Regulation of the firing activity by PKA‐PKC‐Src family kinases in cultured neurons of hypothalamic arcuate nucleus

Abstract The cAMP‐dependent protein kinase A family (PKAs), protein kinase C family (PKCs), and Src family kinases (SFKs) are found to play important roles in pain hypersensitivity. However, more detailed investigations are still needed in order to understand the mechanisms underlying the actions of PKAs, PKCs, and SFKs. Neurons in the hypothalamic arcuate nucleus (ARC) are found to be involved in the regulation of pain hypersensitivity. Here we report that the action potential (AP) firing activity of ARC neurons in culture was up‐regulated by application of the adenylate cyclase activator forskolin or the PKC activator PMA, and that the forskolin or PMA application‐induced up‐regulation of AP firing activity could be blocked by pre‐application of the SFK inhibitor PP2. SFK activation also up‐regulated the AP firing activity and this effect could be prevented by pre‐application of the inhibitors of PKCs, but not of PKAs. Furthermore, we identified that forskolin or PMA application caused increases in the phosphorylation not only in PKAs at T197 or PKCs at S660 and PKCα/βII at T638/641, but also in SFKs at Y416. The forskolin or PMA application‐induced increase in the phosphorylation of PKAs or PKCs was not affected by pre‐treatment with PP2. The regulations of the SFK and AP firing activities by PKCs were independent upon the translocation of either PKCα or PKCβII. Thus, it is demonstrated that PKAs may act as an upstream factor(s) to enhance SFKs while PKCs and SFKs interact reciprocally, and thereby up‐regulate the AP firing activity in hypothalamic ARC neurons.

Neurons in the hypothalamic arcuate nucleus (ARC) are found to be involved in the descending modulation of nociception (Bach, 1997;Sim & Joseph, 1989Wang et al., 2015;Yin, DuanMu, Guo, Yu, & Zhang, 1984). Recent studies (Bu et al., 2015;Peng et al., 2011;Xu et al., 2012;Zheng et al., 2016) have shown that with the development of visceral or peripheral inflammation, increases in the neuronal discharge activity and the expression of active PKCs, SFKs, and the phosphorylated GluN2B subunit at Y1472 occur in the ARC area. Application of inhibitors of PKCs or SFKs into the ARC area blocks the enhancement of the expression of active PKCs, SFKs, and the phosphorylation of GluN2B at Y1472 in this area, and also attenuates the inflammation-induced enhancement of the discharge activity of ARC neurons and pain hypersensitivity (Bu et al., 2015;Peng et al., 2011;Xu et al., 2012;Zheng et al., 2016). Furthermore, we have identified that Src, but not Fyn or Lyn in SFKs, is activated following the development of peripheral inflammation induced by the injection of complete Freund's adjuvant (CFA) into the hind pawl of rats (Ma et al., 2019). It is found that the knockdown of Src in the ARC area blocks the increases in the expression of activated SFKs and the phosphorylation of GluN2B subunit at Y1472, and reduces pain hypersensitivity induced by the CFA injection (Ma et al., 2019).
Potential functional interactions among PKAs, PKCs, and SFKs in the regulation of pain hypersensitivity have been implicated in the spinal cord dorsal horn (Guo et al., 2004;Kawasaki et al., 2004), rostral ventromedial medulla (Guo et al., 2006), and ARC (Bu et al., 2015;Xu et al., 2012;Zheng et al., 2016). Characterizing the functional interactions among these molecules in the CNS is still required in order to understand the detailed mechanisms underlying the regulation of pain hypersensitivity. Therefore, we did this work as the first step, in cultured neurons isolated from the ARC area of rats, to identify the actions of PKCs, PKAs, and SFKs in regulating the action potential (AP) firing activity.

