Leptin regulated calcium channels of neuropeptide Y and proopiomelanocortin neurons by activation of different signal pathways

Neuroscience 156 (2008) 89 –98

LEPTIN REGULATED CALCIUM CHANNELS OF NEUROPEPTIDE Y AND PROOPIOMELANOCORTIN NEURONS BY ACTIVATION OF DIFFERENT SIGNAL PATHWAYS J.-H. WANG,a1 F. WANG,a,b,c1 M.-J. YANG,a D.-F. YU,a W.-N. WU,a J. LIU,a L.-Q. MA,a F. CAIa AND J.-G. CHENa,b,c*

Key words: leptin, patch-clamp, calcium current, POMC, NPY.

Leptin, the protein encoded by the obese (ob) gene (Zhang et al., 1994), is secreted from adipose tissue and is thought to act in the CNS to regulate food intake and energy expenditure (Campfield et al., 1995; Schwartz et al., 1996a). Defects in leptin signaling cause severe obesity in rodents and humans. It is well established that the weight reducing effects of leptin are mediated in part by its ability to modulate hypothalamic functions (Schwartz et al., 1996b). Neuroanatomical studies have suggested that the majority of leptin’s anti-obesity effects are mediated by leptin receptors (LEPRs) in the arcuate nucleus (ARC) of hypothalamus (Saper et al., 2002; Schwartz and Porte, 2005; Spiegelman and Flier, 2001). The signaling form of LEPR is coexpressed with neuropeptide Y (NPY) and agouti-related peptide (AgRP) in a group of orexigenic neurons and with proopiomelanocortin (POMC) and cocaineand amphetamine-regulated transcript (CART) in a group of anorexigenic neurons (Mercer et al., 1996; Erickson et al., 1996; Schwartz et al., 1997; Hahn et al., 1998; Baskin et al., 1999). Increased NPY activity and reduced POMC activity appear to promote feeding and fat deposition, whereas reduced NPY activity and increased POMC activity inhibit feeding and body mass (Lin et al., 2000; Clark et al., 1984; Zarjevski et al., 1993; Huszar et al., 1997; Fan et al., 1997). After its binding to LEPR in the hypothalamus, leptin stimulates a specific signaling cascade that results in the inhibition of NPY neurons (Cowley et al., 2001; van den Top et al., 2004), while stimulating POMC neurons (Cowley et al., 2001; Elias et al., 1999). Despite the well-known differential effects of leptin on NPY and POMC neurons, relatively little is known about the mechanisms especially the electrophysiological mechanisms for actions of leptin. Spanswick et al. (1997) observed that leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels (KATP) using single-channel recording techniques from hypothalamic slices and acutely dissociated neurons. It is well-established that inhibition of KATP channels leads to membrane depolarization and facilitating electrical activity, generating the opening of voltage-gated calcium channels (VGCCs) and accelerating Ca2⫹ influx, and then initiating the release of neurotransmitter (Dunne et al., 1997; Chapman et al., 1999), but the opening of KATP channels leads to membrane hyperpolarization and diminished electrical activity. Previously, Cowley et al. (2001) observed that leptin induces a slow, progressive membrane hyperpolarization

a

Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, #13 Hangkong Road, Wuhan, Hubei 430030, China

b

Key Laboratory of Neurological Diseases (HUST), Ministry of Education of China, Wuhan, Hubei 430030, China

c

Hubei Key Laboratory of Neurological Diseases (HUST), Wuhan, Hubei 430030, China

Abstract—The fat-derived hormone leptin regulates food intake and body weight in part by modulating the activity of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC). To investigate the electrophysiological activity of these neurons and their responses to leptin, we recorded whole-cell calcium currents on NPY and POMC neurons in the ARC of rats, which we identified by morphologic features and immunocytochemical identification at the end of recording. Leptin decreased the peak amplitude of high voltage–activated calcium currents (IHVA) in the isolated neurons from ARC, which were subsequently shown to be immunoreactive for NPY. The inhibition was prevented by pretreatment with inhibitors of Janus kinase 2 (JAK2) and mitogen-activated protein kinases (MAPK). In contrast, leptin increased the amplitude of IHVA in POMC-containing neurons. The stimulations of IHVA were inhibited by blockers of JAK2 and phosphatidylino 3-kinase (PI3-k). Both of these effects were counteracted by the L-type calcium channel antagonist nifedipine, suggesting that Ltype calcium channels were involved in the regulation induced by leptin. These data indicated that leptin exerted opposite effects on these two classes of neurons. Leptin directly inhibited IHVA in NPY neurons via leptin receptor (LEPR) –JAK2–MAPK pathways, whereas evoked IHVA in POMC neurons by LEPR–JAK2–PI3-k pathways. These neural pathways and intracellular signaling mechanisms may play key roles in regulating NPY and POMC neuron activity, anorectic action of leptin and, thereby, feeding. © 2008 Published by Elsevier Ltd on behalf of IBRO. 1

