The Zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function

Abstract

Upon touch, twitch once zebrafish respond with one or two swimming strokes instead of typical full-blown escapes. This use-dependent fatigue is shown to be a consequence of a mutation in the tetratricopeptide domain of muscle rapsyn, inhibiting formation of subsynaptic acetylcholine receptor clusters. Physiological analysis indicates that reduced synaptic strength, attributable to loss of receptors, is augmented by a potent postsynaptic depression not seen at normal neuromuscular junctions. The synergism between these two physiological processes is causal to the use-dependent muscle fatigue. These findings offer insights into the physiological basis of human myasthenic syndrome and reveal the first demonstration of a role for rapsyn in regulating synaptic function.

Fig. 1.

Comparisons of wild-type and two^−/− fish escape responses. Five-day-old wild-type fish (top) andtwo^−/− mutant fish (bottom) were each mechanically prodded to elicit escape responses. The swimming response by each fish was recorded at the indicated times (in milliseconds) after the stimulus. Superimposed images of selected frames are indicated to theright.

Fig. 2.

Spontaneous and evoked synaptic currents in wild-type and two^−/− muscle. The properties of EPCs and mEPCs are compared for wild-type (left) and two^−/−(right) fish. Top, Representative EPCs and mEPCs. The arrow indicates the position of the EPC after the stimulus artifact. Calibration: EPC, 2 msec, 5 nA; mEPC, 100 msec, 100 pA. Middle, Frequency histograms of mEPC amplitude for wild-type and two^−/−fish. Bottom, Scatter plot showing the largest EPC amplitude from each recording in 12 wild-type and 13two^−/− different fish. The overall average ± SD for wild-type andtwo^−/− fish are indicated by thediamonds and bars.

Fig. 3.

Distribution of ACh receptors in the myotomal muscle of wild-type and two^−/−fish. Top, A differential interference contrast image of a section of the tail musculature is shown for orientation of the fluorescence images. This image spans approximately four chevron-shaped segments or myotomes and is are similar to the image area of the accompanying fluorescence photos. Layers of individual muscle cells can be resolved between the myocomata that separate tail segments. Confocal fluorescence image showing the distribution of rhodamine-conjugated α-bungarotoxin labeling in the tail of wild-type (middle) and two^−/−mutant (bottom) fish. Scale bar, 50 μm.

Fig. 4.

Stable expression of a murine rapsyn-GFP fusion protein rescues two^−/−.A, The escape response of atwo^−/− fish expressing murine rapsyn-GFP is recorded at day 5. Sequential images taken at 20 msec intervals are shown after the start of movement. B, Confocal fluorescence images showing the redfluorescence associated with AChRs (left) and thegreen fluorescence associated with rapsyn-GFP (middle) in the fish shown in A. The merged images (right) reveal the striking colocalization of the receptor–rapsyn distribution in thistwo^−/−/rapsyn-GFP fish.

Fig. 5.

Identification of the mutation in thetwitch once rapsyn gene. A, The rapsyn protein motifs are shown, as well as the location of twitch once (two^th26e) mutation in the TPR domain. The 34 residue sequence of the fourth TPR domain is shown. In twitch once rapsyn, the eighth residue in the fourth TPR domain is mutated from glycine to glutamate.B, Raw chromatogram of sequences used to genotype wild-type fish and two^−/−fish.

Fig. 6.

Transient expression of wild-type rapsyn intwo^−/− fish. 1–16 cell stage fish embryos were microinjected with DNA constructs encoding wild-type rapsyn-GFP. Fish embryos were examined by confocal microscopy on day 5 for distribution of rapsyn and receptors. Confocal fluorescence images of red rhodamine-conjugated α-bungarotoxin (A), green rapsyn-GFP (B), and merged images (C). Note the presence of punctate clusters of both receptor and rapsyn that appear to colocalize in the merged image. Scale bar, 50 μm.

Fig. 7.

Transient expression of G130E rapsyn intwo^−/− fish. 1–16 cell stage fish embryos were microinjected with DNA constructs encoding G130E rapsyn-GFP. Fish embryos were examined by confocal microscopy on day 5 for distribution of rapsyn and receptors. A, Low-power confocal fluorescence images of redrhodamine-conjugated α-bungarotoxin (left),green rapsyn-GFP (middle), and merged images (right). Note the absence of localized rapsyn and receptor in the low-power images. Intermediate (B) and higher (C) -magnification images show small clusters of greenrapsyn, with no associated clusters of red receptor, along the edge of muscle. The presence of green clusters on a diffuse red background leads toyellow clusters on the merge. The coincidentgreen and red fluorescence within the muscle represents an optical slice through invaginating T-tubular membrane. Scale bar: A, 50 μm; B, 10 μm; C, 5 μm.

Fig. 8.

Endplate synaptic currents intwo^−/− muscle exhibit marked depression in response to 50 Hz stimulation. A, Ten consecutive endplate currents (top traces) recorded in response to 50 Hz pulse train applied to the spinal cord. The timing of the stimuli relative to the whole-cell muscle recordings is shown in the bottom traces. The spinal cord was stimulated extracellularly using 300 μsec, 30 V pulses. The muscle cells were held at −50 mV to minimize contamination by sodium current. Note the depression of endplate current amplitude in twitch oncemuscle compared with wild type. Calibration: 20 msec, 100 (wild type) or 50 (twitch once) pA. B, The average ± SE ratio of each successive EPC to the amplitude of the first EPC recorded. The EPC ratios for wild-type fish are represented by filled circles (n = 80 from 8 cells), and two^−/− fish are represented by open circles (n = 70 from 7 cells). Each average is based on 10 consecutive traces in a single muscle cell. *p < 0.01; Student'st test. C, The scatter plot relating maximum EPC amplitude recorded from eachtwo^−/− fish versus the amount of depression observed. The depression is indicated by the ratio of the averaged 10th EPC amplitude to the averaged first EPC recorded in the train. Each average is based on 10 consecutive traces in a single muscle cell.