Developing electrical properties of postnatal mouse lumbar motoneurons

Abstract

We studied the rapid changes in electrical properties of lumbar motoneurons between postnatal days 3 and 9 just before mice weight-bear and walk. The input conductance and rheobase significantly increased up to P8. A negative correlation exists between the input resistance (Rin) and rheobase. Both parameters are significantly correlated with the total dendritic surface area of motoneurons, the largest motoneurons having the lowest Rin and the highest rheobase. We classified the motoneurons into three groups according to their discharge firing patterns during current pulse injection (transient, delayed onset, sustained). The delayed onset firing type has the highest rheobase and the fastest action potential (AP) whereas the transient firing group has the lowest rheobase and the less mature AP. We found 32 and 10% of motoneurons with a transient firing at P3-P5 and P8, respectively. About 20% of motoneurons with delayed onset firing were detected at P8. At P9, all motoneurons exhibit a sustained firing. We defined five groups of motoneurons according to their discharge firing patterns in response to ascending and descending current ramps. In addition to the four classical types, we defined a fifth type called transient for the quasi-absence of discharge during the descending phase of the ramp. This transient type represents about 40% between P3-P5 and tends to disappear with age. Types 1 and 2 (linear and clockwise hysteresis) are the most preponderant at P6-P7. Types 3 and 4 (prolonged sustained and counter clockwise hysteresis) emerge at P8-P9. The emergence of types 3 and 4 probably depends on the maturation of L type calcium channels in the dendrites of motoneurons. No correlation was found between groups defined by step or triangular ramp of currents with the exception of transient firing patterns. Our data support the idea that a switch in the electrical properties of lumbar motoneurons might exist in the second postnatal week of life in mice.

Keywords: calcium; dendritic arborization; discharge firing pattern; spinal.

Figure 1

Electrophysiological identification and intracellular staining of lumbar motoneurons in the developing mouse spinal cord. (A) Electrical stimulation of the fifth lumbar (L5) ventral root evoked an anti AP. Asterisk indicates the stimulus artifact. (B) Direct intracellular stimulation of L5 motoneuron giving rise to a train of action potentials (APs; sustained discharge) recorded intracellularly (upper trace) and the propagated spikes in the ventral root (lower trace); rectangular injected current: 1.2 nA (middle trace). (C,D), two fully reconstructed lumbar motoneurons recorded from P8–P9 mice. Depending on the location of the soma in the ventro-lateral part of the spinal cord, dendritic arborisations extended either in all rostro-caudal directions and medially near the central canal (C) or confined in a restricted area into the latero-ventral part of the spinal cord (D). (E,F) Digitized full reconstructions of two motoneurons at postnatal days 3 and 8 (P3 and P8) in the transverse plane. During this period, the total dendritic length increases by 22%, only. Each dendrite is represented by a specific color.

Figure 2

Input resistance (Rin) and rheobase of mouse lumbar motoneurons (n = 53) at different postnatal ages (P3 to P9) and correlations with total dendritic surface area in 12 motoneurons stained with neurobiotin. (A) Mean Rin significantly decreases between the groups P3–5 and P8 (p = 0.028, n = 19 in each group) and then stabilizes at P9 (n = 15). (B) the mean rheobase (minimum current injected into a neuron to elicit an AP in 50% of cases) increases in the same period of time (p = 0.0033, n = 19 for P3–5 and n = 31 for P8–9). (C) significant negative correlation exists between rheobase and Rin in the whole population (n = 32); (D) no significant correlation was found between the Rin and the gain of the motoneurons (n = 32). The gain is the slope of the F-I curves measured in the steady state of the discharge firing elicited during rectangular pulses of currents. (E,F) significant correlations between Rin (E) rheobase (F) and the total dendritic surface area measured using 3D reconstructed motoneurons with neurolucida. Each motoneuron is numbered so that it can be identified from previous publications Amendola and Durand (2008) for Mn n° 1–7 and Filipchuk and Durand (2012) for Mn n° 15, 16, 17. The morphologies of motoneurons n° 22, 23 and 24 were not previously published. The largest motoneurons tend to have the lowest Rin and the highest rheobase. Horizontal bars indicated mean ± sem in the scatter plots of A and B. For statistical significance nonparametric permutation or mann-whitney exact tests and Pearson’s correlation test were used. *p < 0.05; **p < 0.01; ***p < 0.005. Non-significant (ns) p > 0.05.

Figure 3

Discharge firing patterns and distributions with age. Three different types of discharge firing patterns were found in lumbar motoneurons (n = 70) at different postnatal ages in response to rectangular current injection. The transient discharge (A) is characterized by a short burst of spike. The sustained pattern (B) starts by an early spiking followed by a burst and discharge firing maintained during the whole pulse. The third pattern is called delayed onset discharge firing (C) the arrow indicating a late depolarization. At P3–P5, one third of motoneurons still present a transient discharge and the motoneuron is not able to fire APs up to the end of the pulse as illustrated in (A) (see pie chart). In older animals the number of motoneurons presenting this pattern decreases up to P9. At that age, there is no more transient firing but all motoneurons exhibit a sustained discharge firing pattern (B). Between P3 and P8 a fraction of motoneurons has a delayed onset firing pattern which disappears at P9.

Figure 4

Passive and active electrical properties of postnatal mouse lumbar motoneurons (n = 33) in three subgroups defined by their discharge firing pattern. Fourteen electrical properties were compared (12 illustrated). Among them, several electrical properties show significant differences in the medians as indicated on each graph (Kruskal–Wallis test). The delayed subgroup has the highest conductance (A) rheobase (B) and time constant (C) suggesting they represent the largest motoneurons. The resting membrane potential (Em) (D) was hyperpolarized in this subgroup (delayed) but the spike voltage threshold was the lowest (F). The spike voltage threshold and the depolarization to threshold were the highest in the sustained subgroup (E,F). The AP was the shortest in the delayed population (half width in G) and the fastest (H,I) compared to the two others groups. The AP amplitude was similar in sustained and delayed subgroups but lower in the transient group (J). The AHP amplitude was smaller in the transient subgroup and larger in the delayed subgroup (K) whereas the medians of AHP half durations were comparable in the three subgroups (L). Bars indicate median and quartile. Statistical significance for three populations: Kruskal–Wallis exact test.

Figure 5

Five patterns of discharge recorded in response to current ramp stimulation in postnatal lumbar motoneurons (n = 38). The four classical types (A–D) as described in adult motoneurons (Bennett et al., 2001) are present and a fifth type called transient, since no discharge firing, or only a few spikes (<5), could be evoked during the descending phase (E). (A) type 1: linear F-I relationship where the firing frequency curves overlapped on the ascending and descending phases. (B) type 2: clockwise hysteresis pattern where the instantaneous frequency is lower in the descending phase for the same current intensity. (C) type 3: Linear F-I relationship with sustained firing in the descending phase. (D) type 4: Counter clockwise hysteresis where the frequency is higher during the descending phase; (E) type 5: transient discharge during the ascending phase with usually no discharge or only a few spikes in the descending phase. In this case which is frequent before P5 (40%), it was not possible to plot an F-I curve during the descending ramp. (F) distribution of the five discharge patterns according to the postnatal ages. Note that types 1 and 2 are the predominant types before P8 (n = 15/26) whereas the types 3 and 4 are most frequent at P8–P9 (n = 6/12) but rare before P8 (n = 3/26).