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Neuroscience

J Physiol 593.19 (2015) pp 4439–4452

Endogenous casein kinase-1 modulates NMDA receptor activity of hypothalamic presympathetic neurons and sympathetic outflow in hypertension De-Pei Li1 , Jing-Jing Zhou1 and Hui-Lin Pan1,2 1 2

Division of Anesthesiology and Critical Care, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Programs in Neuroscience and Experimental Therapeutics, University of Texas Graduate School of Biomedical Sciences, Houston, TX 77225, USA

Key points

r Increased NMDA receptor activity and excitability of presympathetic neurons in the hypo-

The Journal of Physiology

thalamus can increase sympathetic nerve discharges leading to hypertension.

r In this study, we determined how protein kinases and phosphatases are involved in regulating r r

r

NMDA receptor activity and firing activity of presympathetic neurons in the hypothalamus in normotensive and hypertensive rats. We show that casein kinase-1 inhibition increases NMDA receptor activity and excitability of presympathetic neurons in the hypothalamus and augments sympathetic nerve discharges in normotensive, but not in hypertensive, rats. Our data indicate that casein kinase-1 tonically regulates NMDA receptor activity by interacting with casein kinase-2 and protein phosphatases in the hypothalamus and that imbalance of NMDA receptor phosphorylation can augment the excitability of hypothalamic presympathetic neurons and sympathetic nerve discharges in hypertension. These findings help us understand the neuronal mechanism of hypertension, and reducing the NMDA receptor phosphorylation level may be effective for treating neurogenic hypertension.

Abstract Increased N-methyl-D-aspartate receptor (NMDAR) activity in the paraventricular nucleus (PVN) of the hypothalamus is involved in elevated sympathetic outflow in hypertension. However, the molecular mechanisms underlying augmented NMDAR activity in hypertension remain unclear. In this study, we determined the role of casein kinase-1 (CK1) in regulating NMDAR activity in the PVN. NMDAR-mediated excitatory postsynaptic currents (EPSCs) and puff NMDA-elicited currents were recorded in spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs) and Wistar–Kyoto (WKY) rats. The basal amplitudes of evoked NMDAR-EPSCs and puff NMDA currents were significantly higher in SHRs than in WKY rats. The CK1 inhibitor PF4800567 or PF670462 significantly increased the amplitude of NMDAR-EPSCs and puff NMDA currents in PVN neurons in WKY rats but not in SHRs. PF4800567 caused an NMDAR-dependent increase in the excitability of PVN neurons only in WKY rats. Also, the CK1ε protein level in the PVN was significantly lower in SHRs than in WKY rats. Furthermore, intracerebroventricular infusion of PF4800567 increased blood pressure and lumbar sympathetic nerve activity in WKY rats, and this effect was eliminated by microinjection of the NMDAR antagonist into the PVN. In addition, PF4800567 failed to increase NMDAR activity in brain slices of WKY rats pretreated with the protein phosphatase 1/2A, calcineurin, or casein kinase-2 inhibitor. Our findings suggest that CK1 tonically suppresses NMDAR activity in the PVN by reducing the NMDAR phosphorylation level. Diminished CK1 activity may contribute to potentiated glutamatergic synaptic input to PVN presympathetic neurons and elevated sympathetic vasomotor tone in neurogenic hypertension.

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DOI: 10.1113/JP270831

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(Received 28 April 2015; accepted after revision 6 July 2015; first published online 14 July 2015) Corresponding authors D.-P. Li and H.-L. Pan: Division of Anaesthesiology and Critical Care, Unit 110, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Email: [email protected] and [email protected] Abbreviations ABP, arterial blood pressure; aCSF, artificial cerebrospinal fluid; AMPA, α-amino-3-hydroxy4-isoxazoleproprionic acid; AMPAR, AMPA receptor; CK1, casein kinase-1; CK2, casein kinase-2; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; EPSC, excitatory postsynaptic current; GABA, γ-aminobutyric acid; HR, heart rate; LSNA, lumbar sympathetic nerve activity; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; PVN, paraventricular nucleus; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2B, protein phosphatase 2B; PKC, protein kinase C; RVLM, rostral ventrolateral medulla; SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto.

Introduction Primary (essential) hypertension is the most prevalent type of hypertension, affecting 90–95% of hypertensive patients. However, the root cause of primary hypertension remains poorly understood. Elevated sympathetic outflow is critically involved in the development and maintenance of hypertension (Judy et al. 1976; Allen, 2002). The presympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus control sympathetic outflow through projections to the rostral ventrolateral medulla (RVLM) and preganglionic sympathetic neurons of the intermediolateral cell column in the spinal cord (Ranson et al. 1998). It has been shown that transplantation of embryonic hypothalamic tissues from spontaneously hypertensive rats (SHRs) into the brain of adult normotensive rats leads to the development of hypertension (Eilam et al. 1991). Also, overexpression of potassium channels in the PVN reduces arterial blood pressure in spontaneously hypertensive rats (SHRs) (Geraldes et al. 2014). Although the PVN contributes significantly to the development and maintenance of neurogenic hypertension, the molecular mechanism underlying hyperactivity of PVN presympathetic neurons in hypertension is not fully known. We have shown that enhanced excitatory glutamatergic inputs, especially the N-methyl-D-aspartate receptor (NMDAR) activity, in the PVN are a major source of the excitatory drive to PVN presympathetic neurons and increased sympathetic vasomotor tone in SHRs (Li & Pan, 2007a; Li et al. 2008). NMDAR activity is regulated by casein kinase-2 (CK2), which plays a role in potentiated NMDAR activity in the PVN in SHRs (Ye et al. 2011, 2012). Casein kinase-1 (CK1) is another important serine/threonine protein kinase family (Manning et al. 2002). CK1 isoforms include CK1α, CK1β, CK1γ 1 , CK1γ 2 , CK1γ 3 , CK1δ, and CK1ε in vertebrates (Vielhaber & Virshup, 2001; Knippschild et al. 2005). CK1 can phosphorylate a large number of substrates, including protein phosphatase inhibitor-2 and calcineurin (Zhu et al. 1998; Vielhaber et al. 2000; Knippschild et al. 2005). Notably, the long C-terminal extensions of CK1 are autophosphorylated, and this phosphorylation inhibits

the activity of the kinase domain (Budini et al. 2009). In the hypothalamus (Takano et al. 2004), CK1-induced phosphorylation is involved in the regulation of circadian rhythms (Wang et al. 2007; Meng et al. 2008, 2010). Unlike CK2, CK1 seems to inhibit NMDAR-mediated synaptic transmission in the brain, such as in the striatum (Chergui et al. 2005). In the present study, we used in vitro and in vivo approaches to test the hypothesis that diminished CK1 activity contributes to potentiated NMDAR activity in the PVN and augmented sympathetic outflow in SHRs. Methods Animal models

