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Corticotrophin-Releasing Factor Augments the I_H in Rat Hypothalamic Paraventricular Nucleus Parvocellular Neurons In Vitro

¹Departments of Physiology, ²Public Health, and ³Anesthesiology, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan

Submitted 22 December 2004; accepted in final form 23 March 2005

	ABSTRACT

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The goal of this study was to characterize the effects of corticotrophin-releasing factor (CRF) on rat paraventricular nucleus (PVN) putative parvocellular neurons using whole cell patch-clamp recordings and single-cell reverse transcription-multiplex polymerase chain reaction (single-cell RT-mPCR) techniques. Under current clamp, CRF (10–600 nM) increased the neuronal basal firing rate and depolarized neurons in a dose-dependent manner. CRF-induced depolarization was unaffected by co-perfusion with TTX, 6-cyano-7-nitroquinoxaline-2 3-dione (CNQX), and bicuculline but was completely inhibited by ZD7288. Under voltage clamp, 300 nM CRF significantly increased the hyperpolarization-activated cation current (I_H) in a voltage-dependent manner, shifted the I_H conductance-voltage relationship (V_1/2) toward depolarization by 7.8 mV, and enhanced the I_H kinetics without changing the slope constant (k). Extracellular application of ZD7288 completely blocked I_H and the CRF-induced increase in I_H. Furthermore, CRF-induced effects were completely blocked by extracellular application of 1 µM -helical CRF-(9–14) (-hCRF), a nonselective CRF receptor antagonist, but were not affected by extracellular application of antisauvagine-30, a selective CRF-receptor 2 antagonist. Single-cell RT-mPCR analysis showed that these neurons co-expressed CRF receptor 1 mRNA and CRF receptor 2 mRNA. Furthermore, CRF-sensitive neurons co-expressed HCN1 channel mRNA, HCN2 channel mRNA, and HCN3 channel mRNA, but not HCN4 channel mRNA. These results suggest that CRF modulates the subpopulation of PVN parvocellular neuronal function by CRF-receptor 1–mediated potentiation of HCN ion channel activity.

	INTRODUCTION

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Corticotrophin-releasing factor (CRF) is a 41-amino acid peptide that is synthesized and secreted in many regions of the brain and plays a major role in the coordination of endocrine, autonomic, and behavioral responses to stressful stimuli. CRF is synthesized in the parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and is the primary regulator of adrenocorticotropin hormone (ACTH) release from the anterior pituitary in response to stress (Antoni 1986; Vale et al. 1981). When applied in vivo or in vitro, CRF can directly alter neuronal behaviors in several brain regions. For example, CRF excites neurons of the cortex and the forebrain (Eberly et al. 1983), and the excitability of the hippocampus, Purkinje cells, and the dorsal vagal complex is augmented by CRF-mediated reductions in afterhyperpolarization (AHP) (Aldenhoff et al. 1983; Hollrigel et al. 1998; Lewis et al. 2002; Yamashita et al. 1991).

The effects of CRF are mediated by two G protein–coupled receptors, the type 1 and 2 CRF receptors (Chang et al. 1993; Chen et al. 1993; Lovenberg et al. 1995). The expression of both CRF and CRF-receptor 1 (CRFR-1) mRNA in the parvocellular PVN increases in response to various stimuli, including stress (Luo et al. 1994; Makino et al. 1995) and intracerebroventricularly (ICV) administered CRF (Imaki et al. 1996; Mansi et al. 1996). Other studies have shown that parvocellular PVN neurons showed strong reactivity to an anti-CRFR-1 antibody (Bittencourt and Sawchenco 2000; Chen et al. 2000; Imaki et al. 2001). In contrast, the CRF receptor 2 (CRFR-2) is expressed in discrete regions, including the lateral septum and the ventromedial hypothalamus in the forebrain and the dorsal raphe and nucleus of the solitary tract in the hindbrain (Chalmers et al. 1995; Van Pett et al. 2000). Furthermore, CRF binds with high affinity to the CRFR-1 but has low affinity for CRFR-2 (Dautzenberg and Hauger 2002).

The autonomous beating of the heart and a considerable number of rhythmic activities in the brain are controlled by the hyperpolarization-activated cation current (I_H) (Lüthi and McCormick 1999; Pape 1996; Robinson and Siegelbaum 2003). I_H channels are stimulated by membrane hyperpolarization, gated by cyclic nucleotides (cAMP, cGMP), and blocked by extracellular Cs⁺ and ZD7288 (Ghamari-Langroudi and Bourque 2000; Harris and Constanti 1995; Ludwig et al. 1998). Four different isoforms (HCN1-4) of the I_H channel have been cloned (Ludwig et al. 1998; Monteggia et al. 2000; Santoro et al. 1998), and all four isoforms are expressed in rat PVN (Monteggia et al. 2000). Regulation of these HCN channels may occur via cyclic adenosine monophosphate (cAMP), cyclic guanine monophosphate (cGMP), and/or Ca²⁺ (Biel et al. 2002; Ludwig et al. 1998; Pape 1996).

The PVN comprises magnocellular neurons that secrete oxytocin (OT) and vasopressin (VP), neurosecretory parvocellular neurons that secrete hypophysiotropic hormones, and non-neurosecretory, preautonomic parvocellular neurons (Liposits 1993; Swanson and Sawchenko 1983). Despite the fact that the I_H channels and CRF receptors are both present in the rat PVN neurons, the effect of CRF on activities of the HCN channels is unknown. In this study, we used the whole cell patch-clamp and single-cell reverse transcription-multiplex polymerase chain reaction (RT-mPCR) method to examine the effects of CRF on rat PVN putative parvocellular neurons in vitro.

	METHODS

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Hypothalamic slice preparation

Hypothalamic slices were prepared from P12- to P14-day-old male Wistar rats, as previously described (Qiu et al. 2003). All experiments were approved by the Ethics Committee of the Miyazaki Medical College and were conducted in accordance with international guidelines on the ethical use of animals in a laboratory. Briefly, the brain was quickly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) consisting of (in mM) 140 NaCl, 3 KCl, 1.3 MgSO₄, 1.4 NaH₂PO₄, 11 HEPES, 2.4 CaCl₂, and 3.25 NaOH. The pH was 7.3, the osmolarity was 290–300 mOsm, and the fluid was aerated with 100% O₂. Coronal slices, including the PVN, with a thickness of 250 µm, were generated using a vibrating brain slicer (DSK-2000, Dosaka, Kyoto, Japan). The slices were incubated for 1 h in a chamber filled with equilibrated ACSF at room temperature (24–26°C) before recordings started.

Electrophysiology

Patch pipettes were made with a puller (PB-7, Narishige, Tokyo, Japan) from thick-wall borosilicate glass (GD-1.5, Narishige). They were filled with a solution consisting of (in mM) 130 potassium gluconate, 10 HEPES, 10 KCl, 1 CaCl₂, 5 EGTA, 1 MgCl₂, 2 Na₂ATP, and 0.5 Na₃GTP. The pH was adjusted to 7.2 with KOH. Patch pipette resistances were 5–7 M in the bath, with series of resistance in the range of 10–20 M, compensated by 80%. The liquid junction potential (10 mV) was corrected according to the method described by Neher (1992). Membrane potentials and/or currents were monitored with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), filtered at 1–5 kHz, and acquired through a Digidata 1200 series A/D interface on a personal computer using Clampex 7.0 software (Axon Instruments). Whole cell recordings were made from microscopically identified cells. Once stable recording conditions were obtained, a PVN neuron was 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, whereas type II neurons did not (Luther et al. 2000). To study the effects of CRF on I_H, the PVN neurons were also identified electrophysiologically as inward rectification-expressing neurons or noninward rectification-expressing neurons according to previously established criteria by current clamp in standard ACSF (Luther et al. 2000; Qiu et al. 2003). Only inward rectification-expressing putative parvocellular neurons were included in this study. Selected traces were stored on a computer hard drive, and all data were archived on a 4.7 GB DVD-RAM.

Cytoplasm harvest and reverse transcription

Harvesting of cytoplasm and reverse transcription were carried out as previously described (Liss et al. 1999). After whole cell recording, the cytoplasm was aspirated into the patch pipette by applying gentle negative pressure in the pipette while maintaining a tight seal. The pipette contents (8 µl) were expelled into a 0.5-ml test tube containing the reagents for reverse transcription. First-strand cDNA was synthesized for 1 h at 42°C. The total volume of the reaction was 20 µl, containing 5 µM of a random hexamer primer, 10 mM of dithiothreitol (DTT), and 200 µM each of deoxyNTP (dNTP), a 5x first-strand buffer (3 µl), 40 U RnaseOUT Recombinant Ribonuclease Inhibitor, and 100 U SuperScript Rnase H^–Reverse Transcriptase, all purchased from Invitrogen (Life Technologies). The single-cell cDNA was kept at –70°C until PCR amplification.

