.U. from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, and by a Grant-in Aid for Challenging Exploratory Research, No 23659127 to Y.U. from MEXT, Japan.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Differences in acid-induced currents between oxytocin-mRFP1 and vasopressin-eGFP neurons isolated from the supraoptic and paraventricular nuclei of transgenic rats Jun-ichi Ohkubo a,b , Toyoaki Ohbuchi b , Mitsuhiro Yoshimura a , Takashi Maruyama a , Hirofumi Hashimoto a , Takanori Matsuura a , Hideaki Suzuki b , Yoichi Ueta a,∗ a
Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan b
h i g h l i g h t s • • • •
The differences between OXT-mRFP1 neurons and AVP-eGFP neurons in response to acid. Acid-induced currents were identified in isolated rat SON and PVN MNCs. The currents in the OXT-mRFP1 neurons were smaller than those in the AVP-eGFP neurons. The acid sensitivity of OXT neurons may be lower than that of AVP neurons.
a r t i c l e
i n f o
Article history: Received 27 June 2014 Received in revised form 28 August 2014 Accepted 1 September 2014 Available online 8 September 2014 Keywords: Acid-sensing ion channels Fluorescent protein SON PVN Oxytocin Vasopressin
a b s t r a c t The hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) consists of two types of magnocellular neurosecretory cells, oxytocin (OXT) and arginine vasopressin (AVP). We generated and characterized rats that express an OXT-monomeric red fluorescent protein 1 (mRFP1) and an AVP-enhanced green fluorescent protein (eGFP) fusion transgene. These transgenic rats enable the visualization of OXT or AVP neurons. Taking advantage of this, we examined the differences between OXT-mRFP1 neurons and AVP-eGFP neurons in response to acid. Acid-sensing ion channels (ASICs) are neuronal voltage-insensitive cationic channels that are activated by extracellular acidification. Although functional ASICs have been identified in AVP neurons, differences in acid-induced currents between OXT and AVP neurons in SON have not been reported. In the present study, we used the whole-cell patchclamp technique to investigate differences between OXT-mRFP1 neurons and AVP-eGFP neurons reaction to acid in SON and PVN. In voltage clamp mode, lowering extracellular pH evoked inward currents in both OXT-mRFP1 neurons and AVP-eGFP neurons. In our findings, the acid-induced currents in the OXT-mRFP1 neurons were significantly smaller than those in the AVP-eGFP neurons. These acid-induced currents were inhibited by amiloride, a known blocker of ASICs. Further, to compare the response to acid between OXT-mRFP1 and AVP-eGFP neurons in the same transgenic rat, we used a double transgenic rat by mating an OXT-mRFP1 transgenic rat with an AVP-eGFP transgenic rat. The acid-induced currents of OXT-mRFP1 neurons were significantly smaller than those of AVP-eGFP neurons from the double transgenic rats. These currents were almost completely inhibited by amiloride. The difference of acid-sensitivity between OXT and AVP neurons might contribute to maintaining systematic order in hypothalamic function. © 2014 Elsevier Ireland Ltd. All rights reserved.
∗ Corresponding author at: Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Tel.: +81 93 691 7420; fax: +81 93 692 1711. E-mail address: [email protected] (Y. Ueta). http://dx.doi.org/10.1016/j.neulet.2014.09.004 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Oxytocin (OXT) and arginine vasopressin (AVP) are synthesized and secreted from magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) and in the paraventricular nucleus (PVN) [1]. AVP plays a role in water homeostasis, cardiovascular regulation, controls osmolality, and blood pressure. In recent reports, the centrally released AVP has also been associated with regulation of circadian rhythm and/or social behavior [2,3]. The role
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J.-i. Ohkubo et al. / Neuroscience Letters 583 (2014) 1–5