| ARC neuron culture
ARC tissues isolated acutely from 1-day-old Sprague-Dawley rat pups (both male and female) were used for culture. Total 420 rat pups were used. They were obtained from an in-house breeding colony with parent animals from the laboratory animal center at Soochow University. Animal care and experimental procedures were conducted following the guidelines of Animal Care and Use Committee of the Medical College of Soochow University and approved by Ethics Committee of Soochow University in accordance with the guidelines of the International Association for the Study of Pain. Animals were housed on a 12-hr light/dark cycle. The pups were reared in large cages by their mothers with free access to food and water.
Studies dealing with sex differences, which may produce biological variables, were not performed in this work. Following decapitation the whole brain was quickly removed and transferred to a 100 mm dish filled with ice-cold Hank's balanced salt solution (HBSS,pH 7.4;Gibco,Shanghai,China). Coronal brain sections (≈ 500 μm thick) were performed throughout the hypothalamic region under a stereomicroscope (SMZ455, Olympus, Tokyo, Japan). Tissues of the ARC region (Paxinos & Watson, 2007) (see Figure S1) were then dissected and cut. After wash for twice with HBSS the ARC tissues were treated with papain (2 mg/ml) dissolved in Neurobasal-A medium containing 5 mM of L-cystein (pH 7.4) for 25 min at 37°C.

Significance
The hypothalamic arcuate nucleus (ARC) has been found to be involved in the control of pain hypersensitivity. In order to better understand the actions of PKAs, PKCs, and SFKs in the regulation of neuronal activity, we investigated the regulation of the action potential (AP) firing activity by these kinases in cultured ARC neurons. This work demonstrates that PKAs may act as an upstream factor(s) to enhance SFKs while PKCs and SFKs may interact reciprocally, and thereby up-regulate the AP firing activity in hypothalamic ARC neurons.
humidified air with 5% CO 2 at 37°C for 4-6 days with Neurobasal-A medium supplemented with 2% of B27 and 1% of Gluta-max before use for experiments.

| Electrophysiological recording
Whole cell recordings in the current clamp model were performed for recording current injection-induced APs, as described previously (Wang et al., 2011). In brief, ARC cultures were placed in a recording chamber on an inverted microscope (Ti-DH, Nikon, Tokyo, Japan) equipped with a 40× Varel Relief Contrast System. Recorded cells were monitored during experiments to confirm that the same cells were recorded before and after any treatment. The cultures were continuously perfused with a standard external solution (0.5 ml/min) containing (in mM) 128 NaCl, 2 KCl, 2 CaCl 2 , 2 MgCl 2 , 30 glucose, 25 HEPES, pH was adjusted to 7.4 with NaOH, osmolarity: 305 mOsm.
Recording electrodes pulled from filamented borosilicate glass (Sutter Instruments, Novato, CA) were fire-polished, and filled with an internal solution composed of (in mM): 110 KCl, 10 NaCl, 2 EGTA, 25 HEPES, 4 Mg-ATP, and 0.3 Na 2 GTP, pH was adjusted to 7.3 with KOH, osmolarity: 295 mOsm. The DC resistance of recording electrodes was 7.3 ± 1.6 MΩ (mean ± SD). Recordings were performed at room temperature (23 ± 1°C) with a MultiClamp 700B amplifier (Molecular Devices, San Jose, CA). Electrical signals filtered at 1 kHz were recorded through the amplifier following the subtraction of the capacitive transients and digitized at 10 kHz. Off-line analysis of recorded electrical signals was conducted using the Clampex 10.2 (Molecular Devices). All recorded neurons were clamped at −60 mV. AP firing was induced by injection of depolarizing current pulses (increasing step amplitude: 10 pA; duration: 1 s; injection interval: 600 ms).

| Western blot analysis
Western blotting experiments were performed as described previously (Lei et al., 2002;Xu et al., 2008;Zheng et al., 2016). In brief, ARC cultures were washed three times with ice-cold PBS and scraped into ice-cold RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with EDTA-free cocktails of protease and phosphatase inhibitors (Roche, Basel, Switzerland), DTT (0.5 mM, Beyotime Biotechnology, Shanghai, China) and PMSF (1 mM, Beyotime Biotechnology). ARC cells were lysed via sonication. The homogenates were then centrifuged at 12,000g for 20 min at 4°C. Samples subjected to SDS-PAGE were generated by adding one-third of 4× loading buffer (Thermo, Shanghai, China).
Stripping was considered to be successful if no specific staining signal could be noted by the incubation of the stripped membrane with a secondary antibody. Examples of blots from same full length PVDF membranes, which were stripped and successively probed with antibodies, are shown in Figure S2.
Samples from cultured cells without any treatment (labeled as "naïve" in figures and following text) were examined in each (or each repeat) biochemical experiment in order to control variations which may occur from one experiment to another. Densitometry analysis of all western blots was conducted and the ratio of the band intensity versus that of β-actin was calculated and then normalized to the ratio detected in samples from naïve cells. The normalized ratios were used to show the effects of any treatment. At least five replicates were performed for the Western blot.

