Activation of V_1a Vasopressin Receptors Excite Subicular Pyramidal Neurons by Activating TRPV1 and Depressing GIRK Channels

Abstract

Arginine vasopressin (AVP) is a nonapeptide that serves as a neuromodulator in the brain and a hormone in the periphery that regulates water homeostasis and vasoconstriction. The subiculum is the major output region of the hippocampus and an integral component in the networks that processes sensory and motor cues to form a cognitive map encoding spatial, contextual, and emotional information. Whereas the subiculum expresses high densities of AVP-binding sites and AVP has been shown to increase the synaptic excitability of subicular pyramidal neurons, the underlying cellular and molecular mechanisms have not been determined. We found that activation of V_1a receptors increased the excitability subicular pyramidal neurons via activation of TRPV1 channels and depression of the GIRK channels. V_1a receptor-induced excitation of subicular pyramidal neurons required the function of phospholipase Cβ, but was independent of intracellular Ca²⁺ release. Protein kinase C was responsible for AVP-mediated depression of GIRK channels, whereas degradation of phosphatidylinositol 4,5-bisphosphate was involved in V_1a receptor-elicited activation of TRPV1 channels. Our results may provide one of the cellular and molecular mechanisms to explain the physiological functions of AVP in the brain.

Graphical Abstract

1. Introduction

The neurosecretory cells in the paraventricular and supraoptic nuclei of the hypothalamus synthesize two important hormones: arginine vasopressin (AVP) (also known as antidiuretic hormone) and oxytocin. After synthesis in the hypothalamus, these hormones are transported along the axons of these neurosecretory cells to the posterior pituitary where they are released into the blood stream to play their traditional physiological functions on blood vessels, kidney and uterus (Stoop, 2012). In addition to these neurosecretory functions, AVP and oxytocin also travel along the axonal projections from parvocellular neurons of the hypothalamus to discrete extrahypothalamic limbic brain regions including the hippocampus, subiculum, amygdala and nucleus accumbens (Buijs, 1978; Buijs and Swaab, 1979; DeVries et al., 1985; Hawthorn et al., 1985; Lang et al., 1983). Whereas the hypothalamus is the major source of AVP and oxytocin in the brain, AVP immunoreactivity has also been detected in neurons in the extrahypothalamic structures including the bed nucleus of stria terminalis, septal region, medial amygdala and locus coeruleus (Caffe and van Leeuwen, 1983; Sofroniew, 1985; van Leeuwen and Caffe, 1983) (for a review see (Cilz et al., 2019)). Similar to AVP, neuronal expression of oxytocin has also been found in medial amygdaloid nucleus and the cell group near the anterior commissure extending into the bed nucleus of stria terminalis (Otero-Garcia et al., 2016). Whereas oxytocin interacts primarily with the oxytocin receptors, AVP functions by interacting with 3 types of vasopressin receptors: V_1a, V_1b and V₂ receptors. Whilst V₂ receptors are coupled to G_s proteins resulting in increased activity of adenylyl cyclase to elevate cyclic AMP level, V_1a and V_1b receptors are coupled to G_q/11 proteins enhancing the activity of phospholipase Cβ (PLCβ) which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate 1,4,5-trisphosphate (IP₃) to increase intracellular Ca²⁺ release and diacylglycerol (DAG) to activate protein kinase C (PKC). In addition to its hormonal roles in constricting blood vessels and decreasing urine volume, AVP is also a neuromodulator that regulates a diverse range of functions including anxiety (Caldwell et al., 2008; Neumann and Landgraf, 2012), social behaviors (Cilz et al., 2019; Kompier et al., 2019), learning and memory (Caldwell et al., 2008; de Wied et al., 1993), nociception (Koshimizu and Tsujimoto, 2009) and circadian rhythms (Gizowski et al., 2017). However, the cellular and molecular mechanisms whereby AVP modulates these physiological functions and pathological disorders are not fully determined.

The subiculum is the major output region of the hippocampus and targets a variety of cortical and subcortical areas (O’Mara et al., 2001). Together with CA1 pyramidal neurons, the subiculum processes sensory and motor cues to form a cognitive map encoding spatial, contextual, and emotional information, which is then transmitted throughout the brain. Subiculum is therefore an integral component in the networks underlying memory for spatial tasks (O’Mara et al., 2001). In the subiculum, high densities of AVP-binding sites have been detected (Cilz et al., 2019; Insel et al., 1994; Phillips et al., 1988) and AVP increases the synaptic excitability of subiculum neurons (Chepkova et al., 1995). However, the precise actions of AVP and its underlying cellular and molecular mechanisms in the subiculum have not been determined. In the present study, we probed the effects of AVP on the excitabilities of the pyramidal neurons in the subiculum. Our results demonstrate that AVP enhanced the excitabilities of pyramidal neurons via V_1a receptor-mediated activation TRPV1 channels and depression of the G protein-gated inwardly rectifying potassium (GIRK) channels. AVP-elicited excitation of subicular pyramidal neurons required the function of PLCβ but was independent of intracellular Ca²⁺ release. PKC was responsible for AVP-mediated depression of GIRK channels, whereas PIP₂ depletion was involved in V_1a receptor-induced activation of TRPV1 channels. Our results may provide one of the cellular and molecular mechanisms to explain the physiological functions of AVP in vivo.

2. Materials and Methods

2.1. Slice preparation

Horizontal brain slices (300 μm) were prepared from both male and female Sprague-Dawley rats (20–28 days old) purchased from Envigo RMS, INC. (Indianapolis, IN), TRPV1 knockout (KO) mice (B6.129X1-Trpv1^tm1Jul/J, strain 003770, 1–2 months) and the corresponding age-matched wild-type (WT) mice (C57BL/6J, strain 000664) purchased from The Jackson Laboratory. All animals were housed in the Center for Biomedical Research in the University of North Dakota with food and water available ad libitum. The animal rooms were maintained on a 14/10 h light–dark cycle (lights on at 7:00 a.m.), with a room temperature of 22°C. All procedures and experiments presented in this study were approved by the Institutional Animal Use and Care Committee of the University of North Dakota and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). The number of males and females for each experiment was kept as equal as possible. After being deeply anesthetized with isoflurane, animals were decapitated and their brains were dissected out in ice-cold saline solution that contained (in mM) 130 N-methyl-D-glucamine (NMDG)-Cl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂, 5.0 MgCl₂, and 10 glucose, saturated with 95% O₂ and 5% CO₂ (pH 7.4, adjusted with HCl). Slices were then incubated in the above solution except NMDG-Cl was replaced with NaCl at 35°C for 1 hour for recovery and kept at room temperature (~22°C) until use.

