Mechanisms of orexin 2 receptor-mediated depolarization in the rat paraventricular nucleus of the hypothalamus

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Mechanisms of orexin 2 receptor-mediated depolarization in the rat paraventricular nucleus of the hypothalamus Yu-Wen E. Daia, Yen-Hsien Leeb, Tzu-Ling Lia, Ling-Ling Hwanga,c,∗ a

Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wuxing Street, Taipei, 110, Taiwan Department of Medical Laboratory Science and Biotechnology, Yuanpei University of Medical Technology, Taiwan c Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, 250 Wuxing Street, Taipei, 110, Taiwan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Voltage clamp Rostral ventrolateral medulla Autonomic neuroscience Sympathetic Ion channel Neuronal excitability

The paraventricular nucleus of the hypothalamus (PVN) contains dense orexin 2 (OX2) receptor. We examined the mechanisms of OX2 receptor-mediated excitation on electrophysiologically identified type I (putative magnocellular), low-threshold spikes (LTS)-expressing type II (putative preautonomic), and non-LTS type II (putative parvocellular neuroendocrine) neurons. In the presence of tetrodotoxin, an OX2 receptor agonist, ALOXB (30–1000 nM) depolarized 56% of type I, and 73–75% of type II neurons. In type I neurons, ALOXBinduced inward current displayed increased-conductance current-voltage (I–V) relationship and reversed polarity at −27.5 ± 4.8 mV. A Na+-Ca2+ exchanger (NCX) inhibitor, KBR-7943, attenuated ALOXB responses in the majority of type I neurons, while no attenuation was observed in nearly all type II neurons. Type II neurons exhibited three types of I–V relationships in response to ALOXB, characterized by decreased, increased, and unchanged conductance, respectively. The reversal potential of the decreased-conductance responses was near the equilibrium potential of K+ (Ek+) and became more positive in a high-K+ solution, suggesting that K+ conductance blockade is involved. In a low-Na+ solution, non-reversed I–V curves of increased-conductance responses became decreased-conductance responses and reversed polarity near Ek+, suggesting the involvement of both K+ conductance and non-selective cation conductance (NSCC). Approximately 35% of LTS-expressing type II neurons were vasopressin-immunoreactive and 71% of them responded to ALOXB. In conclusion, orexins may activate OX2 receptor on PVN neurons and cause depolarization by promoting NCX and/or NSCC in magnocellular neurons, and by decreasing K+ conductance and/or increasing NSCC in parvocellular neurons. Furthermore, the majority of vasopressinergic preautonomic neurons are under OX2 receptor regulation.

1. Introduction Orexin A and B (or hypocretin 1 and 2) are hypothalamic neuropeptides (de Lecea et al., 1998; Sakurai et al., 1998). Many studies have highlighted their roles in stress responses, cardiovascular control and autonomic functions, as well as in the pathophysiology of primary hypertension (reviewed by Carrive, 2017). Because of the critical roles orexins play in the pathogenesis of cardiovascular disease, a full understanding of the effects of orexins in the brain regions relevant to cardiovascular functions is imperative. The paraventricular nucleus of the hypothalamus (PVN) is one of these regions (Coote, 2004). The PVN is an integrating center of neuroendocrine and autonomic nervous systems (Coote, 1995; Swanson and Sawchenko, 1980). It consists predominantly of three types of cells: the magnocellular neurons, which project to the posterior pituitary where they secrete either

∗

vasopressin or oxytocin; the parvocellular neuroendocrine neurons, which control hormone secretion from the anterior pituitary; and the parvocellular preautonomic neurons, which project to the brainstem and spinal cord, and are involved in regulating the autonomic nervous system (Badoer, 2001; Liposits, 1993; Sawchenko and Swanson, 1982). Dense orexin fibers (Date et al., 1999; Peyron et al., 1998) are found in the PVN, and orexin 2 (OX2) receptor is the predominant orexin receptor in rat PVN (Marcus et al., 2001; Trivedi et al., 1998). Previous studies have demonstrated excitatory effects of orexin A and B on PVN neurons, but differed in their conclusions regarding whether orexins exerted a direct effect on magnocellular neurons (Follwell and Ferguson, 2002; Shirasaka et al., 2001). These studies agreed upon the fact that orexins directly influence parvocellular neurons; however, none has yet distinguished the reactions of parvocellular neuroendocrine-like and preautonomic neurons to orexinergic ligands. Herein, we

Corresponding author. Department of Physiology, College of Medicine, Taipei Medical University, 250 Wuxing Street, Taipei, 110, Taiwan. E-mail addresses: [email protected] (Y.-W.E. Dai), [email protected] (Y.-H. Lee), [email protected] (T.-L. Li), [email protected] (L.-L. Hwang).

https://doi.org/10.1016/j.ejphar.2019.172802 Received 7 July 2019; Received in revised form 12 November 2019; Accepted 14 November 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Yu-Wen E. Dai, et al., European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2019.172802

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in tetrodotoxin (TTX)-containing (0.5 μM) Krebs solution. The current value was measured at the end of each step. Currents elicited by such voltage commands in control media were subtracted from their counterparts in the presence of ALOXB to yield steady-state I–V curves of ALOXB-induced currents. Some of the recorded neurons were intracellularly stained with a fluorescent dye, Lucifer yellow, which diffused from the patch electrode into the recorded neuron. At the end of recording, the hypothalamic slice was fixed in a solution of 4% paraformaldehyde/0.1 M phosphatebuffered saline (PBS), cryoprotected in 30% sucrose solution, and then cryosectioned into 50-μm sections. Sections were viewed under an epifluorescence microscope and the sections right next to the section containing the Lucifer yellow-labelled neuron were selected for Nissl staining. The locations of the recorded neurons were identified by comparing images of the Lucifer yellow-labeling and the Nissl stain.