of OXT in lactation and parturition has been well characterized. In addition, centrally released OXT is involved in maternal bonding, sexual behavior, and social affiliation [4–7]. Acid-sensing ion channels (ASICs) are widely expressed in the peripheral and central nervous system where they perform physiological functions [8]. The ASICs are voltage-insensitive cation channels that are activated by extracellular acidification. Our recent electrophysiological and biochemical studies showed that functional ASICs are expressed in rat SON AVP neurons identified by the fluorescence generated from an enhanced green fluorescent protein (eGFP) fusion gene in the AVP-producing neurons [9,10]. However, the acid sensitivity of SON OXT neurons has not been assessed until now. In the present study, we used a whole-cell patch-clamp technique to identify the acidmediated currents in isolated rat hypothalamic MNCs that express an OXT-monomeric red fluorescent protein 1 (mRFP1) transgene [11] and/or an AVP-eGFP transgene. Interestingly, acid-induced currents in the OXT-mRFP1 neurons were significantly smaller than those in the AVP-eGFP neurons. All preparations and electrophysiological experiments were performed as previously reported [12]. Experiments were performed on transgenic rats at 3–5 weeks of age (young adults, weighing 80–150 g). These transgenic rats express chimeric OXTmRFP1 [11] and/or AVP-eGFP gene products [9]. The animals were housed in standard plastic cages at 23–25 ◦ C on a 12-h light (07.00–19.00 h)–12-h dark cycle. All experiments in this study were carried out in accordance with the guidelines of the Physiological Society of Japan under the control of the Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health, Japan. A modified Krebs–Henseleit solution (KHB) was used in the experimental steps where SON slice preparations were manipulated; the KHB solution contained: 124 mm NaCl, 5 mm KCl, 1.3 mm MgSO4 , 1.24 mm KH2 PO4 , 2 mm CaCl2 , 25.9 mm NaHCO3 and 10 mm glucose. The solution was continuously oxygenated with a mixture of 95% O2 –5% CO2 . In the experiments with dissociated neurons, HEPES buffered solution (HBS) was used: 140 mm NaCl, 5 mm KCl, 1.2 mm KH2 PO4 , 2 mm CaCl2 , 1.2 mm MgCl2 , 10 mm glucose and 10 mm HEPES, adjusted to pH 7.4 with NaOH. This solution was continuously oxygenated with 100% O2 , and the osmolarity was adjusted to 300 ± 5 mosmol l−1 with mannitol. The pipette solution used in the recording electrodes contained 140 mm potassium gluconate, 1 mm MgCl2 , 1 mm CaCl2 , 10 mm EGTA, 10 mm HEPES, and 2 mm Mg-ATP, adjusted to pH 7.3 with Tris base. Acid solutions were buffered using a combination of 10 mM HEPES and 10 mM 2-(N-morpholino) ethanesulfonic acid (MES) (Sigma). Young adult male transgenic rats were euthanized by decapitation. The brains were quickly removed and cooled in KHB at 4 ◦ C for 1 min. A block containing the hypothalamus was cut from the brain and glued onto the stage of a vibrating blade tissue slicer (Linearslicer Pro 7, DSK, Kyoto, Japan). After removal of the meninges, 500 m coronal slices containing the SON and PVN were sectioned in KHB at 4 ◦ C. SON slices were excised from the coronal brain sections using a circular tissue puncher (inner diameter 1.8 mm) and then incubated in KHB at room temperature (22–24 ◦ C) for 1 h before enzyme digestion. Neurons were dissociated by an enzymatic digestion method [13]. Briefly, slices were incubated in 4 ml HBS containing DNase I (Sigma, 0.5 mg ml−1 ) and papain (20 U ml−1 ; Worthington Biochemical Corp., Lakewood, NJ, USA) in a 50 ml centrifuge tube containing 100% O2 for 50 min at 30 ◦ C. The slices were then transferred to normal HBS and washed for at least 1 h prior to mechanical dissociation by trituration with fire-polished glass pipettes. The cell suspension was plated onto coverslips individually placed into the wells of multiwell culture plates with 2 ml of culture medium and maintained in a humidified incubator at
37 ◦ C with 5% CO2 for 1 day. The culture medium was NeurobasalA medium (Invitrogen) supplemented with 0.5 mm l-glutamine, B27 (Invitrogen), 5 ng ml−1 fibroblast growth factor (Sigma), and penicillin–streptomycin (Invitrogen). The osmolarity of the culture medium was adjusted to 300 ± 5 mosmol l−1 with NaCl. The coverslip with the plated neurons was placed in a glassbottomed chamber and continuously perfused with HBS at a rate of 1.5 ml min−1 using an eight-head peristaltic pump (MP-8, Gilson nucleus, Villiers le Bel, France). The volume of the recording chamber was 1 ml. The solution level was kept constant by a lowpressure aspiration