Limits...
Optical stimulation of zebrafish hair cells expressing channelrhodopsin-2.

Monesson-Olson BD, Browning-Kamins J, Aziz-Bose R, Kreines F, Trapani JG - PLoS ONE (2014)

Bottom Line: These in vivo results support a physiological role for the MET channel in the high fidelity of first spike latency seen during encoding of mechanical sensory stimuli.Finally, we examined whether remote activation of hair cells via ChR2 activation was sufficient to elicit escape responses in free-swimming larvae.Altogether, the myo6b:ChR2 transgenic line provides a platform to investigate hair-cell function and sensory encoding, hair-cell sensory input to the Mauthner cell, and the ability to remotely evoke behavior in free-swimming zebrafish.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Amherst College, Amherst, Massachusetts, United States of America.

ABSTRACT
Vertebrate hair cells are responsible for the high fidelity encoding of mechanical stimuli into trains of action potentials (spikes) in afferent neurons. Here, we generated a transgenic zebrafish line expressing Channelrhodopsin-2 (ChR2) under the control of the hair-cell specific myo6b promoter, in order to examine the role of the mechanoelectrical transduction (MET) channel in sensory encoding in afferent neurons. We performed in vivo recordings from afferent neurons of the zebrafish lateral line while activating hair cells with either mechanical stimuli from a waterjet or optical stimuli from flashes of ∼470-nm light. Comparison of the patterns of encoded spikes during 100-ms stimuli revealed no difference in mean first spike latency between the two modes of activation. However, there was a significant increase in the variability of first spike latency during optical stimulation as well as an increase in the mean number of spikes per stimulus. Next, we compared encoding of spikes during hair-cell stimulation at 10, 20, and 40-Hz. Consistent with the increased variability of first spike latency, we saw a significant decrease in the vector strength of phase-locked spiking during optical stimulation. These in vivo results support a physiological role for the MET channel in the high fidelity of first spike latency seen during encoding of mechanical sensory stimuli. Finally, we examined whether remote activation of hair cells via ChR2 activation was sufficient to elicit escape responses in free-swimming larvae. In transgenic larvae, 100-ms flashes of ∼470-nm light resulted in escape responses that occurred concomitantly with field recordings indicating Mauthner cell activity. Altogether, the myo6b:ChR2 transgenic line provides a platform to investigate hair-cell function and sensory encoding, hair-cell sensory input to the Mauthner cell, and the ability to remotely evoke behavior in free-swimming zebrafish.

Show MeSH

Related in: MedlinePlus

Escape responses from touch and optical stimulation of wild type and myo6b:ChR2 transgenic larvae.(A) Diagram depicting the Mauthner cells (M), a pair of neurons in the hindbrain of teleost fish. The axons of the M-cells project into the spinal cord where they synapse on primary motor neurons and elements of the central pattern generator responsible for left-right tail motions. (B, C) Diagrams of the setup for field recordings of M-cell potentials from larval zebrafish. (B) A waterjet was used to stimulate touch receptors on the head of a larva embedded in low melt agarose. (C) For optical stimuli, field potentials were collected from free-swimming transgenic larvae. (D) In both wild type and myo6b:ChR2 transgenic larvae, the M-cell was activated in response to a 100-ms touch stimulus (onset at arrowhead). (E) In wild-type larvae, the M-cell was not activated by flashes of ∼470-nm light (n = 18). Transgenic myo6b:ChR2 larva responded to ∼470-nm light with both a field potential (n = 55; scale bar 2 ms for D and E) and an escape response (not shown). (F) Increasing the duration of optical flashes increased the percentage of observed field potentials (seen in E) and escape responses in transgenic larvae (n = 12). Note that flashes that were 100-ms or greater resulted in 100% success rate for observed escape responses and field potentials.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4008597&req=5