Multiplex and nested PCR

PCR amplification was performed with a thermal cycler (Gene Amp PCR system 9700, Perkin-Elmer, Norwalk, CT) using a fraction (4.5 µl) of the single-cell cDNA as a template. The first fraction of cDNA was used to screen for GAPDH; the second fraction (4.5 µl) of cDNA was used to screen for CRFR-1 mRNA and CRFR-2 mRNA; and the third fraction (4.5 µl) of cDNA was used to screen for HCN1-4 channel mRNA. The first multiplex-PCR was performed as a hot start in a final volume of 30 µl containing 4.5 µl cDNA, 100 pmol of each primer, and 0.3 mM of each dNTP, a 3 µl 10x PCR buffer, and 3.5 U HotStarTaq DNA Polymerase (Qiagen) in a Gene Amp PCR system 9700 with the following cycling protocol: 1) 15 min at 95°C; 2) 30 cycles of 1 min at 94°C, 1.5 min at 57°C, and 2 min at 72°C; 3) 10 min at 72°C; and 4) holding at 4°C. Nested PCR amplifications were carried out with 2.5 µl of the first PCR product in individual reactions using the following modifications: 3.0 U HotStarTaq DNA Polymerase and 0.2 mM dNTP. The second round was performed as follows: 1) 15 min at 95°C; 2) 35 cycles of 45 s at 94°C, 1 min at 56°C, and 1 min at 72°C; 3) 10 min at 72°C; and 4) holding at 4°C.

The nested primer sequences were as follows: GAPDH (accession no. NM017008) external sense: 5'-GATGGTGAAGGTCGGTGTG (position 849), external antisense: 5'-GGGCTAAGCAGTTGGTGGT (position 1318); GAPDH internal sense: 5'-TACCAGGGCTGCCTTCTCT, internal antisense: 5'-CTCGTGGTTCACACCCATC (361 bp); CRFR-1 (accession no. NM030999) external sense: 5'-GCCGCCTACAATTACTTCCA (position 1299), external antisense: 5'-GGACTGCTTGATGCTGTGAA (position 1958); CRFR-1 internal sense: 5'-GTGGATGTTCGTCTGCATTG, internal antisense: 5'-CACAAAGAAGCCCTGAAAGG (394 bp); CRFR-2 (accession no. NM022714) external sense: 5'-TACTGCAACACGACCTTGGA (position 330), external antisense: 5'-ACCAGCACTGCTCATTCTCA (position 982); CRFR-2 internal sense: 5'-CCCTAGTGGAGAGACCATGC, internal antisense: 5'-AGGTGGTGATGAGGTTCCAG (303 bp); HCN1 (accession no. NM053375) external sense: 5'-CTGACATGCGCCAGAAGATA (position 1315), external antisense: 5'-GATTGGAGGGATCGCTTGTA (position 1998); HCN1 internal sense: 5'-CAACTTCAACTGCCGGAAAC, internal antisense: 5'-CCTTGGTCAGCAGGCATATT (254 bp); HCN2 (accession no. AF247451) external sense: 5'-TCATCGTGGAGAAGGGAATC (position 749), external antisense: 5'-GGCAGTTTGTGGAAGGACAT (position 1310); HCN2 internal sense: 5'-ACTACGCATCGTGCGTTTC, internal antisense: 5'-CGTGCCCAATGAACATAGC (419 bp); HCN3 (accession no. NM053685) external sense: 5'-TCGGACACTTTCTTCCTGCT (position 394), external antisense: 5'-TGACTCATGGCCTTGAACAG (position 920); HCN3 internal sense: 5'-TTCCTGGTGGACCTGATTTC, internal antisense: 5'-CACAGCAGCAACATCATTCC (269 bp); HCN4 (accession no. NM021658) external sense: 5'-ATCGTGGTGGAGGACAACA (position 1179), external antisense: 5'-CCGATGAACATGGCATAGC (position 1759); HCN4 internal sense: 5'-GGAGACTCGCATTGACTCG, internal antisense: 5'-AGCCAGACGTCAGACATGC (405 bp). To investigate the presence and size of the amplified fragments, 10-µl aliquots of PCR products were separated by electrophoresis in an agarose gel (2%) and visualized by ethidium bromide staining. All individual PCR products were verified several times by direct sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit and the Applied Biosystems PRISM 310 Genetic Analyzer (ABI, Foster City, CA). A sequence comparison was performed using the BLAST database.