2.2. Recordings of action potentials, resting membrane potentials and holding currents from subicular pyramidal neurons

Whole-cell recordings using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) in current- or voltage-clamp mode were made from the pyramidal neurons in the subiculum visually identified with infrared video microscopy (Olympus BX51WI) and differential interference contrast optics. During recordings, the bath temperature was maintained at 33–34°C by an in-line heater and an automatic temperature controller (TC-324C, Warner Instruments), unless stated otherwise. The recording electrodes were filled with (in mM) 100 K⁺-gluconate, 8 KCl, 2 MgCl₂, 40 HEPES, 0.6 EGTA, 2 ATPNa₂, 0.4 GTPNa, and 7 phosphocreatine (pH 7.3), unless stated otherwise. The extracellular solution comprised (in mM) 130 NaCl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO4, 2.5 CaCl₂, 1.5 MgCl₂ and 10 glucose, saturated with 95% O₂ and 5% CO₂ (pH 7.4). Data were filtered at 2 kHz, digitized at 10 kHz, acquired on-line and analyzed after-line using pCLAMP 10.7 software (Molecular Devices, Sunnyvale, CA). After the formation of whole-cell configuration, we routinely injected currents from −100 pA to 400 pA (duration: 400 ms) at a step of 50 pA with an interval of 10 s/injection to identify the recorded neurons. Bursting cells (BCs) fire an early burst of 2–4 action potentials (APs) upon injection of depolarizing current of threshold intensity, whereas in regular firing cells (RCs), prolonged depolarization causes either a single AP or trains of single spikes (Hu et al., 2017; Mason, 1993; Mattia et al., 1993). AVP was dissolved in the extracellular solution and bath-applied to the slice. To avoid potential desensitization induced by repeated applications of the agonist, one slice was limited to only one application of AVP. Resting membrane potentials (RMPs) and holding currents (HCs) at −60 mV were recorded in the extracellular solution supplemented with TTX (0.5 μM), kynurenic acid (1 mM) and picrotoxin (100 μM) to block AP firing, glutamatergic and GABAergic transmission, respectively. Pharmacological inhibitors were applied to the cells either extracellularly or intracellularly via the recording pipettes. For extracellular application, slices were pretreated for at least 2 hours to ensure permeation of reagents into the cells in the slices and the extracellular solution continuously contained the same concentration of the reagents, unless stated otherwise. For intracellular application, we waited for >15 min after the formation of whole-cell configuration to ensure the diffusion of the inhibitors into the cells. For the experiments with TRPV1 KO mice and the corresponding WT mice, the experimenters were blind to the identities of the mice.

2.3. Data analysis

Data are presented as the means ± S.E.M. N numbers in the text were the numbers of cells used for each experiment. To minimize potential influences of variation from individual animals, at least 4 animals were used for each experiment. Because the maximal response occurred within 5 min during the application of AVP, we measured the peak response of AVP for statistical analysis. The control data for AVP-induced inward currents were pooled results from the control experiments performed for each individual pharmacological experiment. One-way ANOVA followed by Tukey’s or Dunnett’s multiple comparisons test was used for statistical analysis when the pooled control data were used for comparison. Mann-Whitney test or Wilcoxon matched-pairs signed rank test (abbreviated as Wilcoxon test in the text) or Two-way repeated measures ANOVA followed by Sidak multiple comparison test was used for statistical analysis as appropriate; P values were reported throughout the text and significance was set as P < 0.05.

2.4. Chemicals

The following chemicals were products of R&D Systems: AVP, tetrodotoxin (TTX), kynurenic acid, picrotoxin, SR49059, TASP0390325, tolvaptan, GDP-β-S, U73122, U73343, heparin, thapsigargin, chelerythrine, bisindolylmaleimide II (Bis II), KB-R7943, capsazepine, AMG9810, AMG1629, capsaicin, ML 133, ML 297 and SCH23390. Dioctanoyl phosphatidylinositol 4,5-bisphosphate (dic8-PIP₂) was purchased from Echelon Biosciences. Drugs were initially prepared in stock solution, aliquoted and stored at −20°C. For those chemicals requiring dimethyl sulfoxide (DMSO) as a solvent, the concentration of DMSO was less than 0.1%. This concentration of DMSO either in the recording pipettes or in the bath had no significant effects on neuronal activity.

3. Results

3.1. AVP excites both bursting cells and regular firing cells in the subiculum

Whereas the subiculum has been shown to express AVP-binding sites (Cilz et al., 2019; Insel et al., 1994; Phillips et al., 1988) and bath application of AVP enhances synaptic excitabilities of subicular pyramidal neurons (Chepkova et al., 1995), the precise actions of AVP in subicular pyramidal neurons have not been elucidated. We probed the effects of AVP on the excitabilities of the principal neurons in the subiculum by recording the resting membrane potential (RMP) and potential appearance of action potential (AP) firing from the subicular pyramidal neurons in slices. Subiculum was distinguished from bordering regions by the diffuse distribution of pyramidal cells, compared to the tightly packed pyramidal cell layer of CA1, and the lack of distinct cortical layers observed in the entorhinal cortex (Hu et al., 2017). The extracellular solution contained kynurenic acid (1 mM) to block glutamatergic transmission and picrotoxin (100 μM) to block GABAergic transmission. Under these circumstances, the effects of AVP should be from the recorded neurons. For better comparisons, we used AVP at 0.3 μM which is a near-saturating concentration of AVP (Ramanathan et al., 2012) for each experiment. Because there are two types of pyramidal neurons, i.e., bursting cells (BCs) and regular firing cells (RCs) in the subiculum according to their firing patterns (Graves et al., 2012; Hu et al., 2017; Wozny et al., 2008), we identified the neuronal types by injecting negative and positive currents into the recorded cells initially (Fig. 1). BCs fire an early burst of 2–4 APs upon injection of depolarizing current of threshold intensity, whereas in RCs, prolonged depolarization causes either a single AP or trains of single spikes (Mason, 1993; Mattia et al., 1993). The possible reason for the bursting property of BCs is their selective expression of Cav3.1-containing T-type Ca²⁺ channels (Joksimovic et al., 2017). Of the 40 cells recorded, 28 cells were classified as BCs (~70%) and 12 cells were categorized as RCs (~30%), as reported previously (Mason, 1993; Mattia et al., 1993). We then recorded the RMPs and APs in response to AVP in both cell types. Out of the 28 BCs recorded, 17 cells (~61%) showed transient AP firing (263 ± 33 APs/min, n = 17, P < 0.0001, Fig. 1A₁–A₃) in response to bath application of AVP, whereas 11 BCs (~39%) displayed a subthreshold depolarization (Control: −63.5 ± 1.5 mV, AVP: −60.1 ± 1.5 mV, net depolarization: 3.4 ± 0.4 mV, n = 11, P = 0.001, Fig. 1A₄–A₅). Of the 12 RCs recorded, 6 cells (~50%) demonstrated transient AP firing (297 ± 97 APs/min, n = 6, P = 0.03, Fig. 1B₁–B₃) in response to bath application of AVP, whereas the remaining 6 RCs (~50%) displayed a subthreshold depolarization (Control: −61.9 ± 1.1 mV, AVP: −58.3 ± 0.9 mV, net depolarization: 3.6 ± 0.6 mV, n = 6, P = 0.03, Fig. 1B₄–B₅). There were no significant differences for the AVP-elicited AP-firing (P = 0.85) and subthreshold depolarization (P = 0.66) between BCs and RCs.

Figure 1. AVP excites both bursting cells and regular firing cells.