aimed to clarify the responses of the three types of PVN neurons to OX2 receptor activation. The three types of PVN neurons display different electrophysiological properties. The magnocellular neurons can be differentiated from parvocellular neurons by the presence of a transient outward rectification (Hoffman et al., 1991; Tasker and Dudek, 1991), which is underlain by a large A-type K+ current (Luther and Tasker, 2000). For parvocellular subpopulations, Stern (2001) demonstrated that 88% of dorsal brainstem-projecting (preautonomic) neurons express low-threshold spikes (LTS), while all neurosecretory parvocellular neurons do not (Luther et al., 2002). Using electrophysiological approaches, we examined the effects of OX2 receptor activation on three subgroups of PVN neurons, which respectively express a transient outward rectification, LTS, or none of either. Some of the parvocellular preautonomic neurons express vasopressin. Over 40% of spinal-projecting neurons express vasopressin mRNA (Hallbeck and Blomqvist, 1999), and approximately 15% of rostral ventrolateral medulla (RVLM)-projecting neurons are vasopressin-immunoreactive (Kc et al., 2010). These vasopressinergic neurons are involved in cardiovascular and respiratory regulation (Kc et al., 2002a, 2002b, 2010). In addition, their projections to the RVLM and rostral ventral respiratory column are involved in enhanced cardiorespiratory reactivity in chronic intermittent hypoxia-conditioned animals (Kc et al., 2010). Due to the importance of these neurons, we also examined whether the vasopressinergic preautonomic neurons are regulated through the OX2 receptor mechanism.

2.2. Solutions for electrophysiological experiments

2. Materials and methods

The Krebs solution contained (in mM): 127 NaCl, 1.9 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, and 10 glucose and was gassed with 95% O2 and 5% CO2. A high-K+ (7 mM) Krebs solution was prepared by equimolar replacement of NaCl with KCl. In preparing lowNa+ (26 mM) Krebs solution, NaCl was replaced with Tris-buffer and the pH was adjusted to 7.4 with HCl. The internal solution for patch electrodes was composed of (in mM): 130 potassium gluconate, 1 MgCl2, 2 CaCl2, 4 ATP, 0.3 GTP, 10 EGTA, 10 HEPES, and in some cases, 0.2% Lucifer yellow; the pH of the solution was adjusted to 7.2 with KOH.

2.1. Recording of PVN neurons from hypothalamus slices

2.3. Immunohistochemical staining

We used Sprague-Dawley rats (14–28 days old) of either sex provided by BioLASCO Taiwan (Taipei, Taiwan) for this study. The use of animals was approved by the Institutional Animal Care and Use Committee of the Taipei Medical University (the protocol approval number: LAC-101-0138). Rats were anesthetized with isoflurane (inhalation) and decapitated. The brains were quickly removed and placed in chilled, oxygenated Krebs solution as previously described (Hwang and Dun, 1998, 1999). Coronal hypothalamus slices (500-μm thickness) containing the PVN were prepared with a Vibratome slicer (Vibratome, St. Louis, MO). The tissue was sectioned in the rostral to caudal direction. Two slices were collected for recording, namely the slice containing the caudal end of the optic chiasm and its caudally adjacent slice. The PVN slices were then incubated in oxygenated Krebs solution at room temperature for at least 2 h prior to electrophysiological recordings. Whole-cell patch recordings of the PVN neurons were conducted by the blind patch clamp technique as described previously (Huang et al., 2010). The PVN was recognized in the slice based on the rat brain atlas of Paxinos and Watson (1997). After equilibrium, one slice was transferred to a recording chamber and continuously perfused (2–3 ml/min) with oxygenated Krebs solution at room temperature. Patch electrodes filled with an internal solution (see below for composition) had a resistance of 5–8 MΩ. Signals were recorded with an Axopatch-1D amplifier (Molecular Devices, Sunnyvale, CA), low-pass filtered at 2 kHz and acquired by a personal computer and pClamp software (version 10.0; Molecular Devices) for later analysis. Signals were also monitored on a two-channel chart recorder (Gould Electronics, Eichstetten, Germany). Membrane potentials in the text were corrected for a liquidjunction potential of −8 mV. The access resistance was monitored throughout the experiment and was less than 25 MΩ. The steady-state current-voltage (I–V) relationship of the recorded neurons was obtained in the voltage-clamp mode by applying a series of 100 ms step command every 300 ms from a holding potential of −60mV to different potentials (−120 to 0 mV) with 10 mV increments before and during the application of [Ala11,D-Leu15]-orexin B (ALOXB)

To identify the phenotype of the recorded neurons, the patch electrodes were filled with an internal solution containing 0.2% of a fluorescent dye, Lucifer yellow. During recording, Lucifer yellow diffused from the patch electrode into the recorded neuron. At the end of the recording, the slice was fixed in 4% paraformaldehyde in 0.1 M PBS, cryoprotected with 30% sucrose in PBS, and then cryosectioned into 50μm sections with a cryostat. Sections were examined under an epifluorescence microscope, and the section containing the Lucifer yellowlabelled neuron was selected for vasopressin immunostaining. The section was first blocked with 10% normal goat serum and incubated with rabbit polyclonal vasopressin antiserum (1:2000) for 48 h at 4 °C with gentle agitation. After several washes with PBS, the section was incubated with biotinylated goat anti-rabbit immunoglobulin G (1:200) for 2 h followed by Avidin Texas red (1:200) for 1 h. 2.4. Chemicals ALOXB, TTX and KBR-7943 were obtained from Tocris Bioscience/ Bio-Techne (Ellisville, MO). Lucifer yellow CH dilithium salt was obtained from Sigma-Aldrich (St. Louis, MO). Vasopressin antiserum (LOT: NG1721751 AB1565) was obtained from Millipore Corporation (Billerica, MA), and all other reagents for immunohistochemistry were obtained from Vector Laboratories (Burlingame, CA). 2.5. Data analysis Data were expressed as mean ± S.E.M. Changes of membrane conductance recorded before and during ALOXB treatment were analyzed statistically by Student's t-test. Effects of an inhibitor (KBR-7943) on ALOXB-induced responses were analyzed with repeated-measures analysis of variance (ANOVA) and the difference between two groups at specified time points were determined by Student's t-test. Statistical significance was set at P < 0.05. The half-maximal effective concentration (EC50) of ALOXB was determined by sigmoidal concentration-response curve fitting with 2

