Postnatal emergence of serotonin-induced plateau potentials in commissural interneurons of the mouse spinal cord

Abstract

Most studies of the mouse hindlimb locomotor network have used neonatal (P0-5) mice. In this study, we examine the postnatal development of intrinsic properties and serotonergic modulation of intersegmental commissural interneurons (CINs) from the neonatal period (P0-3) to the time the animals bear weight (P8-10) and begin to show adult walking (P14-16). CINs show an increase in excitability with age, associated with a decrease in action potential halfwidth and appearance of a fast component to the afterhyperpolarization at P14-16. Serotonin (5-HT) depolarizes and increases the excitability of most CINs at all ages. The major developmental difference is that serotonin can induce plateau potential capability in P14-16 CINs, but not at younger ages. These plateau potentials are abolished by nifedipine, suggesting that they are mediated by an L-type calcium current, I(Ca(L)). Voltage-clamp analysis demonstrates that 5-HT increases a nifedipine-sensitive voltage-activated calcium current, I(Ca(V)), in P14-16 CINs but does not increase I(Ca(V)) in P8-10 CINs. These results, together with earlier work on 5-HT effects on neonatal CINs, suggest that 5-HT increases the excitability of CINs at all ages studied, but by opposite effects on calcium currents, decreasing N- and P/Q-type calcium currents and, indirectly, calcium-activated potassium current, at P0-3 but increasing I(Ca(L)) at P14-16.

Fig. 1.

Commisural interneuron (CIN) action potential (AP) properties change during postnatal development. A: representative traces of APs from postnatal day 2 (P2; light gray), P8 (dark gray), and P14 (black) CINs. B: average measurements of AP halfwidth, threshold, and afterhyperpolarization (AHP) minimum for P0–3, P8–10, and P14–16 CINs. *P < 0.05. C: expanded view of the AHP of 2 different P14–16 CINs demonstrating a fast and slow AHP.

Fig. 2.

Effects of 5-HT on excitability of P0–3, P8–10, and P14–16 CINs. A: representative responses to 60-pA depolarizing current injections in P2, P9, and P14 CINs before and during application of 9 μM 5-HT. B: representative frequency-current (f-I) plots from individual P2, P9, and P14 CINs before and during 5-HT application. Average f-I values are shown for P0–3, P8–10, and P14–16 CINs (C), P8–10 CINs before and during 5-HT (D), and P14–16 CINs before and during 5-HT (E). f-I slope values are shown for P0–3, P8–10, and P14–16 CINs (F), P8–10 CINs before and during 5-HT (G), and P14–16 CINs before and during 5-HT (H). *P < 0.05.

Fig. 3.

5-HT modulates P8–10 AP shape. A: representative traces of P8–10 AP shape in control (black), 9 μM 5-HT (dark gray), and washout (light gray). B: a slower time course view of the same APs to demonstrate 5-HT's increase of AP halfwidth. C: 5-HT increased the AP halfwidth in P8–10 CINs. D: 5-HT decreased the absolute (Abs.) AHP amplitude in P8–10 CINs. E: 5-HT had no effect on the AP threshold of P8–10 CINs (*P < 0.05).

Fig. 4.

5-HT imparts plateau potential capability in P14–16 CINs. A: increasing steps of current injected into a P16 CIN under control conditions and during 9 μM 5-HT. Note that during 5-HT, firing continued after the termination of the current pulse at a number of steps. B: an example of a P10 (left) and P16 (right) CIN firing in response to an injected current step in control conditions as well as during 9 μM 5-HT. Only P14–16 CINs demonstrated plateau-potential capability. C: average integral of poststep afterpotentials in P14–16 CINs was increased by 5-HT (*P < 0.05). D: a P16 CIN that demonstrated an afterdepolarization (ADP) in 5-HT.

Fig. 5.

Nifedpine abolishes plateaus in P14–16 CINs. A: a P16 CIN fired in response to an injected current pulse in control, 9 μM 5-HT, and 5-HT + 10 μM nifedipine (NIF). Note that 5-HT induced a plateau potential (middle, arrow) and that addition of NIF abolished the plateau and ADP (bottom, arrow). B: the average integral of afterpotentials in P14–16 CINs was increased by 5-HT and abolished by NIF (*P < 0.05). There was no difference between the control and 5-HT + NIF conditions.

Fig. 6.

5-HT increases barium current (I_Ba) in P14–16 CINs. A and B: current steps in response to 5-mV incremental voltage steps from −50 to 0 mV in control conditions (A) and during application of 9 μM 5-HT (B). C: current-voltage (I-V) plot of peak of current in control (gray) and 5-HT (black).

Fig. 7.

5-HT does not increase NIF-insensitive current in P14–16 CINs. A: calcium current (I_Ca) in response to incremental voltage steps from −50 to 0 mV in a P16 CIN in control conditions, during application of 10 μM NIF, and during application of NIF and 9 μM 5-HT. B: I-V plot from the traces in A showing that 5-HT does not increase the current in the presence of NIF.

Fig. 8.

5-HT does not increase I_Ba in P8–10 CINs. A: I_Ba in response to incremental voltage steps from −50 to 0 mV in control, 9 μM 5-HT, and washout. B: 5-HT did not affect the maximal current (I_max) from a Boltzmann fit of the currents in control, 5-HT, and washout. C: time course of I_Ba amplitude in response to steps from −50 to −10 mV taken every 30 s. D: 5-HT did not have any appreciable effects superimposed on the rundown of the current: a, b, and c are traces taken at the times indicated in C.