Effects of Stresscopin on Rat Hypothalamic Paraventricular Nucleus Neurons In Vitro

The effects of stresscopin (SCP) on rat paraventricular nucleus (PVN) neurons were examined using whole-cell patch-clamp recordings and single-cell reverse-transcription multiplex polymerase chain reaction (SC-RT-mPCR) techniques. Under current-clamp conditions, bath application of SCP (100 nM) induced inhibition in 35.2% (37/105) of putative magnocellular neurons and 24.7% (20/81) of putative parvocellular neurons, and excitation in 5.7% (6/105) of putative magnocellular neurons and 18.5% (15/81) of putative parvocellular neurons. SCP-induced inhibition persisted in the presence of a mixture of TTX, a voltage-gated Na+ channel blocker, CNQX, an AMPA/kainate receptor antagonist and bicuculline, a GABAA receptor antagonist, whereas SCP-induced excitation of PVN neurons was reversed by the mixture. The SCP-induced inhibition of PVN neurons was abolished by bath application of antisauvagine-30, a selective CRF receptor 2 (CRF-R2) antagonist. Under voltage-clamp conditions, SCP evoked outward currents at the holding potential (−60 mV), which reversed near the potassium equilibrium potential. The SCP-evoked membrane currents were completely blocked by bath application of tertiapin-Q, a selective blocker of G protein-activated inwardly rectifying potassium (GIRK) channels. SC-RT-mPCR analysis indicated that all the SCP-sensitive PVN neurons (57 SCP-inhibited neurons, 21 SCP-excited neurons) expressed CRF-R1 and CRF-R2 mRNAs. Among SCP-hyperpolarized PVN neurons, oxytocin (OT) mRNA was detected in 91.8% of putative magnocellular neurons and 45.0% of putative parvocellular neurons. OT mRNA was also detected in 26.6% of SCP-depolarized parvocellular neurons, but not in SCP-depolarized magnocellular neurons. These results indicate that SCP inhibits a subpopulation of PVN neurons, especially OTergic magnocellular neurons, by enhancing the activity of GIRK channels via CRF-R2.