| Immunofluorescence image
For immunofluorescence studies of ARC neurons, cultured cells were fixed and permeabilized by treatment with 4% of polyoxymethylene for 30 min and 0.15% of Triton for 30 min, and then double labeled with NeuN antibody (RRID:AB_2298772; see Table 1) and DAPI. The NeuN antibody staining was visualized by incubation with an Alexa Fluor Donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (RRID:AB_141607; see Table 1).
For studies of PKCα and PKCβII translocations, after treatment with drugs as indicated ARC neurons were fixed and permeabilized as motioned above and then incubated with an antibody of PKCα (RRID:AB_777294) or PKCβII (RRID:AB_779042) (see Table 1 Figure 1a shows examples of ARC cells in culture. In ARC cultures 72.3% ± 7.1% (mean ± SD) of cells stained with DAPI were found to be co-labeled with NeuN staining (Figure 1b). In current clamp recordings depolarizing currents (increasing step amplitude: 10 pA; duration: 1 s; injection interval: 600 ms; see Section 2) were injected into recorded cells to induce APs. All of recorded cells were able to generate multiple APs in response to the current injection (see Figure 1c-g). The resting membrane potentials of recorded cells were −59.3 ± 8.9 mV (mean ± SD, n = 188). The AP thresholds and firing rates were −33.8 ± 5.8 mV and 9.1 ± 3.8 Hz (mean ± SD, n = 188). The AP amplitudes, AP half widths and first spike latencies were 83.0 ± 11.8 mV, 5.0 ± 2.1 ms, and 175.2 ± 77.3 ms (mean ± SD, n = 188). The input resistances were 2.6 ± 1.2 GΩ (mean ± SD, n = 188).

