Melanocortin type 4 receptor–mediated inhibition of A-type K+ current enhances sensory neuronal excitability and mechanical pain sensitivity in rats

α-Melanocyte–stimulating hormone (α-MSH) has been shown to be involved in nociception, but the underlying molecular mechanisms remain largely unknown. In this study, we report that α-MSH suppresses the transient outward A-type K+ current (IA) in trigeminal ganglion (TG) neurons and thereby modulates neuronal excitability and peripheral pain sensitivity in rats. Exposing small-diameter TG neurons to α-MSH concentration-dependently decreased IA. This α-MSH–induced IA decrease was dependent on the melanocortin type 4 receptor (MC4R) and associated with a hyperpolarizing shift in the voltage dependence of A-type K+ channel inactivation. Chemical inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin or of class I PI3Ks with the selective inhibitor CH5132799 prevented the MC4R-mediated IA response. Blocking Gi/o-protein signaling with pertussis toxin or by dialysis of TG neurons with the Gβγ-blocking synthetic peptide QEHA abolished the α-MSH–mediated decrease in IA. Further, α-MSH increased the expression levels of phospho-p38 mitogen-activated protein kinase, and pharmacological or genetic inhibition of p38α abrogated the α-MSH–induced IA response. Additionally, α-MSH significantly increased the action potential firing rate of TG neurons and increased the sensitivity of rats to mechanical stimuli applied to the buccal pad area, and both effects were abrogated by IA blockade. Taken together, our findings suggest that α-MSH suppresses IA by activating MC4R, which is coupled sequentially to the Gβγ complex of the Gi/o-protein and downstream class I PI3K-dependent p38α signaling, thereby increasing TG neuronal excitability and mechanical pain sensitivity in rats.

␣-Melanocyte-stimulating hormone (␣-MSH) has been shown to be involved in nociception, but the underlying molecular mechanisms remain largely unknown. In this study, we report that ␣-MSH suppresses the transient outward A-type K ؉ current (I A ) in trigeminal ganglion (TG) neurons and thereby modulates neuronal excitability and peripheral pain sensitivity in rats. Exposing small-diameter TG neurons to ␣-MSH concentration-dependently decreased I A . This ␣-MSH-induced I A decrease was dependent on the melanocortin type 4 receptor (MC4R) and associated with a hyperpolarizing shift in the voltage dependence of A-type K ؉ channel inactivation. Chemical inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin or of class I PI3Ks with the selective inhibitor CH5132799 prevented the MC4R-mediated I A response. Blocking G i/o -protein signaling with pertussis toxin or by dialysis of TG neurons with the G ␤␥ -blocking synthetic peptide QEHA abolished the ␣-MSH-mediated decrease in I A . Further, ␣-MSH increased the expression levels of phospho-p38 mitogen-activated protein kinase, and pharmacological or genetic inhibition of p38␣ abrogated the ␣-MSH-induced I A response. Additionally, ␣-MSH significantly increased the action potential firing rate of TG neurons and increased the sensitivity of rats to mechanical stimuli applied to the buccal pad area, and both effects were abrogated by I A blockade. Taken together, our findings suggest that ␣-MSH suppresses I A by activating MC4R, which is coupled sequentially to the G ␤␥ complex of the G i/oprotein and downstream class I PI3K-dependent p38␣ signal-ing, thereby increasing TG neuronal excitability and mechanical pain sensitivity in rats.
The ␣-melanocyte-stimulating hormone (␣-MSH) 4 is primarily a pigmentary hormone derived from proopiomelanocortin (POMC), a common precursor protein of all melanocortin peptides, which predominantly expresses in the pituitary gland (1). Five G-protein-coupled melanocortin receptors, named MC1R-MC5R, have been characterized as the endogenous receptors of ␣-MSH (2). These receptors differ in their tissue distribution and in their ability to recognize various melanocortins. Consistent with the distribution of MCRs in the nervous system (mainly MC3R and MC4R) and peripheral organs (3), ␣-MSH exerts a large array of behavioral effects and regulates a variety of physiological functions, including blood pressure regulation, grooming, immunity, and body weight homeostasis (4,5). Additionally, in vivo studies have revealed that ␣-MSH, as well as the melanocortin receptor agonist melanotan II, causes hyperalgesia in various pain models (6 -8). Further support for this hypothesis came from the observation that antagonism of the MC3R and MC4R can effectively alleviate chronic constriction injury (CCI)-induced allodynia (9,10). Nevertheless, the contributing mechanisms underlying ␣-MSH hyperalgesic actions are not well-understood.
Alterations in the excitability of nociceptive sensory neurons can directly affect allodynia and hyperalgesia, the two common symptoms of pain (11). Voltage-gated ion channels, including K ϩ channels (Kv), are pivotal determinants of membrane excitability in peripheral nociceptive neurons (12) and are classified into two major categories, A-type and delayed rectifier chan-nels, which mediate currents of I A and I DR , respectively (13,14). I A currents are defined by their sensitivity to 4-aminopyridine and their rapidly activating and quick inactivating characteristics (15). These channels play critical roles in determining the intrinsic membrane properties and the excitability of nociceptive neurons (12,13), and they have been widely implicated in pain plasticity (15). For instance, peripheral nerve injury was shown to induce the down-regulation of I A , resulting in increased excitability of sensory neurons, which may increase the responsiveness to nociceptive stimulation (16). In addition, genetic and functional analyses have firmly established an important role of A-type channels in amplifying nociceptive signals in the periphery and in contributing to central sensitization in the spinal dorsal horn (15,17,18). Manipulation of A-type channels, therefore, is expected to modulate intrinsic neuronal excitability and subsequent nociceptive transmission, which is considered useful for pain therapy.
In the present study, we determined the role of ␣-MSH in regulating I A and elucidated the detailed mechanisms underlying the actions of ␣-MSH in small-diameter (Ͻ30 m) TG neurons. Our findings demonstrated that ␣-MSH decreased I A via a G ␤␥ -dependent class I PI3K and the downstream p38␣ signaling pathway. This response occurred through the MC4R activation and contributed to the neuronal hyperexcitability of TG neurons and pain hypersensitivity in rats.