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Fig. 1. Electrophysiological properties and locations of magnocellular and parvocellular neurons in the paraventricular nucleus of the hypothalamus (PVN). A, a representative current-clamp protocol used to differentiate the three major types of PVN neurons and a schematic diagram of the subdivisions in the PVN. B-D show representative recording traces of three types of PVN neurons and their locations (black dots) in the PVN at Bregma level −1.8 mm. B, a type I neuron, identified by a prolonged outward rectification (arrow) in response to the depolarizing current. C, a non-LTS type II neuron, recognized by the absence of low-threshold spike (LTS) activity. D, a LTS type II neuron, identified by the existence of a LTS activity (arrow). V, third ventricle; mp, medial parvocellular division; vp, ventral parvocellular division; pm, posterior magnocellular division; dp, dorsal parvocellular division; lp, lateral parvocellular subdivision.

the presence of TTX (0.5 μM). Both OX2 receptor agonists and TTX were applied through a bath perfusion system. TTX, a selective inhibitor of the voltage-gated sodium channel, was used to block action potentialdependent synaptic transmission and, therefore, to help distinguishing a direct action from an indirect action elicited through synaptic transmission from neighboring neurons. Application of either orexin B or ALOXB caused depolarization in all three types of PVN neurons. Orexin B-induced depolarizations were observed in 3 type I, 4 non-LTS type II, and 3 LTS type II neurons. The amplitudes of the orexin B-induced depolarization ranged between 2 and 4 mV at 100 nM and between 6 and 17 mV at 300 nM. The effects of ALOXB were examined in a concentration range from 10 nM to 1 μM. PVN neurons were depolarized by ALOXB at concentrations between 30 nM and 1 μM. Representative responses to 300 nM ALOXB are shown in Fig. 2A. As the responses were observed in the presence of TTX, the effects of orexin B and ALOXB on all three types of PVN neurons were likely direct effects. This excitatory effect was observed in the majority, but not all, of PVN neurons. A more detailed analysis of the effects of OX2 receptor agonists on the three different types of PVN neurons was carried out with the use of ALOXB.

three-parameter logistic regression. The three-parameter logistic equaa tion for curve fitting is y= x b where y is a response measured at 1+(

EC50

)

concentration x, a is the maximal asymptote of response, EC50 is the concentration at half-maximal effect, and b is a slope parameter analogous to the Hill coefficient. 3. Results 3.1. Cell identification and membrane properties of the PVN neurons Before treatments, all cells were classified into three subgroups according to their distinct membrane electrical properties. Under current-clamp recording mode, recorded neurons were hyperpolarized to −100 ~ −120 mV and then depolarized with a series of depolarizing current injections. Putative magnocellular neurons (classified as type I neurons) were differentiated from putative parvocellular neurons, classified as type II neurons, based on the expression of transient outward rectification in response to the depolarizing current (Fig. 1B). Putative parvocellular neurons were further classified into two types, according to the presence of LTS in response to the depolarizing current. Type II neurons exhibiting LTS (Fig. 1D) that generated at most two action potentials were included in the present study as LTS-expressing type II neurons (“LTS type II neurons” for short). They are most likely preautonomic neurons (Luther et al., 2002; Stern, 2001). In a few neurons, the LTS were large and generated more than two action potentials. These neurons were not included for the study because they are likely non-PVN neurons located in the perinuclear area of the PVN (Tasker and Dudek, 1991). Type II neurons lacking LTS activity (nonLTS type II neurons) (Fig. 1C) were predominantly parvocellular neuroendocrine neurons. According to Luther et al.’s (2002) report, approximate 72% of the non-LTS type II neurons are parvocellular neuroendocrine neurons. Resting membrane potentials and whole-cell input resistance of type I (n = 34), non-LTS type II (n = 62), and LTS type II (n = 63) neurons were −56.1 ± 1.2, −55.2 ± 1.0, and −59.0 ± 1.2 mV, and 460.0 ± 30.9, 397.0 ± 27.6, and 409.3 ± 29.9 MΩ, respectively. Representative locations of the three subgroups of PVN neurons at Bregma level −1.8 mm are shown in Fig. 1, which were mapped by Nissl stain or by double labeling of Lucifer yellow and vasopressinimmunostaining (presented later in Fig. 8). Most of the successful recordings are distributed within the medial two-thirds of the mediolateral extent of the PVN at Bregma level −1.4 to −2.0 mm. This might be the reason that less type I neurons were recorded, compared to type II neurons.

3.3. Effects of ALOXB on three types of PVN neurons The numbers of the three types of neurons depolarized by ALOXB at 30 nM to 1 μM are shown in Table 1. Percentages of ALOXB-responsive neurons were approximately 55.9% (19 of 34) in type I neurons, 72.6% (45 of 62) in non-LTS type II neurons, and 74.6% (47 of 63) in LTS type II neurons (Fig. 2B). Therefore, the proportions of ALOXB-responsive neurons were higher in parvocellular neurons and lower in magnocellular neurons. The ALOXB-elicited depolarizations were concentration dependent (Fig. 2C). The EC50 of ALOXB was 25.5 ± 10.8 nM (R2 = 0.96) in type I neurons, 25.5 ± 7.7 nM (R2 = 0.91) in non-LTS type II neurons and 55.9 ± 16.5 nM (R2 = 0.98) in LTS type II neurons. Concentrationresponse curves (Fig. 2C) further revealed that the amplitudes of the ALOXB-induced depolarization were greater in LTS type II neurons than those of the other two types at 300 nM. 3.4. I–V relationships of ALOXB-induced responses To investigate the mechanisms of ALOXB-induced depolarization, I–V relationships of ALOXB-induced current on PVN neurons were studied in voltage-clamp mode in the presence of TTX (0.5 μM). Representative current traces in response to a series of voltage command steps before and during the application of ALOXB (300 nM) are shown in the insets of the left panels of Fig. 3. Values of command voltages and their corresponding currents, measured at the end of each voltage step, were plotted to form steady-state I–V curves, as shown in the left panels of Fig. 3. The I–V curve of ALOXB-induced current, as shown in the right panels of Fig. 3, was obtained by subtracting the I–V curve obtained in a control medium from the I–V curve obtained in the