These authors are contributed to this work equally. *Correspondence to: J.-G. Chen, Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, #13 Hangkong Road, Wuhan, Hubei 430030, China. Tel: ⫹86-2783692636; fax: ⫹86-27-83692847. E-mail address: [email protected] (J.-G. Chen). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; [Ca2⫹]i, cytosolic Ca2⫹ concentration; DMEM, modified Dulbecco’s Eagle’s medium; DMSO, dimethysulfoxide; HVA, high voltage–activated calcium channel; IHVA, high voltage–activated calcium currents; JAK2, Janus kinase 2; KATP, ATP-sensitive potassium; LEPR, leptin receptor; MAPK, mitogen-activated protein kinases; NPY, neuropeptide Y; PI3-k, phosphatidylino 3-kinase; POMC, proopiomelanocortin; STAT, signal transducers and activators of transcription; VGCCs, voltage-gated calcium channels.

0306-4522/08 © 2008 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2008.04.079

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associated with a decrease in membrane resistance and a cessation of all activity of NPY/AgRP neurons in ARC through activation of KATP channel, however, leptin increases the frequency of action potentials in POMC neurons by activating a nonspecific cation channel and reduced inhibition through local orexigenic NPY/GABA neurons. It is widely accepted that the cation channels are voltage- and Ca2⫹-sensitive and, upon patch excision, can be found in several distinguishable modes of gating (Wilson et al., 1998). These observations supported that Ca2⫹ may play a possible role in the effects of leptin on the activities of NPY and POMC neurons. Calcium plays a critical role in mediating neuronal excitability and neuroplasticity. Regulations of calcium contents in NPY and POMC neurons by leptin have been reported in different experimental systems. For instance, Muroya et al. (2004) observed by calcium imaging technique that leptin decreases cytosolic Ca2⫹ concentration ([Ca2⫹]i) in the isolated NPY neurons, but induces longlasting increases of [Ca2⫹]i in POMC neurons. Similarly, it has been reported that leptin could suppress [Ca2⫹]i rise in NPY neurons induced by ghrelin and orexins (Muroya et al., 2004; Kohno et al., 2003, 2007). VGCCs play an important role in the regulation of [Ca2⫹]i (Catterall, 1995), however, the direct effects of leptin on VGCCs in NPY and POMC neurons have not yet been reported. Here, we tested the possible modulations of VGCCs by leptin in primary cultured ARC neurons using patch-clamp techniques, and if so, to specify the signal transduction mechanisms in this modulated actions of leptin.

EXPERIMENTAL PROCEDURES Chemicals Leptin was purchased from ProSpec (Rehovot, Israel), anti-NPY serum and anti-␣-MSH were obtained from Chemicon International (Temecula, CA, USA), AG490 and PD98058 were purchased from Calbiochem (San Diego, CA, USA), wortmannin and nifedipine were obtained from Sigma (St. Louis, MO, USA) and B27 supplement was purchased from Gibco Invitrogen Corporation (Carlsbad, CA, USA). Other common agents were purchased from commercial suppliers. Leptin was prepared freshly with distilled water. AG490, PD98059, wortmannin, and nifedipine were dissolved in dimethysulfoxide (DMSO) and stored at ⫺20 °C. They were diluted to the final concentrations before application. The final concentration of DMSO was ⬍0.05%.

Preparation of single neurons from ARC The research was conducted in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the United National Institutes of Health. All experimental protocols were approved by the Review Committee for the Use of Human or Animal Subjects of Huazhong University of Science and Technology. All efforts were made to minimize the number of animals used and their suffering. Primary cultures of ARC neurons were prepared as previously described (Muroya et al., 2004) with some modifications. Briefly, brain slices containing the entire ARC were prepared from the brains of neonatal Sprague–Dawley rats (days 5–7), and the entire ARC was excised from the left and right sides. The dissected tissues were treated with 0.125% trypsin in Hanks’ balanced salt solution for 25 min at 37 °C and mechanically

dissociated using a fire-polished Pasteur pipettes. Cell suspension was centrifuged for 7 min at 1000⫻g and the cell pellets were re-suspended in the modified Dulbecco’s Eagle’s medium (DMEM) and F-12 supplement (1:1) with 10% fetal bovine serum. For whole-cell patch-clamp recording, cells (20,000 – 40,000) were seeded on poly-D-lysine-coated coverslips and kept at 37 °C in 5% CO2 incubator. After 24 h, the culture medium was changed to DMEM medium supplemented with 2% B27 and the ARC neurons were fed with fresh medium twice weekly. Microscopically, glial cells were not apparent in ARC neurons employing this protocol. The neurons were maintained for 7–10 days in primary culture until used for whole-cell patch-clamp recording.