system. Isolated MNCs expressing OXT-mRFP1 and AVP-eGFP were identified by their red and green fluorescence, respectively. The electrodes used in this study were triple-pulled from a glass capillary with a puller (P-87, Sutter Instrument Co., Novato, CA, USA). The pipettes had a final resistance of 5–8 M when filled. An Axopatch 200B amplifier (Axon Instruments) was used to record ionic currents in the tight-seal, whole-cell configuration. Acidic-solution and the non-specific ASIC blocker, amiloride (Sigma–Aldrich, St. Louis, MO), were pressure-applied via a Y-tube system [14], which is a rapid drug exchange technique that allows the external solution surrounding a neuron to be exchanged within 20 ms. The Y-tube tip was positioned 1.5 mm from the recording electrode in the cell body. Membrane voltages were controlled and recorded with a computer running pCLAMP10.3 software (Molecular Devices, Sunnyvale, CA, USA). Recording commenced at least 3 min after membrane rupture when the currents reached a steady state. To avoid current variances due to differences in cell size, current density (pA/picofarads; pF) was set as peak current amplitude (pA) divided by membrane capacitance (pF) from the same cell [15]. Recordings included in the data analysis were collected during periods of stable series resistance. All experiments were carried out at room temperature (22–24 ◦ C). Results are expressed as the mean ± S.E.M. Statistical comparisons were performed with Student’s t-test or Welch’s t-test. In all cases, p < 0.05 was considered significant. When neurons were held at −70 mV, fast reduction of the extracellular pH from 7.4 to 5.5 induced rapidly desensitizing inward currents in both SON OXT neurons and SON AVP neurons. Peak amplitude of current induced by pH 5.5 in the OXTmRFP1 neurons was 8.79 ± 2.2 pA/pF (n = 18, Fig. 1a and b). On the other hand, peak amplitude of current induced by pH 5.5 in the AVP-eGFP neurons was 73.1 ± 7.6 pA/pF (n = 10, Fig. 1c and d, p < 0.0001 vs. OXT neurons). Co-application of 10 M amiloride significantly reduced pH 5.5-induced current to 2.25 ± 0.62 pA/pF (p = 0.0029) and 14.1 ± 3.9 pA/pF (p < 0.0001) in OXT-mRFP1 neurons and AVP-eGFP neurons, respectively (Fig. 1). Similar results have been observed in double transgenic rats by mating an OXTmRFP1 transgenic rat with an AVP-eGFP transgenic rat (Fig. 2a and b). That is, the peak amplitude of current induced by pH 5.5 in the OXT-mRFP1 neurons and AVP-eGFP neurons were 9.72 ± 2.1 pA/pF (n = 20) and 53.6 ± 5.5 pA/pF (n = 21), respectively (p < 0.0001, Fig. 2c–e). Application of 10 M amiloride significantly reduced pH 5.5-induced current to 2.94 ± 0.56 pA/pF (p = 0.0008) and 8.59 ± 1.5 pA/pF (p < 0.0001) in OXT-mRFP1 and AVP-eGFP neurons, respectively (Fig. 2c–e). We confirmed the reaction to acidic condition in the magnocellular PVN neurons with the same experimental manner of the SON neurons. As a result, the peak amplitude of current induced by pH 5.5 in the OXT-mRFP1 neurons and AVPeGFP neurons were 16.2 ± 3.6 pA/pF (n = 4) and 64.8 ± 10 pA/pF (n = 4), respectively (p = 0.0045). Application of 10 M amiloride significantly reduced pH 5.5-induced current to 4.86 ± 1.5 pA/pF (p = 0.0272) and 8.56 ± 1.8 pA/pF (p = 0.0129) in OXT-mRFP1 and AVP-eGFP neurons, respectively (Fig. 2f). Furthermore, to examine whether the other acid evoked the similar inward currents in both SON OXT neurons and SON AVP neurons, we have been observed the reaction to the other acid
J.-i. Ohkubo et al. / Neuroscience Letters 583 (2014) 1–5
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Fig. 1. Effects of acid and the ASICs antagonists on inward currents in OXT-mRFP1 and AVP-eGFP neurons. Sample traces of the inward currents observed in response to (pH 5.5) and the ASICs antagonist amiloride (10 M) are shown in an OXT-mRFP1 neuron (a). Sample traces of the inward currents observed in response to acid (pH 5.5) and the ASICs antagonist amiloride (10 M) are shown in an AVP-eGFP neuron (c). The effect of acidification and amiloride was compared between OXT-mRFP1 and AVP-eGFP neurons (b, d). All data are expressed as the mean ± S.E.M. (OXT-mRFP1 neurons; n = 18, AVP-eGFP neurons; n = 10) **: pH 5.5 vs. pH 5.5 + amiloride, ††: OXT-mRFP1 neurons vs. AVP-eGFP neurons, p < 0.01.