pone-0096641-g004: Escape responses from touch and optical stimulation of wild type and myo6b:ChR2 transgenic larvae.(A) Diagram depicting the Mauthner cells (M), a pair of neurons in the hindbrain of teleost fish. The axons of the M-cells project into the spinal cord where they synapse on primary motor neurons and elements of the central pattern generator responsible for left-right tail motions. (B, C) Diagrams of the setup for field recordings of M-cell potentials from larval zebrafish. (B) A waterjet was used to stimulate touch receptors on the head of a larva embedded in low melt agarose. (C) For optical stimuli, field potentials were collected from free-swimming transgenic larvae. (D) In both wild type and myo6b:ChR2 transgenic larvae, the M-cell was activated in response to a 100-ms touch stimulus (onset at arrowhead). (E) In wild-type larvae, the M-cell was not activated by flashes of ∼470-nm light (n = 18). Transgenic myo6b:ChR2 larva responded to ∼470-nm light with both a field potential (n = 55; scale bar 2 ms for D and E) and an escape response (not shown). (F) Increasing the duration of optical flashes increased the percentage of observed field potentials (seen in E) and escape responses in transgenic larvae (n = 12). Note that flashes that were 100-ms or greater resulted in 100% success rate for observed escape responses and field potentials.

Mentions: Hair-cell sensory information is vital to the startle and escape responses in vertebrates. We determined whether remote activation of hair cells with optical stimuli could evoke an escape response in transgenic larvae. Delivery of both touch stimuli with a waterjet and optical stimuli with flashes of ∼470-nm light evoked similar escape responses. In addition, we recorded field potentials in order to determine whether a pattern generated by M-cell activity was similar during escape responses from the two modes of stimulation (Fig. 4B–C). Waveforms from field potential recordings indicated initial M-cell responses, as well as activity from other hindbrain neurons and subsequent contraction of axial muscles [29], [30]. Both wild type and myo6b:ChR2 transgenic larvae displayed similar field potentials in response to touch stimuli delivered via a waterjet directed at the head (Fig. 4D). However, wild-type larvae did not respond to a 100-ms flash of ∼470-nm light (Fig. 4E; Video S1; n = 18), while myo6b:ChR2 larvae displayed a robust escape response along with concomitant field potentials (Fig. 4E; Video S2; n = 55). If hair-cell inputs were bringing the M-cell membrane potential to threshold, we predicted that shortening the duration of the optical flash would decrease spiking of afferent neurons and thus lower the probability of an M-cell action potential. Consistent with our prediction, we found that by shortening optical stimulus duration, we reduced the frequency of observed escape responses and coincident field potentials (Fig. 4F).


Optical stimulation of zebrafish hair cells expressing channelrhodopsin-2.

Monesson-Olson BD, Browning-Kamins J, Aziz-Bose R, Kreines F, Trapani JG - PLoS ONE (2014)

Escape responses from touch and optical stimulation of wild type and myo6b:ChR2 transgenic larvae.(A) Diagram depicting the Mauthner cells (M), a pair of neurons in the hindbrain of teleost fish. The axons of the M-cells project into the spinal cord where they synapse on primary motor neurons and elements of the central pattern generator responsible for left-right tail motions. (B, C) Diagrams of the setup for field recordings of M-cell potentials from larval zebrafish. (B) A waterjet was used to stimulate touch receptors on the head of a larva embedded in low melt agarose. (C) For optical stimuli, field potentials were collected from free-swimming transgenic larvae. (D) In both wild type and myo6b:ChR2 transgenic larvae, the M-cell was activated in response to a 100-ms touch stimulus (onset at arrowhead). (E) In wild-type larvae, the M-cell was not activated by flashes of ∼470-nm light (n = 18). Transgenic myo6b:ChR2 larva responded to ∼470-nm light with both a field potential (n = 55; scale bar 2 ms for D and E) and an escape response (not shown). (F) Increasing the duration of optical flashes increased the percentage of observed field potentials (seen in E) and escape responses in transgenic larvae (n = 12). Note that flashes that were 100-ms or greater resulted in 100% success rate for observed escape responses and field potentials.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008597&req=5