Immunocytochemical identification of single ARC neurons The immunocytochemical identification of ARC neurons was prepared as previously reported with slight modifications (Muroya et al., 2004). The ARC neurons were fixed with 4% paraformaldehyde in 0.1 mol/L PBS overnight. They were pretreated with H2O2 in methanol for 1 h. Nonspecific binding sites were then blocked with 10% goat serum in 0.1 mol/L PBS for 1 h at room temperature. Cells were incubated overnight at 4 °C with primary antiserum to NPY or ␣-MSH diluted 1:1000 in PBS containing 1.5% normal goat serum. Cells were subsequently incubated with biotinylated goat anti-rabbit IgG secondary antibody for 1 h at room temperature. The secondary antibody was then rinsed, and the sections were labeled with avidin–peroxidase complex reagent (ABC kit; Vector) for 1 h. The sections were developed with 3,3=-diaminobenzidine (DAB). In control sections, the primary antibodies were replaced by the corresponding nonspecific IgG and processed in parallel.

Whole-cell patch-clamp recording The procedure for whole-cell patch-clamp recording was as that described in our previous reports with minor modification (Chen et al., 2002; Yermolaieva et al., 2001). The bath solution for recording high-voltage activated calcium current (IHVA) contained (in mmol/L): choline-Cl 110, MgCl2 2, CaCl2 10, TEA–Cl 20, Hepes 10, glucose 20, and the pH was adjusted to 7.4 with CsOH. Glass pipettes were used with a resistance of about 2– 4 M⍀ when filled with the following solution (in mmol/L): CsF 64, CsCl 64, CaCl2 0.1, MgCl2 2, EGTA 10.0, Hepes 10.0, Tris–ATP 5.0, and the pH was adjusted to 7.2 with CsOH. After establishing a whole-cell configuration, the adjustment of capacitance compensation and series resistance compensation were done before recording. The current signals were acquired at a sampling rate of 10 kHz and filtered at 3 kHz. Whole-cell patch-clamp recordings were carried out using an EPC-10 amplifier (HEKA, Lambrecht, Germany) driven by Pulse/PulseFit software (HEKA, Southboro, Germany). Drug actions were measured only after steady-state conditions were reached, which were judged by the amplitudes and time courses of currents remaining constant. All the recordings were made at room temperature (20 –22 °C). All experiments were repeated three times using different batches of cells and at least three to four dishes with cells were used for recording in different batches of cells.

Data analysis Dose-response curve was fitted with the Hill equation: I/Imax⫽1/ [1⫹(EC50/C)n], where I is the current amplitude after administration of leptin, Imax is the control current amplitude, C is the concentration of leptin, and n is Hill coefficient. The voltage-dependence of activation was determined using standard protocols. The conductance G was calculated according to G⫽I/(Vm⫺Vrev), where Vrev is the Ca2⫹ reversal potential and Vm is the membrane potential at which the current was recorded.

J.-H. Wang et al. / Neuroscience 156 (2008) 89 –98 Normalized peak conductance (G/Gmax) was then fitted by the following Boltzmann equation: G/Gmax⫽1/{1⫹exp[(V1/2⫺Vm)/k]}, where Gmax is the maximum conductance, V1/2 is the membrane potential of half-maximal activation, and k indicates the slope factor. To investigate the voltage-dependent inactivation, normalized currents (I/Imax) were plotted against the voltages of conditioning voltage, and fitted with a Boltzmann function: I/Imax⫽1/ {1⫹exp[(V1/2⫺Vm)/k]}, where Imax is the maximal current, Vm is the conditioning voltage, V1/2 is the potential of half-maximal inactivation and k is the slope factor.

Statistical analysis Statistical values were presented as means⫾S.E.M. (n⫽number of neurons). The Student’s t-test with paired comparisons was used to evaluate differences. P values less than 0.05 were considered to be statistically significant.

RESULTS Morphological features of ARC NPY and POMC neurons A distinguishing feature of ARC NPY neurons is that they also contain AgRP, a natural antagonist of MC3 and -4 receptors. These NPY neurons are typically small and medium neurons with triangular or spindle-shaped perikaryons. Most of them have one to three slender, poorly ramified primary dendrites (Fig. 1A), which are consistent with the observations by van den Top et al. (2004). Anti-␣-MSH was used to identify POMC-like immunoreactive neurons in this study, as ␣-MSH is the protein-processing product of the precursor protein. POMC neurons are medium and large sizes, displaying a quadrangular or multipolar perikaryon with a variable number of primary dendrites, well-ramified dendrites (Fig. 1B).