Male Wistar–Kyoto (WKY) rats and SHRs (13 weeks old; Harlan, Indianapolis, IN, USA) were used in this study. We used 28 SHRs and 58 WKY rats for the entire study. The surgical procedures and experimental protocols were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center and conformed to the National Institutes of Health guidelines for the ethical use of animals. Retrograde labelling of spinally projecting PVN neurons

Spinally projecting PVN neurons were identified by fluorescent microspheres injected into the intermediolateral cell column in the spinal cord, as described previously (Li et al. 2008; Ye et al. 2011). Briefly, rats were anaesthetized with 2–3% isoflurane in O2 , and the spinal cord at T2–T4 level was exposed through laminectomy. FluoSpheres (0.04 μm; Invitrogen, Eugene, OR, USA) was pressure-ejected bilaterally (Nanojector II, Drummond Scientific, Broomall, PA, USA) through a glass pipette placed in the intermediolateral region of the spinal cord in five or six separate 50 nl injections (Li et al. 2008). After injection, rats were treated prophylactically with enrofloxacin (5 mg kg−1 , subcutaneously daily for 3 days) and buprenorphine (0.5 mg kg−1 , subcutaneously every 12 h for 3 days). The rat was returned to its cage for  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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Casein kinase-1 and synaptic plasticity in hypertension

3–7 days to permit the FluoSpheres to be transported to the PVN.

Electrophysiological recordings in hypothalamic slices

Brain slices containing the PVN were prepared from the FluoSphere-injected rats, as described previously (Li et al. 2008; Ye et al. 2011). Briefly, rats were anaesthetized with 2–3% isoflurane and quickly decapitated. The brain was rapidly removed and placed in ice-cold artificial cerebrospinal fluid (aCSF, saturated by a mixture of 95% O2 and 5% CO2 ) containing (in mM): 124.0 NaCl, 3.0 KCl, 1.3 MgSO4 , 2.4 CaCl2 , 1.4 NaH2 PO4 , 10.0 glucose and 26.0 NaHCO3 . A tissue block containing the PVN was trimmed and glued onto the stage of a vibrating microtome (Technical Products International, St Louis, MO, USA). Coronal slices (300 μm thick) were cut, and the slices were transferred to an incubation chamber filled with aCSF continuously gassed with a mixture of 95% O2 and 5% CO2 at 34°C for at least 1 h before the electrophysiological recording. Whole-cell patch-clamp recordings were performed in FluoSphere-labelled PVN neurons in the hypothalamic slices. The recording chamber was continuously perfused with aCSF (saturated by 95% O2 and 5% CO2 ) at 3.0 ml min−1 . The temperature of the aCSF was maintained at 34°C by using an in-line solution heater. The labelled PVN neurons were identified by using an upright microscope (BX51WI; Olympus, Tokyo, Japan) with a combination of epifluorescence illumination and differential interference contrast optics. The recording electrode was pulled from borosilicate capillaries by using a micropipette puller. The resistance of the pipette was 3–7 M when it was filled with an internal solution containing (in mM): 110.0 Cs2 SO4 , 2.0 MgCl2 , 0.1 CaCl2 , 10.0 Hepes, 1.1 EGTA, 0.3 Na2 -GTP, and 2.0 Mg-ATP, adjusted to pH 7.25 with 1.0 M CsOH (270–290 mosmol l−1 ). Signals were processed with the use of a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA), filtered at 1–2 kHz, and digitized at 10 kHz using a Digidata 1440 digitizer (Molecular Devices). The junction potential was corrected offline on the basis of the composition of the internal and external solutions used for recordings. The evoked excitatory postsynaptic currents (EPSCs) were elicited by electrical simulation (0.2 ms, 0.5–0.8 mA) delivered at a frequency of 0.1 Hz by a bipolar tungsten electrode connected to a stimulator. The tip of the stimulating electrode was placed on the ventral side about 100 μm away from the recorded neuron (Li et al. 2008). Evoked AMPAR-EPSCs were recorded at a holding potential of −60 mV in the presence of 10 μM gabazine, and evoked NMDAR-EPSCs were recorded at a holding potential of +40 mV in the presence of 10 μM gabazine and  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). A sodium channel blocker, lidocaine N-ethyl bromide (QX-314, 10.0 mM), was included in the pipette solution for EPSC recordings. Postsynaptic NMDA or AMPA currents were recorded at a holding potential of −60 mV and elicited by puff application of NMDA or AMPA through a Pressure System IIe (Toohey Company, Fairfield, NJ, USA). The puff pipette (15 μm tip diameter) was placed about 150 μm away from the recorded cells. Positive pressure (3 p.s.i.) was applied (durations of 200 ms) to eject NMDA (100 μM) onto the recorded cell to elicit currents in the presence of 1 μM TTX. In addition, because the NMDA channel is voltage-dependently blocked by Mg2+ at a negative holding potential and co-activated by glycine, the NMDA current was recorded at Mg2+ -free aCSF and in the presence of glycine (10 μM). The spontaneous firing activity of labelled PVN neurons was recorded by using the whole-cell current-clamp technique (Li et al. 2008, 2014). The recording procedures were similar to the recording of EPSCs except that the internal pipette solution contained potassium gluconate (instead of Cs2 SO4 ) and that QX-314 was not used. The spontaneous firing activity was recorded when it reached a steady state. All drugs were freshly prepared in aCSF before the experiments and delivered via syringe pumps at their final concentrations. PF4800567, PF670462, 6cyano-7-nitro-quinoxaline-2,3-dione (CNQX), gabazine, D-(−)-2-amino-5-phosphonopentanoic acid (AP5) and QX-314 were obtained from Abcam (Cambridge, MA, USA).