Introduction
Corticotropin-releasing factor (CRF), a 41-amino acid peptide, is synthesized and secreted in many regions of the central nervous system, and plays a key role in the coordination of endocrine and behavioral responses to stress. Human urocortin III, also known as stresscopin (SCP), is a 38-amino acid peptide of the CRF family [1,2]. Two G-protein-coupled receptors, termed CRF receptor 1 (CRF-R1) and CRF receptor 2 (CRF-R2), have been identified as CRF receptors [3]. SCP is presumed to be a specific ligand for CRF-R2, and binds and activates the receptor [1,3,4]. In vitro binding studies have shown that SCP binds CRF-R2 with high affinity but has minimal affinity for CRF-R1, and it stimulates cAMP production in cells expressing CRF-R2, but not in cells expressing CRF-R1 [1,2]. In contrast, CRF binds with high affinity to CRF-R1 [5]. CRF-R1 is highly expressed in the anterior pituitary, olfactory bulb, neocortex, hippocampus, amygdala and cerebellum [6]. CRF-R2 (a) is expressed mainly in the hypothalamic ventromedial nucleus and paraventricular nucleus (PVN), medial amygdaloid nucleus and lateral septic nucleus of the brain [7], and both CRF-R1 mRNA and CRF-R2 mRNA are expressed in putative parvocellular neurons in the PVN of rats [8].
The distribution of SCP-containing neurons and fibers in the rat brain has been investigated by in situ hybridization and immunohistochemistry [1,2,4]. SCP mRNA is expressed in major regions of the brain, including the rostral perifornical area of the hypothalamus, the lateral septum and the medial amygdaloid nucleus [1,2]. In the hypothalamus, SCP-positive neurons are observed in the Median Preoptic Nucleus of rats [4], which project heavily to neurosecretory neurons of the PVN and the supraoptic nucleus [9,10], suggesting that SCP may regulate the function of PVN neurosecretory neurons.
Central administration of SCP stimulates the hypothalamicpituitary-adrenal axis, elevating levels of plasma adrenocorticotropic hormone. It also suppresses feeding [11][12][13][14], elevates blood glucose levels [11,15], and elicits transient increases in mean arterial blood pressure and heart rate [16,17]. Microinjection of SCP into the PVN induces significant increases in systemic blood pressure, heart rate and renal sympathetic nerve activity via CRF-R2 [18]. Although numerous studies indicate that central SCP modulates neurosecretory and cardiovascular function, the underlying mechanisms are currently unclear.
The PVN consists of magnocellular neurons, neurosecretory parvocellular neurons and non-neurosecretory preautonomic parvocellular neurons, which play a critical role in the regulation of stress responses and neurosecretory and autonomic functions [9,19]. We investigated the effects of SCP on PVN neurons using whole-cell patch-clamp recordings, as well as pharmacological and SC-RT-mPCR techniques in vitro in rats. We found that SCP induced both inhibition and excitation in PVN neurons. SCPinduced inhibition persisted in the presence of a mixture of TTX, CNQX and bicuculline, whereas SCP-induced excitation of PVN neurons was reversed by bath application of the mixture. The SCP-induced inhibition was blocked by a selective CRF-R2 antagonist. The SCP-induced membrane currents reversed near the potassium equilibrium potential, and were blocked by a selective blocker of GIRK channels. SC-RT-mPCR analysis indicated that all the SCP-sensitive PVN neurons expressed CRF-R1 and CRF-R2 mRNA. Oxytocin (OT) mRNA was detected in 91.8% of putative magnocellular neurons, 45.0% of putative parvocellular neurons and 26.6% of SCP-depolarized parvocellular neurons.

