A Nitric Oxide-Dependent Presynaptic LTP at Glutamatergic Synapses of the PVN Magnocellular Neurosecretory Cells in vitro in Rats

Abstract

The magnocellular neurosecretory cells (MNCs) of the hypothalamic paraventricular nucleus (PVN) integrate incoming signals to secrete oxytocin (OT), and vasopressin (VP) from their nerve terminals in the posterior pituitary gland. In the absence of gamma-aminobutyric acid A (GABA_A) and cannabinoids 1 (CB1) receptor activity, we used whole-cell patch-clamp recording, single-cell reverse transcription-multiplex polymerase chain reaction (SC-RT-mPCR), biocytin histochemistry and pharmacological methods to examine the mechanism of high frequency stimulus (HFS, 100 Hz)-induced long-term potentiation (LTP) at glutamatergic synapses in the PVN MNCs of juvenile male rats. Our results showed that HFS-induced LTP at glutamatergic synapses was accompanied by a decrease in the paired-pulse ratio (PPR) of the PVN MNCs. In these MNCs, HFS-induced LTP persisted in the presence of a group 1 metabotropic glutamate receptor (mGluR1) antagonist; however, it was abolished by an N-methyl-D-aspartic acid (NMDA) receptor blocker. Notably, HFS-induced LTP in the PVN MNCs was completely prevented by a nitric oxide synthase (NOS) inhibitor. The application of an NO donor not only induced the LTP of excitatory glutamatergic inputs in the PVN MNCs, but also occluded the HFS-induced LTP in these MNCs. Moreover, HFS-induced LTP in the PVN MNCs was also abolished by a specific protein kinase A (PKA) inhibitor, KT5720. SC-RT-mPCR analysis revealed that 64.5% (62/96) of MNCs expressed OT mRNA. Our results indicate that a HFS can induce an NMDA receptor and NO cascades dependent on presynaptic glutamatergic LTP in the PVN MNCs via a PKA signaling pathway.

Keywords: NMDA receptor; hypothalamic paraventricular nucleus; long-term synaptic plasticity; nitric oxide; protein kinase A; whole-cell recording.

FIGURE 1

Identification of PVN MNCs. (A) Representative membrane potential traces showing a PVN MNC in response to a series of depolarizing injected currents delivered at a hyperpolarized membrane potential, which expressed transient outward rectification (Arrow). (B) OT mRNA was detected in the MNCs. The positive control (Tissue Control +) showed that the mRNAs of OT and GAPDH were detected in the rat hypothalamic tissue total RNA. GAPDH transcripts were analyzed in the same cells as an internal control for the RT reaction. The expected size of the PCR products is indicated. A single cell (–RT) and the rat hypothalamic tissue total RNA (Tissue Control +) were processed without RT. (C) A histological photo illustrates the morphology of the MNC. S denotes stimulation electrode. (D) Enlarged microphotograph of (C) showing the morphology properties of the PVN MNCs.

FIGURE 2

In absence of GABA_A and CB1 receptors activity, HFS induced LTP of excitatory glutamatergic inputs in PVN MNCs. (A) Representative whole-cell recording traces showing paired-stimulation-evoked EPSCs in a PVN the MNC before (Pre) and after (post) delivering high frequency stimulation (HFS; 100 pulses, 3 times, 10 s interval). (B) Summary data showing the time course of the normalized amplitude of N1 under control conditions (No stimulation, open circle; n = 10 cells) and delivery of 100 Hz electrical stimulation (arrow head; filled circles; n = 16 cells). (C) Bar graph (n = 16 cells) showing normalized amplitude of N1 before (Pre sti.), after (Post sti.) delivery of HFS and control conditions (No sti.). (D) Summary of data (n = 16 cells) showing normalized paired-pulse ratio (PPR) before (Pre sti.), after (Post sti.) delivery of 100 Hz stimulation and control conditions (No sti.). Note that electrical stimulation at 100 Hz induced LTP of excitatory glutamatergic inputs accompanied with a decrease in PPR in the PVN MNCs.

