Influence of developmental nicotine exposure on spike-timing precision and reliability in hypoglossal motoneurons

Abstract

Smoothly graded muscle contractions depend in part on the precision and reliability of motoneuron action potential generation. Whether or not a motoneuron generates spikes precisely and reliably depends on both its intrinsic membrane properties and the nature of the synaptic input that it receives. Factors that perturb neuronal intrinsic properties and/or synaptic drive may compromise the temporal precision and the reliability of action potential generation. We have previously shown that developmental nicotine exposure (DNE) alters intrinsic properties and synaptic transmission in hypoglossal motoneurons (XIIMNs). Here we show that the effects of DNE also include alterations in spike-timing precision and reliability, and spike-frequency adaptation, in response to sinusoidal current injection. Current-clamp experiments in brainstem slices from neonatal rats show that DNE lowers the threshold for spike generation but increases the variability of spike-timing mechanisms. DNE is also associated with an increase in spike-frequency adaptation and reductions in both peak and steady-state firing rate in response to brief, square wave current injections. Taken together, our data indicate that DNE causes significant alterations in the input-output efficiency of XIIMNs. These alterations may play a role in the increased frequency of obstructive apneas and altered suckling strength and coordination observed in nicotine-exposed neonatal humans.

Keywords: development; intrinsic properties; motoneuron; nicotine; spike-timing precision; spike-timing reliability.

Fig. 1.

A: measurement of spike-timing precision and reliability. The top trace displays membrane voltage (V_m) of a patch-clamped hypoglossal motoneuron (XIIMN) responding to the injected current profile seen in the bottom trace. Sinusoidal current injections were delivered at amplitudes of 25, 150, or 250 pA. The DC offset (shaded area, bottom trace) was determined before injection of sinusoidal current patterns and varied between cells, as explained in materials and methods. DC offset adjustment was made to position the cells firing threshold at the midpoint of the sinusoidal current amplitudes. The peak of the sinusoidal current injection cycle (point a) was defined as 0° for analysis. A pre-peak position is defined by the diagonal bar shading, and spikes falling here have negative phase angles. Spikes occurring after the peak (defined by the crosshatch shading) would have positive phase angles. The dashed line shows that the 1st spike in the 2nd burst falls before the peak of the sine wave and therefore has a negative phase angle. B: comparison of typical responses to sinusoidal current injection in control and developmental nicotine exposure (DNE) XIIMN. Ba: phase-locked XIIMN from a control animal (top trace), which generates action potentials at the peak of the injected sinusoidal current waveform (middle trace) or just after it (solid vertical lines connect action potential onset to the corresponding sine wave). Bb: phase-locked XIIMN from a DNE animal. Note that the action potentials occur well before the peak of the sine wave (vertical dashed lines), consistent with a negative phase angle.

Fig. 2.

Plasma cotinine levels in nicotine-exposed neonates. As stated in results, cotinine levels were zero in saline-exposed animals, and the data are not shown. ANOVA did not detect any differences in cotinine as a function of postnatal age (F = 2.18, P = 0.1150). Numbers in boxes represent the number of animals studied at each age.

Fig. 3.

Influence of current, input frequency, and treatment on phase angle. Phase angle becomes progressively more positive with increasing input frequency at all 3 amplitudes. At higher current injection amplitudes, DNE neurons (■) fire significantly earlier than the control motoneurons (○). *P < 0.05, DNE vs. control. See results and Table 2.

Fig. 4.

Influence of current, input frequency, and treatment on jitter. Jitter decreases with increasing input frequency at all current amplitudes, and is highest at a current injection amplitude of 25 pA. DNE did not significantly alter jitter at any current amplitude or sine wave frequency. DNE cells, ■; control cells, ○.

Fig. 5.

Influence of current, input frequency, and treatment on phase error. Phase error increases with increasing input frequency and drops markedly as current injection amplitude increases. DNE cells (■) had significantly larger phase error at the lowest current injection amplitude compared with control cells (○). Treatment effects were not observed at the medium and high sinusoidal current injection amplitudes. *P < 0.05, DNE vs. control.

Fig. 6.

Influence of current, input frequency, and treatment on the number of spikes per sine wave cycle. The number of spikes per cycle decreases steeply with increasing input frequency and increases with increasing current injection amplitude. There was a significant treatment effect, with DNE cells (■) generating fewer spikes per cycle than control cells (○), but only at the 2 lowest input frequencies and at the 2 highest levels of injected current (see results). The region of phase locking (1 spike per sine wave cycle, horizontal dashed line) increased progressively with increases in sine wave amplitude.

Fig. 7.

Influence of current, input frequency, and treatment on the probability of a successful cycle. The probability of a successful cycle increases with current injection amplitude in all cells. DNE has complex effects on the probability of successful spiking as a function of current injection frequency and amplitude. There was no overall treatment effect but a significant interaction between treatment and current injection amplitude (Table 2). As explained in text, post hoc analysis showed that at an amplitude of 25 pA, DNE cells (■) are less likely to spike in a given cycle compared with control cells (○). However, at 150 pA DNE cells appear more likely to spike at the higher input frequencies, although this difference is not significant. Points lying above the horizontal dashed line are considered to have a high probability of generating a spike, as explained in results.

Fig. 8.

Influence of current, input frequency and treatment on the number of spikes per successful cycle. When the number of spikes per cycle is normalized to include only successful cycles, DNE cells (■) have slightly fewer spikes than control cells (○). Because there was no treatment:frequency interaction (Table 2), the treatment effect is independent of frequency. However, examination of the data clearly shows that the differences occurred only at the 2 lowest frequencies, and at the 2 highest levels of injected current. See text for further explanation.

Fig. 9.

Spike-frequency adaptation in XIIMNs from control and DNE preparations. A: representative trace showing a motoneuron's response (bottom traces) to square wave current injections (top traces). For each current step, the mean frequency of the 1st and last one-quarter second was measured (f_initial and f_steady-state, respectively), and spike-frequency adaptation was calculated as: adaptation (Hz) = f_initial − f_steady-state. This index was calculated for all current steps that were associated with continuous firing throughout the 1-s step. B: adaptation values (in Hz) for every level of injected current and for all motoneurons studied. DNE motoneurons showed a significantly higher spike-frequency adaptation compared with control cells. The horizontal bars show the median adaptation index in each treatment group. *P = 0.007, DNE vs. control.