Expression and functions of N-type Cav2.2 and T-type Cav3.1 channels in rat vasopressin neurons under normotonic conditions

Arginine vasopressin (AVP) neurons play essential roles in sensing the change in systemic osmolarity and regulating AVP release from their neuronal terminals to maintain the plasma osmolarity. AVP exocytosis depends on the Ca2+ entry via voltage-gated Ca2+ channels (VGCCs) in AVP neurons. In this study, suppression by siRNA-mediated knockdown and pharmacological sensitivity of VGCC currents evidenced molecular and functional expression of N-type Cav2.2 and T-type Cav3.1 in AVP neurons under normotonic conditions. Also, both the Cav2.2 and Cav3.1 currents were found to be sensitive to flufenamic acid (FFA). TTX-insensitive spontaneous action potentials were suppressed by FFA and T-type VGCC blocker Ni2+. However, Cav2.2-selective ω-conotoxin GVIA failed to suppress the firing activity. Taken together, it is concluded that Cav2.2 and Cav3.1 are molecularly and functionally expressed and both are sensitive to FFA in unstimulated rat AVP neurons. Also, it is suggested that Cav3.1 is primarily involved in their action potential generation.


Background
Arginine vasopressin (AVP) neurons that are located at the supraoptic nucleus (SON) and paraventricular nucleus (PVN) in the hypothalamus of the brain are a key player in maintaining the osmolarity of body fluid in a narrow range around 300 mOsm by secreting AVP from their neuronal terminals at the posterior pituitary gland. The action potential firing involves activation of Na + and Ca 2+ conductance in the magnocellular neurosecretory cells (MNCs) consisting of AVP neurons and oxytocin (OXT) neurons [1][2][3]. The amount of vesicular exocytotic release of AVP from neurohypophysial terminals of MNCs is determined by the frequency and pattern of action potential firing [4] and by the entry of Ca 2+ via voltage-gated Ca 2+ channels (VGCCs or Cav channels) [5]. Somatodendritic expression of L-, T-and N-type VGCCs in rat supraoptic AVP neurons was suggested based on pharmacological studies on the increases in the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in response to application of AVP [6] and pituitary adenylate cyclase-activating polypeptide (PACAP) [7]. Besides, nifedipine-sensitive L-type Ca 2+ currents were found to be increased in rat supraoptic MNCs under dehydration conditions produced after water deprivation for 16-24 h [8]. However, it is not known which types of VGCCs are expressed and functioning during the spontaneous action potential firing in unstimulated rat AVP neurons under normotonic conditions. The present study thus aimed to address this question by cytosolic RT-PCR analysis and by whole-cell patch-clamp recordings in dissociated rat AVP neurons identified by transgenic expression of enhanced green fluorescent protein (eGFP) under the control of the AVP promoter [9]. RT-PCR, pharmacology, and gene silencing studies showed that both N-type Cav2.2 and T-type Cav3.1 Ca 2+ channels are molecularly and functionally expressed in dissociated rat AVP neurons under normotonic conditions and also that these VGCC currents are sensitive to an anthranilic acid derivative, flufenamic acid (FFA). Furthermore, pharmacological data suggested that T-type Cav3.1 (but not N-type Cav2.2) channels are primarily involved in tetrodotoxin-insensitive spontaneous action potential firing in dissociated rat AVP neurons under normotonic conditions without any receptor stimulation.

Animals and preparation of acutely dissociated AVP neurons
All procedures involving animals were approved in advance by the Ethics Review Committee for Animal Experimentation of Fukuoka University and were in accordance with the guidelines of the Physiological Society of Japan. Non-transgenic female Wistar rats (Charles River Laboratories Japan, Yokohama, Japan) and heterozygous transgenic male Wistar rats, which express an AVP-enhanced green fluorescent protein (eGFP) fusion gene [9], were bred and housed under standardized conditions (12-h/12-h light/dark cycle) with food and water. For all the experiments, 4-to 5-week-old AVP-eGFP transgenic female rats were used.