␣-MSH selectively decreases I A in small-sized TG neurons
Different subtypes of peripheral sensory neurons are usually examined in in vitro studies of nociceptive and other forms of sensory processing (19 -23). In this study, we sorted adult rat TG neurons into small-sized (Ͻ30 m in soma diameter) and medium-sized (30 -45 m in soma diameter) groups and limited the whole-cell recording to the small ones, as these neurons played pivotal roles in the nociceptive processing (20,24). Kv currents in the small-sized nociceptive neurons are functionally characterized into two main types, including a transient outward, A-type K ϩ current (I A ) and a delayed rectifier K ϩ current (I DR ) (13,25). Therefore, we first separated the two kinetically different whole-cell currents in our patch clamp recordings. A whole-cell outward K ϩ current was elicited by a command potential of ϩ40 mV from a holding potential of Ϫ80 mV in TG neurons (Fig. 1A). The typical current profile observed in these neurons exhibited a component characteristic of fast inactivation and a following sustained portion. After inactivating the transient outward K ϩ currents by a 150-ms-long prepulse, only the I DR was left. Further off-line subtraction of I DR from the total outward current yielded I A (Fig. 1A). The application of 5 mM 4-aminopyridine (4-AP) to TG neurons dramatically inhibited this I A (decrease of 81.9 Ϯ 5.1% at ϩ40 mV, n ϭ 9;  Left, I A was isolated using a two-step voltage protocol. Right, I A was obtained after off-line subtraction of the noninactivating component. Insets in the top panel show the stimulation waveform, which was used for the I A isolation indicated in all figures. B, current-voltage (I/V) relationships of I A current density versus test potential for 5 mM 4-AP effects (n ϭ 9). C and D, representative traces (C) and summary data of current density (D) (n ϭ 8) demonstrating that 0.1 M ␣-MSH selectively decreases I A . E, concentration-response curve plotting the inhibitory effects of ␣-MSH on I A . The curves were fitted with a sigmoidal Hill function (I/I control ϭ 1/(1 ϩ 10 (logEC50 Ϫ log(␣-MSH)) n H ) to compute the effective concentration at which the half-maximum effect occurs (EC 50 ). The number of cells tested at each concentration of ␣-MSH is shown in brackets. F and G, representative current traces (F) and I/V curves (G) (n ϭ 8) indicating the inhibitory effects of 0.1 M ␣-MSH on the current density of I A . The holding potential was Ϫ80 mV, and currents were stimulated with voltage protocols from Ϫ70 to ϩ70 mV with ϩ10-mV increments. H and I, application of 0.1 M ␣-MSH to TG neurons had no significant effect on the voltage dependence of activation potential (H) (n ϭ 12) but leftward-shifted the half-potential of the inactivation curve (I) (n ϭ 12). To measure the voltage dependence of activation, voltage stimuli lasting 400 ms were delivered with voltage steps ranging from Ϫ70 to ϩ70 mV. To determine the steady-state inactivation, prepulses ranging from Ϫ120 to ϩ20 mV were applied with 10-mV increments, followed by a 500-ms voltage step to ϩ40 mV. J, bar graph showing the effects of 0.1 M ␣-MSH on V half of the activation and inactivation curves. *, p Ͻ 0.05; **, p Ͻ 0.01 versus control. Error bars, S.E.