3.2. Excitatory effects of OX2 receptor agonists on PVN neurons To examine the effects of OX2 receptor activation on the three types of PVN neurons, two selective OX2 receptor agonists, orexin B and ALOXB, were used. Effects of OX2 receptor agonists were examined in 3

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Fig. 2. An OX2 receptor agonist, [Ala11,DLeu15]-orexin B (ALOXB) directly excited the three types of PVN neurons in a concentrationdependent manner. Panel A, ALOXB (300 nM) induced depolarization on three types of PVN neurons in hypothalamic tissues slices in the presence of tetrodotoxin (0.5 μM). Recordings of membrane potentials were made under current-clamp mode. Downward deflections are hyperpolarizing electrotonic potential induced by constant-current pulses and were used to monitor membrane resistance. Panel B shows the percentages of the three types of PVN neurons responded to ALOXB. ALOXB depolarized approximately 55.9% (19 of 34) of type I, 72.6% (45 of 62) of non-LTS type II, and 74.6% (47 of 63) of LTS type II neurons. Panel C illustrates the mean change in membrane potential in response to different concentrations of ALOXB (30 nM–1 μM). Data are mean ± S.E.M.

3.5. I–V relationships of ALOXB responses on three types of PVN neurons

Table 1 Numbers of ALOXB-responsive neurons in three types of PVN neurons at concentrations of 0.03–1 μM. ALOXB (μM)

0.03 0.10 0.30 1.00 Total neurons

The steady-state I–V relationship of ALOXB-induced currents was investigated in 8 type I, 41 non-LTS type II and 45 LTS type II neurons. All 8 type I neurons displayed increased conductance in the I–V relationship. Non-LTS type II and LTS type II neurons exhibited all three types of I–V relationships; the majority of these two types of neurons exhibited increased-conductance responses. Table 2 shows the numbers of neurons that displayed increased, decreased and unchanged conductance, respectively in response to ALOXB in the three types of PVN neurons. Table 2 also shows the resting membrane potential and reversal potentials for each category of neurons. The amplitude of ALOXB-induced current at −60 mV and slope conductance before and during ALOXB treatment for each category of neurons are presented in Fig. 4.

Number of responsive/total neurons Type I

Non-LTS Type II

LTS Type II

3/6 6/9 7/13 3/6 19/34

9/11 8/13 20/27 8/11 45/62

10/13 13/14 20/27 4/9 47/63

presence of ALOXB. Therefore, a positive and negative slope of the I–V curve of ALOXB-induced current may respectively imply an increase and decrease in membrane conductance by ALOXB. Three types of I–V relationships were observed. In the first type, the application of ALOXB increased the slope of the I–V curve (Fig. 3A, the left panel). The ALOXB-induced current of this type, therefore, exhibited a positive-slope I–V relationship and often reversed polarity in the voltage range between −40 and 0 mV (Fig. 3A, the right panel). In the second type, ALOXB decreased the slope of the I–V curve (Fig. 3B, the left panel). The ALOXB-induced current of the second type was characterized by a negative-slope I–V relationship and reversed or nearly reversed polarity at potentials more negative than −90 mV (Fig. 3B, the right panel). In the third type, ALOXB did not change the slope of the I–V curve and induced an inward current of similar amplitude in the entire range of the membrane potential tested (Fig. 3C). In summary, according to the changes in the slope conductance of steady-state I–V curves, the I–V relationship of ALOXB-induced current are classified into three types, respectively displaying increased, decreased and unchanged slope conductance.

3.5.1. Type I (magnocellular) neurons ALOXB (300 nM) caused an inward shift of the holding current, with an averaged amplitude of −95.4 ± 22.2 pA at −60 mV in 8 ALOXBresponsive type I neurons (Fig. 4A). Averaged slope conductance of the I–V curves, measured in the potential range between −40 and −70 mV, before and during the application of ALOXB were 4.4 ± 0.9 and 6.1 ± 1.2 nS (P < 0.05), respectively (Fig. 4B). ALOXB-induced current reversed polarity at −27.5 ± 4.8 mV in 5 of the 8 neurons (Table 2) while no reversal was observed in the remaining 3 neurons. 3.5.2. Non-LTS type II (parvocellular neuroendocrine-like) neurons All three types of I–V relationships of ALOXB-induced current were observed in 41 non-LTS type II neurons. The membrane conductance was increased in 22 (53.7%) neurons (3.6 ± 0.6 vs. 7.0 ± 1.2 nS, P < 0.05), decreased in 12 (29.3%) neurons (2.7 ± 0.3 vs. 1.9 ± 0.4 nS, P < 0.05), and unchanged in 7 (17.1%) neurons (2.8 ± 0.4 vs. 2.7 ± 0.5 nS, P > 0.05) during the application of 4