Modification of gene expression and culture of acutely dissociated AVP neurons
To reduce the expression of rat Cav2. 2

Electrophysiology
The patch electrodes had a resistance of around 1-3 MΩ. Currents or voltages were recorded using an Axopatch 200B amplifier (Axon Instruments, CA, USA) coupled to DigiData 1440A A/D and D/A converters (Axon Instruments). Currents or voltage signals were filtered at 5 kHz and digitized at 20 kHz. pClamp software (version 9.0.2: Axon Instruments) was used for command pulse control, data acquisition and analysis. For the measurements of Ca 2+ channel currents, whole-cell voltageclamp recordings were performed at room temperature.
The time course of current activation was monitored by repetitively applying (every 10 s) alternating pulses

Statistical analysis
Data were given as the mean ± SEM of observations (n). Statistical differences of the data were evaluated using one-way ANOVA followed by a Bonferroni-type post hoc multiple comparisons and considered to be significant at P < 0.05.

Chemicals
All chemicals were prepared on the day of the experi-

N-type Cav2.2 and T-type Cav3.1 channels are molecularly and functionally expressed in rat AVP neurons under unstimulated normotonic conditions
First, we performed RT-PCR for VGCC genes on the total RNA extract from pooled cytosols suctioned into patch pipettes from 10 individual eGFP-expressing AVP neurons isolated from the transgenic rat. As shown in Fig. 1 Next, we observed depolarization-induced Ba 2+ currents to examine functional expression of VGCCs by whole-cell patch-clamp recordings in dissociated rat AVP neurons exposed to extracellular solution containing Ba 2+ in place of Ca 2+ . As shown in Fig. 2, in the presence of 2 mM and 50 mM Ba 2+ , voltagegated Ba 2+ currents became activated at > − 60 mV and > − 50 mV, and the peak currents were observed at around − 30 mV and − 10 mV, respectively. Since the currents with 50 mM Ba 2+ were much larger than those with 2 mM Ba 2+ , hereafter, whole-cell VGCC currents were observed in the presence of 50 mM Ba 2+ . As shown in Fig. 3, VGCC currents were partially but significantly suppressed by either application of ω-conotoxin GVIA (ω-CgTx, 0.5 μM: a, c), which is a specific blocker of N-type Ca 2+ channels [11][12][13], or that of Ni 2+ (3 mM: b, c), which is known to exhibit an inhibitory effect on T-type Ca 2+ channels [14,15], and mostly suppressed by simultaneous applications of both ω-CgTx and Ni 2+ (c). As shown in Fig. 3c, it is noted that the peak VGCC currents were observed at − 10 mV in the absence of any blockers (Control) and presence of ω-CgTx (+ ω-CgTx), but those were shifted to + 10 mV and + 20 mV in the presence of Ni 2+ and of Ni 2+ plus ω-CgTx (+ ω-CgTx + Ni 2+ ), respectively. Also, the threshold voltage for VGCC activation was − 50 to − 40 mV for Control and + ω-CgTx, whereas that was positively shifted to − 30 to − 20 mV for + Ni 2+ and + ω-CgTx + Ni 2+ . The inactivation time constant for ω-CgTx-sensitive currents (36.0 ± 4.7 ms, n = 7) was faster than that of Ni 2+ -sensitive currents (70.0 ± 10.4 ms, n = 8). These results indicate that the VGCC currents are composed of Ni 2+ -sensitive lowvoltage-activated and ω-CgTx-sensitive high-voltage-activated ones. Consistently, as summarized in Fig. 3d, the VGCC currents recorded at − 10 mV were almost abolished by Ni 2+ but only partially inhibited by ω-CgTx, whereas those recorded at + 20 mV were less effectively suppressed by Ni 2+ . As seen in Fig. 3  (c, d), there remains a minor part of high-voltage-activated VGCC currents resistant to Ni 2+ and ω-CgTx, presumably L-, P/Q-and/or R-type ones. In addition, as summarized in Fig. 3e, application of 5 μM 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2), which is known as a T-type-selective blocker [16], significantly, though partially, suppressed VGCC currents recorded at − 10 mV (left panel) without significantly affecting the currents recorded at + 20 mV (right panel). The VGCC currents recorded at − 10 mV were markedly inhibited by 10 μM TTA-P2 (Fig. 3e: left panel), but those recorded at + 20 mV were also mildly suppressed (right panel). These results are consistent with the previous observations in dorsal root ganglion cells that TTA-P2 selectively inhibited T-type Ca 2+ currents at < 10 μM, but TTA-P2 at ≥ 10 μM inhibited, though less markedly, high-voltage-activated VGCC currents as well [17]. These pharmacological observations suggest that the VGCC currents are predominantly, though not all, composed of N-type (Cav2.2) and T-type Ca 2+ channel activities in AVP neurons under normotonic conditions. Subsequently, we examined which of the T-type VGCCs of Cav3.1, Cav3.2 and Cav3.3 is functionally expressed in AVP neurons by observing the effects of 10 μM and 3 mM Ni 2+ (Fig. 4a,b). The VGCC currents showed a weak rundown by 11.8 ± 5.1% at − 10 mV and 7.32 ± 4.3% at + 20 mV (Fig. 4c: drug-free) after perfusion of bath solution over several minutes. Peak VGCC currents recorded at − 10 mV were not significantly affected by the application of 10 μM Ni 2+ (Fig. 4a-c), at which the Cav3.2 channel is known to be specifically blocked [14]. However, the currents recorded at − 10 mV almost abolished and those recorded at + 20 mV were less effectively but significantly suppressed by application of 3 mM Ni 2+ (Fig. 4b,c), at which all Cav3.1, Cav3.2 and Cav3.3 channels are known to be inhibited [14,15]. Although 3 mM Ni 2+ may not be precisely specific to T-type Ca 2+ channels, Ni 2+ almost completely suppressed the peak VGCC current observed at − 10 mV at this concentration. Taken together with the TTA-P2 effects (Fig. 3e), it appears that the Ni 2+ -sensitive component observed at − 10 mV represents mainly T-type Ca 2+ currents. In contrast to Ni 2+ and TTA-P2, ML218 (10 μM), which is a known blocker of both Cav3.2 and Cav3.3 channels [18], failed to significantly affect the depolarizationinduced Ba 2+ currents (Fig. 4c), suggesting that only Cav3.1 is functioning as T-type Ca 2+ channels in AVP neurons under normotonic conditions. Fig. 4 Effects of Ni 2+ and ML218 on VGCC currents in unstimulated AVP neurons. a Representative record of currents before and after application of a low concentration (10 μM) or a high concentration (3 mM) of Ni 2+ to AVP neurons exposed to normotonic solution containing 50 mM Ba 2+ . Inset panels given below the current trace represent expanded current responses to step pulses from − 60 to + 70 mV applied at i, ii and iii. b Currentvoltage relationships for peak VGCC currents (n = 8-12). *P < 0.05 vs. Control. c Percent currents at − 10 mV (left panel) and + 20 mV (right panel) after perfusion of control solutions (n = 8) containing no blockers (drug-free: n = 8), 10 μM Ni 2+ (n = 12), 3 mM Ni 2+ (n = 8), and 10 μM ML218 (n = 9) compared to the control currents (Control) recorded before perfusion. *P < 0.05 vs. Control. # P < 0.05 vs. drug-free Sato-Numata et al. J Physiol Sci (2020) 70:49 We then examined the effects of siRNA-mediated single knockdown of Cav2.2 and Cav3.1 in AVP neurons. To do so, we selected yellowish neurons, as shown in Fig. 5a, expressing GFP-tagged AVP (in green) and DY547-tagged siRNAs (in red) for negative control (Control: left panel), Cav2.2 (middle panel), and Cav3.1 (right panel). Knockdown efficacy was confirmed by real-time PCR for Cav2.2 mRNAs (Fig. 5b, left panel) as well as for Cav3.1 mRNAs (Fig. 5b, right panel). As shown in Fig. 5 (c, d), VGCC currents were markedly suppressed by siRNA-mediated knockdown for Cav2.2 (ΔCav2.2) or Cav3.1 (ΔCav3.1). The peak currents for ΔCav2.2 were observed at − 10 to 0 mV, but those for ΔCav3.1 were shifted to + 10 mV (Fig. 5d). On balance, it is concluded that both high-voltage-activated N-type Cav2.2 and low-voltage-activated T-type Cav3.1 channels are not only molecularly, but also functionally expressed in rat AVP neurons under normotonic conditions.