Effects of SCP on membrane potential
A total of 186 PVN neurons (56 rats) were recorded using the whole-cell patch-clamp recording technique. In addition, these cells were screened for GAPDH, CRF-R1, CRF-R2 and OT mRNA using the single-cell RT-mPCR method. Under currentclamp recording conditions, these PVN neurons were classified as either putative magnocellular neurons (105/186; Fig. 1A) or putative parvocellular neurons (81/186; Fig. 2A) according to previously established criteria [20,21]. The effects of bath application of SCP for 90 s on both membrane potential and spike firing frequency of PVN neurons were examined after a 100s stable baseline recording period. The application of SCP in concentrations ranging from 10 nM to 600 nM resulted in hyperpolarization of membrane potential and a decrease in spike firing rate in 35.2% (37/105) of putative magnocellular neurons and 24.7% (20/81) of putative parvocellular neurons, but without changing the characteristics of the action potentials (not shown). The hyperpolarization appeared at approximately 60 s and peaked at approximately 200 s after SCP application, with a mean peak value of 4.3860.42 mV (Fig. 1B, C). The minimum dose of SCP required to elicit a significant effect on membrane potential was 10 nM, and the maximum dose was approximately 600 nM. To determine whether the SCP-induced hyperpolarization was due to a direct effect of SCP on PVN neurons, the amplitude of the SCP-induced hyperpolarization of membrane potential was determined in the absence or presence of a mixture of TTX (0.5 mM), CNQX (10 mM) and bicuculline (10 mM). In six SCP-inhibited PVN neurons, SCP induced a reversible hyperpolarization of membrane potential ( Fig. 1B; upper). Following bath application of the mixture of TTX, CNQX and bicuculline for 10 min, reapplication of SCP induced a hyperpolarization of 4.4160.48 mV, which was similar to SCP-induced hyperpolarization under control conditions (4.7160.44 mV; P = 0.65; n = 6; Fig. 1B, C). The SCP-induced hyperpolarization was dosedependent; the half maximal inhibitory concentration (IC 50 ) was approximately 54 nM (Fig. 1D). These data indicate that bath application of SCP directly hyperpolarizes a subpopulation of PVN neurons. However, SCP (100 nM) induced depolarization of membrane potential (3.1660.15 mV; n = 21) and an increase in spike firing rate in 5.7% (6/105) of putative magnocellular neurons and 18.5% (15/81) of putative parvocellular neurons ( Fig. 2A, B). The frequency of spike firing increased from 0.1960.08 Hz to 0.8160.17 Hz (n = 21; P = 0.002; Fig. 2C). Intriguingly, bath application of a mixture of TTX, CNQX and bicuculline abolished the SCP-elicited depolarization of membrane potential and revealed the SCP-induced hyperpolarization of membrane potential in these neurons. In the presence of TTX, CNQX and bicuculline, SCP induced a hyperpolarization of membrane potential, with a mean value of 3.3160.22 mV (n = 21), which was weaker than the evoked hyperpolarization of membrane potential in SCP-inhibited neurons (4.4160.48 mV; n = 6; P = 0.04; Fig. 2D), suggesting that SCP-induced presynaptic effects overwhelmed the direct effect of SCP on the postsynaptic membrane. These results indicate that the SCP-elicited excitation of a subpopulation of PVN neurons (due to bath application of the peptide) was likely achieved indirectly through a circuitry effect. Indeed, SCP hyperpolarized SCP-excited neurons directly through the postsynaptic membrane.