pone-0096641-g004: Escape responses from touch and optical stimulation of wild type and myo6b:ChR2 transgenic larvae.(A) Diagram depicting the Mauthner cells (M), a pair of neurons in the hindbrain of teleost fish. The axons of the M-cells project into the spinal cord where they synapse on primary motor neurons and elements of the central pattern generator responsible for left-right tail motions. (B, C) Diagrams of the setup for field recordings of M-cell potentials from larval zebrafish. (B) A waterjet was used to stimulate touch receptors on the head of a larva embedded in low melt agarose. (C) For optical stimuli, field potentials were collected from free-swimming transgenic larvae. (D) In both wild type and myo6b:ChR2 transgenic larvae, the M-cell was activated in response to a 100-ms touch stimulus (onset at arrowhead). (E) In wild-type larvae, the M-cell was not activated by flashes of ∼470-nm light (n = 18). Transgenic myo6b:ChR2 larva responded to ∼470-nm light with both a field potential (n = 55; scale bar 2 ms for D and E) and an escape response (not shown). (F) Increasing the duration of optical flashes increased the percentage of observed field potentials (seen in E) and escape responses in transgenic larvae (n = 12). Note that flashes that were 100-ms or greater resulted in 100% success rate for observed escape responses and field potentials.
Mentions: Hair-cell sensory information is vital to the startle and escape responses in vertebrates. We determined whether remote activation of hair cells with optical stimuli could evoke an escape response in transgenic larvae. Delivery of both touch stimuli with a waterjet and optical stimuli with flashes of ∼470-nm light evoked similar escape responses. In addition, we recorded field potentials in order to determine whether a pattern generated by M-cell activity was similar during escape responses from the two modes of stimulation (Fig. 4B–C). Waveforms from field potential recordings indicated initial M-cell responses, as well as activity from other hindbrain neurons and subsequent contraction of axial muscles [29], [30]. Both wild type and myo6b:ChR2 transgenic larvae displayed similar field potentials in response to touch stimuli delivered via a waterjet directed at the head (Fig. 4D). However, wild-type larvae did not respond to a 100-ms flash of ∼470-nm light (Fig. 4E; Video S1; n = 18), while myo6b:ChR2 larvae displayed a robust escape response along with concomitant field potentials (Fig. 4E; Video S2; n = 55). If hair-cell inputs were bringing the M-cell membrane potential to threshold, we predicted that shortening the duration of the optical flash would decrease spiking of afferent neurons and thus lower the probability of an M-cell action potential. Consistent with our prediction, we found that by shortening optical stimulus duration, we reduced the frequency of observed escape responses and coincident field potentials (Fig. 4F).

Bottom Line: These in vivo results support a physiological role for the MET channel in the high fidelity of first spike latency seen during encoding of mechanical sensory stimuli.Finally, we examined whether remote activation of hair cells via ChR2 activation was sufficient to elicit escape responses in free-swimming larvae.Altogether, the myo6b:ChR2 transgenic line provides a platform to investigate hair-cell function and sensory encoding, hair-cell sensory input to the Mauthner cell, and the ability to remotely evoke behavior in free-swimming zebrafish.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Amherst College, Amherst, Massachusetts, United States of America.

ABSTRACT
Vertebrate hair cells are responsible for the high fidelity encoding of mechanical stimuli into trains of action potentials (spikes) in afferent neurons. Here, we generated a transgenic zebrafish line expressing Channelrhodopsin-2 (ChR2) under the control of the hair-cell specific myo6b promoter, in order to examine the role of the mechanoelectrical transduction (MET) channel in sensory encoding in afferent neurons. We performed in vivo recordings from afferent neurons of the zebrafish lateral line while activating hair cells with either mechanical stimuli from a waterjet or optical stimuli from flashes of ∼470-nm light. Comparison of the patterns of encoded spikes during 100-ms stimuli revealed no difference in mean first spike latency between the two modes of activation. However, there was a significant increase in the variability of first spike latency during optical stimulation as well as an increase in the mean number of spikes per stimulus. Next, we compared encoding of spikes during hair-cell stimulation at 10, 20, and 40-Hz. Consistent with the increased variability of first spike latency, we saw a significant decrease in the vector strength of phase-locked spiking during optical stimulation. These in vivo results support a physiological role for the MET channel in the high fidelity of first spike latency seen during encoding of mechanical sensory stimuli. Finally, we examined whether remote activation of hair cells via ChR2 activation was sufficient to elicit escape responses in free-swimming larvae. In transgenic larvae, 100-ms flashes of ∼470-nm light resulted in escape responses that occurred concomitantly with field recordings indicating Mauthner cell activity. Altogether, the myo6b:ChR2 transgenic line provides a platform to investigate hair-cell function and sensory encoding, hair-cell sensory input to the Mauthner cell, and the ability to remotely evoke behavior in free-swimming zebrafish.

Show MeSH
Related in: MedlinePlus