Both Cav2.2 and Cav3.1 channels are sensitive to FFA in rat AVP neurons
We next examined effects of FFA, which was reported to inhibit not only a number of non-selective TRP cation channels in a variety of cell types [19][20][21], but also L-type Ca 2+ channels in smooth muscle cells [22], on VGCC currents in rat supraoptic AVP neurons under conditions where extracellular and intracellular cations are replaced with TEA and NMDG, respectively, and thus non-selective cation channel currents were minimized. As shown in Fig. 6(a, d), to our surprise, FFA (100 μM) partially but significantly suppressed VGCC currents in dissociated AVP neurons under normotonic conditions. The VGCC currents were also found to be significantly suppressed by 70 μM FFA by 59.6 ± 7.7% at − 10 mV and 42.3 ± 8.0% at + 20 mV (n = 5). At 100 μM, FFA markedly inhibited the ω-CgTx-insensitive component of VGCC currents (Fig. 6b) but less markedly the Ni 2+ -insensitive component (Fig. 6c). As summarized in Fig. 6d, FFA significantly suppressed both the ω-CgTx-insensitive components recorded at + 20 mV and − 10 mV, whereas FFA inhibited the Ni 2+ -insensitive component recorded at + 20 mV but not that recorded at − 10 mV. Taken together, it is clear that the FFA-sensitive component of currents are predominantly composed of T-type and N-type VGCC currents. These results show that both N-type Cav2.2 and T-type Cav3.1 Ca 2+ channels functionally expressed in the unstimulated rat AVP neurons are sensitive to FFA.

FFA-sensitive T-type Cav3.1 channels are involved in spontaneous firing activity in unstimulated AVP neurons
To examine whether these FFA-sensitive VGCCs are essentially involved in the spontaneous action potential firing in unstimulated AVP neurons, we observed the effects of FFA on the spontaneous firing in dissociated rat AVP neurons by nystatin-perforated whole-cell current-clamp recordings under normotonic conditions. As shown in Fig. 7, the spontaneous firing was found to be very prominent under normotonic conditions. The spontaneous firing was clearly inhibited by a voltage-gated Na + channel blocker, tetrodotoxin (TTX, 0.5 μM). However, even in the presence of TTX, firing activity was maintained, though it became prominently less frequent. This TTX-insensitive firing activity was almost completely eliminated by additional application of FFA (100 μM: Fig. 7a) or Ni 2+ (3 mM: Fig. 7b), but not affected by ω-CgTx (0.5 μM: Fig. 7c). As summarized in Fig. 7d, the firing frequency was significantly reduced by TTX and virtually nullified by FFA or Ni 2+ added on top of TTX. These results indicate that not only TTX-sensitive Na + channels, but also FFAand Ni 2+ -sensitive Cav3.1 channels are involved in