FIGURE 3

Blockade mGluR1 failed to prevent the LTP induction in the MNCs. (A) In the presence of JNJ16259685 (10 μM), representative traces showing paired-pulse stimulation-evoked EPSCs in a PVN MNC before (Pre) and after (post) delivering HFS. (B) Summary of data show the time course of the normalized amplitude of N1 before and after delivery of HFS (arrow head; n = 12 cells). (C) Bar graph (n = 12 cells) showing the normalized amplitude of N1 before and after delivery of HFS in the presence of JNJ (JNJ-Pre, JNJ-Post) and control condition (Con-post). (D) Summary of data (n = 12 cells) showing the normalized PPR before and after delivery of HFS in the presence of JNJ (JNJ-Pre, JNJ-Post) and control condition (Con-post). Note that blockade mGluR1 failed to prevent the LTP induction in the PVN MNCs.

FIGURE 4

Application of NMDA receptor antagonist abolished the LTP induction in the PVN MNCs. (A) In the presence of NMDA receptor blocker, D-APV (50 μM), representative traces showing the stimulation-evoked EPSCs in a PVN the MNC before (Pre) and after (post) delivering HFS. (B) Summary of data show the time course of the normalized amplitude of N1 before and after delivery of the HFS (arrow head; n = 12 cells). (C) Bar graph (n = 12 cells) showing the normalized amplitude of N1 before (Pre) and after (Post) delivery of the HFS. (D) Summary of data (n = 12 cells) showing the normalized PPR before (Pre) and after (Post) delivery of the HFS. Note that NMDA receptor blocker abolished the induction of LTP in the PVN MNCs. Data points are mean ± S.E.M.

FIGURE 5

Effect of NOS on the LTP induction in the PVN MNCs. (A) Representative traces showing the stimulation-evoked EPSCs in the presence of NOS inhibitor, L-NNA (100 μM), before (Pre) and after (post) delivering the HFS, and before (Pre) and after (post) application of SNAP (100 μM). (B) Summary of data show the time course of the normalized amplitude of N1 before and after delivery of the HFS (arrow head; n = 12 cells). (C) Summary of data show the time course of the normalized amplitude of N1 before (Pre) and after (Post) application of SNAP (100 μM, gray line; 120 s; n = 12 cells). (D) Bar graph (n = 12 cells) showing the normalized amplitude of N1 before (Pre-sti in L-NNA), after delivery of the HFS in L-NNA (Post-sti. in L-NNA), before (Pre SNAP), after application of SNAP (Post SNAP) and after delivery of the HFS in ACSF (Post sti in ACSF). (E) Summary of data (n = 12 cells) showing the normalized PPR of N1 in each treatment. n.s denotes no significant different.

FIGURE 6

Application SNAP occluded the HFS to induce LTP of excitatory glutamatergic inputs in the PVN MNCs. (A) Representative traces showing paired-pulse stimulation-evoked EPSCs in a PVN MNC before (a), after (b) application of SNAP (100 μM), and after delivery of HFS (c). (B) Summary of data (n = 12 cells) show the time course of the normalized amplitude of N1 before, after application of SNAP (gray line; 120 s) and after delivery of HFS (arrow head). The a, b, and c denote baseline, after application of SNAP and after delivery of HFS, respectively. (C) Bar graph (n = 12 cells) showing the normalized amplitude of N1 after application of SNAP (SNAP) (b shown in panel B) and after delivery of HFS (HFS) (c shown in panel B). (D) Summary of data (n = 6 cells) showing the normalized PPR after application of SNAP (SNAP) and after delivery of HFS (HFS). Note that application NO donor could occlude the HFS to induce LTP of excitatory glutamatergic inputs in the PVN MNCs.

FIGURE 7

A specific PKA inhibitor, KT5720 completely prevented the LTP induction in the PVN MNCs. (A) In the presence of a specific PKA inhibitor, KT5720 (1 μM), representative traces showing paired-pulse stimulation-evoked EPSCs in a PVN MNC before (Pre) and after (Post) delivering the HFS. (B) Summary of data show the time course of the normalized amplitude of N1 before and after delivery of the HFS (arrow head; n = 10 cells). (C) Bar graph (n = 10 cells) showing the normalized amplitude of N1 before (Pre) and after (Post) delivery of the HFS. (D) Summary of data (n = 10 cells) showing the normalized PPR before (Pre) and after (Post) delivery of the HFS. Note that blockade of PKA signaling path way abolished the LTP induction in the PVN MNCs. n.s denotes no significant different.