| The regulation of the AP firing activity of ARC neurons by PKAs, PKCs, or SFKs
Previous studies have documented that KT is a potent PKA inhibitor with an IC50 of 3.3 μM (Davies, Reddy, Caivano, & Cohen, 2000;Murray, 2008). Application of 20 nM GF directly to purified PKC proteins leads to a reduction in the activity of PKCs by 50% (Toullec et al., 1991). In cellular experimental models the IC50 of GF may be ranged from 0.1 to 1 μM (Son, Hong, Kim, Firth, & Park, 2011). Although the finding that chelerythrine was a potent inhibitor of PKCs with an IC50 of 0.66 μM (Herbert, Augereau, Gleye, & Maffrand, 1990;Ringvold & Khalil, 2017) has been challenged by a number of studies in which the enzyme activity of PKCs was found not to be affected by application of this compound (Lee et al., 1998;Vieira et al., 2015), it has been demonstrated that CC may inhibit the translocation of PKCs (Chao, Chen, & Cheng, 1998;Siomboing et al., 2001). SFK activity can be inhibited by 50% following 4 nM PP2 application in vitro and 0.6-18 μM in cellular experimental models (Hanke et al., 1996;Karni et al., 2003).
In our present study effects of the PKA inhibitor KT, the PKC inhibitor GF or CC, or the SFK inhibitor PP2 on the electrophysiological properties of cultured ARC neurons were investigated. Table 2 shows summary data demonstrating effects of treatment with vehicle or the inhibitors of PKAs, PKCs, or SFKs for 5 min on the electrophysiological properties of cultured ARC neurons recorded.
In these neurons the effects of FSK were examined in 16 neurons after the test of vehicle application, and in 19 neurons the effects of PMA were examined after the test of vehicle. When compared with those before vehicle application, no significant change in the electrophysiological properties measured could be noted after vehicle application (see Table 2, Figure 1h). Interestingly, no significant change in the AP firing rate could be found following treatment with the inhibitor of PKAs, PKCs, or SFKs while the input resistance after KT treatment, the AP amplitude, and AP half width after GF treatment, and the AP threshold after CC treatment showed statistically significant changes (see Table 2, Figure 1c-h).
We then examined the effects of agents which may enhance the activity of PKAs, PKCs, or SFKs (see Figures 2 and 3).
To examine the effect of PKA activation, in this work FSK at the concentration of 1, 10, 50, or 100 µM was co-applied with 50 μM of IBMX, a non-competitive inhibitor of phosphodiesterase (IC50: had no such effect (see Figure 2c). These data implicate that SFKs may be involved in the regulation of the AP firing activity of ARC neurons by PKAs.
Previous studies have documented that PKC activity can be enhanced by 50% following the application of 0.1-0.2 μM PMA in vitro and in cellular models (Bozou, Rochet, Magnaldo, Vincent, & Kitabgi, 1989;Niedel, Kuhn, & Vandenbark, 1983). In this work a dose-dependent increase was also noted in the AP firing rate following PMA application (see Table 2 and Figure 2d). When compared with that induced by vehicle application, we found that bath application of PMA (≥5 μM) significantly increased the AP firing activity  Figure 2d). We also found that after the application of 5 μM PMA the resting membrane potentials (RMPs) were reduced from −59.7 ± 10.0 to −55.5 ± 8.8 mV (mean ± SD, n = 15), which was statistically significant (p = 0.004, t 14 = 3.48, paired t test, see Table 2). With the exception of application of 10 μM PMA the AP amplitude was significantly reduced following treatments with PMA (see Table 2). Significant increases in the AP half width and decreases in the AP threshold were found in neurons treated with PMA at concentrations of 0.1-5 μM and 0.1-1 μM, respectively (see Table 2).
In comparing with the effect of application of vehicle or PMA (5 µM) alone the increase in the AP firing activity induced by 5 µM PMA was blocked in neurons pre-treated with the PKC inhibitor CC (10 μM) or GF (5 μM), or the SFK inhibitor PP2 (10 μM) for 30 min.
Delivering 1 mM of the peptide into cultured neurons (Yu, Askalan, Keil, & Salter, 1997) or 5 mM of the peptide into hippocampal neurons in brain slice preparations (Chichorro, Porreca, & Sessle, 2017) significantly enhances SFK-mediated neuronal activity. In this work, we investigated effects of the peptide EPQ(pY)EEIPIA delivered into neurons on the firing activity. Since the peptide EPQ(pY)EEIPIA is not membrane-permeant (Liu et al., 1993;Xu et al., 1999), to examine the effect of direct activation of SFKs, the peptide was delivered into ARC neurons through recording electrodes filled with the internal solution containing the EPQ(pY)EEIPIA (1 mM) (Chichorro et al., 2017;Feng et al., 2012;Yu et al., 1997). Figure 3a shows examples of current traces recorded from an ARC neuron at "0" (immediately), 3, 10, and 15 min after breakthrough with electrodes filled with the internal solution containing the EPQ(pY)EEIPIA (1 mM). When compared with those recorded immediately after breakthrough (at "0" min) the AP firing rate and AP threshold recorded at 10 min after breakthrough was significantly increased while the RMP, AP amplitude, and first spike latency were decreased (see Table 2, Figure 3). In contrast, the intracellular application of the non-phosphorylated peptide EPQYEEIPIA (1 mM) produced no such effects but increased the input resistance of neurons recorded (see Table 2 and Figure 3c). In comparing with the ef-