Fig. 1. Characteristics of NPY and POMC neurons in ARC. (A) Immunocytochemical identification of NPY neurons in cultured ARC neurons. (B) Immunocytochemical identification of POMC neurons in cultured ARC neurons. (C) The cell in which IHVA was inhibited by leptin was subsequently proved to be immunoreactive for NPY (arrow). (D) The cell in which IHVA was increased by leptin was proved to be POMC-containing by subsequent immunocytochemical staining with an anti-␣-MSH antiserum (arrow). Scale bar⫽20 ␮m.

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Leptin regulated IHVA in a concentration-dependent manner In the whole-cell patch-clamp recording, we focused on ARC neurons with the characteristics of NPY or ␣-MSH positive neurons. In the voltage-clamp experiments, the cells were stepped from the holding potential of ⫺80 mV to ⫺40 mV (50 ms), and then depolarized to ⫹10 mV (200 ms) after briefly hyperpolarizing the membrane potential for 10 ms to ⫺45 mV. The IHVA was activated by the second depolarization. The protocol was applied every 5 s. Extracellular application of leptin 1–100 nM inhibited IHVA in NPY neurons in a concentration-dependent manner (Fig. 2B). In our observation, leptin produced significant inhibitory effect on the peak amplitude of IHVA only at relative high concentrations. At 100 nM, leptin decreased amplitude of IHVA in NPY neurons by (70.19⫾11.01%) (n⫽6, P⬍0.05). As the concentration of leptin was raised, an increased fraction of presumptive NPY-immunoreactive neurons exhibited reduction of IHVA (Fig. 2C). Among 35 neurons with characteristics of NPY-positive neurons that responded to leptin with IHVA decreases, 30 neurons (86%) were proved to be NPY-immunoreactive neurons (Fig. 1C). IHVA partly returned toward the control level after washing off leptin from NPY neurons with bath solution (Fig. 2A). The concentration of leptin producing 50% effect in IHVA (EC50) was 17.42⫾7.58 nM, and the Hill coefficient was 0.88⫾0.18. Application of 30 nM leptin induced inhibition of IHVA in NPY neurons. This inhibition was detectable in about 30 – 60 s, and rapidly reached a plateau in 90 –150 s (Fig. 2D). Conversely, administration of leptin potentiated IHVA in POMC neurons. The effect of leptin on IHVA was concentration dependent over the range of 1–100 nM (Fig. 3B). The effect of leptin was significant at 10 nM (n⫽6, P⬍0.05), and at a concentration of 100 nM, leptin increased amplitude of IHVA by (68.47⫾11.40%) (n⫽6, P⬍0.05) (Fig. 3A). The concentration producing 50% increase by leptin in IHVA was 33.24⫾4.09 nM, and the Hill coefficient was 0.86⫾0.04. The percent of presumptive POMC neurons that exhibited IHVA increases in response to leptin was increased with the increasing concentration of leptin in bath solution (Fig. 3C). Thirty of 38 neurons that responded to leptin (79%), were found to be immunoreactive for ␣-MSH (Fig. 1D). The augment effect was observed in about 30 – 60 s after POMC neurons were treated with leptin, and maximum leptin effect was obtained after 90 –150 s. To further study the properties or patterns of effects on VGCCs, 30 nM leptin were selected in the following experiments. Effects of leptin on the properties of IHVA activation kinetics To study the activation properties of IHVA, the currents were evoked by a series of 200 ms voltage steps between ⫺50 mV and ⫹50 mV in 10 mV increments preceded by

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Fig. 2. Leptin inhibited IHVA of NPY neurons in the ARC in a dose-dependent manner. (A) Representative traces of IHVA under control condition, application of 30 nM leptin and washout. IHVA was evoked by the protocol shown on top. (B) Dose-response curve for the inhibition of IHVA by 1–100 nM leptin. Each point represents the mean⫾S.E.M (n⫽6). (C) Incidence of responses to increasing concentration of leptin was expressed as the percentage of neurons that exhibit decrease in IHVA. (D) Time-course curve before, during and after 30 nM leptin treatment corresponding to A. * P⬍0.05 vs. control.