In vivo recording of arterial blood pressure, lumbar sympathetic nerve activity, and heart rate

The rats were anaesthetized by intraperitoneal injection of a mixture of α-chloralose (60–75 mg kg−1 ) and urethane (800 mg kg−1 ) after initial anaesthesia with 2% isoflurane in O2 . We confirmed the depth of anaesthesia level before surgery by the absence of both corneal reflexes and paw withdrawal responses to a noxious pinch to the hindpaw. The trachea was cannulated for mechanical ventilation using a rodent ventilator with room air. The arterial blood pressure (ABP) was monitored with a pressure transducer through a catheter placed into the left femoral artery. Heart rate (HR) was counted by triggering from the pulsatile blood pressure. The right femoral vein was cannulated for intravenous administration of drugs. Supplemental doses of urethane and α-chloralose were administered as necessary to maintain an adequate depth of anaesthesia. A small branch of the left lumbar postganglionic sympathetic nerve was isolated under an operating microscope through a retroperitoneal incision. The lumbar sympathetic nerve

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was cut distally to ensure that afferent activity was not recorded. The nerve was then immersed in mineral oil and placed on a stainless steel recording electrode (Li & Pan, 2007a). The nerve signal was amplified and bandpass filtered (100–3000 Hz) by an alternating current amplifier (model P511; Grass Technologies, Warwick, RI, USA), and lumbar sympathetic nerve activity (LSNA) was recorded by using an audio amplifier (Grass Technologies). The LSNA and ABP were recorded by using a 1401-PLUS analog-to-digital converter and Spike2 system (Cambridge Electronic Design, Cambridge, UK). The level of background electrical noise was determined by suppressing the LSNA via intravenous injection of phenylephrine (20 μg kg−1 I.V.) before euthanasia and 5 min after the rats were euthanized by an overdose of pentobarbital sodium (200 mg kg−1 I.V.) at the end of each experiment. The respective electrical noise levels measured using these two methods were similar and were subtracted from the integrated LSNA values, and the percentage change in LSNA from baseline was calculated (Li & Pan, 2007a,b).

decreased the mean ABP by at least 10 mmHg, and the stereotactic coordinates at which the prior GABA microinjection elicited the greatest depressor responses were used in the same rat for the subsequent microinjection of AP5. After microinjection of the drugs, the glass pipette was left in place for 1–2 min to ensure adequate delivery of the drug to the injection site. The location of the pipette tip and diffusion of the drugs in the PVN were determined by including 2% FluoSpheres microspheres (0.04 μm; Invitrogen) in the injection solution (Li & Pan, 2007a,b). The rat brain was removed rapidly at the end of the experiment and fixed in 10% buffered formalin solution overnight. Frozen coronal sections (40 μm thick) were cut on a freezing microtome and mounted on slides. Rhodamine-labelled fluorescent regions were identified using an epifluorescence microscope and plotted on standardized sections from the atlas of Paxinos and Watson (Paxinos & Watson, 1999). Rats were not included for data analysis if they had one misplaced microinjection outside the PVN.

Intracerebroventricular infusion and PVN microinjection

Data analysis

A burr hole was drilled in the skull over the lateral ventricle in the following coordinates: 1.5 mm lateral to the midline and 1.0 mm caudal to the bregma. Intracerebroventricular (I.C.V.) infusion was performed by an injection cannula connected to a microinjection syringe. The injection cannula was advanced 3.5 mm ventral from the surface of the dura (Li & Pan, 2007a; Ye et al. 2011). Each injection consisted of 10 μl of solution delivered over 1 min. The PF4800567 was initially dissolved in DMSO and diluted with aCSF (0.1% DMSO) in a concentration of 1.5 nmol/10 μl. The injection cannula was allowed to remain in place for an additional minute before being removed to ensure that the entire injection was delivered. For PVN microinjections, the rat head was placed in a stereotactic frame and a 4 mm burr hole was drilled around the following coordinates: 1.6–2.0 mm caudal to the bregma, 0.5 mm lateral to the midline to expose the brain. A glass microinjection pipette (tip diameter, 20–30 μm) was advanced into the PVN using the following stereotactic coordinates: 1.6–2.0 mm caudal to the bregma, 0.5 mm lateral to the midline, and 7.0–7.5 mm ventral to the dura (Li & Pan, 2007a,b). The injection sites of the PVN were initially identified by the depressor responses to microinjection of 5.0 nmol GABA (20 nl, 250 mM). The microinjection was done by using a calibrated microinjection system (Nanojector II; Drumond Scientific, Broomall, PA, USA) and monitored using an operating microscope. GABA microinjections were separated by 10 min intervals to allow for recovery of the depressor response. The PVN vasomotor site was located when the GABA injection

Data are presented as the mean ± SEM. The firing activity was analysed off-line using a peak detection program (Mini Analysis, Synaptosoft, Fort Lee, NJ, USA). The peak amplitude of evoked EPSCs was determined and analysed using pCLAMP 10 (Molecular Devices). Only one neuron was recorded in each brain slice, and at least four rats were used in each group. The LSNA, ABP and HR were analysed using the Spike2 software program. LSNA was rectified and integrated offline after subtracting the background noise. Control values were obtained by averaging the signal over a 60 s period immediately before each treatment. Response values after each intervention were averaged over 30 s when the maximal responses occurred. For comparisons of two groups, statistical significance was tested using Student’s t test. For comparisons of more than two groups, the repeated measures ANOVA with Dunnett’s post hoc test or two-way ANOVA with Bonferroni’s post hoc test was performed to compare responses within or between experimental groups. P < 0.05 was considered statistically significant.

Results CK1 inhibition increases NMDAR activity in PVN presympathetic neurons in WKY rats but not in SHRs

We first determined the role of endogenous CK1 in regulation of glutamatergic synaptic transmission in the PVN. CK1 is generally considered to be constitutively active (Rivers et al. 1998; Hutchinson et al. 2011). Because CK1ε is highly expressed in the hypothalamus (Wang  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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et al. 2007; Meng et al. 2008, 2010), we determined the effect of PF4800567, a specific inhibitor for CK1ε (Walton et al. 2009; Meng et al. 2010), on evoked EPSCs in labelled PVN neurons. Incubation of brain slices from WKY rats with PF4800567 (10 and 50 μM, 30 min) (Meng et al. 2010) similarly increased the peak amplitude of evoked NMDAR-EPSCs in labelled PVN neurons compared with that in vehicle-treated slice (Fig. 1A and B). However, PF4800567 had no significant effect on the peak amplitude of evoked α-amino-3-hydroxy-4-isoxazoleproprionic acid receptor (AMPAR)-EPSCs in PVN neurons in WKY rats (Fig. 1A and B). The ratio of NMDAR-EPSCs to AMPAR-EPSCs was significantly higher in PF4800567-treated than in vehicle-treated brain slices (0.54 ± 0.13 vs. 0.21 ± 0.03, P < 0.05, Fig. 1C). We also tested the effect of PF670462, which inhibits both CK1δ and CK1ε (Badura et al. 2007). Incubation of brain slices from WKY rats with PF670462 (10 μM, 30 min) (Badura et al. 2007) significantly increased the amplitude of evoked NMDAR-EPSCs, but not AMPAR-EPSCs, in seven labelled PVN neurons (Fig. 1A–C). To determine the role of CK1 in the regulation of postsynaptic NMDAR activity, we recorded NMDAR currents elicited by puff application of NMDA (100 μM) to labelled