| Phosphorylation regulation of PKAs, PKCs, or SFKs in cultured ARC cells
The phosphorylation of PKAs at the residue T197 (Montenegro, Masgrau, Gonzalez-Lafont, Lluch, & Garcia-Viloca, 2012;Seifert et al., 2002), PKCs at the residues equivalent to S660 of PKCβII or PKCα/βII at T638/641 (Antal, Callender, Kornev, Taylor, & Newton, 2015;Freeley, Kelleher, & Long, 2011;Keranen, Dutil, & Newton, 1995) or SFKs at the residues equivalent to Y416 of chicken c-Src Salter & Kalia, 2004;Thomas & Brugge, 1997; has been found to be related to the functional status of these kinases. Furthermore, we investigated the phosphorylation of these kinases in ARC cells to understand  Note: Summary data showing changes in the electrophysiological properties of 188 neurons (which did not include those neurons pre-treated with antagonists of PKAs, PKCs, or SFKs) following treatment as indicated for 5 min are presented. In these neurons, the effects of FSK were examined in 16 neurons after the test of vehicle application, and in 19 neurons the effects of PMA were examined after the test of vehicle. The input resistance of whole cells was determined by ΔV/I injection (ΔV: changes in the steady state membrane potential in response to the injection of −10 pA currents; I injection : currents (−10 pA) injected into neurons. With the exception of those data indicated with ^ (p < 0.05, KS normality test), all the data of the resting membrane potential (RMP), firing rate, action potential (AP) amplitudes, AP half width, AP threshold and first spike of latency, and input resistance of ARC neurons in each group before treatment passed normality test (p > 0.05, KS normality test). Relative values to those before treatment (= 1) are shown in brackets. FSK was co-applied with IBMX (50 μM). The data following intracellular application of EPQ(pY)EEIPIA or EPQYEEIPIA shown in this table were recorded at "0" min (pre-, immediately after breakthrough) and 10 min after breakthrough.  Table 1). Compared with those in naïve cells treated only with culture medium, no significant change in the We then examined effects of bath application of FSK or PMA on the phosphorylation of PKAs, PKCs, or SFKs in cultured ARC cells.
However, no increase in pSFKs was induced by FSK application in cells pre-treated with PP2 (p = 0.63, t 10 = 0.5, unpaired t test; see Figure 5). In cells pre-treated with PP3 (10 μM) the FSK application induced a significant increase in the expression of pSFKs (p = 0.011, t 11 = 2.05, unpaired t test; see Figure 5).
When compared to that found in naïve cells, we found that PMA (10 μM for 30 min) significantly increased the expression of both pPKCs and pPKCα/βII (pPKCs: p = 0.003, t 12 = 3.7; pPKCα/ βII: p = 0.028, t 12 = 2.5, unpaired t test; see Figure 6). While the The gels were loaded with lysates prepared from cultured ARC cells without any treatment (naïve) or cells which were treated only with 50 or 100 μM FSK. Each group of blots was cropped from the same PVDF membrane, stripped, and successively probed with pPKCα/βII (RRID:AB_2284224), pPKCpan (RRID:AB_2168219), and β-actin (RRID:AB_2687938) antibodies as indicated on the left of blots. Examples of the blots from the same full length PVDF membranes were shown in Figure S2. The ratio of band intensities versus that of β-actin was normalized to the ratio in naïve cells (= 1, dashed line) for determining relative changes. Values on the right side of blots indicate the molecular mass (Kd). (c) Summary data (mean ± SD) of the relative changes in pPKAs, pSFKs, pPKCα/βII, and pPKCpan following treatment with 50 or 100 μΜ FSK. $, $$, $$$: p < 0.05, 0.01, 0.001, unpaired t test in comparing with that in naïve cells (= 1, dashed line). Values in brackets indicate the number of experimental repeats t 12 = 1.6, unpaired t test), pPKCα/βII (GF: p = 0.57, t 12 = 0.58; CC: p = 0.98, t 12 = 0.03, unpaired t test) or pSFKs (GF: p = 0.48, t 12 = 0.72; CC: p = 0.36, t 12 = 0.94, unpaired t test) was found following the application of 10 μM PMA for 30 min in cells pre-treated with GF (5 μM) or CC (10 μM) for 30 min (see Figure 6). In cells pre-treated with PP2 (10 μM) for 30 min the effect of PMA on the expression of pSFKs was prevented (p = 0.67, t 12 = 0.43, unpaired t test) while no change was found in the PMA application-induced increase in the expression of pPKCs or pPKCα/βII ( Figure 6). Pre-treatment with PP3 (10 μM) for 30 min produced no such effects ( Figure 6). Since the peptide EPQ(pY)EEIPIA is not membrane-permeant (Liu et al., 1993;Xu et al., 1999), effects of the direct activation of SFKs on PKAs or PKCs still need to be clarified. Despite this limitation, our data have identified that the activation of PKAs or PKCs may enhance the activity of SFKs and thereby up-regulate the AP firing activity in ARC neurons.