SCP-induced inhibition of PVN neurons is mediated by CRF-R2
CRF receptors are classified as CRF-R1 or CRF-R2 [3]. CRF has high affinity for CRF-R1, whereas SCP is presumed to be a specific ligand for CRF-R2 [1,3,4]. Our results show that bath application of SCP directly hyperpolarizes a subpopulation of PVN neurons, and this effect may be mediated by CRF receptors, especially CRF-R2. To examine the pharmacological profile of SCP-induced inhibition mediated by CRF receptors, a CRF receptor nonselective antagonist, a-helical CRF-(9-14), or a selective and competitive CRF-R2 antagonist, antisauvagine-30, was applied to the SCP-inhibited neurons under current-clamp conditions [22,23]. Bath application of a-helical CRF prevented the SCP-induced hyperpolarization of membrane potential in SCP-inhibited neurons (SCP: 24.1160.42 mV; SCP + a-helical CRF: 0.6160.39 mV; P = 0.004; n = 8; not shown). Since SCP is presumed to be a specific ligand for CRF-R2 [1,3,4], we hypothesized that SCP-elicited inhibition of PVN neurons is mediated by CRF-R2. To test this, we further examined the effects of SCP on neuronal excitability in the presence or absence of antisauvagine-30 (

SCP enhances GIRK channel activity under voltage-clamp
Under voltage-clamp, the effects of SCP (300 nM) on SCPsensitive PVN neurons were examined using steady-state (2140 to 40 mV, 20 mV/s; V hold = 260 mV) whole-cell current-voltage (I-V) relationships. The I-V relationships revealed that 300 nM SCP evoked an outward current at the holding potential (260 mV) that was associated with an increase in membrane conductance (Fig. 4A). The reversal potential of the evoked currents was 295.662.31 mV (n = 6; Fig. 4A, subtraction), which was near the potassium equilibrium potential calculated using the Nernst equation (E k = 298.7 mV). When neurons were held at 260 mV, SCP induced an increase in outward current; the normalized membrane current was 130.563.7% of baseline (P = 0.023; n = 12). These results suggest that bath application of SCP induces an increase in membrane current through potassium channels.
Since CRF receptors are G protein-coupled receptors and SCP induces hyperpolarization by enhancing the activity of potassium channels, it is possible that SCP could hyperpolarize PVN neurons by activating G protein-activated inwardly rectifying potassium (GIRK) channels. To test this hypothesis, we applied a nonspecific blocker of GIRKs, BaCl 2 , or a specific blocker, tertiapin-Q [24]. When neurons were held at 260 mV, SCP (300 nM) induced an increase in outward current, elevating it to 129.664.1% of baseline (P = 0.036; n = 6). Bath application of Ba 2+ (0.2 mM) for 5 min completely blocked the SCP-induced increase in outward current (data not shown). Since Ba 2+ is a relatively specific blocker of inwardly rectifying K + channels (IRKs) and GIRKs at a concentration of 0.2 mM [24], we employed a selective blocker of GIRK channels, tertiapin-Q (200 nM). In neurons, tertiapin-Q blocks GIRK channels, with  little effect on IRK channels [25]. Under voltage-clamp, the I-V relationships revealed that tertiapin-Q blocked the SCP-evoked membrane current (Fig. 4B, subtraction). When the neurons were held at 260 mV, bath application of tertiapin-Q induced a decrease in membrane current by 92.961.6% of baseline (P = 0.042; n = 6) and completely blocked the SCP-induced increase in the outward current (SCP: 131.365.0% of baseline; tertiapin-Q + SCP: 94.462.1% of baseline; n = 6; P = 0.007; Fig. 4C). These results indicate that extracellular blockade of GIRK channels prevents the SCP-induced increase in membrane current, suggesting that SCP-induced hyperpolarization of membrane potential was mediated by the activation of GIRK channels. In addition, application of tertiapin-Q blocked the activity of GIRK channels, resulting in a significant decrease in membrane current. This indicates that GIRK channels are constitutively activated at a holding potential of 260 mV [26,27].
CRF augments I H via CRF-R1 [21,28,29]. Therefore we examined the effects of SCP on I H under voltage clamp conditions. In the presence of TTX (0.5 mM), bath application of SCP (300 nM) produced an outward current (30.864.6 pA; n = 7) at a holding potential of 260 mV. When a series of 1-s hyperpolarizing voltage steps from 260 mV to 2130 mV were applied, SCP induced an increase in instantaneous current (I Ins ) and steady-state current (I SS ), which were reversed at 92.464.36 mV (I Ins ; n = 7; Fig. 5A, B) and 94.363.11 mV (I SS ; n = 7; Fig. 5A, C), respectively. The reversal potentials of I Ins and I SS were near the potassium equilibrium potential calculated by the Nernst equation (E k = 298.7 mV). However, SCP did not change the amplitude of the I H (I SS -I Ins ) current (Fig. 5D); the normalized values of I H   (Fig. 5E). These results indicate that SCP augments the activity of GIRK channels without affecting the I H current.

SCP-inhibited PVN neurons express OT mRNA
After completion of electrophysiological recordings, the cytoplasm was aspirated into the patch pipette by the application of a gentle negative pressure, and first-strand cDNA was synthesized. All the SCP-sensitive neurons were screened for GAPDH (positive control), CRF-R1, CRF-R2 and OT mRNA using the SC-RT-mPCR technique. Screening of rat hypothalamic total RNA (positive control) resulted in detection of all the specific mRNAs, each corresponding to the size predicted by its mRNA sequence. The SC-RT-mPCR analysis indicated that all the SCP-sensitive neurons expressed CRF-R1 and CRF-R2 mRNAs (Fig. 6). Among the PVN neurons directly hyperpolarized by SCP, OT mRNA was detected in 91.8% (34/37) of putative magnocellular neurons and 45.0% (9/20) of putative parvocellular neurons (Fig. 6). In comparison, OT mRNA was detected in only 26.6% (4/15) of SCP-depolarized parvocellular neurons, and not at all in SCPdepolarized magnocellular neurons.