MC4R inhibits A-type K ؉ currents
␣-MSH (0.1 M) significantly reduced the peak current amplitude of I A by 33.2 Ϯ 2.8% in small-sized TG neurons (n ϭ 8), whereas I DR was not significantly affected (decrease of 2.1 Ϯ 1.6%, n ϭ 8) (Fig. 1, C and D). The inhibition of I A was partially reversible within 5 min of washout of ␣-MSH (Fig. 1D). Further investigation showed that the ␣-MSH-induced I A suppression was concentration-dependent and had a median effective concentration (EC 50 ) of 36.9 nM (Fig. 1E).
We then investigated whether the biophysical properties of A-type channels was also affected by ␣-MSH. The currentvoltage relationships (I/V curves) indicated that at each test potential above Ϫ20 mV, 0.1 M ␣-MSH significantly reduced the peak current amplitude of I A (Fig. 1, F and G), and at ϩ40 mV, the peak current density of I A decreased from 134.7 Ϯ 21.7 to 78.3 Ϯ 15.8 pA/picofarad (n ϭ 8; Fig. 1G). Next, we analyzed the voltage dependence of activation and inactivation potentials following the ␣-MSH application. Whereas the potential of voltage-dependent activation did not change significantly (V half from 8.9 Ϯ 2.4 to 9.7 Ϯ 3.3 mV; n ϭ 12; Fig. 1, H and J), application of 0.1 M ␣-MSH to TG neurons significantly shifted the inactivation curve toward the hyperpolarizing direction by ϳ10.7 mV (V half from Ϫ52.6 Ϯ 1.9 to Ϫ63.3 Ϯ 1.4 mV; n ϭ 12; Fig. 1, I and J). These results reveal that the increased proportion of A-type channels in the steady-state inactivation might contribute to the ␣-MSH-induced I A decrease.

MC4R mediates the ␣-MSH-induced I A decrease
Five types of melanocortin receptors in the mammalian genome have been identified as the endogenous receptors for ␣-MSH, among which only MC3R and MC4R prominently expressed in the spinal cord and dorsal root ganglia (3,7,26,27). We determined the participation of these two receptors in ␣-MSH-induced I A decrease by first examining the expression profile of MC3R and MC4R. RT-PCR analysis demonstrated that only the MC4R transcripts (the predicted size is 923 bp), and not the MC3R (the predicted size is 307 bp), were detected in rat TG tissues ( Fig. 2A). MC4R protein expression in TGs was confirmed by Western blotting (Fig. 2B). Next, we analyzed the subcellular localization of MC4R protein by immunofluorescent staining. In rat TG sections, 32.5 Ϯ 6.3% of MC4Rpositive neurons were co-stained with IB 4 , and 43.9 Ϯ 5.2% of MC4R-positive neurons exhibited immunoreactivity for CGRP (

MC4R mediates I A decrease via G ␤␥ -dependent PI3K signaling
The signaling of MC4R activation in a variety of primary cells and tissues is known to act via heterotrimeric G s -proteins (28).

