Endogenous patterns of activity are required for the maturation of a motor network

Abstract

Many parts of the nervous system become active before development is complete, including the embryonic spinal cord. Remarkably, although the subject has been debated for over a century (Harrison, 1904), it is still unclear whether such activity is required for normal development of motor circuitry. In Drosophila, embryonic motor output is initially poorly organized, and coordinated crawling-like behavior gradually emerges over the subsequent phase of development. We show that reversibly blocking synaptic transmission during this phase severely delays the first appearance of coordinated movements. When we interfere with the pattern of neuronal firing during this period, coordination is also delayed or blocked. We conclude that there is a period during which endogenous patterns of neuronal activity are required for the normal development of motor circuits in Drosophila.

Figure 1.

Embryonic motor output. A, Ventral view of embryo carrying GFP traps expressed at muscle Z-lines. Muscles (VL, ventral longitudinal muscles; VO, ventral oblique muscles) can be individually identified (Bate, 1993) in abdominal segments A1–A7. Muscle contractions are recorded by monitoring Z-line displacement. B, Hatched larvae move by peristaltic waves of contraction, spreading from posterior to anterior. C, The developmental sequence of motor output in the embryo. Before 75 min BTF, muscle contractions are myogenic, spreading along the right or left side of the embryo (Ci). Neurally driven bursts of uncoordinated contractions begin 75 min BTF (Cii). Subsequent bursts contain partial waves of contraction (Ciii, arrowhead). Fifteen minutes BTF, the first complete sequences of posterior-to-anterior contraction occur (Civ, arrowheads).

Figure 2.

Blocking synaptic transmission delays the onset of coordinated movements. Top panels indicate time of heating to 30°C for each experimental group. A, Time to first coordinated sequence in embryos expressing shi^ts1 in all neurons (elav-GAL4;UAS-shi^ts1; n = 5 in each group) after return to permissive temperature. B, Time of first coordinated sequence of contractions in embryos expressing shi^ts1 in all neurons (n = 5 in each group) compared with controls (n = 5 for UAS-shi^ts1 alone; n = 8 for w;G203;ZCL2144). C, Time of first coordinated sequence in embryos expressing shi^ts1 in motor neurons (n = 5). Data are shown as box-and-whisker plots; whiskers, smallest and largest observation; box, 1st and 3rd quartile; middle bar, median; circles, outliers.

Figure 3.

Sensory input influences the onset of coordination. Time of first coordinated sequence is shown. Two control groups were imaged: (i) embryos carrying UAS-ChR2 but no GAL4 driver, exposed to 488 nm light at 1 Hz, (ii) PO163-GAL4;UAS-ChR2 embryos exposed to longer wavelengths of light of comparable intensity. iii, Experimental embryos with stimulated (488 nm, 1 Hz) ChR2 expressed in sensory neurons (n = 7). Insert shows wavelength of light used in each experiment (black, 488 nm; gray, longer wavelength light).

Figure 4.

Stimulation during the sensitive period delays or prevents the onset of coordinated movements. A, Two control groups were imaged: (i) UAS-ChR2, no GAL4 driver (n = 6), (ii) elav-GAL4;UAS-ChR2 long wavelength light. Stimulation before (iii) the sensitive period has no effect. iv, Two of 8 embryos stimulated during the sensitive period showed no coordinated sequences and did not hatch. The remaining 6 embryos displayed severely delayed onset of coordinated muscle contraction. v, Embryos execute coordinated sequences throughout stimulation after the sensitive period (TF-60 min ATF). B, UAS-ChR2 and UAS-shi^ts1 in all neurons. Embryos were (i) stimulated with 488 nm light, (ii) held at restrictive temperature, or (iii) received 488 nm stimulus at restrictive temperature 90–30 min BTF (n = 7).

Figure 5.

Timeline of motor system development in Drosophila. Summary diagram showing sequence of electrical (Baines and Bate, 1998), morphological (Tripodi et al., 2008), and behavioral development (Crisp et al., 2008). Times are relative to tracheal filling (TF = 0). NT, neurotransmitter. Vertical bars indicate episodic activity in the motor system as revealed by muscle contractions.