A₁-A₅, Bath application of AVP excited bursting cells by eliciting AP firing or subthreshold depolarization. A₁, Voltage responses (Upper panel) elicited by a negative (−100 pA) and a positive (+100 pA) current injection (Lower panel). Note the initial bursting response evoked by the positive current injection. A₂, Application of AVP elicited transient AP firing recorded from the RMP of the same neuron. A₃, Average of the AP numbers pooled from 17 bursting cells. A₄, Subthreshold depolarization evoked by AVP in a bursting cell. A₅, Summary graph showing subthreshold depolarization elicited by AVP in 11 bursting cells. B₁-B₅, Bath application of AVP excited regular firing cells by eliciting AP firing or subthreshold depolarization. B₁, Voltage responses (Upper panel) elicited by a negative (−100 pA) and a positive (+100 pA) current injection (Lower panel). Note the regular AP firing evoked by the positive current injection. B₂, Application of AVP elicited transient AP firing recorded from the RMP of the same neuron. B₃, Average of the AP numbers pooled from 6 regular firing cells. B₄, Subthreshold depolarization evoked by AVP in a regular firing cell. B₅, Summary graph showing subthreshold depolarization elicited by AVP in 6 regular firing cells.

Because application of AVP elicited only subthreshold depolarization in at least 50% subicular pyramidal neurons, we then assessed the effect of AVP on neuronal excitability by injecting a serious of incremental positive currents (from 30 pA to 480 pA, at an interval of 30 pA with a duration of 600 ms) before and during the application of AVP (Fig. 2). Application of AVP significantly increased the number of APs recorded from both BCs (n = 12, F_(1,11) = 125.9, P < 0.0001, Two-way repeated measures ANOVA followed by Sidak multiple comparison test, Fig. 2A₁–A₂) and RCs (n = 6, F_(1,5) = 9.71, P = 0.026, Two-way repeated measures ANOVA followed by Sidak multiple comparison test, Fig. 2B₁–B₂), further supporting the notion that AVP excites both BCs and RCs in the subiculum.

Figure 2. AVP increases the excitability of subicular pyramidal neurons evoked by injecting a series of positive currents.

A₁-A₂, Bath application of AVP increased the excitability of BCs evoked by injecting a series of positive currents from 30 pA to 480 pA at an interval of 30 pA and duration of 600 ms. The experiment was performed in the extracellular solution containing 1 mM kynurenic acid and 100 μM picrotoxin to block synaptic transmission. A₁, Current traces recorded from a BC before (left) and during (right) the application of AVP. A₂, Current-AP relationship constructed from 12 BCs before and during the application of AVP. ** P < 0.01. B₁-B₂, Bath application of AVP excited RCs elicited by injecting a series of positive currents from 30 pA to 480 pA at an interval of 30 pA and duration of 600 ms. The experiment was performed in the extracellular solution containing 1 mM kynurenic acid and 100 μM picrotoxin to block synaptic transmission. B₁, Current traces recorded from a RC before (left) and during (right) the application of AVP. B₂, Current-AP relationship constructed from 6 RCs before and during the application of AVP. * P < 0.05, ** P < 0.01. C₁-C₂, Bath application of AVP increased the excitability of BCs evoked by the same current injection protocol in normal extracellular solution. C₁, Current traces recorded from a BC before (left) and during (right) the application of AVP in normal extracellular solution. C₂, Current-AP relationship constructed from 16 BCs before and during the application of AVP in normal extracellular solution. ** P < 0.01. D₁-D₂, Bath application of AVP excited RCs elicited by the positive current injection protocol in normal extracellular solution. D₁, Current traces recorded from a RC before (left) and during (right) the application of AVP in normal extracellular solution. D₂, Current-AP relationship constructed from 8 RCs before and during the application of AVP in normal extracellular solution. ** P < 0.01.

The above experiments were performed in the extracellular solution containing kynurenic acid (1 mM) to block glutamatergic transmission and picrotoxin (100 μM) to block GABAergic transmission. However, in physiological condition, both glutamatergic transmission and GABAergic transmission are functional. We therefore repeated the experiments in the normal extracellular solution without adding any blockers. Under these circumstances, application of AVP still significantly enhanced the number of APs elicited by the positive current injection protocol in both BCs (n = 16, F_(1,15) = 69.85, P <0.0001, Two-way repeated measures ANOVA followed by Sidak multiple comparison test, Fig. 2C₁–C₂) and RCs (n = 8, F_(1,7) = 23.96, P = 0.002, Two-way repeated measures ANOVA followed by Sidak multiple comparison test, Fig. 2D₁–D₂). These results suggest that AVP-mediated increases in AP firing frequency should be physiologically functional. Because the effects of AVP in the subiculum were not cell-specific, we used both cell types for the remaining experiments.

3.2. AVP-mediated excitation of subicular pyramidal neurons is mediated by V_1a receptors

We then used voltage clamp and recorded holding currents at −60 mV in the abovementioned extracellular solution supplemented with TTX (0.5 μM) to further block AP firing. Under these circumstances, application of AVP induced an inward current (−55.1 ± 9.6 pA, n = 14, P = 0.0001, Fig. 3A, 3E). We denoted the AVP-induced inward currents in control condition as I_AVP thereafter. We tested the roles of AVP receptors in AVP-induced excitation of subicular pyramidal neurons. Pretreatment of slices with and continuous bath application of the selective V_1a receptor antagonist, SR49059 (1 μM), completely blocked I_AVP (−1.9 ± 1.8 pA, n = 10, P = 0.49 vs. baseline, Fig. 3B, 3E), demonstrating the involvement of V_1a receptors. Conversely, I_AVP was not significantly altered by application of the selective V_1b antagonist, TASP0390325 (TASP, 0.5 μM, −58.5 ± 4.8 pA, n = 11, P = 0.001 vs. baseline; P = 0.47 vs. control I_AVP, Fig. 3C, 3E) or the selective V₂ receptor antagonist, tolvaptan (50 nM, −54.7 ± 10.5 pA, n = 6, P = 0.03 vs. baseline; P = 0.97 vs. control I_AVP, Fig. 3D–E). These results together demonstrate that AVP-induced excitation of subicular pyramidal neurons is mediated by activation of V_1a receptors.

Figure 3. AVP excites subicular pyramidal neurons by activation of V_1a receptors.

A, Bath application of AVP generated an inward current recorded at −60 mV in a subicular pyramidal neuron. B, Pretreatment of slices with and continuous bath application of the selective V_1a receptor antagonist, SR49059 (1 μM), blocked AVP-elicited inward currents. C, Pretreatment of slices with and continuous bath application of the selective V_1b antagonist, TASP0390325 (TASP, 0.5 μM), had no effect on AVP-mediated inward currents. D, Pretreatment of slices with and continuous bath application of the selective V₂ antagonist, tolvaptan (50 nM), did not affect AVP-induced inward currents. E, Summary graph. Empty symbols represented values from individual cells and bar graphs were their averages. ** P <0.0001 vs. AVP alone.