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conductance (Fig. 3A), and at −99.5 ± 5.6 mV (Table 2) in 8 of the 12 neurons that showed decreased conductance (Fig. 3B). No current reversal was observed in the remaining neurons. 3.5.3. LTS type II (parvocellular preautonomic) neurons Three types of I–V relationships were also observed in 45 ALOXBresponsive LTS type II neurons. The slope conductance was increased in 26 (57.8%) neurons (4.2 ± 0.4 vs. 7.8 ± 1.1 nS, P < 0.05), decreased in 11 (24.4%) neurons (3.7 ± 0.9 vs. 2.4 ± 0.9 nS, P < 0.05), and unchanged in the remaining 8 (17.8%) neurons (3.2 ± 0.6 vs. 3.3 ± 0.7 nS, P < 0.05) (Fig. 4B). The amplitudes of responses were −167.1 ± 39.5, −46.2 ± 9.8, and −74.9 ± 30.7 pA at −60 mV, respectively (Fig. 4A). ALOXB-induced current reversed polarity at −25.1 ± 2.7 mV in 16 of the 26 neurons that showed increased conductance while it reversed polarity at −95.1 ± 3.6 mV in 10 out of the 11 neurons that showed decreased conductance (Table 2). No current reversal was observed in the remaining neurons. It is worth noting that the increased-conductance response was the major type of response in the type II (parvocellular) neurons, and it had much bigger amplitude than the decreased- and unchanged-conductance responses. 3.6. Mechanism of negative-slope (or decreased-conductance) response As mentioned above, the decreased-conductance ALOXB response (Fig. 3B) reversed polarity at −99.5 ± 5.6 mV in non-LTS type II neurons and at −95.1 ± 3.6 mV in LTS type II neurons, which were close to the equilibrium potential of K+ (EK+; −94 mV), implying the involvement of K+ conductance. To confirm this notion, the I–V relationship of ALOXB-induced currents was examined in a high-K+ solution containing 7 mM K+. Compared to the response in 3.1 mM K+-containing Krebs solution, ALOXB-induced currents reversed polarity at a more positive voltage range in the high-K+ solution (Fig. 5), as predicted by the Nernst equation for K+ conductance. In other words, the reversal potential shifted in the same direction as EK+ would have shifted in a high-K+ solution. This phenomenon was observed in 6 type II neurons, including 4 non-LTS type II and 2 LTS type II neurons, and the averaged reversal potential was −66.2 ± 3.0 mV. The EK+ in the high-K+ solution was −74 mV. Therefore, a decrease in K+ conductance might be the primary mechanism underlying the negative-slope (or decreased-conductance) ALOXB response.

Fig. 3. Three types of current-voltage (I–V) relationship of ALOXB-induced current in PVN neurons. Panels A, B, and C show representative I–V curves recorded from three individual cells to demonstrate three types of I–V relation of ALOXB-induced current, which exhibited increased (A), decreased (B), and unchanged (C) conductance. Left panels show the steady-state I–V curves before (open circles) and during (filled circles) the superfusion of ALOXB (300 nM). The difference between two curves is shown in the right panels (filled triangles). Representative current traces in response to a series of voltage command steps (from −120 to 0 mV) before and during the application of ALOXB were shown in the insets in the left panels.

ALOXB (Fig. 4B). Amplitudes of ALOXB-induced inward currents at −60 mV were −157.8 ± 39.0, −30.1 ± 6.6, and −36.2 ± 17.2 pA, respectively (Fig. 4A). ALOXB-induced current reversed polarity at −23.5 ± 4.2 mV (Table 2) in 13 out of the 22 neurons that showed increased

3.7. Mechanisms of the positive-slope response Two possible mechanisms have been proposed to mediate orexins-

Table 2 The numbers and properties of three subtypes of PVN neurons showing specified conductance change by ALOXB. Neuron Type

Type I Number of neuron RMP (mV) Reversal potential (mV) Non-LTS Type II Number of neuron RMP (mV) Reversal potential (mV) LTS Type II Number of neuron RMP (mV) Reversal potential (mV)

ALOXB-induced conductance change Increased

Decreased

Unchanged

8 −56.5 ± 3.4 −27.5 ± 4.8 (n = 5)

0 NA NA

0 NA NA

22 −55.7 ± 1.5 −23.5 ± 4.2 (n = 13)

12 −54.4 ± 2.3 −99.5 ± 5.6 (n = 8)

7 −54.4 ± 3.5 NA

26 −57.1 ± 1.7 −25.1 ± 2.7 (n = 16)

11 −57.3 ± 3.1 −95.1 ± 3.6 (n = 10)

8 −58.4 ± 2.0 NA

Data are mean ± S.E.M. RMP: resting membrane potential; NA: not applicable. 5

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Fig. 4. Amplitudes of ALOXB-induced currents and the corresponding changes in slope conductance in different subgroups of PVN neurons. Each of the three major types of PVN neurons (type I, non-LTS type II, and LTS type II) was further classified into subgroups according to their conductance changes in response to ALOXB, including increased-, decreased-, and unchanged-conductance subgroups. Panel A presents the amplitude of ALOXB-induced current of each subgroup of PVN neurons. Negative currents indicate inward currents. Number of cells examined for each subgroup is displayed on the top of each bar. Panel B demonstrates the slope conductance recorded before (open bars) and during (filled bars) ALOXB treatment from each subgroup (referred by an arrow) of PVN neurons. * indicates significant difference at P < 0.05, determined by Student's t-test.

neurons. The results show that the NCX inhibitor did not suppress ALOXB responses in the majority (12 out of 13, 92%) of type II neurons. Together with the finding that over half (54–58%) of type II neurons exhibited a positive-slope I–V relationship in response to ALOXB, it is likely that the NCX activation might not be the primary mechanism in type II neurons. Instead, an increase in non-selective cation current might be the primary mechanism of the ALOXB responses characterized by a positive-slope I–V relationship in type II neurons. On the other hand, both NCX and non-selective cation conductance were likely involved in ALOXB responses in type I neurons.

Fig. 5. Changing extracellular [K+] shifted the reversal potential of ALOXB-induced currents characterized with decreased conductance. Steady-state current-voltage (I–V) curves of ALOXB-induced responses in a normal Krebs solution (containing 3.1 mM K+) and a 7 mM-K+ Krebs solution are shown in left and right panels, respectively.

