Motor Behavior Selectively Inhibits Hair Cells Activated by Forward Motion in the Lateral Line of Zebrafish

Abstract

How do sensory systems disambiguate events in the external world from signals generated by the animal's own motor actions? One strategy is to use an "efference copy" of the motor command to inhibit the sensory input caused by active behavior [1]. But does inhibition of self-generated inputs also block transmission of external stimuli? We investigated this question in the lateral line, a sensory system that allows fish and amphibians to detect water currents and that contributes to behaviors such as rheotaxis [2] and predator avoidance [3, 4]. This mechanical sense begins in hair cells grouped into neuromasts dotted along the animal's body [5]. Each neuromast contains two populations of hair cells, activated by deflection in either the anterior or posterior direction [6], as well as efferent fibers that are active during motor behavior to suppress afferents projecting to the brain [7-12]. To test how far the efference copy signal modulates responses to external stimuli, we imaged neural and synaptic activity in larval zebrafish during fictive swimming. We find that efferents transmit a precise copy of the motor signal and a single spike in the motor nerve can be associated with ∼50% inhibition of glutamate release. The efference copy signal acted with high selectivity on hair cells polarized to be activated by posterior deflections, as would occur during forward motion. During swimming, therefore, "push-pull" encoding of stimulus direction by afferents of opposite polarity is disrupted while still allowing a subset of hair cells to detect stimuli originating in the external world.

Keywords: corollary discharge; efference copy; hair cell; lateral line; zebrafish.

Graphical abstract

Figure 1

The Efferent Signal Is an Almost Exact Copy of the Motor Signal during Fictive Swimming (A) At 7 dpf, the posterior lateral line of larval zebrafish consists of 14 neuromasts on each side (red dots). Each neuromast is innervated by at least two afferent neurons (yellow) and a single cholinergic efferent (blue). The hair bundles of all hair cells are confined by a gelatinous structure called cupula (red in right panel). We imaged glutamate release of individual hair cells in a neuromast while measuring motor neuron activity through a suction pipette. A second pipette applied pressure steps to the neuromast. (B and C) Average projections of the afferent synapses in the hindbrain (B) and a neuromast (C) of a larva expressing iGluSnFR under transcriptional control of the Sill promoter (Tg(Sill2, UAS:iGluSnFR)). (D) Average projection of a neuromast in a larva expressing GCaMP6f under the transcriptional control of the HuC (elavl3) promoter (Tg(HuC:GCaMP6f)), in which afferents and efferents (but not hair cells) are labeled. Red dots indicate efferent regions of interest (ROIs) identified based on their firing pattern. (E) Top trace (red): “spontaneous” calcium transients in efferent synapses observed in the absence of mechanical stimulation (from D) over a 5-min period. The lower traces (black) depict the raw motor activity and the spike rate. The asterisk indicates a signal in the efferent synapses that correlates to six spikes in the motor nerve. (F) Magnified view of the dashed area in (E), showing that efferent synapses in the neuromast are activated at each swim bout. (G) The number of spikes per swimming bout and the integral of the fluorescent signal during that episode were strongly correlated (r = 0.9; n = 155 bouts from 4 neuromasts, each depicted in a different color). See also Figures S1 and S2.

Figure 2

Spontaneous Release of Glutamate from Hair Cells Is Suppressed during Fictive Swimming (A) Experiments were carried out in “relaxed” mutants that express the glutamate reporter iGluSnFR in afferent neurons (Tg(Sill2, UAS:iGluSnFR), cacnb^ts25/ts25) at 5 dpf. The dotted line represents the plane of imaging. (B) A representative hair cell synapse outlined in red. (C) Spontaneous glutamate release from the synapse in (B) (top) and motor neuron activity measured simultaneously (bottom). Blue areas indicate bursts of fictive swimming. (D) Magnified view of boxed area in (C). The maximum suppression of glutamate release was similar for each burst of motor activity. (E) Relationship between the number of spikes in a burst and the negative integral of the iGluSnFR signal from the neuromast depicted in (B) (n = 28 swimming episodes; r = −0.95). (F) Cross-correlation of iGlusnFR signal and the spike train in the motor nerve (down sampled to match imaging frequency). The inset shows that the maximum degree of anti-correlation occurred at a delay of 50 ms, indicating that the iGluSnFR signal fell within one frame interval of a spike in the motor nerve. See also Figure S3.