Discussion
SCP, also known as human urocortin III, is considered a specific ligand for CRF-R2. In vitro studies have shown that SCP binds CRF-R2 with high affinity [1,2]. Although the actions of SCP have been investigated for some time, this is the first study, to our knowledge, examining the effects of the peptide on PVN neuronal membrane potential and currents. In this study, we found that bath application of SCP induced inhibition in 35.2% of putative magnocellular neurons and 24.7% of putative parvocellular neurons, and excitation in 5.7% of putative magnocellular neurons and 18.5% of putative parvocellular neurons. SCP-induced inhibition, but not excitation, persisted in the presence of a mixture of TTX, CNQX and bicuculline. The SCP-mediated inhibition was blocked by a selective CRF-R2 antagonist. Under voltage-clamp, SCP evoked outward currents that reversed near the potassium equilibrium potential and that could be blocked by the selective GIRK channel blocker. SC-RT-mPCR analysis indicated that all SCP-sensitive PVN neurons expressed CRF-R1 and CRF-R2 mRNA. Approximately 70% of SCP-hyperpolarized neurons expressed OT mRNA. These results indicate that SCP inhibits a subpopulation of PVN neurons, especially OTergic magnocellular neurons, by enhancing the activity of GIRK channels via CRF-R2.
Both mouse and human SCP are highly selective for CRF-R2 and exhibit low affinities for CRF-R1 [1,3,4]. In the brain, CRF-R2 (a) is expressed mainly in the hypothalamic PVN, medial amygdaloid nucleus and lateral septic nucleus of the brain [7]. We previously found that PVN neurons express CRF-R1 and CRF-R2  mRNAs, and that CRF depolarizes a subpopulation of PVN parvocellular neurons via CRF-R1 [8]. In this study, our SC-RT-mPCR results confirmed that SCP-sensitive neurons co-express CRF-R1 and CRF-R2 mRNAs, suggesting that CRF-R1 and CRF-R2 receptors are both present in SCP-inhibited PVN neurons. Furthermore, the SCP-elicited hyperpolarization of membrane potential in PVN neurons was blocked by the CRF-R1 and CRF-R2 non-selective antagonist, a-helical CRF [22], and by a selective CRF-R2 antagonist, antisauvagine-30 [23]. This indicates that the SCP-induced hyperpolarization was mediated by CRF-R2. Our results are consistent with in vitro binding studies showing that SCP binds CRF-R2 with high affinity, but has minimal affinity for CRF-R1 [1,2]. This suggests that the direct inhibition of the subpopulation of PVN neurons by SCP is mainly mediated through CRF-R2.
PVN neurons receive glutamatergic and GABAergic afferents from a number of forebrain structures, including the Median Preoptic Nucleus, the subfornical organ, the Organum Vasculosum of the Lamina Terminal, as well as brainstem nuclei [19] and other intrahypothalamic nuclei [30]. In the present study, SCP induced excitation in 5.7% of putative magnocellular neurons and 18.5% of putative parvocellular neurons. Interestingly, the SCPinduced depolarization was reversed by the mixture of TTX, CNQX and bicuculline, suggesting that SCP might excite a subpopulation of PVN neurons through an indirect pathway; for example, through activation of glutamatergic interneurons or inhibition of GABAergic interneurons. Activation of glutamatergic interneurons can increase glutamate release onto PVN neurons, resulting in membrane depolarization and an increase in spike firing rate. However, the vast majority of local synaptic inputs onto PVN neurons are GABAergic [31]. Despite additional excitatory glutamatergic inputs [32], PVN neurons receive tonic GABAergic inhibition, which maintain low rates of spontaneous firing [33,34]. Inhibition of GABAergic interneurons can reduce tonic and phasic inhibition of PVN neurons, resulting in membrane depolarization and an increase in firing rate.