3.8. Mechanisms of unchanged-slope response: co-expression of two mechanisms The concomitant increased non-selective cation conductance, which mainly contributed by Na+ and K+, and decreased K+ conductance underlying orexins’ excitatory effects have been reported for other brain regions (Hwang et al., 2001; Kolaj et al., 2007; Murai and Akaike, 2005; Yang and Ferguson, 2003). Co-expression of these two mechanisms at different ratios results in different shapes of I–V curves, including the unchanged-slope I–V curve shown in Fig. 3C, and the positive- or negative-slope I–V curves that did not reverse polarity in the potential range examined (Hwang et al., 2001). To confirm these mechanisms, the I–V relationships of the ALOXB responses were examined in normal Krebs (containing 153 mM Na+ and 3.1 mM K+), low-Na+ (26 mM Na+/3.1 mM K+) and high-K+ (153 mM Na+/7.0 mM K+) solutions. I–V relationships of the ALOXB responses were examined in the Krebs solution and then in the low-Na+ or high-K+ solution in each neuron. Fig. 7A shows an increased-conductance I–V curve of ALOXB (300 nM)-induced current in the Krebs solution (black triangle). This I–V curve did not reverse polarity in the entire potential range examined. In a low-Na+ solution, the I–V curve (open triangle) became decreased-conductance and reversed polarity near Ek+. Lowering extracellular concentration of Na+ ([Na+]o) from 153 to 26 mM should largely reduce the magnitude of the non-selective cation conductance, but should not affect K+ conductance. Therefore, this result revealed that the two mechanisms, increasing non-selective cation conductance

induced inward currents characterized by a positive-slope I–V curve: an increase in non-selective cation current, and an activation of an electrogenic Na+-Ca2+ exchanger (NCX) (reviewed by Leonard and Kukkonen, 2014). Involvement of NCX in this type of response was evaluated by examining the effect of an NCX inhibitor, KBR-7943, on ALOXB-induced depolarization. Sixteen ALOXB-responsive PVN neurons were examined including 3 type I, 7 LTS type II, and 6 non-LTS type II neurons. Concentration of KBR-7943 used here is similar to those used in brain slice recordings (Burdakov et al., 2003; Zhang et al., 2011). In 13 of the 16 (81%) neurons, including 1 type I, 7 LTS type II and 5 non-LTS type II neurons, KBR-7943 (70 μM) did not suppress ALOXB (300 nM)-induced depolarization (Fig. 6A). In the remaining 3 neurons, including 2 type I and 1 non-LTS type II neurons, ALOXB-induced depolarization was suppressed by KBR-7943 (Fig. 6B). Fig. 6C shows average changes of membrane potential of the 13 and 3 neurons. A significant interaction between time and different response groups was found [F (2.312, 32.366) = 6.078, P = 0.004, determined by repeatedmeasures ANOVA] and significant difference between groups was detected during the treatment of KBR-7943 (Fig. 6C). In other words, ALOXB responses were suppressed by the NCX inhibitor in 2 out of the 3 type I neurons and 1 out of the 6 non-LTS type II neurons. The NCX inhibitor did not suppress ALOXB responses in any of the 7 LTS type II 6

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Fig. 6. A Na+-Ca2+ exchanger inhibitor, KBR7943, suppressed ALOXB-induced depolarization in the minority of PVN neurons. Panel A, a representative recording trace, observed in 13 of 16 neurons, shows that KBR-7943 (70 μM) did not suppress ALOXB (300 nM)-induced depolarization. Arrows indicate the start of reagent application. Two solid lines below the recording trace indicate the time periods of ALOXB and KBR-7943 application, respectively. A dash line labels the before-treatment level of membrane potential. Panel B, a representative recording trace, observed from the rest 3 neurons, shows the suppression of ALOXBinduced depolarization by KBR-7943. Panel C illustrates average changes of membrane potentials measured from KBR-7943 unaffected (open circles, n = 13) and KBR-7943 affected neurons (filled circles, n = 3). Data are mean ± S.E.M. To analyze differential effects of KBR-7943 on ALOXB responses in the two groups of cells, the data points afterward the reach of the plateau of ALOXB response were analyzed (i.e. the last 11 data points) with repeated-measures ANOVA. Student's t-test was used to determine between-group difference at each time point. * indicates significant difference at P < 0.05.

preautonomic neurons, neurons were intracellularly labelled with Lucifer yellow during patch-clamp recording and then examined for their vasopressinergic phenotypes with immunostaining. Twenty LTS type II neurons were examined. Seven of the 20 (35.0%) neurons were vasopressin-immunoreactive (vasopressin-IR). Fig. 8 shows representative images of a vasopressinergic neuron (upper panel), which was double-labelled with Lucifer yellow and vasopressin-IR, and a nonvasopressinergic neuron (lower panel), which was not double-labelled.

Fig. 7. Effects of extracellular [Na+] and [K+] on ALOXB-induced currents characterized with increased conductance. A, representative increased-conductance I–V curves of ALOXB-induced current in a normal (filled triangles) and a low-Na+ (open triangles) Krebs solutions. Data of the two curves in panel A were recorded from a single neuron. B, representative increased-conductance I–V curves of ALOXB-induced current in a normal (filled triangles) and a high-K+ (open triangles) Krebs solutions. Data of the two curves in panel B were recorded from a single neuron.

(namely the cation mechanism for short) and decreasing K+ conductance (the K+ mechanism), might underlie the ALOXB response in this neuron. The existence of the K+ mechanism might be the reason of no reversal in the I–V curve in the Krebs solution. Similar observations were detected in one non-LTS type II and two LTS type II neurons. Fig. 7B shows an increased-conductance I–V curve of the ALOXBinduced current, which had a reversal potential around −10 mV in the Krebs solution. The I–V relationship of the ALOXB-induced current in a high-K+ solution was similar to the one obtained in the Krebs solution, except for a smaller amplitude. The results suggest that the cation mechanism was the major mechanism of the ALOXB response in this neuron. Similar observations were made in one non-LTS type II and one LTS type II neurons. Changing extracellular concentration of K+ ([K+]o) affects the reversal potentials of both the K+ and cation mechanisms. The effects of increasing [K+]o on the I–V relationship of the ALOXB responses underlain by the co-expression of both mechanisms are complicated, and therefore not evaluated.

