GlyR perturbations during development impair motor network function. (A) In situ hybridization of GlyR mRNA transcripts in longitudinal optical sections of the spinal cord. GlyR α2 mRNA transcripts are detected at 1 day (1d α2) in development (arrowheads in i) but not at 2 days (2d α2; ii). GlyR α1 mRNA expression is absent at 1 day in development (1d α1; iii) and is widely expressed by 2 days (2d α1; iv). Broken lines demarcate dorsal and ventral limits of the spinal cord. (B) mAb4a-stained cryosections of embryos 24 h after fertilization reveal GlyR expression on cells of wild-type fish (WT 1d; i) and α1 AMO-injected fish (α1 k/d 1d; iii) but not α2 AMO-injected fish (α2 k/d 1d; ii). At 2 days mAb4a staining is increased in wild-type larvae (WT 2d; iv) and α2 knockdown larvae (v) but not α1 knockdown larvae (vi). mAb4a staining is observed in mismatch α2 and mismatch α1 AMO-injected embryos (vii and viii) and larvae (ix and x). Larvae coinjected with α1 and α2 AMOs have no detectable mAb4a staining (xi). (Scale bar: 20 μm.) (xii) Western blots showing mAb4a labeling in wild-type and GlyR α2 mismatch (MM) embryos (1d) and larvae (2d) and α2 knockdown (α2 k/d) larvae but not α2 knockdown embryos. (C) Twenty-four-hour embryo motoneuron receives glycinergic synaptic bursts (GB) and electrically mediated periodic discharges (PD; n = 9). (D) Twenty-five-hour GlyR α2 knockdown embryo reveals loss of glycinergic bursts (n = 5/5). (E) Motoneuron of a 27-h embryo injected with α1 AMO receives glycinergic synaptic bursts (GB) and electrically mediated periodic discharges (PD). (F) Motoneuron of a 25-h embryo injected with the GlyR α2 mismatch AMO receives glycinergic synaptic bursts (GB) and electrically mediated periodic discharges (PD). (G) Three-day motoneurons are rhythmically active. (H) Arrhythmic motoneuron activity elicited by touching in a 3-day GlyR α2 knockdown zebrafish (n = 18/29). (I) Motoneurons of 3-day fish injected with GlyR α2 mismatch AMO are rhythmically active (n = 6/6). (J) Motoneurons of 3-day fish injected with the GlyR α1 AMO are rhythmically active (n = 51/51). Voltage clamp holding potential in C–F was -60 mV. Insets in G–J depict enlarged portions of traces underlined with bars.
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