Motoneuron activity patterns related to the earliest behavior of the zebrafish embryo

Abstract

As a first step in the study of the developing motor circuitry of the embryonic zebrafish spinal cord, we obtained patch-clamp recordings in vivo from identified motoneurons in curarized embryos from the onset of the first motor behavior. At an early developmental stage in which embryos showed slow and repetitive spontaneous contractions of the trunk, motoneurons showed periodic depolarizations that triggered rhythmic bursts of action potentials with a frequency and duration that were consistent with those of the spontaneous contractions. The periodic depolarizations were blocked by tetrodotoxin or Cd(2+). Surprisingly, the contractions and periodic depolarizations were insensitive to general blockade of synaptic transmission (by elevated Mg(2+) and reduced Ca(2+), or by Co(2+)) and to selective blockade of the major neurotransmitter receptors of the mature spinal cord (acetylcholine, GABA(A), NMDA, AMPA/kainate, and glycine). The periodic depolarizations were suppressed by heptanol or by intracellular acidification, treatments that are known to uncouple gap junctions, indicating that electrotonic synapses could underlie the earliest motor behavior. A few hours later, most motoneurons already showed a new pattern of repetitive activity consisting of bursts of glycinergic synaptic events, but these were not necessary for the spontaneous contractions. Transecting the spinal cord at the hindbrain border did not affect the rhythmic activity patterns of the motoneurons. We suggest that spontaneous contractions of the zebrafish embryo are mediated by an early spinal circuit that is independent of the main neurotransmitter systems and descending hindbrain projections that are required for locomotion in the mature vertebrate spinal cord.

Fig. 1.

Cell-attached recordings from motoneurons in embryos aged 19–24 hr. A, Examples of recordings at 19, 21, and 24 hr. Note the different amplitude scales, and as the embryos matured the bursts contained more spikes. B, The average frequency of cell-attached bursts (●) had similar values and showed the same decline as the frequency of contractions in freely moving embryos (○) (from Saint-Amant and Drapeau, 1998). C, The number of spikes per burst increased dramatically during this short time span of development. D, The mean instantaneous frequency of spikes within bursts increased significantly from 19 to 24 hr. The asterisks show a significant increase from 19 hr (p < 0.01). In this and all other figures, *p < 0.05 and **p < 0.01.

Fig. 2.

Examples of the three types of motoneurons recorded from. A–C, Fluorescence images (Lucifer yellow). In all cases the axons reached out of the plane of focus to project between the two overlying muscle layers. D–F, The same cells photographed in bright-field with Hoffman modulation optics. A, D, A RoP motoneuron at 24 hr. The axon projected caudally (to the right) from the cell body toward the ventral border of the spinal cord (dotted line) and then projected out of the spinal cord via the ventral root to the midline muscle mass. B, E, A MiP motoneuron at 22 hr. The axon projected caudally, exited the ventral root, and projected to the dorsal muscle mass.C, F, A CaP at 20 hr. The axon projected directly to the ventral root, exited, and headed for the ventral muscle. Scale bar, 25 μm.

Fig. 3.

Whole-cell voltage-clamp recordings of motoneurons at 19 and 24 hr. A, Recording at 19 hr showing only periodic inward currents (PIC). B, A recording at 24 hr showing both PIC and synaptic bursts (SB). C, D, Segments from the recording in B are shown on an expanded time scale. PICs were composed of a slow and low amplitude inward current followed by a smaller, transient outward current (seen more clearly inA and B). SBs were composed of synaptic currents of large amplitude, and each event had a rapid time course.E, Age of onset of each activity pattern. The graphs display the percentage of the cells expressing each type of activity versus the age at which the recording was made. PICs (●) were present in all of the earliest recordings. SBs (▿) appeared later than the waves but were nevertheless present in most of the cells by 21 hr. Thenumbers above each point indicate the number of cells recorded from for each age group.

Fig. 4.

Properties and maturation of periodic inward currents. A, Current-clamp recording from a motoneuron at 20 hr showing only periodic depolarizations. The baseline was at −55 mV, and spikes were generated during the episodes of periodic depolarizations. B, Voltage-clamp recordings from a 20 hr motoneuron at different holding potentials. C, Normalized amplitude versus membrane voltage (n = 7 cells). The amplitude of episodes of periodic inward currents changed little over a substantial voltage range. D, The amplitude of the periodic inward currents (at a holding potential of −60 mV) did not change significantly from 19 to 24 hr and was approximately −10 pA. E, Average frequency of periodic inward currents sampled at different ages. As for the behavior, there was a gradual decrease in the average frequency over time.F, The input resistance of the motoneurons decreased significantly from 20 to 24 hr.

Fig. 5.

Properties and maturation of synaptic bursts.A, Current-clamp recording from a motoneuron at 24 hr.B, Recordings from a 22 hr motoneuron at different holding potentials. C, Normalized amplitude versus membrane potential (n = 8 cells). The mean amplitude of synaptic potentials decreased with increasing voltage and reversed near −40 mV. D, The amplitude of synaptic events was low (<20 pA) at 20 and 21 hr but increased dramatically by 22 hr. E shows that the mean instantaneous frequency increased dramatically from 11 Hz at 20 hr to 51 Hz. F, In some cell-attached recordings, synaptic bursts could be detected (SB, top trace) and were found to result from a current with the same polarity as the sodium current during action potential bursts (APB, bottom trace; note the different scale).

Fig. 6.

Pharmacology of activity patterns.A, CNQX, kynurenate, and APV (5 min) had no effect on the rhythmic activity. B, Strychnine blocked the synaptic bursts but not the periodic inward currents. C, Cobalt (Co⁺⁺, 2 mm) blocked synaptic bursts but not periodic depolarizations.D, Similarly, the low Ca²⁺/high Mg²⁺ solution blocked synaptic bursts but not periodic depolarizations.

Fig. 7.

Effect of acidification on motoneuron activity patterns. NH₄Cl was transiently applied to modify the intracellular pH of motoneurons. A shows a current-clamp recording in which a is the preapplication control,b shows the application of NH₄Cl,c shows the block of activity caused by the early wash-out, and d is the late wash-out and return to control values. B shows excerpts from Aon an expanded time scale. C, Graph showing the results for an average of six cells.