3.3. AVP-elicited excitation of subicular pyramidal neurons is mediated by activation of a cationic conductance and depression of an inwardly rectifying K⁺ channel

We examined the ionic mechanisms underlying AVP-induced excitation of subicular pyramidal neurons by constructing the current-voltage (I-V) relationship of the currents generated by AVP. The abovementioned extracellular solution contained TTX (0.5 μM) to block voltage-gated Na⁺ channels. Cells were held at −60 mV and stepped from −120 mV to −40 mV for 400 ms at a voltage interval of 10 mV every 5 s. Steady-state currents were measured within 5 ms prior to the end of the step voltage protocol. In 12 of the 20 cells recorded, AVP elicited a persistent inward current at the voltage range between −120 mV and −40 mV (Fig. 4A₁–A₃), suggestive of the involvement of cation channels (Fig. 4A₃). The I-V curve of the I_AVP in the remaining 8 cells resembled that of the inwardly rectifying K⁺ (Kir) channels with a reversal potential of −85.0 ± 4.7 mV (n = 8, Fig. 4B₁–B₃), close to the calculated K⁺ reversal potential (−90.3 mV). These results demonstrate that activation of V_1a receptors excites ~60% (12/20) subicular pyramidal neurons primarily by activating a cationic conductance and ~40% (8/20) subicular pyramidal neurons mainly by depressing a Kir channel. AVP-elicited activation of cation channels and depression of K⁺ channels were not correlated with the neuronal types in the subiculum, because the 12 cells showing an I-V of cation channels included 8 BCs and 4 RCs whereas the 8 cells displaying an I-V of Kir channels consisted of 6 BCs and 2 RCs.

Figure 4. AVP excites subicular pyramidal neurons by activating a cationic channel and depressing a GIRK channel.

A₁-A₃: Application of AVP excited a subpopulation of subicular pyramidal neurons by activating a cationic conductance. A₁, Currents recorded from a subicular pyramidal neuron in response to the voltage-step protocol before (left) and during (middle) the application of AVP and the net current obtained by subtraction (right). The dash line was the zero current level. A₂, I-V curves of the currents elicited by the voltage-step protocol before and during the application of AVP (n = 12, * P < 0.05, ** P < 0.01). A₃, Net currents obtained by subtracting the currents in control condition from those recorded from the same cells during the application of AVP. B₁-B₃: Application of AVP excited a subpopulation of subicular pyramidal neurons by depressing Kir channels. B₁, Currents recorded from a subicular pyramidal neuron in response to the voltage-step protocol before (left) and during (middle) the application of AVP and the net current obtained by subtraction (right). The dash line was the zero current level. B₂, I-V curves of the currents elicited by the voltage-step protocol before and during the application of AVP (n = 8, * P < 0.05, ** P < 0.01). B₃, Net currents obtained by subtracting the currents in control condition from those recorded from the same cells during the application of AVP. C₁-C₃, bath application of Ba²⁺ (300 μM) by itself inhibited Kir currents and significantly reduced AVP-elicited inward currents. C₁, Current trace recorded from a subicular pyramidal neuron in response to bath application of Ba²⁺ and Ba²⁺ plus AVP. C₂, Summary graph. Note that bath application of Ba²⁺ induced an inward current (** P < 0.01 vs. baseline) and significantly reduced AVP-elicited inward currents. C₃, I-V curve of the net currents elicited by Ba²⁺ (300 μM) showed inward rectification (n = 5). D₁-D₃, In the continuous presence of Ba²⁺ (300 μM), AVP elicited a cationic channel current with a reversal potential at about −15 mV. D₁, Current traces evoked by a voltage step protocol (from −120 mV to +40 mV for 400 ms at a voltage interval of 10 mV every 10 s) before (left) and during (middle) the application of AVP and the net current obtained by subtraction (right). The dash line was the zero current level. Steady-state currents were measured within 5 ms before the end of the step voltage protocols. D₂, I-V curves of the currents elicited by the voltage-step protocol before and during the application of AVP in the continuous presence of Ba²⁺ (n = 11). D₃, net currents obtained by subtracting the currents in the presence of Ba²⁺ alone from those recorded from the same cells in the presence of both Ba²⁺ and AVP (n = 11).

Because micromolar concentration of Ba²⁺ (100–300 μM) has been shown to block Kir channels by at least 80% (Hu et al., 2017; Lacey et al., 1988; Li et al., 2019), we included 300 μM Ba²⁺ in the extracellular solution to inhibit Kir channels. Bath application of 300 μM Ba²⁺ by itself induced an inward current (−40.6 ± 6.2 pA, n = 12, P < 0.001 vs. baseline, Fig. 4C₁–C₂) and the I-V curve of the Ba²⁺-induced net currents showed inward rectification (n = 5, Fig. 4C₃), suggesting that subicular pyramidal neurons express Kir channels. In the presence of Ba²⁺, application of AVP induced a significantly smaller inward current (−23.0 ± 3.4 pA, n = 12, P < 0.001 vs. baseline; P = 0.006 vs. control I_AVP, Fig. 4C₁–C₂), further confirming the involvement of Kir channels. We further extended the voltage range from −120 mV to +40 mV to measure the reversal potential of the cationic currents elicited by AVP in the continuous presence of Ba²⁺ (300 μM) to block the contamination of the Kir channels. In addition to 300 μM BaCl₂, the extracellular solution also contained TTX (0.5 μM) to block voltage-gated Na⁺ channels, CdCl₂ (200 μM) and NiCl₂ (1 mM) to block the contamination of voltage-gated Ca²⁺ channels. Under these circumstances, AVP-elicited net currents showed a reversal potential of −15.9 ± 1.5 mV with outward rectification (n = 11, Fig. 4D₁–D₃), further confirming the involvement of cationic channels.

3.4. TRPV1 channels are necessary for AVP-mediated excitation of subicular pyramidal neurons

Na⁺/Ca²⁺ exchanger moves 3 Na⁺ ions in and 1 Ca²⁺ ion out, therefore generating an electrogenic potential. We therefore tested whether AVP excites subicular pyramidal neurons by increasing the function of Na⁺/Ca²⁺ exchanger. Pretreatment of slices with and continuous bath application of the selective Na⁺/Ca²⁺ exchanger inhibitor, KB-R7943 (70 μM), did not significantly alter I_AVP (−47.6 ± 7.7 pA, n = 10, P = 0.002 vs. baseline; P = 0.93 vs. control I_AVP, Fig. 5A, 5C), suggesting that the functions of Na⁺/Ca²⁺ exchangers are not involved in AVP-induced excitation of subicular pyramidal neurons.

Figure 5. TRPV1 channels are involved in AVP-mediated excitation of subicular pyramidal neurons.

A, Application of the selective Na⁺/Ca²⁺ exchanger inhibitor, KB-R7943 (70 μM), did not block AVP-induced inward currents. B, Bath application AVP induced a smaller inward current of subicular pyramidal neuron at room temperature (22°C). C, Summary graph. D, Bath application of the TRPV1 blocker, capsazepine (10 μM), reduced I_AVP. E, Co-application of the TRPV1 blocker, capsazepine (10 μM) and the Kir channel blocker, Ba²⁺ (300 μM), blocked AVP-elicited inward currents. F, Bath application of the selective TRPV1 blocker, AMG9810 (10 μM), reduced I_AVP. G, Bath application of the selective TRPV1 blocker, AMG21629 (3 μM), reduced I_AVP. H, Bath application of the TRPV1 agonist, capsaicin (50 μM), evoked an inward current. I, Application of the TRPV1 channel blocker, capsazepine (10 μM), blocked capsaicin-evoked inward currents in a subicular pyramidal neuron. J, Summary graph. * P < 0.05, ** P < 0.01 vs. control I_AVP. K, Application of AVP induced a small inward current recorded from a subicular pyramidal neuron in a slice cut from a TRPV1 KO mouse. L, Application of AVP still evoked a comparable inward current recorded from a subicular pyramidal neuron in a slice cut from a WT mouse. M, Bath application of the selective TRPV1 agonist, capsaicin (50 μM), evoked an inward current. N, Bath application of capsaicin (50 μM) did not elicit inward currents recorded from a subicular pyramidal neuron in a slice cut from a TRPV1 KO mouse. O, Bath application of Ba²⁺ (300 μM) blocked AVP-mediated inward current recorded from a subicular pyramidal neuron in a slice cut from a TRPV1 KO mouse. P, Summary graph.

