Protein kinase G-dependent mechanisms modulate hypoglossal motoneuronal excitability and long-term facilitation

Abstract

Since protein kinase-dependent modulation of motoneuronal excitability contributes to adaptive changes in breathing, we hypothesized that cGMP-dependent pathways activating protein kinase G (PKG) modulate motoneuronal inspiratory drive currents and long-term plasticity. In a medullary slice preparation from neonatal rat (postnatal days 0-4) generating spontaneous respiratory-related rhythm, hypoglossal (XII) motoneuronal inspiratory drive currents and respiratory-related XII nerve activity were recorded. Focal application of a PKG activator, 8-bromoguanosine-3',5'-cyclomonophosphate (8-Br-cGMP), to voltage-clamped XII motoneurones decreased inspiratory drive currents. In the presence of tetrodotoxin (TTX), 8-Br-cGMP decreased the exogenous postsynaptic inward currents induced by focal application of AMPA. Intracellular dialysis of XII motoneurones with an inhibitory peptide to PKG (PKGI) increased endogenous inspiratory-drive currents and exogenous AMPA-induced currents. Application of 8-Br-cGMP with PKGI had no further effect on spontaneous or evoked currents, confirming that the observed effects were induced by PKG. However, PKG differentially increased longer-term plasticity. Three 3 min applications (separated by 5 min) of the α(1)-adrenergic agonist phenylephrine (PE) in combination with 8-Br-cGMP yielded greater in vitro long-term facilitation than PE alone. These data indicate the presence of a cGMP/PKG-dependent signalling pathway in XII motoneurones that modulates inspiratory drive currents and plasticity of XII motoneurones, possibly contributing to their adaptation during physiological challenges, such as sleep and exercise.

Figure 1. Focal application of 8-Br-cGMP depresses inspiratory drive currents

A, endogenous glutamatergic inspiratory drive currents in a XII motoneurone before (black line) and ∼1 min after (red line) focal application of 100 μm 8-Br-cGMP. Traces are averages of 10 individual currents. B, peak endogenous current amplitude decreased following the focal application of 8-Br-cGMP (n = 6; P < 0.01 (**); repeated measures t test) returning to control within 5 min.

Figure 2. Postsynaptic exogenous AMPA-induced currents are depressed by 8-Br-cGMP

A, continuous recording showing effect of 100 μm 8-Br-cGMP bath application on exogenous AMPA-induced currents. Whole-cell currents were generated by focal application of 10 μm AMPA after bath application of 1 μm TTX. B, AMPA-induced current before (black line) and several minutes after (red line) initiation of bath application of 8-Br-cGMP. AMPA ejection at arrow. C, peak AMPA-induced current amplitude following focal application of 8-Br-cGMP (n = 7; P < 0.01 (**); paired t test).

Figure 3. Potentiation of endogenous excitatory drive by inhibition of PKG activity

A, endogenous glutamatergic inspiratory drive current right after (black line) and 30 min after (red line) establishing whole-cell patch on a XII motoneurone with electrode filled with 100 μm PKGI. B, time course of effect of dialysis with PKGI on endogenous motoneuronal currents. Current increased to its maximal value and stabilized within 15 min after break-in. Example control trace (green) demonstrates the stability typical of endogenous motoneuronal currents in untreated cells following break-in. C, increase in endogenous peak current amplitude 30 min after establishing whole-cell patch conditions (n = 5; P < 0.01 (**); paired t test).

Figure 4. PKG-dependent mechanisms directly depress AMPA receptor currents

A, single AMPA-induced currents in a motoneurone dialysed with PKGI for at least 30 min before (black line) and several minutes after (red line) 100 μm 8-Br-cGMP bath application. B, no change in the peak AMPA-induced current amplitude following bath application of 8-Br-cGMP. The current amplitude was 99 ± 5% (n = 4; n.s.).

Figure 5. Activation of PKG facilitates induction of ivLTF

A, integrated XII (∫XII) nerve activity in response to episodic PE application (10 μm; upper trace), 8-Br-cGMP (100 μm; middle trace) or PE and 8-Br-cGMP together (10 μm and 100 μm, respectively, lower trace). B, co-application of 8-Br-cGMP with PE significantly increased ivLTF relative to PE alone (P < 0.05 (*); two-way repeated measures ANOVA). Filled circles, PE alone; filled squares, co-application of PE and 8-Br-cGMP. Horizontal bars show group mean responses. C, episodic application of 8-Br-cGMP alone did not significantly increase ∫XII nerve activity (105 ± 12% at 60 min; n = 9 slices; n.s.; one-way repeated measures ANOVA). D, power analysis for the probability of failing to detect a long-term effect of episodically applied 8-Br-cGMP on XII nerve activity plotted as a function of the size of the effect.