| The regulation of PKCα and PKCβII distributions in ARC neurons
Previous studies have shown that the activity of PKCs may be regulated by their subcellular localization Farrar, Thomas, & Anderson, 1985;Mochly-Rosen, 1995;Nishizuka, 1992), and that stimulating PKCs by application of PKC activators such as PMA may induce PKC translocation from the cytoplasm to the plasma membrane (Chao et al., 1998;Mochly-Rosen, 1995;Nishizuka, 1992;Siomboing et al., 2001). It has also been found that PKC translocation may play an important role in the regulation of nociception (Gu et al., 2016;He & Wang, 2015). Since the expression of pPKCα/βII was found to be increased following PMA treatment (see Figure 6) 80%-100% regions along the straight line across a neuron. If the peak intensities in both the 0%-20% and 80%-100% regions were higher than that in the 20%-80% region, an "enriched expression on the plasma membrane region" was then defined in this work. Since in some cases the line for measurement appeared in the perinucleus region, as indicated by DAPI staining (see Figure 7b), we conducted a correlation analysis between the intensities of PKCα or PKCβII antibody staining and DAPI staining for each of these cases. Since no statistically significant negative co-relation was found in these cases We found that in randomly examined untreated naïve neurons, 7.6% ± 1.8% (n = 17 fields; 208 neurons) and 8.6% ± 1.0%

| D ISCUSS I ON
This study has identified that PKAs may act as an upstream factor(s) to enhance SFKs (see Figure 8) and that PKCs and SFKs may interact reciprocally-PKCs and SFKs may activate each other and the inhibition of either may abolish the up-regulation of the AP firing activity induced by PKC-SFK signaling in cultured ARC neurons (see Figure 8).
We have previously found that the inhibition of PKCs or SFKs in the ARC area not only blocks the enhancement of expressions of active PKCs, SFKs, and phosphorylated NMDA GluN2B at Y1472 in this area, but also attenuates the inflammation-induced increases in the discharge activity of ARC neurons and pain hypersensitivity (Bu et al., 2015;Peng et al., 2011;Xu et al., 2012;Zheng et al., 2016). Taken together with our present findings, it has been implicated that the functional interactions among these enzymes may be novel mechanisms involved in the regulation of neuronal excitability.
We have identified that in the ARC area Src, but not Fyn or Lyn in SFKs, is activated following the development of peripheral inflammation, and that Src knockdown in this area blocks the inflammation-induced increases in the expressions of activated SFKs and the phosphorylated GluN2B subunit at Y1472 in the ARC area, and reduces pain hypersensitivity (Ma et al., 2019). Thus, studies focusing on whether and how the functional interactions among PKAs, PKCs, and SFKs identified in cultured ARC neurons are involved in the regulation of pain hypersensitivity in vivo are essential for us to understand mechanisms underlying the formation and maintenance of pain hypersensitivity. The findings that PKCs and SFKs may activate each other and the inhibition of either may abolish the up-regulation of the AP firing activity by the PKC-SFK signaling in cultured ARC neurons suggest a new avenue for developing novel approaches to treat increased excitability associated with pain hypersensitivity.

ACK N OWLED G M ENTS
We are grateful to comments of Drs. B.R. Groveman, W.K. Xin, and X.Q. Fang on this work. In memory of Dr. Q.Z. Yin for his great support and directions.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

AUTH O R CO NTR I B UTI O N S
All authors have full access to all the data in the study and take re-

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S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found online in the Supporting Information section at the end of the article.