The I-V curve of AVP-sensitive currents in the presence of Ba²⁺ to block the contamination of Kir channels showed outward rectification (Fig. 4D₃), resembling that of TRPV1 channels (Wu et al., 2010). Because TRPV1 channels are sensitive to temperature and the recording temperature in the current study was at 33–34°C, we therefore recorded I_AVP at room temperature (22–23°C). Application of AVP at room temperature induced a much smaller inward current (−9.3 ± 2.5 pA, n = 13, P < 0.001 vs. baseline; P < 0.0001 vs. control I_AVP, Fig. 5B–C), demonstrating that AVP-elicited inward currents are temperature-dependent. We further probed the roles of TRPV1 channels in AVP-mediated excitation of subicular pyramidal neurons. Bath application of the TRPV1 blocker, capsazepine (10 μM), significantly reduced I_AVP (−18.5 ± 4.6 pA, n = 11, P = 0.002 vs. baseline; P = 0.002 vs. control I_AVP, Fig. 5D, 5J). Co-application of capsazepine with Ba²⁺ (300 μM) to block Kir channels annulled I_AVP (0.15 ± 0.54 pA, n = 11, P = 0.78 vs. baseline, Fig. 5E, 5J), suggesting that TRPV1 and Kir channels are the two principal conductances involved in AVP-induced excitation of subicular pyramidal neurons. Furthermore, I_AVP was significantly reduced by bath applications of other two TRPV1 blockers, AMG9810 (10 μM, −29.0 ± 3.9 pA, n = 12, P = 0.0001 vs. baseline; P = 0.02 vs. control I_AVP, Fig. 5F, 5J) and AMG21629 (3 μM, −25.0 ± 2.8 pA, n = 10, P = 0.002 vs. baseline; P = 0.009 vs. control I_AVP, Fig. 5G, 5J), suggesting the involvement of TRPV1 channels. If TRPV1 channels are required for AVP-elicited excitation, application of the TRPV1 agonist, capsaicin, should elicit an inward current in the subicular pyramidal neurons. Consistent with our anticipation, bath application of capsaicin (50 μM) elicited an inward current in the subicular pyramidal neurons (−69.3 ± 3.8 pA, n = 6, P = 0.03 vs. baseline, Fig. 5H, 5J) and co-application of capsazepine blocked capsaicin-induced inward currents (1.20 ± 0.92 pA, n = 8, P = 0.23 vs. baseline, Fig. 5I–J), further confirming the expression of TRPV1 channels in the subiculum. Consistent with the results of the pharmacological experiments, application of AVP elicited a significantly smaller inward current in slices cut from TRPV1 KO mice (−10.4 ± 2.2 pA, n = 21, Fig. 5K, 5P), compared with the WT mice (−46.6 ± 5.8 pA, n = 15, P < 0.0001, Fig. 5L, 5P). Moreover, application of capsaicin elicited an inward current recorded from subicular pyramidal neurons in slices cut from WT mice (−59.1 ± 6.9 pA, n = 12, P = 0.0005, Fig. 5M, 5P), but failed to evoke inward currents in slices cut from TRPV1 KO mice (0.45 ± 1.77 pA, n = 12, P = 0.85, Fig. 5N, 5P). In the slices cut from TRPV1 KO mice, co-application of Ba²⁺ (300 μM) blocked AVP-induced inward currents (−0.64 ± 2.01 pA, n = 12, P = 0.79, Fig. 5O–P). These data together indicate that activation of V_1a receptors excites subicular pyramidal neurons by activating TRPV1 channels and depressing Kir channels.

3.5. GIRK channels are involved in AVP-elicited excitation of subicular pyramidal neurons

We further characterized the subtypes of the Kir channels involved in AVP-induced excitation. There are seven Kir channel subfamilies that can be classified into four functional groups: i) Kir2 subfamily comprising Kir2.1, Kir2.2, Kir2.3 and Kir2.4 form the classical Kir channels and are constitutively active; ii) Kir3 subfamily including Kir3.1 (GIRK1), Kir3.2 (GIRK2), Kir3.3 (GIRK3) and Kir3.4 (GIRK4) encoded by the genes KCNJ3, KCNJ6, KCNJ9, and KCNJ5, respectively, constitute the G protein-gated GIRK channels; iii) Kir6 subfamily encompassing Kir6.1 and Kir6.2 form the ATP-sensitive K⁺ (K_ATP) channels; iv) K⁺ transport channels include Kir1.1, Kir4.1, Kir4.2 and Kir7.1 (Hibino et al., 2010). Whereas AVP has been shown to depress K_ATP channels (Shi et al., 2007; Wakatsuki et al., 1992), K_ATP channels are unlikely to be involved because our recording solution in the pipettes contained 2 mM ATP which blocked K_ATP channels. We used ML 133, a specific antagonist for Kir2 subfamily (Ford and Baccei, 2016; Huang et al., 2018; Kim et al., 2015; Sonkusare et al., 2016; Wang et al., 2011) to test the roles of the Kir2 subfamily in AVP-elicited excitation of subicular pyramidal neurons. Bath application of ML 133 (30 μM) by itself did not change the holding currents significantly (−4.6 ± 3.1 pA, n = 14, P = 0.24, Fig. 6A). In the presence of ML 133, application of AVP still elicited a comparable inward current (−50.3 ± 11.1 pA, n = 14, P < 0.0001 vs. baseline; P = 0.73 vs. control I_AVP, Fig. 6A, 6E), suggesting that Kir2 subfamily is not the Kir channels involved in AVP-elicited excitation. We then tested the roles of GIRK (Kir3) subfamily in AVP-elicited excitation. Application of SCH23390 (20 μM), a GIRK channel inhibitor (Chee et al., 2011; Kuzhikandathil and Oxford, 2002), induced an inward current by itself (−24.4 ± 3.8 pA, n = 13, P = 0.0002, Fig. 6B), suggesting the expression of GIRK channels in the subicular pyramidal neurons. In the continuous presence of SCH23390, application of AVP induced a significantly smaller inward current (−24.0 ± 2.8 pA, n = 13, P = 0.0002 vs. baseline; P = 0.02 vs. control I_AVP, Fig. 6B, 6E), suggesting that GIRK channels are involved in AVP-mediated excitation of subicular pyramidal neurons. Furthermore, co-application of SCH23390 (20 μM) and capsazepine (10 μM) blocked I_AVP (−3.7 ± 1.9 pA, n = 12, P = 0.13 vs. baseline; P < 0.001 vs. control I_AVP, Fig. 6C, 6E). Consistent with the involvement of GIRK channels, application of ML 297 (10 μM), an activator of GIRK1-containing channels (Kaufmann et al., 2013), induced an outward current (58.7 ± 10.2 pA, n = 12, P = 0.0005, Fig. 6D–E), suggesting that GIRK1 channels are functionally expressed in the subiculum and they may be involved in AVP-elicited excitation of subicular pyramidal neurons.