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PVN neurons (Li et al. 2008; Ye et al. 2011). PF4800567 significantly increased NMDA-elicited currents in PVN neurons in WKY rats (n = 8, Fig. 1D and F). Inhibition of CK1 with PF670462 (10 μM, 30 min) also significantly increased the amplitude of puff NMDA currents (n = 7, Fig. 1D and E). These data suggest that the tonic activity of CK1 regulates NMDAR activity in PVN presympathetic neurons. Because both CK1 inhibitors produced a similar effect on NMDAR activity, we used PF4800567 in the following experiments. We next determined whether CK1 regulates glutamatergic synaptic transmission in the PVN in SHRs. While the amplitude of AMPAR-EPSCs of PVN neurons in SHRs was similar to that in WKY rats, the amplitude of evoked NMDAR-EPSCs was significantly greater in SHRs than in WKY rats (159.9 ± 10.7 vs. 61.2 ± 13.9 pA, n = 7 in each group; Fig. 2A–C). PF4800567 at 10 or 50 μM did not significantly alter the amplitude of evoked NMDAR-EPSCs or AMPAR-EPSCs in eight labelled PVN neurons in SHRs (Fig. 2A–C). Also, the amplitude of puff NMDA-elicited currents in labelled PVN neurons was significantly higher in SHRs (145.7 ± 8.1 pA, n = 8) than in WKY rats (52.3 ± 8.3 pA, n = 7). However, PF4800567 had no significant effect on the amplitude of the already increased puff NMDA

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Figure 1. CK1 inhibition increases NMDAR activity of spinally projecting PVN neurons in WKY rats A, representative current traces showing evoked AMPAR- and NMDAR-EPSCs in labelled PVN neurons in brain slices treated with vehicle, PF4800567 (10 μM), or PF670462 (10 μM) in WKY rats. The holding potential is indicated at the left. B and C, summary data showing the amplitudes of evoked AMPAR-EPSCs and NMDAR-EPSCs (B) and the ratio of NMDAR-EPSCs to AMPAR-EPSCs (C) in brain slices treated with vehicle (n = 7 neurons), PF4800567 (n = 8 neurons), or PF670462 (n = 7 neurons) in WKY rats. D, original recordings showing currents elicited by puff application of NMDA (100 μM, indicated by arrows) to labelled PVN neurons in brain slices treated with vehicle, PF4800567, or PF670462 in WKY rats. E, summary data showing that PF4800567 (n = 8 neurons) or PF670462 (n = 7) treatment significantly increased the amplitude of puff NMDA-elicited currents in labelled PVN neurons, compared with vehicle treatment (n = 7 neurons) in WKY rats. Data are presented as means ± SEM. ∗ P < 0.05, compared with the vehicle group.  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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currents in PVN neurons in SHRs (Fig. 2D and E). These results suggest that increased NMDAR activity in the PVN in SHRs may results from diminished CK1 activity or the maximum phosphorylation level of NMDARs.

CK1 inhibition increases the firing activity of PVN presympathetic neurons in WKY rats but not in SHRs

We next determined the role of CK1 in the regulation of the firing activity of spinally projecting PVN neurons in WKY rats and SHRs. CK1 inhibition with PF4800567 (10 μM, 30 min) significantly increased the firing rate of labelled PVN neurons compared with that in vehicle-treated brain slices in WKY rats (Fig. 3A and B). Furthermore, bath application of the specific NMDAR antagonist D-(−)-2-amino-5-phosphonopentanoic acid (AP5, 50 μM) significantly decreased the firing rate of labelled PVN neurons treated with PF4800567 in WKY rats (Fig. 3A and B). Blocking NMDARs with AP5 alone decreases the firing activity of PVN neurons in SHRs (Li et al. 2008; Ye et al. 2011). The basal firing rate of labelled PVN neurons was significantly greater in SHRs than that in WKY rats (2.7 ± 0.5 vs. 0.97 ± 0.1 Hz, n = 8 in each group, P < 0.05, Fig. 3A and B). Although PF4800567 treatment did not significantly change the already elevated firing activity of PVN neurons in SHRs, subsequent application of AP5 significantly decreased the firing rate in these PVN neurons (Fig. 3A and B). The lack of effect of PF4800567 on the firing activity of PVN neurons in SHRs was unlikely to be due to the ceiling effect because activation of metabotropic glutamate receptor-5 increases

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the firing activity of spinally projecting PVN neurons to 4 Hz in SHRs (Li et al. 2014). The CK1 inhibitor did not significantly alter the electrophysiological properties of labelled neurons in WKY rats and SHRs (Table 1). These data suggest that CK1 tonically inhibits the excitability of PVN presympathetic neurons by reducing the NMDAR activity in the normotensive condition and that this action is diminished in SHRs.

CK1 protein level in the PVN is reduced in SHRs

We performed Western blot analysis to measure CK1ε or CK1δ protein levels in micropunched PVN tissues from WKY rats and SHRs. The CK1ε protein level in the PVN was significantly lower in SHRs than in WKY rats (Fig. 3C and D). However, the CK1δ protein band in the PVN was not detectable in either WKY rats or SHRs.

Differential responses of LSNA, ABP and HR to CK1 inhibition and bilateral microinjection of AP5 into the PVN in WKY rats and SHRs

We then determined the role of CK1 and NMDARs in controlling sympathetic vasomotor tone in WKY rats and SHRs. Because of the technical difficulty of performing precise microinjections of the NMDAR antagonist into the PVN and recording high quality LSNA signal in conscious rats, the in vivo experiments were performed in anaesthetized rats. To minimize tissue damage caused by repeated microinjections into the PVN, PF4800567 was administered I.C.V. Also, the solution of PF4800567 at the high concentration required for microinjection

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Figure 2. Lack of an effect of CK1 inhibition on increased NMDAR activity of PVN neurons in SHRs A, representative traces showing evoked AMPARand NMDAR-EPSCs in labelled PVN neurons in brain slices treated with vehicle or PF4800567 (10 μM) in SHRs. B and C, mean amplitudes of evoked AMPAR-EPSCs and NMDAR-EPSCs (B) and the ratio of NMDAR-EPSCs to AMPAR-EPSCs (C) in brain slices treated with vehicle (n = 7 neurons) or PF4800567 (n = 8 neurons) in SHRs. D, raw recording traces showing puff NMDA-elicited currents in PVN neurons in brain slice treated with vehicle or PF4800567 in SHRs. E, summary data showing that PF4800567 or vehicle treatment had no effect on puff NMDA-elicited currents in PVN neurons in SHRs (n = 8 neurons in each group). Data are presented as means ± SEM.

