PLCβ-Mediated Depletion of PIP2 and ATP-Sensitive K+ Channels Are Involved in Arginine Vasopressin-Induced Facilitation of Neuronal Excitability and LTP in the Dentate Gyrus

Abstract

Arginine vasopressin (AVP) serves as a neuromodulator in the brain. The hippocampus is one of the major targets for AVP, as it has been demonstrated that the hippocampus receives vasopressinergic innervation and expresses AVP receptors. The dentate gyrus (DG) granule cells (GCs) serve as a gate governing the inflow of information to the hippocampus. High densities of AVP receptors are expressed in the DG GCs. However, the roles and the underlying cellular and molecular mechanisms of AVP in the DG GCs have not been determined. We addressed this question by recording from the DG GCs in rat hippocampal slices. Our results showed that application of AVP concentration-dependently evoked an inward holding current recorded from the DG GCs. AVP depolarized the DG GCs and increased their action potential firing frequency. The excitatory effects of AVP were mediated by activation of V_1a receptors and required the function of phospholipase Cβ (PLCβ). Whereas intracellular Ca²⁺ release and protein kinase C activity were unnecessary, PLCβ-induced depletion of phosphatidylinositol 4,5-bisphosphate was involved in AVP-evoked excitation of the DG GCs. AVP excited the DG GCs by depression of the ATP-sensitive K⁺ channels, which were required for AVP-elicited facilitation of long-term potentiation at the perforant path-GC synapses. Our results may provide a cellular and molecular mechanism to explain the physiological functions of AVP, such as learning and memory, and pathologic disorders like anxiety.

Keywords: action potential; depolarization; hippocampus; peptide; receptors; signal transduction.

Figure 1.

Bath application of AVP elicits an inward current in DG GCs. A, Current trace recorded from a DG GC in response to bath application of AVP (0.3 μm). B, Current trace recorded from a DG GC in a slice pretreated with the selective V_1a receptor antagonist SR49095 (1 μm). The extracellular solution continuously contained the same concentration of SR49095. C, Summary graph showing AVP-induced inward currents in control condition or in the presence of SR49095 (1 μm). The circles represent the values from individual cells, and the bars are their averages. D, Concentration–response curve of AVP constructed by measuring AVP-induced inward currents. The numbers within the parentheses were the numbers of cells recorded at each concentration.

Figure 2.

AVP-elicited inward currents are mediated by depression of Kir channels. A, B, AVP increased the input resistance of DG GCs. A, Voltage responses evoked by the injection of negative currents from 0 to −75 pA at an interval of 25 pA before (left) and during (right) the application of AVP from a DG GC. B, The current–voltage relationship averaged from nine cells. Input resistance was obtained by linear fitting of the current–voltage relationship. C, Currents elicited by a voltage step protocol before (left) and during (middle) bath application of AVP and the net current obtained by subtraction (right) from a GC. Cells were held at −60 mV and stepped from −140 to −40 mV for 400 ms at a voltage interval of 10 mV every 10 s. Steady-state currents were measured within 5 ms before the end of the step voltage protocols. Note the differences in the scale bars. The dashed line was the zero current level. D, I–V curve averaged from 12 GCs before and during the application of AVP. E, I–V curve of the net current obtained by subtracting the currents in control condition from those during the application of AVP. Note that the net currents showed inward rectification, suggesting the involvement of Kir channels.

Figure 3.

Effects of Kir channel blockers on AVP-elicited inward currents recorded from DG GCs. A, Current trace recorded from a DG GC in response to bath application of Ba²⁺ (500 μm) alone and concomitant application of AVP. B, Current trace recorded from a DG GC in response to bath application of ML 133 (30 μm) alone and coapplication of AVP. C, Current trace recorded from a DG GC in response to bath application of SCH23390 (40 μm) alone and coapplication of AVP. D, Current trace recorded from a DG GC in response to bath application of glibenclamide (100 μm) alone and coapplication of AVP. E, Summary graph showing the effects of Kir channel blockers on AVP-induced inward currents. The shaded bar was the averaged inward currents evoked by AVP in control condition pooled from the control experiment conducted for each individual pharmacological experiment. *p = 0.018, ****p < 0.0001 versus AVP alone, one-way ANOVA followed by Dunnett’s test.