Figure 6. GIRK channels are involved in AVP-elicited excitation of subicular pyramidal neurons.

A, Bath application of the Kir2 subfamily inhibitor, ML 133 (30 μM), did not alter AVP-induced inward current. B, Bath application of SCH23390 (20 μM), a GIRK channel blocker, induced an inward current by itself and reduced AVP-elicited inward currents. C, Co-application of SCH23390 (20 μM) and the TRPV1 blocker, capsazepine (10 μM) blocked I_AVP. D, Bath application of ML 297 (10 μM), the GIRK1 activator, induced an outward current. E, Summary graph. * P < 0.05, ** P < 0.01. Empty symbols represented values from individual cells and bar graphs were their averages.

3.6. AVP-mediated excitation of subicular pyramidal neurons requires the functions of G proteins and PLCβ, but is independent of intracellular Ca²⁺ release

Because V_1a receptors are coupled to Gα_q/11, we next examined the roles of G proteins in AVP-induced excitation of subicular pyramidal neurons. Intracellular application of the G protein inactivator, GDP-β-S (0.5 mM), via the recording pipettes significantly reduced I_AVP (−6.4 ± 1.5 pA, n = 12, P = 0.003 vs. baseline; P < 0.0001 vs. control I_AVP, Fig. 7A, F), demonstrating that G proteins are required for AVP-induced excitation of subicular pyramidal neurons. Activation of V_1a receptors increases the function of PLCβ resulting in the hydrolysis of PIP₂ to generate IP₃ to increase intracellular Ca²⁺ release and DAG to activate PKC. We next examined the roles of this pathway in AVP-elicited excitation of subicular pyramidal neurons. Slices were pretreated with the selective PLCβ inhibitor, U73122 (5 μM), for >2 h. The same concentration of the inactive analog U73343 was used as a control. Under these circumstances, application of AVP induced a significantly smaller I_AVP in the slices treated with U73122 (−12.1 ± 3.2 pA, n = 15, P = 0.004, Fig. 7B, 7F), compared with the slices pretreated with U73343 (−59.0 ± 7.4 pA, n = 12, P < 0.001 vs. baseline; P < 0.001 vs. U73122, Fig. 7C, F). These results demonstrate that the function of PLC is required for AVP-elicited excitation of subicular pyramidal neurons.

Figure 7. AVP-elicited excitation of subicular pyramidal neurons is dependent on G proteins and PLCβ but independent of intracellular Ca²⁺ release.

A, Intracellular application of GDP-β-S (0.5 mM) via the recording pipettes depressed I_AVP. B, Pretreatment of slices with U73122 (5 μM) inhibited I_AVP. C, Pretreatment of slices with the inactive analog, U73343 (5 μM), in the same fashion, did not block I_AVP. D, Intracellular perfusion of heparin (2 mg/ml) via the recording pipette did not block I_AVP. E, Intracellular dialysis of thapsigargin (10 μM) did not block I_AVP. F, Summary graph. Empty symbols were the values from individual cells and bar graphs were their averages. ** P < 0.01 vs. control I_AVP.

We further tested the roles of intracellular Ca²⁺ release in AVP-elicited excitation of subicular pyramidal neurons. Application of the IP₃ receptor blocker, heparin (2 mg/ml), via the recording pipettes, failed to significantly alter I_AVP (−52.5 ± 5.7 pA, n = 12, P < 0.001 vs. baseline; P = 0.90 vs. control I_AVP, Fig. 7D, F), suggesting that IP₃ receptors are not required for AVP-induced increases in neuronal excitability in the subiculum. Likewise, intracellular application of the sarco-endoplasmic reticulum Ca²⁺-ATPase inhibitor, thapsigargin (10 μM), via the recording pipettes did not significantly change I_AVP (−56.1 ± 10.4 pA, n = 12, P < 0.001 vs. baseline; P = 0.94 vs. control I_AVP, Fig. 7E–F). These results demonstrate that intracellular Ca²⁺ release is not required for AVP-induced excitation of subicular pyramidal neurons.

3.7. PKC is required for the depression of GIRK channels

We tested the roles of PKC in AVP-mediated excitation of subicular pyramidal neurons. Pretreatment of slices with the selective PKC inhibitor, chelerythrine (10 μM), significantly reduced I_AVP (−26.4 ± 3.2 pA, n = 17, P < 0.0001 vs. baseline; P <0.001 vs. control I_AVP, Fig. 8A, 8E). Similarly, pretreatment of slices with bisindolylmaleimide II (Bis II, 1 μM), another PKC inhibitor, significantly reduced I_AVP (−29.0 ± 3.2 pA, n = 12, P = 0.0005 vs. baseline; P < 0.01 vs. control I_AVP, Fig. 8B, 8E). Whilst application of the PKC inhibitors significantly reduced AVP-induced inward currents, there was a large amount of current resistant to PKC inhibition. Because our results demonstrated that V_1a receptor activation excited subicular pyramidal neurons by activating TRPV1 channels and depressing GIRK channels, we further probed which type of channels is the target for PKC. We included 20 μM SCH23390 in the extracellular solution to block GIRK channels. In the presence of SCH23390, AVP-induced inward currents should be mediated by TRPV1 channels. Nonetheless, AVP-induced inward currents in the presence of both SCH23390 and Bis II were not significantly different from those when Bis II was applied alone (P > 0.05, Fig. 8C, E), suggesting that GIRK channels are the target for PKC. We used the TRPV1 KO mice to further probe the target of PKC. Application of Bis II completely blocked AVP-induced inward currents in slices cut from TRPV1 KO mice (0.19 ± 1.75 pA, n = 12, P = 0.91 vs. baseline, Fig. 8D–E), which is significantly smaller compared with the control effect of AVP in TRPV1 KO mice (P = 0.001, Fig. 8E). These data together demonstrate that the PKC target is GIRK channels.

Figure 8. PKC is required for V_1a receptor-mediated depression of GIRK channels.

A, Pretreatment slices with and continuous bath application of the selective PKC inhibitor, chelerythrine, reduced AVP-mediated increases in inward currents. B, AVP elicited a smaller inward current in slices treated with the selective PKC inhibitor, Bis II. C, Blockade of GIRK channels with SCH23390 in the extracellular solution did not alter Bis II-mediated reduction of I_AVP. D, Treatment of slices with Bis II blocked AVP-induced inward currents in slices cut from TRPV1 KO mice. E, Summary graph. ** P < 0.01, *** P < 0.001 vs. AVP alone.