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became viscous, which is difficult for microinjections via a glass pipette with a small tip. In WKY rats, PF4800567 (1.5 nmol in 10 μl aCSF) was I.C.V. infused initially, which significantly increased the arterial blood pressure (ABP), lumbar sympathetic nerve activity (LSNA), and heart rate (HR) by 19.0 ± 1.7 mmHg, 26.5 ± 3.8%, and 28.4 ± 5.8 bpm, respectively (n = 10, Fig. 4). The LSNA and ABP started to increase at 27.7 ± 5 min after PF4800567 injection, and these effects lasted for another 32 ± 3 min. I.C.V. infusion of the vehicle (0.5% DMSO)

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had no significant effect on ABP, LSNA, and HR in another nine WKY rats (Fig. 4). AP5 (1.0 nmol, 50 nl) microinjection was performed 30 min after PF4800567 injection. Subsequent bilateral microinjection of AP5 into the PVN did not significantly affect the ABP, LSNA and HR in vehicle-treated rats. However, AP5 microinjection markedly reduced LSNA, ABP and HR in WKY rats receiving prior PF4800567 injection (Fig. 4). The mean integrated LSNA (averaged over 60 s) under the basal condition was significantly higher in SHRs than in WKY rats (0.13 ± 0.1 vs. 0.07 ± 0.1 μV s, P < 0.05). In SHRs, I.C.V. infusion of PF4800567 on the vehicle had no significant effect on the already elevated ABP, LSNA and HR (n = 8, Fig. 5). Subsequent bilateral microinjection of AP5 into the PVN significantly decreased LSNA, ABP and HR in both vehicle-treated and PF4800567-treated SHRs (Fig. 5).

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Figure 3. CK1 inhibition increases the firing activity of labelled PVN neurons in WKY rats but not in SHRs A, representative recordings showing the firing activity of labelled PVN neuron treated with vehicle, PF4800567, or PF4800567 plus AP5 (50 μM) in brain slices from a WKY rat and an SHR. B, summary data showing that PF4800567 treatment increased the firing activity of labelled PVN neurons in WKY rats (n = 8 neurons in each group), an effect that was blocked by bath application of AP5. However, PF4800567 had no effect on the firing activity of PVN neurons in SHRs (n = 8 neurons in each group). C and D, representative gel images (C) and group data (D, n = 5) showing the protein level of CK1ε in the PVN in SHRs and WKY rats. The molecular mass is indicated on the right side of the gel image. Data presented as means ± SEM. ∗ P < 0.05, compared with the vehicle group or WKY group. # P < 0.05, compared with the PF4800567 effect alone.

 C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

Signalling mechanisms involved in the increase in NMDAR activity by CK1 inhibition in the PVN

Because the protein phosphatase PP1/PP2A controls NMDAR phosphorylation and activity (Chatterjea et al. 2010; Farinelli et al. 2012) and may mediate the effect of CK1 inhibition, we determined whether PP1/PP2A is involved in the effect of CK1 inhibition on NMDAR activity in the PVN. Treatment of brain slices with tautomycetin (1 μM, Tocris Bioscience, Bristol, UK), the specific PP1/2A inhibitor (Mitsuhashi et al. 2001), significantly increased the amplitude of evoked NMDAR-EPSCs of PVN neurons in WKY rats (n = 6, Fig. 6). However, treatment with PF4800567 did not further increase the amplitude of NMDAR-EPSCs in the presence of tautomycetin (n = 7, Fig. 6). The amplitude of AMPAR-EPSCs of PVN neurons did not differ significantly between vehicle-, PF4800567-, and PF4800567 plus tautomycetin-treaded brain slices (Fig. 6). Also, incubation of PF4800567 (n = 7) or tautomycetin (n = 7) similarly increased the amplitude of puff NMDA currents of labelled PVN neurons in WKY rats. However, treatment with PF4800567 did not further increase the amplitude of puff NMDA currents in the presence of tautomycetin (n = 6, Fig. 6). NMDAR phosphorylation and activity are also regulated by the protein phosphatase 2B (PP2B, calcineurin) (Lieberman & Mody, 1994; Tong et al. 1995). We thus determined whether calcineurin is also involved in enhanced NMDAR activity by CK1 inhibition in the PVN. Inhibition of calcineurin activity with FK506 (1 μM, 1–2 h) (Chen et al. 2014) significantly increased the amplitude of evoked NMDAR-EPSCs and puff NMDA-elicited currents without changing the amplitude of evoked AMPAR-EPSCs of labelled PVN neurons (n = 8, Fig. 7A). Incubation of

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J Physiol 593.19

Table 1. Electrophysiological property of spinally projecting PVN neurons WKY rat

Membrane potential (mV) Input resistance (M) Access resistance (M) Cell capacitance (pF)

SHR

Basal

PF4800567

AP5+ PF4800567

Basal

PF4800567

AP5+ PF4800567

−59.7 ± 1.3 491.3 ± 71.5 22.4 ± 1.7 41.0 ± 1.7

−56.4 ± 1.9 517.0 ± 61.2 21.2 ± 2.6 41.0 ± 1.9

−59.3 ± 1.6 490.1 ± 45.6 22.4 ± 1.5 41.1 ± 1.8

−54.8 ± 2.8 503.7 ± 55.9 21.9 ± 2.7 40.5 ± 1.8

−54.0 ± 2.1 509.4 ± 34.8 23.2 ± 2.1 42.6 ± 1.9

−60.5 ± 3.3 490.4 ± 52.7 21.5 ± 1.7 42.8 ± 2.8

Values are means ± S.E.M. n = 8 in each group.

Protein kinase C (PKC) can affect NMDAR trafficking and gating in the brain (Lan et al. 2001). We used a cell-permeant PKC inhibitor, chelerythrine (Abcam) (Herbert et al. 1990), to determine the role of PKC in the

brain slices with PF4800567 did not further increase the amplitude of NMDAR-EPSCs and puff NMDA currents in PVN neurons of WKY rats in the presence of FK506 (n = 7, Fig. 7A).