Figure 4.

AVP-elicited inward currents depend on PLCβ and depletion of PIP₂, but do not require the functions of intracellular Ca²⁺ release and PKC activity. A, Current trace recorded from a DG GC in response to bath application of AVP alone. B, Current trace recorded from a DG GC in response to bath application of AVP in a slice pretreated with the PLC inhibitor U73122 (5 μm). C, Current trace recorded from a DG GC before, during, and after bath application of AVP in the intracellular solution containing heparin (0.5 mg/ml). D, Current trace recorded from a DG GC in response to bath application of AVP in the intracellular solution supplemented with thapsigargin (10 μm). E, AVP-induced inward current recorded from a DG GC in a slice pretreated with Bis II (2 μm). The extracellular solution continuously contained the same concentration of Bis II. F, AVP-elicited inward current trace recorded from a DG GC in a slice pretreated with RHC 80267 (25 μm), a DAG lipase inhibitor. The extracellular solution contained the same concentration of RHC 80267. G, Current trace recorded from a DG GC dialyzed with the intracellular solution containing diC8-PIP₂ (50 μm). H, Summary graph. The shaded bar was the averaged inward currents evoked by AVP in control condition pooled from the control experiment conducted for each individual pharmacological experiment. **p = 0.002, ***p = 0.0002 versus AVP alone, one-way ANOVA followed by Dunnett’s test.

Figure 5.

AVP depolarizes GCs and increases the number of APs elicited by injection of a series of positive currents. A, Resting membrane potential recorded from a GC before, during, and after the application of AVP. B, Summary data for AVP-induced depolarization. The empty circles represented the values from individual cells, and the solid symbols were their averages. C, APs elicited by injections of a series of positive currents from 25 to 400 pA in a GC before (left) and during (right) the application of AVP. D, Relationship between the injected currents and the elicited AP numbers from 13 GCs. *p < 0.05, **p < 0.001, two-way repeated-measures ANOVA followed by Sidak’s multiple-comparisons test.

Figure 6.

AVP does not modulate basal glutamatergic transmission but enhances LTP at the PP–GC synapses. A, Bath application of AVP (0.3 μm) did not alter significantly AMPA EPSCs recorded at the PP–GC synapses at −65 mV. The stimulation frequency was 0.1 Hz. The extracellular solution contained 10 μm bicuculline, and the intracellular solution was the K⁺-gluconate solution supplemented with 1 mm QX-314. The current traces were the averages of 1 min indicated at the time points shown in the figure. The stimulation artifacts were blanked. B, Bath application of AVP (0.3 μm) significantly enhanced LTP induced by pairing presynaptic stimulation (1 Hz, 40 pulses) with postsynaptic depolarization (−30 mV) recorded with K⁺-gluconate-containing intracellular solution. After recording basal AMPA EPSCs at −65 mV with the stimulation frequency of 0.1 Hz for 5 min, the bath was perfused with the extracellular solution containing AVP (0.3 μm) or saline (0.9% NaCl used to dissolve AVP) for 3 min, and the pairing protocol (1 Hz, 40 pulses, postsynaptic depolarization to −30 mV) was applied in the presence of AVP or saline. Recordings of AMPA EPSCs (−65 mV, 0.1 Hz) were resumed in the extracellular solution to observe the expression of LTP. Current traces were the averages in 1 min at the time points indicated in the figure. C, Application of AVP failed to enhance LTP when Cs⁺-gluconate-intracellular solution was used. D, Application of AVP did not augment LTP in the extracellular solution containing glibenclamide (100 μm) when K⁺-gluconate-intracellular solution was used.