Purinergic P2 and glutamate NMDA receptor coupling contributes to osmotically driven excitability in hypothalamic magnocellular neurosecretory neurons.

KEY POINTS: Purinergic and glutamatergic signalling pathways play a key role in regulating the activity of hypothalamic magnocellular neurosecretory neurons (MNNs). However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Here, we report that ATP acts on purinergic P2 receptors in MNNs to potentiate in a Ca2+ -dependent manner extrasynaptic NMDAR function. The P2-NMDAR coupling is engaged in response to an acute hyperosmotic stimulation, contributing to osmotically driven firing activity in MNNs. These results help us to better understand the precise mechanisms contributing to the osmotic regulation of firing activity and hormone release from MNNs. ABSTRACT: The firing activity of hypothalamic magnocellular neurosecretory neurons (MNNs) located in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) is coordinated by the combined, fine-tuned action of intrinsic membrane properties, synaptic and extrasynaptic signalling. Among these, purinergic and glutamatergic signalling pathways have been shown to play a key role regulating the activity of MNNs. However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Whole-cell patch-clamp recordings obtained from MNNs showed that ATP (100 µM) induced an increase in firing rate, an effect that was blocked by either 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]2-pyridinyl]azo]1,3-benzenedisulfonic acid tetrasodium salt (PPADS) (10 µM) or kynurenic acid (1 mm). While ATP did not affect the frequency or magnitude of glutamatergic excitatory postsynaptic currents (EPSCs), it induced an inward shift in the holding current that was prevented by PPADS or kynurenic acid treatment, suggesting that ATP enhances a tonic extrasynaptic glutamatergic excitatory current. We observed that ATP-potentiated glutamatergic receptor-mediated currents were evoked by focal application of L-glu (1 mm) and NMDA (50 µM), but not AMPA (50 µM). ATP potentiation of NMDA-evoked currents was blocked by PPADS (10 µM) and by chelation of intracellular Ca2+ with BAPTA (10 mm). Finally, we report that a hyperosmotic stimulus (mannitol 1%, +55 mOsm/kgH2 O) potentiated NMDA-evoked currents and increased MNN firing activity, effects that were blocked by PPADS. Taken together, our data support a functional excitatory coupling between P2 and extrasynaptic NMDA receptors in MNNs, which is engaged in response to an acute hyperosmotic stimulus.

Purinergic P2 receptors in the paraventricular nucleus of the hypothalamus are involved in hyperosmotic-induced sympathoexcitation.

Increases in plasma osmolality activates the paraventricular nucleus of the hypothalamus (PVN) which in turn mounts a physiological response by increasing the release of arginine vasopressin and sympathetic nerve activity to end organs such as the kidney. The PVN expresses an abundance of purinergic receptors including P2X2 receptors. In the present study, we sought to determine (1) whether P2X2-expressing PVN neurons are activated by hypertonic saline or hypertonic mannitol and (2) what effects P2X receptor blockade has on sympathetic nerve activation mediated by a hyperosmotic stimulus. Male Wistar rats were randomly assigned to three groups and intravenously infused with either isotonic saline (0.154M, 0.5mL), hypertonic saline (3M, 0.5mL) or hypertonic mannitol (10% w/v, 0.5mL). Significantly greater numbers of Fos-positive cells were observed in the hypertonic saline (393±29)- and hypertonic mannitol (141±11)-infused rats compared with control, saline-treated, rats (47±2 neurons/PVN section). Furthermore, there was a significant increase in the number of activated (Fos-positive) P2X2 expressing PVN neurons in the hypertonic saline (65±7) and hypertonic mannitol (37±7)-treated rats compared with controls (16±2). Microinjection of a P2X receptor antagonist, PPADS, within the PVN significantly attenuated sympathetic nerve activation driven by a hyperosmotic stimulus. The hyperosmotically induced increase in lumbar sympathetic nerve activity was significantly blunted after PPADS pre-treatment. Collectively, our findings indicate that hyperosmotic stimulation activates a subset of P2X2 expressing PVN neurons that might facilitate increased sympathetic drive.

Angiotensin II acting on PVN induces sympathoexcitation and pressor responses via the PI3K-dependent pathway.

In vitro studies have shown that angiotensin II (ANG II), via activation of ANG II type 1 (AT1) receptor plays an important role on the neural control of the blood pressure (BP), through an intracellular signalling pathway involving PI3K in the paraventricular nucleus of the hypothalamus (PVN). However, to the best of our knowledge, no in vivo study has been performed yet to unravel the functional role of ANG II and its interaction with PI3K pathways in the neural control of circulation of non-anesthetized animals. Here, we demonstrate that exogenous ANG II microinjected into the PVN in anaesthetic-free animals evokes an increase in sympathetic nerve activity and BP in a PI3K-dependent manner.

Commissural nucleus of the solitary tract regulates the antihypertensive effects elicited by moxonidine.

The rostral ventrolateral medulla (RVLM) contains the presympathetic neurons involved in cardiovascular regulation that has been implicated as one of the most important central sites for the antihypertensive action of moxonidine (an α2-adrenergic and imidazoline agonist). Here, we sought to evaluate the cardiovascular effects produced by moxonidine injected into another important brainstem site, the commissural nucleus of the solitary tract (commNTS). Mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and activity of putative sympathoexcitatory vasomotor neurons of the RVLM were recorded in conscious or urethane-anesthetized, and artificial ventilated male Wistar rats. In conscious or anesthetized rats, moxonidine (2.5 and 5 nmol/50 nl) injected into the commNTS reduced MAP, HR and sSNA. The injection of moxonidine into the commNTS also elicited a reduction of 28% in the activity of sympathoexcitatory vasomotor neurons of the RVLM. To further assess the notion that moxonidine could act in another brainstem area to elicit the antihypertensive effects, a group with electrolytic lesions of the commNTS or sham and with stainless steel guide-cannulas implanted into the 4th V were used. In the sham group, moxonidine (20 nmol/1 µl) injected into 4th V decreased MAP and HR. The hypotension but not the bradycardia produced by moxonidine into the 4th V was reduced in acute (1 day) commNTS-lesioned rats. These data suggest that moxonidine can certainly act in other brainstem regions, such as commNTS to produce its beneficial therapeutic effects, such as hypotension and reduction in sympathetic nerve activity.

Purinergic and glutamatergic interactions in the hypothalamic paraventricular nucleus modulate sympathetic outflow.

P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5'-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ±13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow.