Dynamic interactions of excitatory and inhibitory inputs in hypoglossal motoneurones: respiratory phasing and modulation by PKA

Abstract

The balance of excitation and inhibition converging upon a neurone is a principal determinant of neuronal output. We investigated the role of inhibition in shaping and gating inspiratory drive to hypoglossal (XII) motoneuronal activity. In neonatal rat medullary slices that generate a spontaneous respiratory rhythm, patch-clamp recordings were made from XII motoneurones, which were divided into three populations according to their inhibitory inputs: non-inhibited, inspiratory-inhibited and late-inspiratory-inhibited. In late-inspiratory-inhibited motoneurones, blockade of GABA(A) receptors with bicuculline abolished inspiratory-phased inhibition and increased the duration of inspiratory drive currents. In inspiratory-inhibited motoneurones, bicuculline abolished phasic inhibition, which frequently revealed excitatory inspiratory drive currents. In non-inhibited motoneurones, neither bicuculline nor strychnine markedly changed inspiratory drive currents. Inhibitory currents in XII motoneurones were potentiated by protein kinase A (PKA) activity. Intracellular dialysis of the catalytic subunit of PKA or bath application of the PKA activator Sp-cAMP significantly increased the amplitude of expiratory-phased IPSCs without any change in IPSP frequency. Inspiratory-phased inhibition in inspiratory-inhibited motoneurones was potentiated by Sp-cAMP. We conclude that inspiratory-phased inhibition is prevalent in neonatal XII motoneurones and plays an important role in shaping motoneuronal output. These inhibitory inputs are modulated by PKA, which also modulates excitatory inputs.

Figure 1. Examples of the three populations of motoneurones

A–C, examples of intracellular recordings from motoneurones. Upper traces (∫XII), integrated discharge of the XII nerve; lower trace (Im) voltage-clamp recordings from XII motoneurones. A, a non-inhibited motoneurone; note the absence of positive-going inhibitory currents. B, a late-inspiratory-inhibited motoneurone; note the predominance of upward inhibitory currents toward the latter portion of the inspiratory period as defined by the duration of the integrated XII nerve discharge. C, an inspiratory-inhibited motoneurone; note the positive-going inhibitory currents in phase with the inspiratory period as defined by the XII nerve discharge. Inset on right shows an example of an individual respiratory cycle.

Figure 2. Phasic inhibition during the late inspiratory period

A, upper trace, ∫XII nerve discharge (90 sweeps). B, average of voltage-clamp recordings (V_h=−70 mV) from a late-inspiratory-inhibited motoneurone illustrating the extensive inhibition occurring in the second half of the Inspiratory period (90 sweeps). C, inspiratory drive currents pre- and postbicuculline application (10 μm) from a different late-inspiratory-inhibited motoneurone (V_h=−70 mV). Bicuculline has negligible effect on the amplitude of the drive current but markedly increased its duration.

Figure 3. Bicuculline-sensitive inhibition during inspiration and the postinspiratory period

A, overlays of 10 consecutive voltage-clamp recordings from a late-inspiratory-inhibited motoneurone that received strong inhibition throughout all phases of the respiratory cycle. IPSCs are positive-going deflections. B, reversal of the IPSCs by changing the holding potential to –100 mV (10 consecutive recordings). Note that the current characteristics now resemble those of a non-inhibited motoneurone. IPSCs become negative going. C, application of bicuculline (10 μm) reduced the number of IPSCs during the inspiratory period and abolished IPSCs during the postinspiratory period (10 consecutive recordings). Note that although reduced in amplitude IPSCs persist during the expiratory period indicating respiratory phased inhibition.

Figure 4. Comparison of the characteristics of non-inhibited and late inspiratory-inhibited motoneurones

Bar charts representing the respective timing of various events within the inspiratory period. The inspiratory period for 6 late-inspiratory-inhibited motoneurones and 6 non-inhibited motoneurones was normalized to the duration of inspiration as defined by the XII nerve activity. Inset: a typical recording from a non-inhibited motoneurone; upper trace, ∫XIIn discharge (∫XII); lower trace, inspiratory current (Im), illustrating the duration of inspiration (shaded area). The time of occurrence of onset of inspiratory current (¶), time of peak integrated XII nerve discharge (‡), time of peak inspiratory current (*) and termination of inspiratory drive currents (§) were measured and plotted as the percentage time of occurrence in the inspiratory cycle. Negative values indicate events occurring before the onset of the inspiratory burst. Statistical significance was assessed using unpaired t tests and a value of P≤ 0.05 was considered significant; ns, not significant.

Figure 5. Inspiratory phased inhibition occludes excitatory postsynaptic currents

A, voltage-clamp recordings of inspiratory phased IPSCs in an inspiratory-inhibited motoneurone (30 sweeps). Averages show a clear inspiratory phased inhibition (Control) that is abolished by application of bicuculline (10 μm). B, voltage-clamp recording of inspiratory phased IPSCs in a second inspiratory-inhibited motoneurone. Upper trace, inspiratory triggered acquisitions of membrane current showing inspiratory phased IPSCs. Lower trace, inspiratory triggered averages (Control) failed to reveal inhibitory currents (100 sweeps). Despite the increased incidence of IPSCs during the inspiratory period (top trace) there is no apparent synchrony of events within the inspiratory period precluding averaging. Bicuculline application (10 μm) revealed an excitatory inspiratory drive current (30 sweeps).

Figure 6. PKA potentiated expiratory phased IPSCs via a postsynaptic mechanism

A, expiratory IPSCs recorded within the first few minutes of patch formation. Note that these currents are clearly outward. B, expiratory IPSCs recorded from the same neurone 20 min after patch-formation with cPKA (250 units ml⁻¹) included within the patch-clamp electrode. C, upper histogram, cumulative histogram of IPSC amplitude pre- and postdialysis of the motoneurone with cPKA; a rightward shift in the curves indicates a statistically significant (P < 0.01) potentiation of the IPSCs. Lower histogram, cumulative histogram of the interevent interval. No statistical difference in the curves indicates that the effects are limited to the postsynaptic site. D, upper histogram, cumulative histogram of IPSC amplitude pre- and postapplication of Sp-cAMP (100 μm). A rightward shift in the curves indicates a statistically significant (P < 0.01) potentiation of the IPSCs. Lower histogram, cumulative histogram of the interevent interval; non-significant change in the curves indicates that the effects are limited to the postsynaptic site.

Figure 7. PKA potentiated inspiratory phased GABA_A currents

A, voltage-clamp recordings from an inspiratory-inhibited motoneurone. Averages of membrane current revealed inspiratory phased inhibition (Control, 70 sweeps). Activation of PKA by Sp-cAMP (100 μm) potentiated inspiratory phased inhibition (Post-Sp-cAMP, 80 sweeps). Bicuculline (10 μm) blocked this inhibition and revealed excitatory drive currents (Post-Sp-cAMP-bicuculline, 45 sweeps); subsequent blockade of glycinergic inhibition with strychnine (1 μm) has no further effect (Post-Sp-cAMP-bicuculline & strychnine, 20 sweeps) (n= 4). B, graph of changes in charge transfer due to activation of PKA by Sp-cAMP and blockade of inhibition with bicuculline.