RNA isolation and cDNA preparation for control reactions

Poly(A)+ RNA was prepared from fresh hypothalamus tissue of 13-day-old Wistar rats using the Micro-to-Midi Total RNA Purification System (Invitrogen). Reverse transcription was performed with 250 µg of the poly(A)+ RNA, as described above. The RNA was diluted and used as a positive (+ reverse transcriptase [+RT]) or negative (–RT) control for the PCRs. All nine PCR fragments were detected routinely in the positive control when using the PCR protocol described above. The negative controls of single cells were carried out in parallel with single-cell experiments, excluding only the harvesting procedure, resulting in no detectable bands (n = 10).

Chemicals

Reagents included human/rat CRF (Peptide Institute), -helical CRF- (9–14) (Sigma-Aldrich, St. Louis, MO), anti-sauvagine (11–40) (Bachem AG, Bubendorf, Switzerland), ZD7288 (Tocris Cookson, Ballwin, MO), TTX (Sigma-Aldrich), 6-cyano-7-nitroquinoxaline-2 3-dione (CNQX), bicuculline, and CsCl (Sigma, Ballwin, MO). ZD7288 was prepared as a 50 mM stock solution (in H₂O) and stored at –20°C until use. All other drugs were dissolved in ACSF. In voltage clamp, TTX (0.5 µM) and BaCl₂ (100 µM) were included in the external recording solutions to block the voltage-gated Na⁺ channels and the Ba²⁺-sensitive K⁺ current and express the I_H current (Cardenas et al. 1999).

Data analysis

Data were analyzed using Clampfit 8.0 (Axon Instruments) and are expressed as means ± SE. I_H was determined by subtracting I_Ins from I_ss at each hyperpolarizing voltage step using the following equation

(1)

In addition, I_H conductance (G_H) was estimated as the amplitude of I_H measured at various potentials (V) divided by the driving force (V –E_H), where E_H is the reversal potential of I_H (Ghamari-Langroudi and Bourque 2000) as follows

(2)

The value of E_H was arbitrarily set at –33 mV, which reflects the median E_H reported in our previous study (Qiu et al. 2003).

Differences between mean values recorded under control and test conditions were evaluated using one-way ANOVA with Tukey's posthoc test with the SPSS Medical Pack. Differences were considered statistically significant at P < 0.05.

	RESULTS

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Effects of CRF on the PVN parvocellular neurons in current clamp

A total of 231 PVN neurons were characterized as type 2 neurons under whole cell current clamp (Luther et al. 2000; Qiu et al. 2003). Responsive neurons exhibited a lack of transient outward rectification in response to a series of depolarizing current pulses delivered at a hyperpolarized membrane potential (Fig. 1A). Eighty percent (185/231) of type 2 neurons displayed time-dependent inward rectification during the hyperpolarizing pulses (Fig. 1, A and B), and this response was blocked by 70 µM ZD7288 (Fig. 1B) or by 3 mM Cs⁺ (data not shown). Under voltage clamp, these neurons exhibited a hyperpolarization-activated ZD7288-sensitive inward current (Fig. 1, C and D). These properties are consistent with hyperpolarization-activated inward current (I_H) conductance (Ludwig et al. 1998; Qiu et al. 2003; Santoro et al. 1998), indicating that hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels exist in these neurons.

View larger version (42K): [in this window] [in a new window]   FIG. 1. Electrophysiological properties of paraventricular nucleus (PVN) corticotrophin-releasing factor (CRF) on rat-sensitive neurons. A: neuron displayed time-dependent inward rectification and lacked transient outward rectification (black arrow) in response to a series of depolarizing current pulses delivered at a hyperpolarized membrane potential. B: neuron displayed inward rectification (sag), which was blocked by 70 µM ZD7288 in response to a –60 pA hyperpolarizing current pulse. C: current traces elicited by 1-s, 120-mV hyperpolarizing voltage steps (Vh = –50 mV) in the absence and presence of 70 µM ZD7288. The neuron displayed IH, which was blocked by ZD7288 in voltage clamp. D: summary of the hyperpolarizing voltage steps vs. the IH current amplitude in artificial cerebrospinal fluid (ACSF; ) and in the presence of 70 µM ZD7288 (; n = 6).