the holding potential of ⫺80 mV and briefly depolarizing to ⫺40 mV (50 ms). The current amplitudes in dependence of the potential can be shown as an I–V curve (Fig. 4B). Leptin at 30 nM inhibited the maximum amplitude of IHVA in NPY neurons, but had no effect on the reversal potential of IHVA. At ⫹10 mV the current amplitude declined from (⫺608⫾63) pA to (⫺429⫾67) pA (n⫽6, P⬍0.05) in the presence of leptin at 30 nM. As shown in Fig. 4C, the steady-state activation curve of IHVA was plotted as normalized conductance as a function of membrane potential. The resulting curves were fitted by the Boltzmann function (see Experimental Procedures). Leptin at 30 nM shifted the activation curve of IHVA positively with V1/2⫽⫺5.41⫾1.25 mV, k⫽5.61⫾1.04 in control group, and V1/2⫽⫺1.62⫾1.00 mV, k⫽3.83⫾0.97 by 30 nM leptin (Fig. 3C, n⫽6, P⬍0.05). In contrast, 30 nM leptin increased the maximum amplitude of IHVA in POMC neurons, but the voltage dependence of activation of the IHVA was unchanged, as illustrated by the I–V curves before and after leptin application (Fig. 4D). The steady-state activation curve was shifted slightly to more hyperpolarized potentials. The half-activation voltage and slope factor in control conditions were ⫺1.02⫾0.62 mV (n⫽6) and 3.51⫾0.97 (n⫽6), respectively, and ⫺4.59⫾1.19 mV (n⫽6) and 5.82⫾1.05 (n⫽6) in the presence of 30 nM leptin (Fig. 4E).

Effects of leptin on the properties of IHVA inactivation kinetics To investigate the steady-state inactivation properties of IHVA, 300 ms conditioning prepulses from ⫺105 mV to 30 mV in 10 mV increments were applied before step depolarizations to the fixed potential of ⫹15 mV (Fig. 5). The normalized current was plotted against the membrane potential, and fitted with a Boltzmann function. Leptin at 30 nM did not significantly alter the voltage-dependence of inactivation kinetics of IHVA in NPY neurons (Fig. 5B), with V1/2⫽⫺24.26⫾6.08mV, k⫽⫺37.82⫾4.39 in leptin group and V1/2⫽⫺24.81⫾7.43 mV, k⫽⫺45.67⫾5.07 in control group (n⫽6, P⬎0.05). Similarly, the voltage dependence of inactivation of IHVA in POMC neurons was also unaffected, having a half-inactivation voltage of ⫺33.67⫾1.98 mV and slope factor of ⫺24.33⫾2.40 in control and a half-inactivation voltage of ⫺30.66⫾1.37 mV and slope factor of ⫺21.14⫾1.51 in the presence of 30 nM leptin (Fig. 5C, n⫽6). Leptin modulated IHVA in NPY and POMC neurons by different signal pathways To identify the mechanism by which leptin modulated IHVA, a Janus kinase 2 (JAK2) inhibitor AG 490 50 ␮M was used. A combination of AG 490 with 30 nM leptin decreased IHVA in NPY neurons to 88.1⫾9.0% of control (Fig. 6A, n⫽6, P⬍0.05

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Fig. 3. Leptin increased IHVA of POMC neurons in the ARC in a dose-dependent manner. (A) Representative traces of IHVA under control condition, application of 30 nM leptin and washout. IHVA was evoked by the protocol shown on top. (B) Dose-response curve for the potentiation of IHVA by 1–100 nM leptin. Each point represents the mean⫾S.E.M (n⫽6). (C) Incidence of responses to increasing concentration of leptin was expressed as the percentage of neurons that exhibit increase in IHVA. (D) Time-course curve before, during and after 30 nM leptin treatment corresponding to A. * P⬍0.05, vs. control.

vs. leptin alone), and increased the current amplitude of IHVA in POMC neurons by 15.6⫾9.3% (Fig. 6B, n⫽6, P⬍0.05 vs. leptin alone), indicating that JAK2 activation contributed to the regulation of calcium current by leptin. To test whether leptin regulated VGCCs via activation of mitogen-activated protein kinase (MAPK), we employed MAPK inhibitor PD98059 in bath solution. As shown in Fig. 6C, application of MAPK inhibitors PD98059 (100 nM, 10 min) partly antagonized the inhibition effect of leptin on IHVA in NPY neurons (from 52.1⫾11.0% to 83.3⫾10.2%, n⫽6, P⬍0.05), but had no effect on the increase of IHVA by leptin in POMC neurons. However, phosphatidylino 3-kinase (PI3-k) inhibitor wortmannin (200 nM, 10 min) failed to affect the inhibitory effect of leptin on IHVA in NPY neurons, but significantly blocked the leptin-elicited IHVA elevation in POMC neurons (Fig. 6D). The maximal stimulation of IHVA by 30 nM leptin in the presence of wortmannin was 16.3⫾5.4% (n⫽6, P⬍0.05, vs. leptin alone 44.2⫾10.2%). Taken together, our data clearly indicate that MAPK-dependent pathway is likely to be involved in leptin-induced IHVA inhibition in NPY neurons, however, leptin-stimulated IHVA elevation in POMC neurons is mediated via a PI3-k dependent pathway. Effects of nifedipine on regulation of IHVA by leptin In the following experiments, presumptive NPY and POMC neurons were characterized by morphology and responsiveness to leptin. To further demonstrate which type of calcium channel was modulated by leptin, the effects of