3.8. Degradation of PIP₂ is involved in V_1a receptor-elicited activation of TRPV1 channels in subicular pyramidal neurons

Because PLCβ-mediated depletion of PIP₂ has been demonstrated to be a mechanism underlying the modulation of numerous ion channels (Rodriguez-Menchaca et al., 2012; Suh and Hille, 2008), we next probed the roles of PIP₂ depletion induced by activation of PLCβ. Inclusion of the short-chain, water-soluble analog dioctanoyl (dic8)-PIP₂ (20 μM) in the recording pipettes significantly reduced AVP-induced inward currents (−6.4 ± 3.9 pA, n = 18, P < 0.0001 vs. baseline; P <0.001 vs. control I_AVP, Fig. 9A, D), suggesting that PIP₂ depletion is involved in AVP-mediated excitation of subicular pyramidal neurons. We further tested whether PIP₂ depletion is involved in V_1a receptor-mediated activation of TRPV1 channels or depression of GIRK channels. Bath application of AVP did not elicit significant inward currents when SCH23390 was included in the extracellular solution to block GIRK channels and dic8-PIP₂ was included in the intracellular solution to compensate for PIP₂ depletion (−0.92 ± 0.82 pA, n = 16, P = 0.35 vs. baseline, Fig. 9B, 9D), which was significantly smaller compared with the effect of AVP when dic8-PIP₂ was applied intracellularly alone (P < 0.01, Fig. 9D). Nevertheless, intracellular perfusion of dic8-PIP₂ failed to further reduce AVP-elicited inward currents in slices cut from TRPV1 KO mice (−11.7 ± 1.8 pA, n = 18, P < 0.0001 vs. baseline; P = 0.79 vs. AVP alone, Fig. 9C–D). These results collectively demonstrate that PIP₂ depletion is responsible for V_1a receptor-mediated activation of TRPV1 channels.

Figure 9. PIP₂ depletion is responsible for V_1a receptor-mediated activation of TRPV1 channels.

A, Intracellular perfusion of dic8-PIP₂ reduced AVP-elicited inward currents. B, AVP did not induce inward currents in the extracellular solution containing the GIRK channel blocker, SCH23390, and intracellular solution containing dic8-PIP₂. C, Intracellular perfusion of dic8-PIP₂ did not change AVP-mediated inward currents in slices cut from TRPV1 KO mice. D, Summary graph. *** P < 0.001 vs. AVP alone.

4. Discussion

Our results demonstrate that activation of V_1a receptors increases the excitability of pyramidal neurons in the subiculum by activation of TRPV1 channels and inhibition of GIRK channels. The V_1a receptor-elicited excitation of subicular pyramidal neurons is mediated by activation of PLCβ but independent of intracellular Ca²⁺ release. PKC is required for V_1a receptor-mediated depression of GIRK channels, whereas PIP₂ depletion is involved in V_1a receptor-elicited activation of TRPV1 channels. Our results may therefore provide one of the cellular and molecular mechanisms to explain the physiological functions of AVP in vivo.

We found that in about 60% of the subicular pyramidal neurons activation of V_1a receptors increases neuronal excitability primarily by activating TRPV1 channels. TRPV1 channels showed outward rectification (Wu et al., 2010) and the I-V curve of the AVP-elicited currents displayed outward rectification. The involvement of TRPV1 channels was further supported by the pharmacological experiments because application of the TRPV1 channel blockers significantly reduced AVP-elicited currents. Moreover, application of AVP induced a significantly smaller current in slices cut from TRPV1 KO mice compared with wild-type mice.

How does activation of V_1a receptors result in opening of TRPV1 channels? V_1a receptors are coupled to G_q/11 resulting in increases in the function of PLCβ which hydrolyses PIP₂ to generate IP₃ to elevate intracellular Ca²⁺ release and DAG to activate PKC. Our results demonstrate that V_1a receptor-mediated excitation of subicular pyramidal neurons requires the function of PLCβ, but is independent of intracellular Ca²⁺ release. We further demonstrate that PIP₂ depletion is responsible for V_1a receptor-elicited activation of TRPV1 channels. Consistent with our results, TRPV1 channels are activated by many Gα_q-coupled receptors including oxytocin receptors (Hu et al., 2020a), bradykinin receptor 2 (Shin et al., 2002), prostaglandin receptor (Moriyama et al., 2005), protease activated receptor 2 (Amadesi et al., 2004), histamine receptor 1 (Shim et al., 2007), endothelin-1 receptors (Plant et al., 2007), neurokinin receptors (Sculptoreanu et al., 2008; Zhang et al., 2007) and sensory neuron-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) (Solinski et al., 2012). TRPV1 channels are inhibited by PIP₂ and PLCβ-mediated degradation of PIP₂ relieves TRPV1 from the inhibitory effects of PIP₂, thereby activating the ion channels (Chuang et al., 2001; Prescott and Julius, 2003; Suh and Hille, 2008). Consistent with this scenario, application of oxytocin, which differs from AVP in only two amino acids, activates TRPV1 channels in the subiculum via PIP₂ degradation (Hu et al., 2020a), and TRPV2 channels in the hypothalamic neurons via phosphoinositide 3-kinase (van den Burg et al., 2015). Because phosphoinositide 3-kinase converts PIP₂ to phosphatidylinositol-3, 4, 5-triphosphate (PIP₃), and PIP₂ interacts with a group of positive residues downstream the TRPbox of TRPV1 and TRPV2 (Taberner et al., 2015; Ufret-Vincenty et al., 2011), it is plausible that PIP₂ depletion catalyzed by phosphoinositide 3-kinase could be involved in oxytocin receptor-mediated facilitation of TRPV2 channels as well.

Our results also indicate that AVP excites about 40% subicular pyramidal neurons majorly via inhibition of Kir channels because the I-V curves of the AVP-elicited currents displayed the characteristics of Kir channels and application of Ba²⁺ at 300 μM significantly reduced AVP-induced inward currents. Consistent with our results, AVP has been shown to suppress Kir channels in porcine cerebral capillary endothelial cells (Hoyer et al., 1991). Furthermore, activation of oxytocin receptors inhibits Kir channels in the immortalized gonadotropin-releasing hormone-positive GN11 cell line (Gravati et al., 2010), the lateral nucleus of central amygdala neurons (Hu et al., 2020b) and subicular pyramidal neurons (Hu et al., 2020a). There are many subtypes of Kir channels and K_ATP channels are one of them. Whilst activation of V_1a receptors has been shown to depress K_ATP channels (Shi et al., 2007; Wakatsuki et al., 1992), it is unlikely that K_ATP channels are responsible for AVP-elicited excitation of subicular pyramidal neurons because our recording pipettes contained 2 mM ATP which inhibited K_ATP channels. Application of ML 133, the selective inhibitor for Kir2 subfamily (Ford and Baccei, 2016; Huang et al., 2018; Kim et al., 2015; Sonkusare et al., 2016; Wang et al., 2011) by itself failed to consistently and significantly elicit an inward current, although Kir2 channels are tonically active (Hibino et al., 2010). This may be due to the scarce expression of Kir2 subfamily in the subiculum (Karschin et al., 1996). Consistently, application of AVP still elicited a comparable inward current in the continuous presence of ML 133, demonstrating that V_1a receptor-elicited excitation of subicular pyramidal neurons is not mediated by depressing Kir2 subfamily. Our results indicate that GIRK channels are the subfamily involved in AVP-mediated excitation of subicular pyramidal neurons because application of SCH23390, the selective GIRK channel blocker, significantly reduced AVP-induced inward currents. If activation of V_1a receptors excites subicular pyramidal neurons by depression of GIRK channels, GIRK channels must be constitutively active. Consistent with this prerequisite, GIRK channels are constitutively active (Chen and Johnston, 2005; Gonzalez et al., 2018). In line with this scenario, our results demonstrate that application of SCH23390 alone induced in inward current, suggesting that GIRK channels in the subicular pyramidal neurons are tonically active. GIRK channels contain 4 subunits including GIRK1, GIRK2, GIRK3 and GIRK4 and exist as predominantly heterotetramers of GIRK1, GIRK2 and/or GIRK3, or as homotetramers of the GIRK2 subunit (Hibino et al., 2010; Luscher and Slesinger, 2010). GIRK4 is restrictedly expressed in certain neuronal populations, such as Purkinje cells and neurons of the globus pallidus and the ventral pallidum (Murer et al., 1997). In the subiculum, the major GIRK subunits are GIRK1, GIRK2 and GIRK3 (Karschin et al., 1996) with much less expression of GIRK4 (Karschin et al., 1996; Spauschus et al., 1996). Whilst the GIRK subunits involved in V_1a receptor-elicited excitation of subicular pyramidal neurons remain to be determined, GIRK1 seems to be a candidate because we have shown that application of the selective GIRK1 activator, ML 297 (Kaufmann et al., 2013), induced an outward current. Consistent with our results, another neuropeptide, gastrin-releasing peptide excites rat paraventricular thalamic neurons by simultaneous suppression of Kir channels and activation of TRPV1 channels (Hermes et al., 2013), although the subtype of the Kir channels is Kir2 subfamily not GIRK channels as we demonstrated in the present study. Congruously, AVP excites motoneurons by activating a nonselective cationic conductance and suppressing a K⁺ conductance (Kolaj and Renaud, 1998; Ogier et al., 2006; Raggenbass, 2008), although the identities of the cationic and K⁺ channels have not been determined. Our study identified that the cationic and K⁺ channels underlying V_1a receptor-mediated excitation of subicular pyramidal neurons are TRPV1 and GIRK channels, respectively.