Discussion
Release of AVP and oxytocin (OXT) from MNCs in the hypothalamus is evoked by Ca 2+ influx through VGCCs [5,23,24]. Electrophysiological studies on VGCC currents showed that the soma of rat supraoptic MNCs functionally express T-, N-, L-, P/Q-and R-type Ca 2+ channels [25][26][27][28][29]. However, increasing evidence has shown that there are some differences in important properties including ion channel activities and firing patterns between AVP and OXT neurons [30]. Thus, further studies have been warranted to be performed on VGCC activities in AVP neurons by distinguishing from those in OXT neurons, and vice versa. In both rat supraoptic AVP and OXT neurons, molecular expression of mRNAs for non-T-type, high-voltage-activated Ca 2+ channels were first reported by Glasgow et al. (1999) [31]. On the other hand, functional expression of T-type Ca 2+ channels was observed in guinea pig supraoptic neurons, which likely represent AVP neurons displaying a depolarizing potential fired phasically [32]. Expression of T-, N-and L-type Ca 2+ channels in rat supraoptic AVP neurons was indirectly suggested by observing sensitivity to VGCC blockers of the increases in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in response to AVP [6] and PACAP [7]. Direct electrophysiological studies showed functional expression of L-, N-and P/Q-type of Ca 2+ channels in the neurohypophysial nerve terminals isolated from rats under unstimulated normotonic conditions [5]. However, these studies have not directly addressed the expression of VGCCs in AVP neurons largely devoid of their nerve   (Fig. 1). In dissociated rat AVP neurons, whole-cell VGCC currents were significantly inhibited, in an additive manner (Fig. 3), by ω-CgTx, which is a known blocker specific for N-type Cav2.2 channels [11][12][13], and by a high concentration (3 mM) of Ni 2+ , which is known to predominantly block T-type Cav3.1, Cav3.2 and Cav3.3 channels [14,15]. The VGCC currents sensitive to ω-CgTx and to Ni 2+ exhibited rapid and moderate inactivation rates (with τ of 36 and 70 ms), which match the inactivation properties of T-and N-type Ca 2+ channels, respectively [33]. Expression of T-type Ca 2+ channels in unstimulated rat AVP neurons is consistent with that of Ni 2+ -sensitive Ca 2+ channels observed in unstimulated guinea pig AVP-like magnocellular neurons [32]. In contrast, Ni 2+ failed to suppress the VGCC currents at 10 μM (Fig. 4), the concentration of which was reported to block Cav3.2, but not Cav3.1 and Cav3.3, channels [14]. Also, the VGCC currents were insensitive to ML218 (Fig. 4c), which blocks Cav3.2 and Cav3.3 channels [18]. Taken together, it is concluded that Cav2.2 and Cav3.1 channels are predominantly functioning in the plasma membrane of unstimulated rat AVP neurons under normotonic conditions. Low-voltage-activated or low-threshold T-type Ca 2+ channels are activated at lower voltages than high-threshold L-, P/Q-, N-and R-type Ca 2+ channels. Under physiological recording conditions, the apparent activation threshold for T-type Ca 2+ channels is − 50 to − 70 mV (see Reviews [33,34]). However, when the extracellular Ba 2+ concentration was increased from 1 and 2 mM to 10 and 40 mM, the threshold activation voltage for T-type Ca 2+ channels was shown to be shifted by around 10 and 20 mV, respectively, to a positive direction, because of the effect of such high concentrations of Ba 2+ on surface charge screening [35,36]. In agreement with these facts, in the present study, the Ba 2+ currents exhibited a threshold activation voltage of > − 60 and > − 50 mV in the presence of 2 and 50 mM Ba 2+ , respectively (Fig. 2). As a consequence, the voltage at which the peak currents were observed was also shifted from around − 30 mV to − 10 mV, when extracellular Ba 2+ , was increased from 2 to 50 mM (Fig. 2), suggesting that such a high concentration of Ba 2+ caused formation of a positive surface potential, thereby shifted the voltage actually subjected to the Ca 2+ channel protein toward a more negative one in the present study as well.
The present study also, for the first time, demonstrated that both N-type Cav2.2 and T-type Cav3.1 Ca 2+ channels in AVP neurons are sensitive to FFA (Fig. 6). FFA has long been used therapeutically as one of the top prescribed non-steroidal anti-inflammatory drugs (NSAIDs) which exhibit anti-inflammatory, analgesic and antipyretic effects [37]. When 200 mg FFA was orally administered to young healthy persons, the peak plasma concentration was reported to reach 6 to 20 μg mL −1 , or 21 to 71 μM, within 1.5 h [38]. Since FFA was shown to largely suppress both T-and N-type VGCC currents at 70 and 100 μM in the present study in vitro, it is feasible that the endogenous VGCC activities, especially T-type one, in the axon terminal in situ are sometimes partially suppressed by the plasma FFA after oral administration, because the posterior pituitary region exists outside the blood-brain barrier [39].
Somatodendritic action potentials of rat supraoptic MCNs were previously shown to arise from co-activation of Na + and Ca 2+ conductances [2]. However, it has not been known whether this is the case for AVP neurons distinguished from OXT neurons. In the present study, it was shown that the spontaneous firing in rat AVP neurons under unstimulated normotonic conditions is caused by the activities both of TTX-sensitive Na + channels and of FFA-and Ni 2+ -sensitive T-type Cav3.1 Ca 2+ channels (Fig. 7). The time interval between spikes of around 490 ms may be sufficient to attain ≥ 90% recovery of T-type Ca 2+ channel activity from inactivation, in light of previous data of around 90% recovery from short inactivation at 400 ms after firing of neuronal T-type Ca 2+ channels [40]. When FFA was applied to AVP neurons in the presence of TTX, the membrane became hyperpolarized (Fig. 7a), whereas the resting potential was not much affected by Ni 2+ and ω-CgTx (Fig. 7b, c). Since FFA is known to suppress a number of the cation-permeable TRP channel family [19][20][21], it is suggested that FFAinduced hyperpolarization was caused by its inhibitory action to some of TRP cation channels in AVP neurons. Further studies are required to elucidate the contribution of each type of VGCC to the firing activity under hypertonic or prolonged dehydration/hydration conditions.

Conclusions
In dissociated rat AVP neurons under normotonic conditions, N-type Cav2.2 and T-type Cav3.1 VGCCs were found to be expressed and predominantly functioning, and be sensitive to FFA. Also, it is suggested that T-type Cav3.1 VGCC is primarily involved in their action potential generation in AVP neurons under normotonic conditions.