leptin on IHVA in these neurons were observed in the presence of 10 ␮M nifedipine, a dihydropyridine calcium channel blocker. Nifedipine alone reduced the IHVA amplitude by (41.91⫾6.98%) from (513⫾41) pA to (298⫾23) pA in presumptive NPY neurons when applied in the extracellular solution (10 ␮M, n⫽22, P⬍0.05). Pretreatment with 10 ␮M nifedipine, leptin failed to affect the current amplitude of IHVA in presumptive NPY neurons (Fig. 7A and 7C). Similarly, nifedipine also significantly reduced the peak amplitude of IHVA to (52.84⫾10.32%) of control in presumptive POMC neurons (10 ␮M, n⫽20, P⬍0.05). As shown in Fig. 7B and 7D, leptin-induced IHVA increase in presumptive POMC neurons was blunted while 10 ␮M nifedipine was applied firstly, nifedipine reduced IHVA to (56.19⫾10.24%) (n⫽20, P⬎0.05 vs. nifedipine alone) of the control, a value similar to that without leptin treatment.

DISCUSSION In this study, we revealed that leptin directly inhibits IHVA of NPY neurons in the ARC but increases IHVA of POMC neurons in a dose-dependent manner, which concentration falls within the circulating concentration range in animals and humans (Tartaglia et al., 1995; Rock et al., 1996; Glaum et al., 1996). Our work provided direct electrophysiological evidence for mechanisms underlying the differential effects of leptin on NPY and POMC neurons. First, we demonstrated that leptin attenuates the amplitude of IHVA

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Fig. 4. Effects of 30 nM leptin on I–V relationship and activation kinetics of IHVA in ARC NPY and POMC neurons. (A) Representative recording of IHVA evoked by a series of depolarizing steps (inset). (B) I–V curve for IHVA in NPY neurons. The amplitude of IHVA was decreased by 30 nM leptin. (C) The steady-state activation curve of IHVA in NPY neurons under control condition and during application of 30 nM leptin. (D) Effects of 30 nM leptin on the I–V curves of IHVA in POMC neurons. (E) Effects on 30 nM leptin on steady-state activation curves of IHVA in POMC neurons. The normalized conductance is plotted against the membrane potential, and fitted with the Boltzmann equation. Each point represents the mean⫾S.E.M. of six neurons.

in NPY neurons and shifts the steady-state activation curve to positive, but does not change its steady-state inactivation kinetics. Whereas the amplitude of IHVA in POMC neurons was enhanced by leptin, the steady-state activation curve was shifted to more negative potentials, however, the steady-state inactivation curve was not altered. Second, the key signaling cascades in these responses were determined. We found that leptin-induced decrease in IHVA in NPY neurons is dependent on JAK2 and MAPK activation, but not on PI3-k activation, consis-

tent with the observations of Jo et al. (2005) on perifornical lateral hypothalamus (LH) neurons. In contrast, our reports indicated that leptin stimulates increase in IHVA in POMC neurons is dependent on JAK2 and PI3-k activation. It is in agreement with the report that leptin mediates PI3-k activation may contribute to the well-documented stimulatory effect of leptin on POMC neurons (Cowley et al., 2001; Ibrahim et al., 2003). Finally, leptin regulated VGCCs in presumptive NPY and POMC neurons via L-type calcium channels. In support of our suggestion, earlier report

Fig. 5. Effects of 30 nM leptin on steady-state inactivation of IHVA in NPY and POMC neurons. (A) The original traces of IHVA induced by a series of depolarizing steps (inset). (B) Comparison of steady-state inactivation of IHVA in NPY neurons with or without leptin (C) Effects of leptin on steady-state inactivation IHVA curves in POMC neurons. The inactivation curves were fitted with the Boltzmann equation. Each point represents the mean⫾S.E.M. of six neurons.