View larger version (35K): [in this window] [in a new window]   FIG. 2. Effects of CRF on PVN CRF-sensitive neurons in current clamp. A1–A3: responses to 30, 100, and 600 nM CRF, respectively (bar, Vh = –60 mV). B1–B3: instantaneous spike rates of the neurons in A1, A2, and A3, respectively. CRF elicited increases in the firing-action potential in a dose-dependent manner. C: 300 nM CRF (bar) provoked a reversible membrane depolarization accompanied by an increase in the firing rate (top), and CRF-induced depolarization was unaffected by preperfusion with 0.5 µM TTX. D: summary of data showing 300 nM CRF-induced depolarization in the absence and presence of 0.5 µM TTX + 6-cyano-7-nitroquinoxaline-2 3-dione (CNQX; 10 µM) + bicuculine (10 µM) (P > 0.05, n = 8). E: concentration-response curve for the CRF-induced depolarization. Number of neurons tested for each concentration is indicated near the bars.

View larger version (30K): [in this window] [in a new window]   FIG. 3. CRF enhanced the activation of IH after reduced afterhyperpolarization (AHP) in current clamp. A: representative traces of AHP evoked in ACSF and in the presence of 300 nM CRF. To evoke single-action potentials, neurons were maintained at –60 mV before passing a short (10 ms) depolarizing current pulse of sufficient intensity to evoke an action potential at the offset of the pulse (top). B: neuron displayed inward rectification (sag) in response to a –60 pA hyperpolarizing current pulse in the absence and presence of 300 nM CRF. C: representative traces of AHP evoked in ZD7288 (70 µM) and ZD7288 co-perfused CRF. D: ZD7288 (70 µM) abolished the sag and blocked the CRF (300 nM)-induced effect. E: summary of data showing AHP amplitude in the ACSF, CRF, ZD7288 (70 µM), and ZD7288 co-perfused with CRF (#P < 0.05, n = 7). F: bar graph of the depolarizing sag in ACSF, CRF, ZD7288, and ZD7288 co-perfused with CRF (#P < 0.05,n = 7).

In the presence of TTX (0.5 µM) and Ba²⁺ (100 µM), application of 300 nM CRF to CRF-sensitive neurons with voltage clamp at –60 mV produced a negligible inward current (8.2 ± 2.3 pA, n = 10). However, when neurons were held at –50 mV and a series of 1-s hyperpolarizing voltage steps from –50 to –120 mV was applied, CRF induced a significant increment in instantaneous current (I_Ins) at step potentials less than –90 mV (Fig. 4, A and B; *P < 0.05, n = 7) and a steady-state current (I_SS) at step potentials less than –60 mV (Fig. 4, A and C; *P < 0.05). In addition, the I_H (I_SS – I_Ins) current was increased at step potentials less than –60 mV (Fig. 4D). Furthermore, we estimated the effect of CRF on I_H conductance (G_H) (see METHODS). The mean G_H –V relations are shown in Fig. 4E. Note that CRF enhanced the I_H conductance at step potentials less than –60 mV (*P < 0.05 vs. control). The modified Boltzmann equation was used as follows

(3)

where G_H(V) is the fraction of maximal G_H observed at V, k is the slope factor, and V_1/2 is the half-maximal voltage. The mean values of the Boltzmann equation were as follows: V_1/2 = –93.3 ± 2.4 mV, k = 11.5 ± 1.8 in the control and V_1/2 = –85.5 ± 2.8 mV, k = 12.91 ± 1.8 during the application of CRF (*P < 0.05 vs. control). These data suggest that CRF produced a significant shift in V_1/2 to a more depolarized potential (7.8 ± 1.2 mV) and that the slope factor values were not altered by CRF (P > 0.05 vs. control).