Figure 3

Motor Behavior Blocks Synaptic Transmission from a Subset of Hair Cells Experiments were carried out in relaxed mutants that express the glutamate reporter iGluSnFR in afferent neurons (Tg(Sill2, UAS:iGluSnFR), cacnb^ts25/ts25) at 5 dpf. (A and B) Image of iGluSnFR expression in afferents of neuromast 1 (A). Two representative synaptic inputs are highlighted in red (activated by anterior deflection) and blue (activated by posterior deflection). The responses of these synapses to mechanical stimuli are shown in (B), together with motor nerve activity (black traces) and pressure steps applied to the neuromast. Positive pressure steps correspond to posterior deflections of the cupula and negative steps to anterior deflections. Blue shading indicates periods of motor nerve activity, and numbered boxes indicate the stimulation episodes that are magnified in (E). (C and D) A corresponding representation of hair cell activity in neuromast 2. (E) Expansion of records in boxes 1–4 in (B) and (D). The superimposed dashed red and blue traces indicate the average mechanically induced response of that synapse in the absence of motor nerve activity. Shaded areas represent the SEM. In example 2, inhibition of glutamate release is almost complete within 50 ms of the beginning of the motor burst. In example 3, suppression is complete within 50 ms, and further motor activity reduces glutamate release below resting levels. In example 4, glutamate release begins to recover within 50 ms of the end of the motor burst. See also Figure S4.

Figure 4

Motor Behavior Selectively Modulates Hair Cells Activated by Deflection in the Posterior Direction (A–C) Three examples of synapses whose response was (A) unaffected, (B) suppressed during the entire stimulation episode, and (C) suppressed only during the initial part of the stimulus (shaded areas represent the SEM). (D) The suppression index (SI), calculated on a point-by-point basis during mechanical stimulation (Equation 1). The red, blue, and green traces show synapses from three different hair cells, with the corresponding motor activity shown below. Glutamate release from the green synapse (A) was not significantly suppressed, with an SI ∼0 (negative values occur whenever the response during motor activity is larger than the average response in the absence of motor activity). Stimulated release from the red synapse (C) was nulled during motor activity (SI ∼1) but then recovered at the end of the burst of spikes. Glutamate release from the blue synapse (B) was reduced to below resting levels (SI > 1). (E) Plot of the relation between the SI at each time point during a mechanical stimulus and the number of spikes in the motor nerve in the preceding 50-ms time interval. Only synapses classified as suppressed were analyzed. Collected results from 29 synapses in 6 fish are shown. The data could be described by a Hill equation of the form SI(N_s) = (SI_max^∗N_s)/(N_s + N_1/2), where N_s is the number of spikes, SI_max is the maximum SI (1.05 ± 0.08), and N_1/2 is the number of spikes coinciding with half-maximal suppression (1.12 ± 0.42). Error bars show SEM. (F) The effects of motor activity on synaptic transmission from hair cells of opposing polarity. Column 1: the number of synapses activated by deflection in the posterior and anterior directions is shown. Measurements were made in a total of 41 synapses in 8 neuromasts in 6 fish. Column 2: the number of synapses suppressed during motor activity, classified as described in the STAR Methods, is shown. Column 3: the number of synapses unaffected by motor activity is shown. (G) Comparison of the average magnitude of the suppression index during a swimming bout in hair cells polarized for anterior and posterior deflection. We also compared the maximal SI values during a swimming bout: these were also significantly different in hair cells of opposite polarity (p < 0.001; Mann-Whitney U test). Bars show SEM.