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Fig. 6. Leptin inhibited IHVA of NPY neurons via MAPK-dependent pathway and increased IHVA of POMC neurons by PI3-k-dependent pathway, but JAK2 pathway contributed to leptin-induced responses in both two kinds of neurons. (A) Examples of IHVA in the presence of leptin and AG490 in NPY neurons. (B) Examples of IHVA in the presence of leptin and AG490 in POMC neurons. (C) Summary data of the results with different kinase inhibitors on leptin-mediated inhibition of IHVA in NPY neurons. The JAK2 inhibitor AG490 and MAPK inhibitor PD98059 attenuated the inhibitory effect on IHVA by leptin. However, PI-3k inhibitor wortmannin failed to have any effect on this response. (D) Summary of the maximal response of 30 nM leptin on IHVA in POMC neurons. AG490 and wortmannin attenuated the stimulation effect on IHVA by leptin. MAPK inhibitor PD98059 did not reduce the leptin-stimulated IHVA elevation. Each bar represents the mean⫾S.E.M (n⫽6). * P⬍0.05 vs. leptin group.

showed that L-type calcium channels are modulated by phosphorylation and N, P/Q and R-type channels are modulated by G-proteins (Gomperts et al., 2003). POMC and NPY neurons in ARC of the hypothalamus are both principal sites of LEPR expression (Hâkansson et al., 1998) and the source of potent neuropeptide modulators, and they exert opposing effects on feeding and metabolism (Elias et al., 1999; Cone, 1999). Prior studies of the arcuate nucleus demonstrated differential effects of leptin on the firing properties of NPY neurons versus POMC neurons (Schwartz et al., 2000). POMC neurons may play a key role in reducing food intake and are stimulated by leptin (Cowley et al., 2001; van den Top et al., 2004). In contrast, NPY neurons are thought to enhance food intake and positive caloric balance and are inhibited by leptin (Cowley et al., 2001; Elias et al., 1999). These neurons are therefore ideal for characterizing action of leptin. However, their diffuse distribution makes them difficult to study. Previous study identified these neurons by targeted expression of green fluorescent protein in transgenic mice (Cowley et al., 2001) or single-cell RT-PCR (van den Top et al., 2004). Immunocytochemical identification of these neurons was adopted in recent research work (Wilson et al., 1998; Muroya et al., 2004; Kohno et al., 2007). In this study, morphological observation in combination with subsequent immunocytochemical determination was employed to identify NPY and POMC neurons. At the end of whole-cell patch-clamp recording, we took photographs of all cells in the microscopic field. Based on these photographs,

the cells in which IHVA was measured were correlated with the corresponding immunocytochemical results. The leptin-mediated anti-obesity actions could be the result of long-term effects on gene expression secondary to activation of JAK2 and signal transducers and activators of transcription (STAT) pathway via specific intracellular docking sites (Bates et al., 2003), rapid effects on membrane potential and firing rates with changed release of neurotransmitters and/or neuropeptides (Cowley et al., 2001; van den Top et al., 2004; Glaum et al., 1996), or the combined effects of both. Although leptin has been shown to inhibit NPY neurons and activate POMC neurons in ARC, the electrophysiological mechanisms remain relatively unexplored. Previous studies have painted an intricate but complex picture of hypothalamic energy balance circuitry. PI3-k may contribute to the activation of both NPY and POMC neurons, but the direction of its regulation by leptin depends on whether the action is mediated directly or indirectly. Leptin directly activated PI3-k in POMC neurons, but the effect of leptin withdrawal activating PI3-k in NPY neurons required synaptic transmission (Xu et al., 2005). In another model, the GABA-mediated inputs to POMC neurons came from NPY neurons, and leptin directly depolarized the POMC neurons while simultaneously hyperpolarized the somata of NPY/GABA terminals. This diminished GABA release disinhibited the POMC neurons. In addition, both POMC and NPY neurons express autoreceptors for some of their respective neuropeptide products, and activation of these autoreceptors

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Fig. 7. Effects of nifedipine on the leptin-induced IHVA response in presumptive NPY and POMC neurons. (A) Addition of 30 nM leptin after exposure to nifedipine (10 ␮M, 10 min) had no significant effect on IHVA in presumptive NPY neurons. (B) Application 10 ␮M nifedipine 10 min prior to addition of leptin significantly reduced the action of leptin in presumptive POMC neurons. (C) Summary of effects of nifedipine (n⫽22) and leptin (n⫽20) on IHVA in presumptive NPY neurons. (D) Summary of effects of nifedipine (n⫽20) and leptin (n⫽20) on IHVA in presumptive POMC neurons. * P⬍0.05 vs. control.