View larger version (38K): [in this window] [in a new window]   FIG. 4. Effects of CRF on PVN neurons in voltage clamp. A: in the presence of BaCl2 (100 µM), current traces were elicited by a series of 1-s hyperpolarizing voltage step CRF-sensitive decrements (10-mV decrements; Vh = –50 mV) in ACSF and during the application of 300 nM CRF. B: plots of instantaneous current in the control () and during the application of 300 nM CRF () against the membrane potential ( shown in A). C: plots of the steady-state current in the control () and during the application of CRF () against the membrane potential ( shown in A). D: plots of the IH (ISS –IIns) in the control () and during the application of CRF () against the membrane potential. E: current (IH) data shown in D were converted into conductance (GH) using the equation GH = IH/(V +33) (Qiu et al. 2003; V is the test voltage). Solid lines are the best fit through the data points using the Boltzmann equation (ACSF, ; 300 nM CRF, ; n = 7). The mean values were as follows: V1/2 = –93.3 ± 2.4 mV, k = 11.5 ± 1.8 in the control and V1/2 = –85.5 ± 2.8 mV, k = 12.91 ± 1.8 during the application of CRF. *P < 0.05 vs. ACSF.

View larger version (39K): [in this window] [in a new window]   FIG. 5. Acceleration of IH activation kinetics by CRF. A: in the presence of TTX (0.5 µM) and BaCl2 (100 µM), current traces were elicited by hyperpolarizing steps to –120 mV from a holding potential of –50 mV under control conditions, during addition of CRF, and after CRF washout. B: time constant of exponential fit () for the same experiment plotted vs. time. Each circle shows value for a single response evoked at 0.05 Hz. Black bar, 300 nM CRF application. C: IH traces evoked by steps to various voltages in the ACSF and during the application of 300 nM CRF. Superimposed on each trace is a monoexponential fit of the data points (a solid line extending to the right). Time constant used in the fits () is indicated beside each trace. D: plots of mean IH activation time constants (n = 7) against the voltage steps. *P < 0.05 vs. ACSF.

View larger version (38K): [in this window] [in a new window]   FIG. 6. ZD7288 blocked the CRF-induced responses. A: CRF (100 nM, bar) provoked a reversible membrane depolarization accompanied by an increase in the firing rate, whereas 70 µM ZD7288 blocked CRF-induced responses. B: summary of data showing neuronal membrane potential in the presence of 100 nM CRF, 70 µM ZD7288, and 100 nM CRF +70 µM ZD7288 (*P < 0.05 vs. 100 nM CRF, n = 5, Vh = –60 mV). C: in the presence of TTX (0.5 µM) and BaCl2 (100 µM), current traces were elicited by 1-s, –120-mV hyperpolarizing voltage steps (Vh = –50 mV) under ACSF, 300 nM CRF, 70 µM ZD7288, and 70 µM ZD7288 + 300 nM CRF. D: plots of steady-state current in the control () and during the application of CRF () against the membrane potential ( shown in C, n = 4). E: plots of the steady-state current in the ZD7288 () and co-perfusion of ZD7288 and CRF () against the membrane potential ( shown in C, n = 4).

To establish the pharmacological profile of CRF receptors that mediated the CRF-enhanced I_H, the CRFR-1 and CRFR-2 nonselective antagonist -helical CRF- (9–14) (-helCRF, 1 µM) and a selective CRFR-2 antagonist, anti-sauvagine-30 (aSvg, 200 nM), were applied to PVN CRF-sensitive neurons. The effects of CRF (300 nM) on I_SS and I_H in the absence and presence of -helCRF and aSvg (after perfusion -helCRF or aSvg for 10 min) were examined under voltage clamp. CRF (300 nM) significantly increased the I_SS and I_H, and -helCRF completely blocked the CRF-induced augmentation of I_SS and I_H (Fig. 7, A, B, and E; n = 6). However, aSvg (200 nM) did not prevent the CRF-induced augmentation of I_SS and I_H (Fig. 7, C, D, and F; n = 6). Even the high concentration of aSvg (1 µM) did not prevent the CRF-induced augmentation of I_H (n = 2, data not shown). These data indicate that CRF-induced increase in I_H was not mediated by CRFR-2.