ICV Vehicle

ICV PF4800567 Int. LSNA (%) LSNA (µV) HR (bpm) ABP (mmHg)

AP5 (PVN)

100 50 400 300 200 20 0

200

AP5 (PVN) 20 min

150 100 50 350 300 250 200 20.0 0.0

-20.0 200 150 100 50 0

-20 150 100 50 0

a

150

50

200

0

0

0

PVN 3V Bregma -2.1mm

ICV Vehicle

ICV PF4800567

ICV Vehicle

ICV PF4800567

ICV Vehicle

B PF ase 48 line PF 005 48 67 00 5 +A 67 P5

AH VMH

60 se li Ve ne hi cle AP 5

Bregma -1.8 mm

250

se li Ve ne hi cle AP 5

AH

3V

80

Ba

PVN

AH O T

300

100

B PF ase 48 line PF 005 48 67 00 5 +A 67 P5

Bregma -1.4 mm

350

HR (bpm)

100

E

B PF ase 48 line PF 005 48 67 00 5 +A 67 P5

PVN

D

120

se li Ve ne hi cle AP 5

AH

C

LSNA (%)

0 .5µ

Fx

Mean ABP (mmHg)

3 V

b

1 min

1 min

Ba

B

Ba

Int. LSNA (%)

20 min

200 150

LSNA (µV)

HR (bpm) ABP (mmHg)

A

ICV PF4800567

Figure 4. Effects of intracerebroventricular infusion of PF4800567 and PVN microinjection of AP5 on sympathetic outflow in WKY rats A, representative recordings showing the effect of I.C.V. injection of PF4800567 (1.5 nmol, 10 μl) and bilateral microinjection of AP5 (1.0 nmol, 50 nl) into the PVN on ABP, HR and LSNA in one WKY rat. Ba, a representative photomicrograph showing the FluoSphere injection sites in the PVN. b, schematic drawings showing the microinjection sites for AP5 in the PVN in rats treated with I.C.V. PF4800567 (•) or vehicle (◦) in WKY rats. C–E, summary data showing changes in mean ABP, LSNA and HR in response to microinjection of AP5 into the PVN after I.C.V. infusion of PF4800567 (n = 9 rats) or vehicle (n = 8 rats) in WKY rats. ∗ P < 0.05, compared with the baseline control. # P < 0.05, compared with the prior PF4800567 effect. AH, anterior hypothalamus; 3V, third ventricle.  C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

Casein kinase-1 and synaptic plasticity in hypertension

J Physiol 593.19

significant effect on NMDAR- and AMPAR-EPSCs and puff NMDA-elicited currents in labelled PVN neurons (Fig. 7C). PF4800567 failed to significantly alter NMDAR-EPSCs and puff NMDA-elicited currents in the presence of DRB (n = 7, Fig. 7C). We did not test PP1/2A/B inhibitors in SHRs, because CK1 inhibition had no effect on NMDAR activity in SHRs. Collectively, these findings suggest that CK1 regulates NMDAR activity in the PVN through protein phosphorylation, which is also controlled by PP1/2A, calcineurin and CK2.

increase in NMDAR activity induced by CK1 inhibition. Treatment of brain slices with chelerythrine (1 μM, 1–2 h) did not significantly alter the amplitude of NMDAR- and AMPAR-EPSCs and puff NMDA currents in labelled PVN neurons of WKY rats (Fig. 7B). However, treatment with PF4800567 still significantly increased the amplitude of evoked NMDAR-EPSCs and puff NMDA currents in the presence of chelerythrine (Fig. 7B). We have shown that increased CK2 activity plays a role in potentiated NMDAR activity in the PVN in SHRs (Ye et al. 2011, 2012). Therefore, we also determined whether CK2 is involved in CK1 inhibitor-induced increases in NMDAR activity. In brain slices obtained from WKY rats, treatment with a specific CK2 inhibitor, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) (100 μM, 1–2 h) (Ye et al. 2011, 2012), had no

ICV Vehicle

20 min

The PVN is a major source of the excitatory drive causing increased sympathetic vasomotor tone in neurogenic

ICV PF4800567

AP5 (PVN)

160 120 80 450 350 250 20.0 0.0

-20.0 150 100 50 0

B

Discussion

Int. LSNA (%) LSNA (µV) HR (bpm) ABP (mmHg)

Int. LSNA (%) LSNA (µV) HR (bpm) ABP (mmHg)

A

4447

20 min

AP5 (PVN)

160 120 80 450 350 250 20.0 0.0 -20.0 150 100 50 0

1 min

1 min

Fx AH

VMH

3V

Bregma -2.1mm

HR (bpm)

50

200

0

0

0

Ba

PVN

50

ICV Vehicle

ICV PF4800567

ICV Vehicle ICV PF4800567

se l Ve ine PV hic N le AP 5 Ba PF se 4 lin PF 800 e 4 56 +A 800 7 P5 56 7

AH

300

Ba

Bregma -1.8 mm

100

E 400

se l Ve ine PV hic N le AP 5 B PF ase 4 lin PF 800 e 4 5 +A 800 67 P5 56 7

3V

100

150

Ba

PVN AH

AH O

D

150

LSNA (%)

Bregma -1.4 mm

C

se l Ve ine PV hic N le AP 5 Ba PF se 4 lin PF 800 e 4 56 +A 800 7 P5 56 7

3V

Mean ABP (mmHg)

PVN

ICV Vehicle ICV PF4800567

Figure 5. CK1 inhibition has no effect on sympathetic vasomotor tone in SHRs A, representative recordings showing the effect of I.C.V. injection of PF4800567 (1.5 nmol, 10 μl) and bilateral microinjection of AP5 (1.0 nmol, 50 nl) into the PVN on ABP, HR and LSNA in one SHR. B, schematic drawings showing the AP5 microinjection sites in the PVN in rats treated with I.C.V. PF4800567 (•) or vehicle (◦) in SHRs. C–E, summary data showing changes in mean ABP (B), LSNA (C) and HR (D) in response to microinjection of AP5 into the PVN after I.C.V. infusion of PF4800567 (n = 7 rats) or vehicle (n = 6 rats) in SHRs. ∗ P < 0.05, compared with the prior PF4800567 effect. AH, anterior hypothalamus; 3V, third ventricle.

 C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

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hypertension (Allen, 2002; Li & Pan, 2007a). CK1 is involved in the regulation of many physiological functions. For example, CK1 plays a role in the control of DNA repair, cellular morphology, Wnt signalling (Peters et al.