MC4R inhibits A-type K ؉ currents
Pre-incubating TG neurons with 0.5 g/ml cholera toxin (CTX), which effectively inactivated by G␣ s (24,29), did not affect the ␣-MSH-induced I A response (decrease of 29.1 Ϯ 3.7%, n ϭ 10; Fig. 3, A and C). Contrastingly, inhibition of G␣ i/o by pretreatment with 0.2 g/ml pertussis toxin (PTX) for 16 h abolished the inhibitory effect of ␣-MSH on I A (decrease of 1.2 Ϯ 3.9%, n ϭ 8; Fig. 3, B and C). Dialysis of small-sized TG neurons with 10 M QEHA, a synthetic peptide that competitively binds G ␤␥ and blocks the G ␤␥ -mediated signaling (30), prevented the ␣-MSH-induced response in I A (decrease of 2.1 Ϯ 1.8%, n ϭ 8; Fig. 3D), whereas intracellular application of SKEE (10 M), the scrambled peptide of QEHA, did not elicit such effects (decrease of 34.1 Ϯ 4.6%, n ϭ 11; Fig. 3D). These results suggest that the G ␤␥ complex of G i/o -protein participates in the MC4R-induced I A decrease. Previous studies showed that protein kinase A (PKA) is downstream of G ␤␥ activation (31); however, dialyzing small TG neurons with 1 M PKI 6-22, a synthetic peptide inhibitor of PKA, did not alter the ability of ␣-MSH to decrease I A (decrease of 31.2 Ϯ 3.9%, n ϭ 9; Fig. 3D). The PKI 6-22 at 1 M was effective in this assay because the intracellular administration of PKI 6-22 resulted in a nearly complete blockade of 20 M forskolin-induced I A response (decrease of 1.5 Ϯ 3.1%, n ϭ 7; Fig. 3E). PI3K activation has been shown to modulate I A and act as a downstream effector of G ␤␥ activation (32). Application of either 0.1 M ␣-MSH or the selective MC4R agonist Ro 27-3225 at 0.2 M significantly enhanced the PI3K activity, and the pretreatment of TG neurons with the selective MC4R antagonist HS024 at 0.5 M completely abolished these effects (Fig. 3F). Further, pretreating TG neurons with 0.5 M wortmannin, a PI3K inhibitor

The ␣-MSH-induced I A decrease requires p38 MAPK
Akt is a common and major downstream target of PI3K. Therefore, we examined whether the ␣-MSH action in TG neurons was through the Akt activation. Although the phosphorylated Akt (p-Akt) level was significantly increased following the ␣-MSH treatment (Fig. 4A), the inhibition of Akt activity by 10 M Akt inhibitor III showed no influence on 0.1 M ␣-MSH-induced I A decrease (decrease of 31.9 Ϯ 7.6%, n ϭ 11; Fig. 4B). This finding suggests that the ␣-MSHinduced I A response is independent of Akt. It has been shown that mitogen-activated protein kinase (MAPK) cascades play pivotal roles in pain sensation (33), and the MAPK family molecules are involved in the regulation of I A (34). Therefore, subsequently, we investigated in TG neurons whether MAPK participated in the MC4R-mediated decrease in I A . Immunoblot analysis indicated that exposure of TG cells to 0.1 M ␣-MSH markedly increased phosphorylated p38 (p-p38), whereas the total p38 (t-p38), as well as phospho-JNK and phospho-ERK (p-ERK) expression levels, remained unaffected (Fig. 4C). Pre-incubation of TG cells with the PI3K antagonist wortmannin at 0.5 M eliminated the ␣-MSH-induced p38 activation (Fig. 4D), indicating the involvement of PI3K-dependent p38 MAPK activation. Furthermore, the pretreatment of TG neurons with the p38

MC4R inhibits A-type K ؉ currents
MAPK inhibitor SB203580 at 10 M abrogated the ␣-MSHinduced decrease in I A (decrease of 2.9 Ϯ 2.1%, n ϭ 9; Fig. 4, E and G). Contrastingly, the negative control compound SB202474, which is structurally related to SB203580 but does not inhibit p38 MAPK activity, elicited no such effects at the same concentration (decrease of 30.9 Ϯ 4.9%, n ϭ 7;  Fig. 4, F and G). Thus, the Akt-independent p38 MAPK activation is required for the ␣-MSH-induced I A decrease in TG neurons.

p38␣ mediates the ␣-MSH-induced I A response
In the nervous system, such as in rat spinal cord, two major isoforms of p38 exist, p38␣ and p38␤ (Fig. 5A). We further dissected the exact p38 isoform involved in the ␣-MSHinduced I A decrease in rat TG neurons. Western blot analysis revealed that only p38␣, and not p38␤, was endogenously expressed in adult rat TGs (Fig. 5A). Pretreating TG neurons with 50 nM JX-401, a potent inhibitor specific to the p38␣ isoform, completely abolished the 0.1 M ␣-MSH-mediated decrease in I A (decrease of 2.9 Ϯ 3.5%, n ϭ 9; Fig. 5B). To further confirm this specific p38␣-mediated signaling pathway, we utilized an adenovirus-based shRNA approach to knock down p38␣ in TG cells. In contrast to the substantial expression of p38␣ in the cells transduced with negative control shRNA (NC-shRNA), the protein expression level of p38␣ was markedly reduced in TG cells transduced with the p38␣-specific shRNA (p38␣-shRNA; Fig. 5C). Knockdown of p38␣ in TG neurons led to the attenuation of the ␣-MSH-mediated decrease in I A (decrease of 2.3 Ϯ 2.1%, n ϭ 17; Fig. 5D), indicating that p38␣ is specifically involved in the ␣-MSH-induced I A decrease.