View larger version (34K): [in this window] [in a new window]   FIG. 7. CRF-induced augmentation in IH was blocked by -helical CRF-(9–14) (-helCRF) but not by anti-sauvagine-30 (aSvg-30). A: in the presence of BaCl2 (100 µM), current traces were elicited by 1-s, –120-mV hyperpolarizing voltage steps (Vh = –50 mV) under ACSF, 300 nM CRF, and 1 µM -helCRF co-perfused with 300 nM CRF. B: plots of Iss in ACSF (), during the application of CRF (), and during the co-application of -helCRF and CRF () against the membrane potential ( shown in A, n = 6). C: in the presence of BaCl2 (100 µM), current traces are shown in A under ACSF, 300 nM CRF, and 200 nM aSvg co-perfused with 300 nM CRF. D: plots of Iss in ACSF (), during the application of CRF (), and during co-perfusion of aSvg-30 and 300 nM CRF () against the membrane potential ( shown in C, n = 6). E: plots of increased IH in the application of 300 nM CRF () and the co-perfusion of 1 µM -helCRF and CRF (; n = 7). F: plots of the increased IH in the application of 300 nM CRF () and 200 nM aSvg-30 co-perfused with CRF (; n = 6). It should be noted that CRF-induced increase in IH was abolished by -helCRF but not attenuated by the CRFR-2 selective antagonist, aSvg-30.

After completion of the electrophysiological recording, the CRF-sensitive neurons (n = 20) were screened for GAPDH, CRFR-1, CRFR-2, and HCN1-4 channels mRNA using the single-cell RT-mPCR technique. Screening of rat hypothalamic total RNA (positive control) resulted in the detection of all the specific mRNAs, each corresponding to the size predicted by its mRNA sequence (Fig. 8). The identities of all PCR fragments were verified by direct sequencing. All the neurons (20/20) expressed GAPDH, CRFR-1, CRFR-2, HCN1, HCN2, and HCN3 channel mRNAs, but not HCN4 channel mRNA (Fig. 8).

View larger version (51K): [in this window] [in a new window]   FIG. 8. Identification of CRFR-1, CRFR-2, HCN1, HCN2, HCN3, and HCN4 channel mRNA in CRF-sensitive neurons by single-cell reverse transcription-multiplex polymerase chain reaction (RT-mPCR) analysis. The positive control (RT+) showed that the mRNAs of CRFR-1, CRFR-2, HCN1, HCN2, HCN3, HCN4, and GAPDH were detected from the rat hypothalamic tissue total RNA. GAPDH transcripts were analyzed in the same cells as an internal control for the RT reaction. The expected size of the PCR products is indicated, and the single-cell PCR products were verified with sequencing. In addition, a single cell and the rat hypothalamic tissue total RNA were processed without RT (–RT), but no PCR product was obtained (no. 7). All 6 of the CRF-sensitive neurons (cell nos. 03101403, 03101604, 03111802, 03112104, 03112701, and 0421202) expressed CRFR-1, CRFR-2, HCN1, HCN2, and HCN3 channel mRNA but not HCN4 channel mRNA.

	DISCUSSION

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The previous studies showed that CRF has a depolarizing effect on the majority of the CNS neurons, most likely due to a decrease in the amplitude of AHP (Eberly et al. 1983; Haug and Storm 2000; Lewis et al. 2002; Yamashita et al. 1991). However, our data showed that CRF evoked a depolarization and increased the neuronal excitability in PVN CRF-sensitive neurons by enhanced I_H channels activities but not directly by attenuated Ca²⁺-activated K⁺ channels activities. This is supported by several findings. First, the PVN CRF-sensitive parvocellular neurons displayed a ZD7288-sensitive, time-dependent inward rectification during the hyperpolarizing pulses, and the single-cell RT-PCR result indicated that these neurons expressed HCN1, HCN2, and HCN3 channels mRNA. The HCN channels were partially active at the holding potential (Qiu et al. 2003; Yaji and Sumino 1998), and CRF enhanced I_H at the holding potentials, inducing a depolarization in the CRF-sensitive neurons in current-clamp. Under voltage clamp, CRF enhanced HCN channels kinetics at membrane potentials below –50 mV and produced a significant shift in V_1/2 to a more depolarized potential. Previous reports showed that I_H plays a significant role in setting both the resting membrane potential (RMP) and the baseline level of excitability of hippocampal GABAergic interneurons found in the stratum oriens of area CA1 (Lupica et al. 2001) and that the presence of I_H in magnocellular neurosecretory cells of the rat supraoptic nucleus provides an excitatory drive that contributes to phasic and tonic firing (Ghamari-Langroudi and Bourque 2000). Furthermore, ZD7288 completely blocked the CRF-induced depolarization and the increments in I_Ins and I_SS, which reflect an increase in tonically activated I_H conductance (Mayer and Westbrook 1983; Qiu et al. 2003; Yaji and Sumino 1998). Moreover, ZD7288 completely abolished the CRF-induced augmentation in I_H and prevented a CRF-induced decrease in the amplitude of AHP in current clamp, which indicated that the enhancement of HCN channels activities induced a decrement of the Ca²⁺-activated K⁺ channels activities and attenuated the amplitude of the AHP (Galligan et al. 1990; Linden et al. 2003).