How does V_1a receptor activation lead to depression of GIRK channels? Our results further demonstrate that PKC is required for AVP-mediated depression of GIRK channels in subicular pyramidal neurons. Consistent with our results, GIRK channels are inhibited by PKC-mediated phosphorylation (Adney et al., 2015; Mao et al., 2004; Stevens et al., 1999) and PKC-elicited suppression of GIRK channels is responsible for oxytocin receptor-mediated excitation of the lateral nucleus of central amygdala neurons (Hu et al., 2020b) and subicular pyramidal neurons (Hu et al., 2020a) as well.

The subiculum is the major output pathway of hippocampus and targets a variety of cortical areas and subcortical structures (O’Mara et al., 2001). This divergent output makes subiculum an integral component in networks underlying diverse functions and behaviors, such as regulation of spatial memory (O’Mara et al., 2001; O’Mara et al., 2009), the functions of hypothalamic-pituitary axis (O’Mara, 2005) and stress responses (Mueller et al., 2004). Additionally, dysregulation of subicular function has been implicated in pathological conditions such as epilepsy (Stafstrom, 2005; Wozny et al., 2005) and drug addiction (O’Mara et al., 2009). Vasopressinergic fibers project from the parvocellular neurons of the hypothalamus to discrete extrahypothalamic limbic brain regions including the hippocampus, subiculum, amygdala and nucleus accumbens (Buijs, 1978; Buijs and Swaab, 1979; DeVries et al., 1985; Hawthorn et al., 1985; Lang et al., 1983), although AVP immunoreactive cells have also been detected in many extrahypothalamic structures including the bed nucleus of stria terminalis, septal region, medial amygdala and locus coeruleus (Cilz et al., 2019). In the brain, AVP modulates a variety of physiological functions including emotional responses (Caldwell et al., 2008; Neumann and Landgraf, 2012), social behaviors (Cilz et al., 2019; Kompier et al., 2019), learning and memory (Caldwell et al., 2008; de Wied et al., 1993), nociception (Koshimizu and Tsujimoto, 2009) and circadian rhythms (Gizowski et al., 2017). Many of these functions are associated with the limbic structures including the subiculum. Because many structures in the limbic system are reciprocally connected, functional alteration of one component such as the subiculum would likely affect the general function of the limbic system. The cellular and molecular effects of AVP should contribute to its in vivo physiological functions. To date, at least two cellular and molecular mechanisms for AVP in the subiculum have been detected. First, bath application of AVP induced a long-term enhancement of EPSP amplitude recorded from subiculum neurons and the maximal effect could be observed 30–45 min after AVP application (Chepkova et al., 1995). Second, the current study suggested that activation of V_1a receptors exerted short-term (within 3–5 min) excitation of subicular pyramidal neurons. AVP-induced short- and long-term excitation of subiculum should contribute to its in vivo physiological functions such as learning and memory as well as emotional control. Whilst the cellular and molecular mechanisms underlying AVP-induced long-term facilitation of EPSP amplitudes in the subiculum have not been determined (Chepkova et al., 1995), our results demonstrate that activation of V_1a receptors excites subicular pyramidal neurons via PLCβ-, PKC- and PIP₂-mediated depression of GIRK channels and activation of TRPV1 channels. Knowing the molecular targets of V_1a receptors in the brain would shed light on understanding the functions of AVP in vivo. For example, activation of V_1a receptors exerts anxiogenic effects (Bielsky et al., 2005; Bielsky et al., 2004; Egashira et al., 2007; Landgraf et al., 1995; Simon et al., 2008), although the underlying cellular and molecular mechanisms have not been determined. Because TRPV1 channels (Ho et al., 2012; Marsch et al., 2007), GIRK channels (Pravetoni and Wickman, 2008; Victoria et al., 2016; Wydeven et al., 2014), PLCβ (McOmish et al., 2008; Xiao et al., 2012) and PKC (Bowers et al., 2000; Lesscher et al., 2008; Liu et al., 2014) are involved in the modulation of anxiety, our results may provide an ionic and signaling mechanisms to explain the anxiogenic effects of AVP. Furthermore, activation of V_1a receptors facilitates learning and memory (Bielsky et al., 2005; Nephew and Bridges, 2008) and both TRPV1 (Bannazadeh et al., 2017; Bashiri et al., 2018; Edwards, 2014; Genro et al., 2012) and GIRK channels (Mayfield et al., 2015) are involved in cognitive functions. Our results may therefore provide one of the cellular and molecular mechanisms to explain the effects of AVP on memory as well.

5. Conclusion

Whereas the subiculum receives vasopressinergic innervation and expresses vasopressin receptors, the effects of AVP in the subiculum and the underlying cellular and molecular mechanisms have not been determined. We demonstrate that activation of V_1a receptors excites subicular pyramidal neurons via activation of TRPV1 channels and depression of GIRK channels. V_1a receptor-elicited excitation requires the function of PLCβ but is independent of intracellular Ca²⁺ release. PKC is responsible for V_1a receptor-mediated depression of GIRK channels whereas PIP₂ depletion is involved in V_1a receptor-elicited activation of TRPV1 channels. Our results may provide one of the cellular and molecular mechanisms to explain the physiological functions of AVP in vivo.

Highlights.

• Activation of V_1a receptors excites subicular pyramidal neurons

• V_1a receptor-induced excitation is mediated primarily by activation of TRPV1 channels in ~60% subicular pyramidal neurons

• Inhibition of GIRK channels is responsible for V_1a receptor-elicited excitation in ~40% subicular pyramidal neurons

• PLCβ not intracellular Ca²⁺ is necessary for V_1a receptor-mediated excitation of subicular pyramidal neurons

• PKC is responsible for V_1a receptor-elicited inhibition of GIRK channels, whereas PIP₂ degradation is involved in V_1a receptor-mediated activation of TRPV1 channels