␣-MSH enhances TG neuronal excitability through I A modulation
I A encoded by Kv channels plays pivotal roles in modulating membrane excitability in multiple excitable cell types, including TG neurons (15,23). To determine the functional roles of the I A decrease mediated by MC4R activation, we investigated whether ␣-MSH application might affect the neuronal excitability. The application of 0.1 M ␣-MSH to small-sized TG neurons had no effect on the whole-cell Nav currents (decrease of Ϫ2.7 Ϯ 1.6% at Ϫ10 mV, n ϭ 10; Fig. 6A). It has been shown that the activation of MC4R inhibits presynaptic N-type Ca 2ϩ channels in amygdaloid complex neurons (35). To exclude the possible influence of ␣-MSH regulation of N-type as well as other types of Ca 2ϩ channels in TG neurons, we applied CdCl 2 (100 M) in the external solution to block voltage-gated Ca 2ϩ channels during current clamp recordings. We observed that the application of ␣-MSH at 0.1 M significantly increased action potential (AP) firing frequency by 61.8 Ϯ 5.3% (n ϭ 15; Fig. 6, B and C). Additionally, 0.1 M ␣-MSH significantly shortened first spike latency and increased AP amplitude (n ϭ 15; Fig. 6D). Other membrane properties of neuronal excitability, such as resting membrane potential and input resistance, were not significantly changed by the ␣-MSH application (not shown). Pretreating TG neurons with the MC4R antagonist HS024 (0.5 M) prevented the increased AP firing rate induced by 0.1 M ␣-MSH, indicating the MC4R involvement (n ϭ 11; Fig. 6E). To further verify that the MC4R-mediated neuronal hyperexcitability occurred through the decrease of I A , we applied 4-AP in the external solution to block I A and found that application of 5 mM 4-AP mimicked the MC4R-mediated neuronal hyperexcitability (n ϭ 10; Fig. 6, F and G). Notably, application of ␣-MSH during the 4-AP-induced response failed to produce any further increase in firing frequency (Fig. 6G). These findings suggest that the MC4R-mediated I A decrease contributes to the ␣-MSH-induced TG neuronal hyperexcitability.

␣-MSH-mediated I A decrease induces pain hypersensitivity in vivo
To further determine the functional implications of MC4R at the behavioral level, we investigated whether ␣-MSH affects peripheral pain sensitivity in rats. The escape threshold to mechanical facial nociception was determined by an ascending series of von Frey filaments, which were applied to the buccal pad region. Intra-TG injection of ␣-MSH at 2 nmol induced a marked decrease in the escape threshold 1-2 h post-administration (Fig. 7A), and this effect recovered after 3 h. The ␣-MSH-mediated mechanical pain hypersensitivity was abrogated by intra-TG injection of the MC4R inhibitor HS024 at 5 nmol (Fig. 7B). The participation of I A in the ␣-MSH-mediated mechanical hypersensitivity was further examined using the specific A-type channel blocker 4-AP. Intra-TG application of 4-AP at 25 nmol exhibited a significant increase in mechanical sensitivity as compared with the control animals that received a saline injection (Fig. 7C). Sensitivity assessed after intra-TG injection of ␣-MSH showed that ␣-MSH had no additive effect to 4-AP on mechanical sensitivity in rats (Fig. 7C), which sug-

MC4R inhibits A-type K ؉ currents
gests that the effect of ␣-MSH and 4-AP can be mediated through the same signaling pathway in vivo. Collectively, our data provide evidence that the ␣-MSH-induced I A decrease contributes to the MC4R-mediated mechanical pain hypersensitivity in rats.