Potential mechanisms of CRF-regulated HCN channels

CRFR-1 mRNA levels in the PVN increase in response to stress (Luo et al. 1994; Makino et al. 1995) and ICV-administered CRF (Imaki et al. 1996; Mansi et al. 1996). CRFR-2 is also expressed in the PVN of the hypothalamus (Lovenberg et al. 1995). Our single-cell RT-mPCR results showed that the CRF-sensitive neurons co-expressed CRFR-1 mRNA and CRFR-2 mRNA, indicating that CRFR-1 and CRFR-2 co-existed in the CRF-sensitive neurons. The CRFR-1 and CRFR-2 nonselective antagonist, -helCRF (De Souza 1987), attenuated the CRF-induced augmentation of I_H and suggested that CRF-mediated effects on PVN neurons were mediated by CRF receptors. However, the selective CRFR-2 antagonist, aSvg (Lawrence et al. 2002), did not attenuate the CRF-induced augmentation of I_H in PVN neurons, suggesting that the CRF-induced increase in excitation and the activities of I_H in PVN neurons were not mediated by CRFR-2. A previous study showed that the CRF binds with high affinity to CRFR-1 and with low affinity to CRFR-2 (Dautzenberg and Hauger 2002). Thus these data suggest that the augmentation of I_H and the increase of neuronal excitability are mediated via the CRFR-1 rather than via the CRFR-2 receptor.

CRF is a potent activator of adenylate cyclase and cAMP production (Facci et al. 2003). After binding to CRFR-1, CRF couples to the stimulatory G protein (Gs), leading to the stimulation of adenylate cyclase and the activation of protein kinase A (PKA) and other cAMP pathway events, increasing the production of cAMP (Dautzenberg and Hauger 2002; Dautzenberg et al. 2000; Grammatopoulos and Chrousos 2002). A key property of neuronal HCN channels is their regulation by neurotransmitters and hormones that act via cAMP, cGMP, or intracellular Ca²⁺ (Pape 1996); cAMP and cGMP modulate HCN channel activity via direct interaction with the cyclic nucleotide-binding domain protein of the C-terminus (Ludwig et al. 1998). A recent report showed that multiple neurotransmitter receptor systems coupled both positively and negatively to the cAMP synthesis (via the Gs- and Gi-proteins, respectively) and steadily up- and down-regulated HCN channels activities (Frere and Luthi 2004; Pape 1996).

Physiological significance

CRF is a key neurotransmitter that mediates the endocrine, autonomic, and behavioral responses to a variety of stressors (Smagin et al. 2001; Vale et al. 1981). CRF is synthesized in the parvocellular neurons of the hypothalamic PVN and is the primary regulator of the release of ACTH from the anterior pituitary in response to stress (Antoni 1986; Vale et al. 1981). The PVN contains putative preautonomic neurons that directly project to the intermediolateral cell column of the spinal cord (Badoer 2001). The increased endogenesis CRF may directly modulate the PVN putative preautonomic neurons by enhancing HCN channels activities via CRFR-1, thus activating the sympathetic nervous center in the spinal cord, which results in increases in blood pressure, heart rate, and plasma norepinephrine (Chu et al. 2004).

Collectively, we propose that CRF binds to CRFR-1, resulting in increased intracellular camp and leading to an increment in HCN channels activity and neuronal excitation. This response may contribute to the activation of autonomic centers in the brain stem and spinal cord that regulate blood pressure, heart rate, and plasma norepinephrine.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was partially supported by a Grant-in-Aid for Scientific Research (14370024) from the Ministry of Education, Science, Sports, and Culture, Japan. This study was also performed as a part of the Japanese Center of Excellence Program (Section of Life Science).

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. Kannan, Dept. of Physiology, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki 889-1692, Japan (E-mail: kannanh{at}med.miyazaki-u.ac.jp)

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