A

Vehicle

PF4800567

Tautomycetin

PF4800567+ Tautomycetin

+40 mV

100 pA

-60 mV

Vehicle

0.5

0

PF4800567

0.0

Tautomycetin

PF4800567+ Tautomycetin

50 pA

C

0

NMDAR/AMPAR ratio

125

250

1.0

PF Veh Ta 480 icle ut 0 PF om 567 y Ta 48 cet ut 00 in om 56 yc 7+ et in

NMDAR-EPSCs (pA)

250

500

PF Veh Ta 480 icle ut 0 PF om 567 y Ta 48 cet ut 00 in om 56 yc 7+ et in

AMPAR-EPSCs (pA)

20 ms

PF Veh Ta 480 icle ut 0 PF om 567 y Ta 48 cet ut 00 in om 56 yc 7+ et in

B

2s

200 150 100

tin PF Ta 48 ut 0 om 05 yc 67 et + in

to m yc e

7 56 00 48

Ve h

PF

Ta u

0

e

50 icl

NMDAR currents (pA)

D

Figure 6. CK1 inhibitor increases NMDAR activity of spinally projecting PVN neurons in WKY rats through interacting with PP1/2A A and B, representative traces (A) and summary data (B) showing evoked AMPAR- and NMDAR-EPSCs in labelled PVN neurons in brain slices treated with vehicle, PF4800567 (n = 7), tautomycetin (1 μM, n = 6), or PF4800567 plus tautomycetin (n = 7) in WKY rats. C and D, original recordings (C) and summary data (D) showing puff NMDA currents in labelled PVN neurons from brain slices treated with vehicle (n = 6), PF4800567 (n = 7), tautomycetin (n = 7), or PF4800567 plus tautomycetin (n = 6) in WKY rats. Data presented as means ± SEM. ∗ P < 0.05, compared with the vehicle group.

J Physiol 593.19

1999), nuclear import of NFAT factors (Zhu et al. 1998), and circadian rhythms in adulthood (Ebisawa, 2007). However, little is known about the role of CK1 in regulating the autonomic nervous system. In this study, we recorded evoked glutamatergic EPSCs in PVN presympathetic neurons in a brain slice preparation. Although some of the physiological inputs to PVN presympathetic neurons are likely to be severed during the slice cutting, the synaptic inputs to PVN presympathetic neurons remained largely intact. We have shown that NMDAR activity of presympathetic PVN neurons is increased in both pre- and postsynaptic sites in SHRs and that the NMDAR activity stimulated by endogenous glutamate release is increased in SHRs (Li et al. 2008). CK1 is widely expressed in the brain including the hypothalamus (Takano et al. 2000, 2004; Yasojima et al. 2000). Because NMDARs in the PVN play a major role in increased sympathetic outflow in hypertension (Li & Pan, 2007a; Li et al. 2008), we determined the role of CK1 in the regulation of NMDAR activity of PVN presympathetic neurons and sympathetic vasomotor tone. We found that CK1 inhibition increased the activity of NMDARs, but not AMPAR, in spinally projecting PVN neurons in WKY rats. Consistent with our finding, CK1 inhibition increases the NMDAR activity through phosphorylation of NMDAR subunits in striatum neurons (Chergui et al. 2005). However, CK1 inhibition had no effect on the already increased NMDAR activity in spinally projecting PVN neurons in SHRs. These data suggest that the basal NMDAR activity is tonically suppressed by CK1 in the PVN in the normotensive condition. Increased NMDAR activity contributes to the hyperactivity of PVN presympathetic neurons in SHRs (Li et al. 2008). In the present study, we found that CK1 inhibition significantly increased the firing rate of spinally projecting PVN neurons in WKY rats but not in SHRs. Because CK1 inhibition significantly increased the NMDAR activity, we reasoned that CK1 inhibition increases the firing activity of PVN neurons through its potentiating effect on NMDARs. We showed that blocking NMDARs with AP5 significantly decreased the firing activity of PVN neurons caused by CK1 inhibition in WKY rats. Notably, CK1 inhibition did not further increase the elevated firing rate of PVN neurons in SHRs, although subsequent treatment with the NMDAR antagonist reduced the firing activity of these PVN neurons. Our findings suggest that CK1 regulates the excitability of PVN presympathetic neurons through tonic control of the NMDAR activity. The lack of an effect of CK1 inhibition on NMDAR activity and excitability of PVN neurons in SHRs could be due to diminished CK1 activity or the maximum phosphorylation level of NMDARs in the PVN. In hypertension, the sympathetic vasomotor tone is tightly regulated by PVN neuronal activity (Li & Pan,

 C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

Casein kinase-1 and synaptic plasticity in hypertension

NMDAR currents (pA) NMDAR currents (pA) NMDAR currents (pA)

200

48 6 +F 00 K5 567 06

le

50

ic

PF

FK

Ve h Ve h

ic

0

le r PF yth r 4 i C 8 he 0 ne le 05 ry 67 th + rin e

100

he

100

48 RB 0 +D 05 R 67 B

PF

D

ic

le

0 Ve h

PF 48 RB +D 005 R 67 B

0.0

0

C

he C

0.5

100

le

Ve h

1.0

le

48 B +D005 R 67 B

R D

ic

le

0

PF

0.0

ic

50

NMDAR/AMPAR ratio

100

200

48 6 +F 00 K5 56 06 7

le ic

0.5

le he C

150

FK

Ve h

1.0

200

le icle r PF yth 4 C 8 rine he 0 le 05 ry 67 th + rin e

PF

0.0

ic

0

le r PF yth r 4 i C 8 he 0 ne le 05 ry 67 th + rin e

50

Ve h

48 RB +D005 R 67 B

PF

Ve h

D

le

0

48 6 +F 00 K5 56 06 7

ic

100

Ve h

Ve h

200

FK

Ve h

150

le he C

400

ic

AMPAR-EPSCs (pA)

C

50

le

0

0.5

D

50

1.0

50

NMDAR/AMPAR ratio

100

PF

PF

150

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in SHRs. In supporting of this hypothesis, we found that the CK1ε protein level in the PVN was significantly decreased in SHRs, which may explain the diminished CK1 inhibitor effect in SHRs. Nevertheless, we recognize that I.C.V. infusion of PF4800567 can potentially affect CK1 activity in other brain regions, such as the dorsal medial hypothalamus and anterior hypothalamus. It is unclear whether the CK1 activity is altered in other brain regions involved in the control of sympathetic vasomotor tone in SHRs. We cannot rule out the possibility that the increases in LSNA and ABP by I.C.V. administration of PF4800567 may result from CK1 inhibition in other brain regions regulating sympathetic outflow.