SCP promotes the activation of GIRK channels
The GIRK channels, which are members of the IRK channel family, are directly activated by G proteins and are considered to play a critical role in the inhibitory regulation of neural activity. Electrophysiological studies have shown that a variety of G protein-coupled receptors, including M 2 -muscarinic, D 2 -dopamine, GABA B , opioid and somatostatin receptors in the brain, interact with GIRK channels to induce inhibitory postsynaptic potentials [35]. In the hypothalamic PVN, pharmacological activation of GABA B receptors produces inhibition of magnocellular neurons via activation of GIRK channel conductance [36]. Bath application of the a 2 -adrenoceptor agonist dexmedetomidine inhibits PVN magnocellular neurons through activation of GIRKs and suppression of hyperpolarization-activated currents [37]. In this study, I-V relationships revealed that SCP evoked outward currents when membrane potential was held at 260 mV, and the reversal potential of the evoked currents was near the potassium equilibrium potential. These results indicate that bath application of SCP induces an increase in outward currents through potassium channels. Furthermore, the SCP-evoked increase in outward currents was completely blocked by bath application of Ba 2+ , a non-selective blocker of IRK and GIRK channels [24,37]. Since both CRF-R1 and CRF-R2 are G protein-coupled receptors, it is most likely that SCP hyperpolarizes PVN neurons by enhancing GIRK channel activity. Collectively, our results show that blockade of GIRK channels prevents the SCP-induced increase in membrane currents, suggesting that SCP activates GIRK channels via CRF-R2, resulting in the hyperpolarization of membrane potential. Further studies are required to clarify the signaling pathways and molecular mechanisms underlying the SCP-induced activation of GIRK channels.

Physiological significance
The PVN is a complex heterogeneous region consisting of magnocellular neurons and parvocellular neurons. It has the critical task of integrating endocrine and autonomic functions, including the stress response and autonomic control of cardiovascular activity [19]. PVN magnocellular neurons integrate incoming information and secret OT and vasopressin from their nerve terminals in the posterior pituitary, whereas PVN parvocellular neurons comprise neuroendocrine neurons and pre-autonomic neurons [9]. In the hypothalamus, SCP-positive neurons are observed in the Median Preoptic Nucleus of rats, suggesting that SCP secreted from this region may regulate the neurosecretory function of PVN neurons via CRF-R2. Our present results show that SCP mediates inhibition of OTergic neurons by enhancing GIRK channel activity via CRF-R2. OT plays an important role in mediating stress responses. Various stressors, such as force swimming stress, shaker stress and chronic homotypic stress, increase OT mRNA expression or OT secretion in the PVN [38][39][40]. Increased levels of OT might modulate the stress response by attenuating stress-induced activation of the hypothalamus-pituitary-adrenal axis [41,42]. Central administration of OT has been shown to attenuate stress-induced corticosterone release and create an anxiolytic effect in rats [42,43]. Intracerebroventricular injection of OT significantly attenuates the increase in CRF mRNA expression in the PVN induced by restraint stress [40]. In the present study, we found that SCP inhibits OTergic neuronal activity via CRF-R2, whereas CRF depolarizes OTergic neurons by enhancing I H channel activity via CRF-R1 [21].
PVN parvocellular neurons comprise neuroendocrine neurons and pre-autonomic neurons, including CRF-secreting neurons and spinally projecting neurons, which play a critical role in the regulation of endocrine and autonomic functions [9,19]. Approximately 25% of PVN parvocellular neurons are directly inhibited by SCP through enhancement of GIRK channel activity via CRF-R2, suggesting that SCP might attenuate stress and autonomic responses. In addition, the vast majority of local synaptic inputs to PVN neurons are GABAergic [31]. PVN neurons receive tonic GABAergic inhibition, which maintain their low rates of spontaneous spiking [33,34]. Our results demonstrate that SCP indirectly depolarizes a subpopulation of PVN neurons, suggesting that SCP also directly inhibits GABAergic interneurons via CRF-R2, resulting in a decrease in tonic and phasic inhibition of PVN neurons. The decrease in tonic inhibition of spinally projecting sympathetic pre-autonomic neurons likely results in an increase in sympathetic outflow [44]. Consequently, central administration of SCP transiently increases mean arterial blood pressure, heart rate and epinephrine release [16]. Furthermore, microinjection of SCP into the PVN significantly increases systemic blood pressure, heart rate and renal sympathetic nerve activity via CRF-R2 [18].