Discussion
This study reveals a novel functional role of ␣-MSH in the regulation of I A in rat TG neurons. We identify a signaling path-way by through ␣-MSH attenuates I A through stimulation of MC4R that couples to a G ␤␥ -dependent class I PI3K and the downstream p38␣ signaling (Fig. 8). One of the immediate outcomes is the induction of sensory neuronal hyperexcitability and mechanical pain hypersensitivity.
Studies examining the PKA-dependent modulation of I A lead to conflicting conclusions. In hippocampal pyramidal neurons, activation of PKA by 8-bromo-cAMP significantly decreased I A (36). Similarly, 3-isobutyl-1-methylxanthine stimulation of PKA down-regulated the peak current density of I A (37). In contrast, PKA inducer mimicked the response of serotonin 1D receptor activator and was shown to stimulate I A in TG neurons (38). In the current study, the decrease of I A induced by ␣-MSH was not affected by the PKA inhibitor PKI 6-22, suggesting that other mechanisms rather than PKA participated in the MC4Rmediated I A response. We observed that in small-sized TG neurons, the selective inhibition of I A by MC4R activation was mediated by the class I PI3K and then relayed to the downstream p38-dependent signaling. Our data are consistent with previous studies conducted in early hippocampal neurons, which demonstrate that the PI3K/Akt signaling mediated the inhibition of I A induced by amyloid-␤ (39). Similarly, Kv currents, including I A , recorded from pancreatic ␤ cells decrease in response to PI3K activation (40). Interestingly, the activation of PI3K has been shown to increase I A in cultured rat cerebellar granule cells (41), and PI3K-induced activation of the Kv4.3 channel through glucocorticoid-inducible kinase-1 has also

MC4R inhibits A-type K ؉ currents
been identified (42). Although the discrepancy requires further investigation, the regulatory effects of different PI3K subtypes, including class I through class III, might be variable in distinct tissue/cell types expressing Kv channels.
Akt is the most common component downstream of PI3K. Previous studies suggested that Akt may regulate the activity of Kv4 channels in a cell type-and signal-specific manner. For instance, in cultured cerebellar granule cells of rats, Akt has been shown to down-regulate Kv4.2 channels (43), whereas the Akt-dependent signaling stimulated Kv4 in the arcuate nucleus (44). In the present study, the MC4R-mediated I A decrease appears not to be mediated by Akt in small TG neurons. Accumulating evidence has suggested that the extracellular signalregulated kinase-1 and -2 (ERK1/2) signaling is involved in the modulation of pain (45). Additionally, ERK was shown to directly phosphorylate the pore-forming subunit of Kv4.2 channels (46), and activation of ERK attenuated I A in neurons in superficial laminae of the spinal dorsal horn (34). In contrast, ERK signaling mediating the dopamine-induced I A increase in lateral pyloric neurons was also reported (47). However, in this study, the stimulation of MC4R in small TG neurons resulted in an elevated level of phospho-p38, but not phospho-ERK or phospho-JNK, excluding the possible involvement of ERK in the MC4R-induced I A response. Our findings are in line with previous studies conducted in dorsal root ganglion neurons and heart cells, in which the p38 activation led to a decrease of Kv currents (48) by phosphorylation of the residues Tyr-124 of Kv channels (49). On the contrary, p38 has been shown to stimulate Kv currents in transfected Chinese hamster ovary cells (50). This discrepancy could be explained by distinct p38␣ phosphorylation sites, by different Kv subtypes encoding I A in TG neurons and in modified Chinese hamster ovary cells (51), or by distinct splice variants of Kv channel-interacting protein (52).
Changes of peripheral neuronal excitability can directly influence painful conditions such as hyperalgesia and allodynia in vivo (11,12). A-type channels, the key components influencing neuronal excitability, have been implicated in controlling both the delay of first-spike latency and the decrease in spike frequency (15,53), which are two important determinants in the release time course of neurotransmitter and hence the nociceptive transmission (54). An important consequence of peripheral A-type channel modulation is to influence somatic and visceral nociceptive inputs, and the decrease of I A results in significant nociception in a variety of animal neuropathic pain models (17). In the current study, consistent with the ␣-MSHinduced decrease in I A in small-sized TG neurons, activation of MC4R significantly increased TG neuronal excitability along with the shorter first-spike latency and increased firing frequency; blockade of I A by 4-AP prevented this effect. Moreover, the acute mechanical hypersensitivity induced by ␣-MSH was occluded by the A-type channel blocker application. As such, it is reasonable to infer that the nociceptive effects of MC4R activation are mediated, partially if not completely, through its inhibition of I A . Indeed, our present results are in line with previous in vivo studies indicating that antagonism of MC4R reduces the CCI-induced allodynia in rodents (9,55,56), whereas activation of MC4R causes hyperalgesia in various pain models (6 -8). Nevertheless, potential channel targets other than A-type channels can also be activated by the MC4R pathway. It has been shown that the activation of MC4R with its agonist melanotan II inhibits presynaptic N-type Ca 2ϩ channels in amygdaloid complex neurons (35). In addition, MC4R constitutive activity inhibits Cav1.2 channel currents in transiently transfected HEK293 cells (57).
In summary, we present new insights underlying the effect of ␣-MSH on I A in small-sized TG neurons. Our findings indicated that ␣-MSH decreases I A through stimulation of a G i/oprotein-coupled MC4R and downstream class I PI3K-mediated p38␣ signaling. The identified signaling pathway mediates the functional effects of ␣-MSH on induced sensory neuronal hyperexcitability and mechanical pain hypersensitivity. It would be interesting to further examine the effects of MC4R signaling on other types of pain (e.g. pain caused by thermal injuries) in future work. Moreover, previous studies have revealed that the expression of p38␣ is up-regulated in ipsilateral TGs in an orofacial inflammatory pain model induced by complete Freund's adjuvant (58) and that pharmacological blockade of p38 activation reduces the CCI-induced thermal hyperalgesia (56). Therefore, the present findings regarding the MC4R-mediated p38␣ pathway in TG neurons may be reproduced in other sensory neurons, including dorsal root ganglia, and may pave the way for MC4R to be developed as a potential therapeutic target for the clinical management of chronic pain.