Fig. 8. Photomicrographs of a vasopressinergic and a non-vasopressinergic LTS-expressing type II neurons in the PVN. LTS-expressing type II neurons were intracellularly labelled with Lucifer yellow during electrophysiological recording and later processed for vasopressin immunostaining. Left panel shows low-magnification micrographs of two Lucifer yellow-labelled neurons (green) in the PVN. Middle panel illustrates vasopressin-immunoreactive (vasopressin-IR) neurons (red) of the same area shown in the left panel. Higher magnification micrographs of the Lucifer yellow-filled and vasopressin-IR neurons are shown in the right panels. The upper and lower panels illustrates a double-labelled and a non-double-labelled neurons, respectively. V, third ventricle. Dash lines denote approximate ranges of the PVN. Arrowheads indicate the locations of Lucifer yellow-filled neurons.

3.9. Vasopressinergic preautonomic neurons To examine whether ALOXB affects the activity of vasopressinergic 7

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Ferguson, 2002) of type I neurons. The percentage of orexins-responsive type I neurons from these two studies were higher than the ratio in the present study (55.9%). We did not explore reactions of PVN neurons to ALOXB in the absence of TTX, but speculate that orexins may excite PVN type I neurons through both presynaptic and postsynaptic actions, which echoes Follwell and Ferguson's (2002) implication of orexin A-mediated depolarization in type I neurons. In terms of parvocellular neurons, results from two previous studies (Follwell and Ferguson, 2002; Shirasaka et al., 2001) and our present study agree upon the postsynaptic effects of orexins. However, we further explored the orexin-induced effects on classified non-LTS type II neurons and LTS type II neurons. Our result showed that ALOXB excites preautonomic neurons directly with a high response rate and large amplitudes of membrane depolarization, suggesting a robust influence of orexins in regulating the autonomic functions through the PVN. In addition to the postsynaptic effects of orexins on PVN parvocellular neurons, the effect of orexins on presynaptic neurotransmitter release cannot be ignored. Regulation of the activity of PVN neurons highly depends on GABAergic and glutamatergic innervations, as well as angiotensin and nitric oxide signals (see Ferguson et al., 2008 for a review). Blockage of Ca2+ channel with Cd2+ in the bath solution significantly reduced orexin B-induced depolarization in parvocellular neurons (Shirasaka et al., 2001), indicating that orexin B-evoked excitation was caused in part by Cd2+-sensitive Ca2+ channels, which may contribute to the release of neurotransmitters from presynaptic nerve terminals.

Five of the 7 (71.4%) vasopressinergic LTS type II neurons were depolarized by ALOXB (300 nM) while the other 2 neurons were not responsive to ALOXB. 4. Discussion The present study demonstrates that OX2 receptor ligands depolarized the three types of PVN neurons through direct actions on these neurons. The type II (putative parvocellular) neurons responded to ALOXB in a higher ratio (73–75%) than the type I (putative magnocellular) neurons did (56%), and the LTS type II (putative parvocellular preautonomic) neurons had a greater change of membrane potential when compared with the other two types. The depolarizations were mediated by promoting NCX activity and non-selective cation conductance in magnocellular neurons. In parvocellular neurons, enhanced non-selective cation conductance and decreased K+ conductance were likely the primary mechanism. In addition, about 35.0% of LTS type II (putative preautonomic) neurons were vasopressinergic. Approximate 71% of vasopressinergic LTS type II neurons responded to ALOXB (300 nM), indicating that the majority of vasopressinergic preautonomic neurons are under OX2 receptor regulation. 4.1. Cell identification Hoffman et al. (1991) reported that the majority (80%) of type I neurons are neurophysin-immunoreactive (i.e. vasopressin- or oxytocin-expressing) magnocellular neurons. In line with this finding, Luther et al. (2002) found that 89% of type I neurons were neuroendocrine neurons, confirmed by retrograde labeling. We examined the phenotypes of 2 and 4 type I neurons for oxytocin and vasopressin, respectively. Two ALOXB-responsive type I neurons were identified as oxytocin-immunoreactive, while 2 of 4 ALOXB-responsive type I neurons were vasopressin-immunoreactive (data not shown). The type II neurons represent a more diverse combination of subpopulations of neurons, including neuroendocrine neurons and neurons that project to the medulla and/or the spinal cord (Armstrong et al., 1980; Swanson and Kuypers, 1980). Stern (2001) identified PVN preautonomic neurons by combing in vivo retrograde tracing techniques with in vitro patch-clamp recordings of the labelled neurons. His result demonstrated that 88% of PVN preautonomic neurons express LTS. Luther et al. (2002) used intravenous injection of the retrograde tracer, fluoro-gold, to label all PVN neuroendocrine neurons. They found that all labelled type II neurons (i.e. parvocellular neuroendocrine neurons) showed no LTS, while about 74% of non-labelled type II neurons (i.e. non-neuroendocrine neurons) displayed LTS, consistent with the findings from Stern (2001). Accordingly, LTS type II neurons are preautonomic neurons while the majority of non-LTS type II neurons are neuroendocrine neurons. The locations of the three types of PVN neurons recorded in the present study are consistent with locations of magnocellular neurosecretory neurons, parvocellular preautonomic neurons, and parvocellular neurosecretory neurons reported in the literature (Armstrong et al., 1980; Luther et al., 2002; Sawchenko and Swanson, 1982; Swanson and Kuypers, 1980).