Hypothalamic slice preparation
Hypothalamic slices were prepared from P12-14 male Wistar rats, as previously described [21,45]. All experiments were approved by the Ethics Committee of Miyazaki University or the Animal Care and Use Committee of Jilin University, and were in accordance with international guidelines on the ethical use of animals in laboratory experiments. In brief, the brain was immediately placed into ice-cold oxygenated ACSF containing the following (in mM): 140 NaCl, 3 KCl, 1.3 MgSO 4 , 1.4 NaH 2 PO 4 , 5 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 11 D-glucose, 2.4 CaCl 2 and 3.25 NaOH. The pH of the ACSF was 7.3, the osmolarity was 290-300 mOsm/L, and the solution was bubbled with 100% O 2 . Coronal hypothalamic slices (250 mm thick) were prepared using a vibrating brain slicer (DSK-2000; Dosaka, Kyoto, Japan). The slices were incubated for at least 1 hour in a chamber filled with equilibrated ACSF at room temperature (24-26uC) before electrophysiological recordings were started.

Electrophysiology
Patch pipettes were made from thick-wall borosilicate glass (GD-1.5; Narishige) using a puller (PB-7; Narishige, Tokyo, Japan). They were filled with a solution consisting of (in mM) 130 potassium gluconate, 10 HEPES, 10 KCl, 5 EGTA, 1 CaCl 2 , 1 MgCl 2 , 2 Na 2 ATP and 0.5 Na 3 GTP. The pH was adjusted to 7.2 with KOH. Patch pipette resistances were 5-7 MV in the bath, with series resistances in the range of 10-20 MV. Membrane potentials and/or currents were monitored with an Axopatch 200B amplifier or an Axopatch 21D amplifier (Molecular Devices, Foster City, CA, USA), filtered at 5 kHz, and acquired through a Digidata 1200 series analog-to-digital interface on a personal computer using Clampex 7.0 or 8.1 software (Molecular Devices). Whole-cell recordings from PVN neurons were made from microscopically identified cells. Once stable recording conditions were obtained, PVN neurons were identified electrophysiologically as type I (magnocellular) or type II (parvocellular), according to previously established criteria by current-clamp in standard ACSF; type-I neurons displayed transient outward rectification, while type-II neurons did not [20,21]. All the SCPsensitive neurons were recorded from individual PVN slices.

Cytoplasm harvesting and reverse transcription
Harvesting of cytoplasm and reverse transcription were carried out as previously described [21,45]. After whole-cell recording, the cytoplasm was aspirated into the patch pipette by the application of a gentle negative pressure in the pipette while maintaining the tight seal. First strand cDNA was synthesized for 1 h at 42uC. The single-cell cDNA was kept at 270uC until PCR amplification.

Multiplex and nested PCR
PCR amplification was performed with a thermal cycler (Gene Amp PCR system 9700; PerkinElmer, Norwalk, CT, USA) using a fraction (4 ml) of the single-cell cDNA as template. First multiplex-PCR was performed as a hot start in a final volume of 30 ml containing 4 ml cDNA, 100 pmol of each primer, 0.3 mM each dNTP, 3 ml 106 PCR buffer and 3.5 U HotStarTaq DNA Polymerase (Qiagen K.K., Tokyo, Japan) in a Gene Amp PCR system 9700 with the following cycling protocol: (1) 15 min at 95uC; (2) 35 cycles of 1 min at 94uC, 1.5 min at 57uC and 2 min at 72uC; (3) 10 min at 72uC; and (4) final hold at 4uC. The nested-PCR amplifications were carried out using 2.5 ml of the first PCR product with the following modification: 2.5 U HotStarTaq DNA Polymerase and 0.2 mM dNTPs. Thermocycling for the second round was as follows: (1) 15 min at 95uC; (2) 35 cycles of 45 s at