solution (HEPES buffered solution adjusted to pH 5.5 with HCl) in the double transgenic rats SON OXT neurons and AVP neurons. Similar results have been observed. That is, the peak amplitude of current induced by pH 5.5 in the OXT-mRFP1 neurons and AVPeGFP neurons were 6.51 ± 0.52 pA/pF (n = 5) and 64.8 ± 6.2 pA/pF (n = 5), respectively (p = 0.0007). Application of 10 M amiloride significantly reduced pH 5.5-induced current to 2.44 ± 0.72 pA/pF (p = 0.0018) and 8.71 ± 1.8 pA/pF (p = 0.0003) in OXT-mRFP1 and AVP-eGFP neurons, respectively. In the present study, we revealed that the acid sensitivity was significantly lower in the OXT neurons than in the AVP neurons. A pH of 6.3 did not evoke any changes in SON OXT-mRFP1 neurons (data not shown), whereas SON AVP-eGFP neurons were potentiated by pH 6.3, a putative physiological/pathophysiological pH-range as determined by a previous study [10]. These results indicate that there is negligible acid sensitivity in OXT neurons, with respect to physiological/pathophysiological pH conditions, and pH 5.5-induced currents appear to play only a minor role in potentiating OXT neurons. Our previous study demonstrated that the activity of ASICs expressed in SON AVP neurons is modulated by lactate, which is increased by SON hypoxia that results from
the stimulation of perivascular V1a -receptors by AVP that is locally released from dendrites in the rat SON and causes the constriction of local arterioles [10]. The difference in the magnitude of acid-induced currents in the OXT and AVP neurons may attribute to differences in ASICs density, unitary current, and/or affinity to acidic condition due to composition of the ASIC subunits. Previous findings suggest that OXT dose-dependently decreased the amplitude of ASIC currents in DRG neurons and altered acidosisevoked membrane excitability of DRG neurons and significantly decreased the amplitude of the depolarization and number of action potentials induced by acid stimuli [16]. This report may support our conclusion in our present study and may be associated with the difference in the magnitude of acid-induced currents in the OXT and AVP neurons. Our current findings convincingly validate that AVP is closely related to the local SON hypoxia that follows vasoconstriction, but not OXT, which has only a minor effect on vasoconstriction. From a technical point of view, the present study proved the utility of fluorescence tagging OXT with mRFP1 and AVP with eGFP. The double transgenic rats were also helpful for identifying living OXT and AVP neurons confirming the usefulness of these techniques.
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J.-i. Ohkubo et al. / Neuroscience Letters 583 (2014) 1–5
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Fig. 2. Effects of acid and the ASICs antagonist on inward currents in OXT-mRFP1 and AVP-eGFP neurons obtained from the SON and magnocellular PVN of a double transgenic rat. The OXT-mRFP1-expressing magnocellular neurosecretory cells (MNCs) were identified by red fluorescence, and the AVP-eGFP-expressing MNCs were identified by green fluorescence in the SON. The merged view of fluorescence of mRFP1 and eGFP (a). MNCs isolated from the SON (b). Scale bar indicates 50 m. MNCs isolated from SON of the double transgenic rats were examined in response to acid and the ASICs antagonist amiloride (c, d, e). MNCs isolated from the magnocellular PVN of the double transgenic rats were examined in response to acid and the ASICs antagonist amiloride (f). All data are expressed as the mean ± S.E.M. (SON: OXT-mRFP1 neurons; n = 20, AVP-eGFP neurons; n = 21, PVN: OXT-mRFP1 neurons; n = 4, AVP-eGFP neurons; n = 4) **: pH 5.5 vs. pH 5.5 + amiloride, ††: OXT-mRFP1 neurons vs. AVP-eGFP neurons, p < 0.01, *: pH 5.5 vs. pH 5.5 + amiloride, p < 0.05.
Certain issues remain unresolved in this study. We cannot definitively affirm that acid-induced currents in OXT-mRFP1 and AVP-eGFP neurons are modulated by ASICs since amiloride does not necessarily exhibit strict pharmacological specificity for ASICs. In addition, we cannot identify the ASIC subtypes in OXT-mRFP1 neurons. Further biochemical evidence is needed to confirm the molecular composition of these channels. Despite these limitations, our results strongly suggest that the hypothalamic MNCs are divided into the acid-insensitive OXT neurons and the acidsensitive AVP neurons, which might contribute to maintaining systematic order in hypothalamus functions. Acknowledgements We thank Ms. Kanako Shoguchi (University of Occupational and Environmental Health, Kitakyushu, Japan) for her technical assistance. This study was supported by a UOEH Grant for Advanced Research from the University of Occupational and Environmental Health for Y.U., by a Grant-in-Aid for Scientific Research (B), No 22390044 to Y.U. from the Japan Society for the Promotion of Science (JSPS), by a Grant-in-Aid for Scientific Research on Innovative Area, No 23113518 to Y.U. from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, and by a Grant-in Aid for Challenging Exploratory Research, No 23659127 to Y.U. from MEXT, Japan.
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