The cellular mechanisms underlying orexins-induced depolarization have been reviewed by Leonard and Kukkonen (2014). The orexin-induced depolarization can be attributed to three categories: decreased K+ conductance, activation of an electrogenic NCX and enhanced nonselective cation conductance. All three mechanisms were detected in the PVN. However, the primary mechanisms of OX2 receptor responses were different in the magnocellular and parvocellular neurons. In all type I (putative magnocellular) neurons, the ALOXB response exhibited an increased-conductance I–V relationship. The ALOXB response was attenuated by the NCX inhibitor in 2 of 3 type I neurons, implying that both NCX and non-selective cation conductance are involved in OX2 receptor-mediated membrane depolarization in putative magnocellular PVN neurons. Based on the mechanisms of the OX2 receptor-mediated excitatory responses, both LTS type II (putative preautonomic) and non-LTS type II (putative parvocellular neuroendocrine) neurons should be classified into three subpopulations. The percentages of the three subpopulations are similar between preautonomic and neuroendocrine neurons. Increasing non-selective cation conductance mediated the response in 54–58% of parvocellular neurons, while decreasing K+ conductance is likely to be the primary mechanism of the OX2 receptor-mediated responses in approximately 17–18% of parvocellular neurons. Co-exist of two mechanisms may underlie the responses in the rest of neurons. The functional roles of the three subpopulations of neurons remain to be determined.

4.2. Excitatory effects of selective OX2 receptor agonists on PVN neurons

4.4. Vasopressinergic preautonomic neurons

Our results indicate direct effects of ALOXB on PVN type I neurons, consistent with the results form Shirasaka et al. (2001) that the orexin B-induced excitatory effects were generated postsynaptically. Conversely, Follwell and Ferguson's study (2002) suggested that orexin Amediated depolarization in type I neurons was an indirect effect, resulting from an increase in glutamatergic synaptic input and the modulation of a postsynaptic receptor causing an increase in EPSC amplitude. Interestingly, in the absence of TTX, orexin B depolarized 80.8% (Shirasaka et al., 2001), and orexin A depolarized 67.0% (Follwell and

PVN preautonomic neurons could be peptidergic, expressing multiple neurotransmitters (Hallbeck and Blomqvist, 1999; Hallbeck et al., 2001; Palkovits, 1999). Among these neurotransmitters, vasopressinergic projections to the brainstem and the spinal cord have been implicated in modulating cardiovascular functions (Kc et al., 2010; Malpas and Coote, 1994). Injections of vasopressin into the RVLM induced a pressor response and increased heart rate (Andreatta-Van Leyen et al., 1990). About 15% of the RVLM-projecting PVN neurons are vasopressinergic, and vasopressin released from PVN-RVLM projections

4.3. Mechanisms of OX2 receptor activation on PVN neurons

8

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Acknowledgements

modulates cardiorespiratory output (Kc et al., 2010). As for the spinally projecting neurons, more than 40% of them express vasopressin mRNA (Hallbeck and Blomqvist, 1999). Intrathecal administration of vasopressin increased renal sympathetic nerve activity, blood pressure, and heart rate (Malpas and Coote, 1994; Porter and Brody, 1986; Riphagen and Pittman, 1986; Yang et al., 2002). Intriguingly, intrathecal administration of vasopressin V1 receptor antagonist blocks the sympathoexcitatory and hemodynamic responses induced by either intrathecal vasopressin or by the stimulation of PVN (Malpas and Coote, 1994). Further studies demonstrate that PVN stimulation-caused sympathoexcitatory responses are mainly mediated by glutamatergic or vasopressinergic transmission in the spinal cord (Yang et al., 2002). In the present study, about 35.0% of the LTS type II (preautonomic) neurons were vasopressinergic, which might include both brainstemand spinal cord-projecting neurons. The percentage is reasonable when compared with the findings that 15% of PVN-RVLM projecting neurons (Kc et al., 2010) and 40% of PVN-spinal cord projecting neurons (Hallbeck and Blomqvist, 1999) are vasopressinergic, as we took both PVN-brainstem and PVN-spinal cord projecting neurons into account. We also found that the majority (71.4%) of vasopressinergic LTS type II neurons responded to ALOXB, implying the importance of the OX2 receptor-mediated regulation on PVN vasopressinergic outflows to control autonomic and cardiovascular functions.

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5. Conclusion and perspectives In conclusion, orexins may directly excite the three major types of PVN neurons through OX2 receptor, by promoting NCX activity and non-selective cation conductance in magnocellular neurons and by affecting non-selective cation conductance and K+ conductance in parvocellular neurons. The PVN plays essential roles in the homeostatic control of neuroendocrine and autonomic functions (Guyenet, 2006; Swanson and Sawchenko, 1980). Accordingly, OX2 receptor activation may initiate cellular signals on different PVN neurons and result in diverse physiological consequences. In line with this notion, previous studies have demonstrated that orexins participate in regulating the hypothalamic-pituitary-adrenal axis, PVN vasopressin expression (AlBarazanji et al., 2001; Kuru et al., 2000; Samson and Taylor, 2001), activities of PVN oxytocin neurons (Maejima et al., 2017), and sympathetic activity (Fan et al., 2018). Although not all of these studies validate the PVN as the action site of orexin's regulatory effects, anatomical and functional connections between orexinergic terminals and spinally projecting PVN neurons have been validated (Dergacheva et al., 2017). The fact that changes of the orexinergic activities in the PVN are associated with prevalent cardiovascular diseases, such as hypertension (Huber et al., 2017; Zhou et al., 2015, 2017) and heart failure (Kang et al., 2014) further highlight the importance of orexinergic regulation in the PVN. Little is known about the underlying mechanisms of orexins’ actions on functional subpopulations of PVN magnocellular and parvocellular neurons. It is of interest that the mechanisms of OX2 receptor effects are different between magnocellular and parvocellular neurons; furthermore, there appears to be subsets of neurons regulated by orexins through different mechanisms in each types of PVN neurons. Whether these subsets of neurons represent different functional subpopulations of PVN neurons remains to be elucidated. Funding This study was supported by the Ministry of Science and Technology, Taiwan (NSC102-2320-B-038-034-MY3; MOST105-2320B-038-060; MOST 107-2320-B-038-053). Declaration of competing interest None. 9

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