NMDAR/AMPAR ratio

200

le r PF yth r 4 i C 8 he 0 ne le 05 ry 67 th + rin e

200

0

200

FK

Ve h

400

ic

AMPAR-EPSCs (pA)

B

50 4 6 +F800 5 K5 6 06 7

le

0

NMDAR-EPSCs (pA)

200

200

NMDAR-EPSCs (pA)

400

ic

AMPAR-EPSCs (pA)

A

NMDAR-EPSCs (pA)

2007a,b; Li et al. 2008; Ye et al. 2011). Because CK1 inhibition increased the NMDAR and firing activity of PVN presympathetic neurons, we examined the role of CK1 in controlling sympathetic outflow in vivo. We found that I.C.V. infusion of PF4800567 significantly altered the sympathoexcitatory responses to blocking NMDARs in the PVN in control rats, but not in SHRs. These data suggest that tonic CK1 activity may restrain the NMDAR activity in the PVN involved in the control of sympathetic vasomotor tone in the physiological condition. Our in vivo data provide further evidence that diminished CK1 activity may contribute to elevated sympathetic outflow through potentiation of NMDAR activity in the PVN

Ve h

J Physiol 593.19

Figure 7. CK1 inhibition increases NMDAR activity of spinally projecting PVN neurons through interactions with calcineurin and CK2 A, summary data showing evoked AMPAR-EPSCs, NMDAR-EPSCs, ratio of NMDAR-EPSCs to AMPAR-EPSCs, and puff NMDA currents of labelled PVN neurons in brain slices treated with vehicle (n = 7 neurons), FK506 (1 μM, n = 8 neurons), or PF4800567 plus FK506 (n = 7 neurons) in WKY rats. B, mean data showing evoked AMPAR-EPSCs, NMDAR-EPSCs, ratio of NMDAR-EPSCs to AMPAR-EPSCs, and puff NMDA currents of labelled PVN neurons in brain slices treated with vehicle (n = 6 neurons), DRB (100 μM, n = 7 neurons), or PF4800567 plus DRB (n = 7) in WKY rats. C, summary data showing evoked AMPAR-EPSCs, NMDAR-EPSCs, ratio of NMDAR-EPSCs to AMPAR-EPSCs, and puff NMDA currents of labelled PVN neurons in brain slices treated with vehicle (n = 6 neurons), chelerythrine (1 μM, n = 6 neurons), or PF4800567 plus chelerythrine (n = 7 neurons) in WKY rats. Data presented as means ± SEM. ∗ P < 0.05, compared with the vehicle group.

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The precise signalling pathways involved in enhanced NMDAR activity by CK1 inhibition in the PVN are not clear. Because CK1 is a serine/threonine kinase and the known substrates do not include NMDARs (Venerando et al. 2014), CK1 may regulate NMDAR activity indirectly by affecting the activity of protein phosphatases such as PP1/2A (Wang et al. 1994; Westphal et al. 1999; Chan & Sucher, 2001). For example, CK1 may increase PP1/2A activity through phosphorylation of PP1 inhibitor-2 (Agostinis et al. 1992; Marin et al. 1994). We found in this study that inhibition of PP1/2A with tautomycetin mimicked the effect of CK1 inhibition on the NMDAR activity and that CK1 inhibition had no further effect on NMDAR currents in PVN neurons in the presence of the PP1/2A inhibitor. Another protein phosphatase, calcineurin, is also a CK1 substrate (Singh & Wang, 1987). Calcineurin reduces the activity of synaptic NMDARs through shortening the opening duration of NMDAR channels in the dentate gyrus (Tong et al. 1995; Lieberman & Mody, 1999). We observed that calcineurin inhibition with FK506 significantly increased NMDAR currents of PVN neurons in WKY rats and that CK1 inhibition did not further increase NMDAR currents in the presence of FK506. The phosphorylation level of NMDARs is dynamically controlled by protein phosphatases and protein kinases. NMDAR function is also regulated by protein kinases such as CK2 (Ye et al. 2011, 2012) and PKC (Lan et al. 2001). We found that the CK2 inhibitor DRB did not significantly alter the NMDAR activity but eliminated the effect of CK1 inhibition on NMDAR currents of spinally projecting PVN

Normotension

J Physiol 593.19

neurons in WKY rats. Our findings reinforce the notion that CK1 regulates NMDAR activity by interacting with certain protein phosphatases (PP1/2A and calcineurin) and kinases (CK2) to alter the phosphorylation level of NMDARs and/or related proteins in the PVN. In SHRs, diminished CK1 activity may lead to reduced function of phosphatases, which is unable to control the phosphorylation level of NMDARs and/or associated proteins in the PVN via active dephosphorylation (Fig. 8). Therefore, the imbalance of phosphorylation and dephosphorylation plays a key role in increased NMDAR activity in the PVN and elevated sympathetic vasomotor tone, which contribute to the development of neurogenic hypertension. In conclusion, our study provides important new information that tonic CK1 activity in the PVN is involved in the regulation of NMDAR activity and sympathetic vasomotor tone. PVN neurons receive inputs from the subfornical organ (Tanaka et al. 1986; Miyakubo et al. 2002), which may also increase the excitability of PVN neurons projecting to spinal intermediolateral cell column and RVLM to augment sympathetic outflow in hypertension. Our findings suggest that diminished CK1 activity may contribute to hyperactivity of PVN presympathetic neurons and elevated sympathetic outflow in hypertension. These findings provide new insights into the molecular mechanisms underlying the elevated sympathetic vasomotor tone in neurogenic hypertension. Therapeutic interventions that can increase CK1 activity and reduce NMDAR phosphorylation levels may improve the treatment of neurogenic hypertension.

Hypertension

NMDAR

NMDAR

p

p

p PP1/2A, PP2B

CK2

PP1/2A, PP2B

p p

CK2

p CK1

CK1

Figure 8. Diagram showing the proposed signalling pathways involved in regulation of NMDAR activity of presympathetic neurons in the PVN by protein kinases (CK1 and CK2) and phosphatases (PP1/2A and PP2B) In the normotensive condition, NMDAR phosphorylation state is low and balanced by tonically active CK2 (promoting phosphorylation) and CK1 (reducing phosphorylation by increasing PP1/2A and PP2B activity). In the hypertensive condition, diminished CK1 activity leads to an increase in NMDAR phosphorylation due to reduced activity of PP1/2A and PP2B (unable to dephosphorylate NMDARs). Also, increased CK2 activity potentiates the phosphorylation state of NMDARs in hypertension. This phosphorylation imbalance contributes to the hyperactivity of PVN presympathetic neurons and elevated sympathetic outflow in hypertension.

 C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society

J Physiol 593.19

Casein kinase-1 and synaptic plasticity in hypertension

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Additional information Competing interests The authors declare that they have no competing interests.

Author contributions Conception and design of the experiments: D.-P.L. and H.-L.P. Collection, assembly, analysis and interpretation of data: D.-P.L., J.-J.Z. and H.-L.P. Drafting the article or revising it critically for intellectual content: D.-P.L. and H.-L.P. All authors approved the final version of the manuscript and all persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding This work was supported by the National Institutes of Health (Grants HL077400 and MH096086) and the N. G. and Helen T. Hawkins endowment (to H.-L.P.).

 C 2015 The Authors. The Journal of Physiology  C 2015 The Physiological Society