may provide ultrashort feedback loops that further modulate the effects of leptin on these neurons (Cowley et al., 2001). However, as single cells were quite sparsely plated in our preparation, it is unlikely that neurons frequently have synaptic contacts to each other. Thus cultured primary neurons provided an invaluable and simple tool for determining the direct action of leptin on these important neurons. As calcium entry through VGCCs is essential for control of membrane excitability, synaptic function, cellular homeostasis, gene transcription, development and growth (Hille, 1986; e Silva and Lewis, 1995; Overholt and Prabhakar, 1997), leptin-sensitive inhibition or stimulation of VGCCs would be responsible for the decreased or increased excitability in corresponding neurons following leptin application. We found that leptin directly decreases IHVA in NPY neurons and increases IHVA in POMC neurons, which, at least in part, contributed to the regulation of neuron activity by leptin. In addition to the different effects on IHVA in NPY and POMC by leptin, the signal pathways underlying these effects were also different. Leptin interaction with LEPR might activate JAK2, which in turn can lead to the other downstream kinase cascades, including STAT transcription factors, MAPK, PI3-k and insulin receptor substrates (IRS-1 and IRS-2) (Carvalheira et al.,2005), whereas MAPK and PI3-k appear to play the more significant roles in the modulation of membrane potential and firing rates. Thus, AG490, PD98059 and wortmannin were used to investigate whether these mechanisms were involved in the action of leptin. AG490, PD98059 markedly

abolished the decrease of IHVA in NPY neurons by leptin, but wortmannin failed to inhibit the effect of leptin, which indicated that leptin-induced inhibitory effect on IHVA in NPY neurons possibly resulted from activation of LEPRs and MAPK signal pathways. However, AG490 and wortmannin reversed the potentiation effect on IHVA in POMC neurons by leptin, suggesting that activation of LEPRs and PI3-k signal pathways must contribute to the action on IHVA in POMC neurons by leptin (Fig. 8).

Fig. 8. Summary diagram of current studies and proposed model. Schematic of ARC in hypothalamus illustrating NPY/POMC neurons were the focus of the current study. The activation of LEPR inhibited VGCCs via activation of JAK2 and MAPK in NPY neurons, whereas evoked VGCCs by JAK2 and PI3-k in POMC neurons.

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According to the difference of activation threshold, VGCCs are divided into high voltage–activated channels (HVA) and low voltage–activated channels (LVA). Physiological and pharmacological data illustrated that HVA can be further divided into different types such as L-, N-, P/Q-, R-type et al., with L- and N- type being the major component of HVA channels (Tsien et al., 1995). Different subtypes of VGCCs make different contribution to neuronal function. Several lines of evidence suggested that N-type Ca2⫹ channels are involved in the release of NPY stimulated by high concentration of potassium, and N-type Ca2⫹ channels could play a pivotal role in the regulation of Ca2⫹ influx and [Ca2⫹]i in NPY neurons (King et al., 1999; Kohno et al., 2003). To examine whether a specific subtype of HVA channels is modulated by leptin in presumptive NPY and POMC neurons, nifedipine, a blocker of L-type Ca2⫹ channels was used. In this experiment, the modulation of IHVA by leptin was reversed by nifedipine, indicating that leptin selectively decreased or increased L-type Ca2⫹ channels in presumptive NPY and POMC neurons. It has been demonstrated that L-type calcium channels play a crucial role in the regulation of gene transcription (Catterall, 1998). In contrast to this, in our experiments, the effects of leptin were rapid and could be partially reversed, which suggested a direct modulation of channel activity rather than relying on gene expression.

CONCLUSION In summary, these data strongly supported the concept that leptin directly inhibits the L-type calcium currents in presumptive NPY neurons by activation of MAPK-mediated signals, and evokes L-type calcium currents in presumptive POMC neurons by PI3-k signal pathways. The regulation of VGCCs mediated the alteration of activity in NPY and POMC neurons, at least in part, the anorectic action of leptin. Our findings provided a new perspective with respect to the nature of the response, in which activation of LEPRs may contribute to the regulation of IHVA in both NPY and POMC neurons, but the reciprocal regulation by leptin depends on which downstream kinase was activated. Thus, different downstream kinase pathways provide a unifying hypothesis to explain how this hormone exerts opposing effects on the activity of these two key subsets of neurons. Acknowledgments—This work was supported by the grants from the National Basic Research Program of China (973 Program) (No. 2007CB507404), the National Science Fund for Distinguished Young Scholars of China (No. 30425024), and the National Science Foundation of China (No. 30570556) to Dr. JianGuo Chen, and Joint Research Fund for Overseas Chinese Young Scholars to Dr. Yong Xia and Dr. Jian-Guo Chen (No. 30728010).

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(Accepted 8 April 2008) (Available online 5 June 2008)