Nitric oxide enhances inhibitory synaptic transmission and neuronal excitability in Guinea-pig submucous plexus

Abstract

Varicosities immunoreactive for nitric oxide synthase (NOS) make synaptic connections with submucosal neurons in the guinea-pig small intestine, but the effects of nitric oxide (NO) on these neurons are unknown. We used intracellular recording to characterize effects of sodium nitroprusside (SNP, NO donor) and nitro-l-arginine (NOLA, NOS inhibitor), on inhibitory synaptic potentials (IPSPs), slow excitatory synaptic potentials (EPSPs) and action potential firing in submucosal neurons of guinea-pig ileum in vitro. Recordings were made from neurons with the characteristic IPSPs of non-cholinergic secretomotor neurons. SNP (100 muM) markedly enhanced IPSPs evoked by single stimuli applied to intermodal strands and IPSPs evoked by trains of 2-10 pulses (30 Hz). Both noradrenergic (idazoxan-sensitive) and non-adrenergic (idazoxan-insensitive) IPSPs were affected. SNP enhanced hyperpolarizations evoked by locally applied noradrenaline or somatostatin. SNP did not affect slow EPSPs evoked by single stimuli, but depressed slow EPSPs evoked by stimulus trains. NOLA (100 muM) depressed IPSPs evoked by one to three stimulus pulses and enhanced slow EPSPs evoked by trains of two to three stimuli (30 Hz). SNP also increased the number of action potentials and the duration of firing evoked by prolonged (500 or 1000 ms) depolarizing current pulses, but NOLA had no consistent effect on action potential firing. We conclude that neurally released NO acts post-synaptically to enhance IPSPs and depress slow EPSPs, but may enhance the intrinsic excitability of these neurons. Thus, NOS neurons may locally regulate several secretomotor pathways ending on common neurons.

Figure 1

SNP enhances IPSPs but suppresses some SEPSPs. (A) IPSPs evoked by a 3-pulse stimulus in control (black) and SNP (100 μM) (red). (B,C) Quantitative data for the effects of SNP on amplitudes and durations respectively of 1-, 2-, 3-, 5- and 10-pulse evoked IPSPs (control solid black line, SNP solid red line). (D) SNP reduced the amplitudes of 2-, 3-, 5- and 10-pulse slow EPSPs, but had no effect on single stimulus evoked SEPSPs. *P < 0.05.

Figure 2

Inhibition of endogenous NOS depresses IPSPs. (A) Three-pulse IPSP in control (black) and in the presence of NOLA (100 μM) (red) (B) Quantitative data; NOLA significantly reduced the amplitudes of 1-, 2- and 3-pulse IPSPs. *P < 0.05 (single-tailed t test).

Figure 3

Sodium nitroprusside enhances responses evoked by noradrenaline and somatostatin. (A) Hyperpolarization evoked by local application of noradrenaline (NA, at arrow, green), (B) response evoked by NA in presence of SNP (red). (C) Quantitative data (n – number of neurons). (D,E) Hyperpolarizations evoked by SOM in control (blue) and SNP (red). (F) Quantitative data. *P < 0.05.

Figure 4

Sodium nitroprusside enhances non-adrenergic IPSPs. (A) Ten-pulse non-adrenergic IPSP (idazoxan present, black control, red in SNP and idazoxan). (B) Quantitative data showing the effects of SNP (red line) on the amplitudes of 1-, 2-, 3-, 5- and 10-pulse non-adrenergic IPSPs. *P < 0.05.

Figure 5

Sodium nitroprusside increased the excitability of submucosal neurons displaying IPSPs. (A) Action potentials (APs) evoked by a 500-ms duration depolarizing current pulse in control (black), and in SNP (red). Quantitative data over a range of current amplitudes for AP number and firing duration (B,C). *P < 0.05 (two-tailed t test).

Figure 6

Schematic diagram of inputs to submucosal VIP neurons VIP neurons receive synaptic inputs from many sources; sympathetic neurons (Symp) are responsible for noradrenergic IPSPs; myenteric and submucosal intrinsic sensory neurons (ISN) are excitatory; myenteric interneurons immunoreactive for serotonin (5-HT) or SOM are likely to mediate the non-noradrenergic IPSPs and myenteric neurons immunoreactive for VIP/nNOS are the probably source of endogenous NO released by electrical stimuli. SMP submucosal plexus, CM circular muscle, MP myenteric plexus, ACh acetylcholine, < denotes an excitatory synapse, denotes an inhibitory synapse.