Dissociation of TG neurons
Rats were maintained in specific pathogen-free facilities on a 12-h/12-h light/dark cycle at a room temperature (22 Ϯ 1°C) and housed in cages with access to food and water ad libitum. All procedures in the animal studies were approved by the Animal Studies Committee of Soochow University and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. TG neurons were dissociated from Sprague-Dawley rats (220 -250 g, Figure 8. Scheme of the proposed mechanisms of MC4R on I A . ␣-MSH acts through MC4R, which is coupled to the G-protein G i/o , causing it to release the G ␤␥ subunits. The released G ␤␥ causes an increase in class I PI3K activity in TG neurons. Stimulation of PI3K signaling may phosphorylate p38␣ to regulate I A and induces TG neuronal hyperexcitability and pain hypersensitivity. PI3K can catalyze the conversion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to phosphatidylinositol 4,5-trisphosphate (PIP 3 ), which serves as a second messenger that helps to activate Akt. However, neither Akt nor PKA is necessary for the MC4R-mediated decrease of I A . Determination of whether p38␣ in small-sized TG neurons directly phosphorylates I A channel subtypes or acts through some intermediate signal molecules requires further investigation.

MC4R inhibits A-type K ؉ currents
regardless of gender) according to the established protocol (38,39). Briefly, the TGs were dissected out bilaterally, and the connective tissue was removed. After they were minced into 8 -10 pieces, the tissues were enzymatically digested first with 1.5 mg/ml collagenase II (Worthington) for 30 min and then with 20 units/ml papain for 20 min (Worthington). After papain treatment, the tissue was mechanically dissociated by trituration with sterile fire-polished glass pipettes. After centrifugation, the pellet was resuspended in minimum essential medium (Invitrogen) containing 10% fetal bovine serum (Hyclone, GE Healthcare), 2% B27 supplements (Invitrogen), 1% GlutaMAX, and 1% penicillin/streptomycin (Invitrogen) and then plated onto glass coverslips coated with Matrigel. The cells were maintained at 37°C in humidified incubators with 5% CO 2 and 95% air. Electrophysiological recordings were performed 2-6 h after plating.

Whole-cell patch clamp recordings
Electrophysiological procedures were performed at room temperature (23 Ϯ 1°C) as described previously (22,24). We chose only small-sized TG neurons that were less than 30 m in soma diameter for patch clamp experiments. Pipettes In experiments in which neurons were dialyzed with compounds, patch pipette resistance ranged from 2 to 3 megaohms for infusion, and currents were measured at least 5 min after breaking into the whole-cell configuration.

Detection of gene expression
RT-PCR was performed as described previously (30). Briefly, total RNA from rat entire TG tissues was extracted using an RNeasy kit (Qiagen, Germantown, MD) according to the manufacturer's protocol. RNA was treated with DNase (Promega Corp., Madison, WI) and then reverse-transcribed with Super-Script TM II (Thermo Fisher Scientific). The primers, designed in Primer version 5.0 software (Premier Biosoft International, Palo Alto, CA), were derived from a partial genomic sequence: MC3R, accession number NM_001025270.3, 5Ј-TCTGCT-GTGCTGTGGGGGTG-3Ј (forward) and 5Ј-CGGTTAGG-CGGGTCGGGATC-3Ј (reverse); MC4R, accession number NM_013099.3, 5Ј-CCACAAGAGAAGCACCTAGA-3Ј (forward) and 5Ј-GTTGCCGTTCCTCACCACAG-3Ј (reverse). Experiments included non-reverse-transcribed DNase-treated RNA samples as negative controls. PCR of these RT control groups never showed amplification, indicating that the RNA was genomic DNA-free. The assays were carried out in duplicate using the same samples to confirm the reproducibility of the results.

MC4R inhibits A-type K ؉ currents
Measurement of PI3K activity PI3K activity was determined as described previously (32). In brief, after stimulation of cells with ␣-MSH (0.1 M) or Ro 27-3225 (0.2 M) for 20 min, PI3K activity in homogenates was determined using a PI3K HTRF TM assay kit (Millipore Corp., Bedford, MA) according to the manufacturer's protocol. HTRF was then measured with an excitation wavelength of 335 nm and emission wavelengths of 620 and 665 nm with a spectrofluorometer (Tecan, Infinite M1000, Salzburg, Austria).

Adenovirus transduction
Adenovirus-mediated gene silencing was performed following a procedure described previously (22,32). Three candidate shRNAs targeting p38␣ (GenBank TM accession number NM_031020.2) were designed, and the shRNA sequence (5Ј-GGACCTCCTTATAGACGAATG-3Ј) with the best knockdown efficiency were selected. The nonsense sequence was designed as the negative control (NC-shRNA, 5Ј-ACCTGACT-GTGTCAGGAATCA-3Ј). The pAdTrack-CMV-GFP vector carrying p38␣ shRNA (p38␣-shRNA) plasmid was packaged by Genechem Co., Ltd. (Shanghai, China). After 48 h of infection, the efficiency of shRNA knockdown was determined by Western blot analysis as described above. For electrophysiological analysis, small TG neurons expressing GFP were subjected to whole-cell recordings.

Escape threshold for mechanical stimulation
Animals (Sprague-Dawley rats, 220 -250 g, regardless of gender) were housed under standard conditions (22 Ϯ 1°C, 50 -70% humidity, 12-h/12-h light/dark cycle, with ad libitum access to food and water) and were allowed to habituate to laboratory conditions for at least 3 days prior to the experiments. The escape threshold to mechanical stimulation was determined by an ascending series of von Frey filaments (Ugo Basile) as described previously (59,60). Von Frey stimuli were applied to the buccal pad region and were applied three times in each series of trials. ␣-MSH (2 nmol), HS024 (5 nmol), or 4-AP (25 nmol) was intra-TG-injected into the trigeminal ganglia with a 30-gauge needle inserted through the infraorbital foramen, infraorbital canal, and foramen rotundum. The needle tip was positioned in the medial part of the ganglia, and the treatment agent was slowly delivered in a volume of 5 l.

Drug application
CdCl 2 , 4-aminopyridine, cholera toxin, and pertussis toxin were purchased from Sigma-Aldrich; ␣-MSH was from Abcam; PKI 6-22 was from Tocris Bioscience; and Akt inhibitor III was from Santa Cruz Biotechnology, all of which were prepared in distilled deionized water as stock solutions. Forskolin, wortmannin, Ro 27-3225, CH5132799, and SB203580 were obtained from Sigma-Aldrich; HS024 was from Tocris Bioscience; SB202474 was from Merck Millipore; and JX-401 was from R&D systems, all of which were prepared as concentrated stock solutions in DMSO. The final concentration of DMSO in the bath solution was less than 0.01%, and this compound had no functional effects on I A (not shown).

Data analysis
Data acquisition and analysis were done using Clampfit version 10.2 software (Molecular Devices) and GraphPad Prism version 5.0 (GraphPad Software). Data are expressed as original traces or presented as means Ϯ S.E. The amplitude of I A was measured at the peak. The voltage dependence of activation and inactivation of I A was fitted with standard Boltzmann equations. A paired or two-sample t test was used when comparisons were restricted to two means. Treatment effects were statistically analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's test or two-way ANOVA followed by Bonferroni's test. Error probabilities of p Ͻ 0.05 were considered statistically significant.