Optogenetic activation of excitatory premotor interneurons is sufficient to generate coordinated locomotor activity in larval zebrafish - PubMed (original) (raw)
Optogenetic activation of excitatory premotor interneurons is sufficient to generate coordinated locomotor activity in larval zebrafish
Emma Eklöf Ljunggren et al. J Neurosci. 2014.
Abstract
Neural networks in the spinal cord can generate locomotion in the absence of rhythmic input from higher brain structures or sensory feedback because they contain an intrinsic source of excitation. However, the molecular identity of the spinal interneurons underlying the excitatory drive within the locomotor circuit has remained unclear. Using optogenetics, we show that activation of a molecularly defined class of ipsilateral premotor interneurons elicits locomotion. These interneurons represent the excitatory module of the locomotor networks and are sufficient to produce a coordinated swimming pattern in zebrafish. They correspond to the V2a interneuron class and express the transcription factor Chx10. They produce sufficient excitatory drive within the spinal networks to generate coordinated locomotor activity. Therefore, our results define the V2a interneurons as the excitatory module within the spinal locomotor networks that is sufficient to initiate and maintain locomotor activity.
Figures
Figure 1.
Specific expression of Gal4 in V2a interneurons. A, Interneurons expressing Gal4 display the characteristic morphological features of V2a interneurons. B, Expression of Gal4 is not detected in motoneurons. C, Interneurons expressing Gal4 also express the transcription factor Chx10. Confocal reconstruction of a part of the spinal cord in the mid-body region of a double-transgenic larvae zebrafish, Gal4s1011t;UAS:Kaede, in which immunohistochemistry with a Chx10 antibody was performed. D, Enlarged picture of the white box in C showing two V2a interneurons expressing Kaede. E, Same area as in D showing neurons immunoreactive against Chx10. F, Overlay of D and E showing that V2a interneurons were double labeled for Kaede and Chx10.
Figure 2.
Optogenetic activation of interconnected V2a interneurons elicits rhythmic burst activity. A, Optical stimulation of a V2a interneuron expressing ChR2 induced a membrane potential depolarization that could reach the threshold for firing action potentials. B, Optogenetic activation of V2a interneurons induced a ChR2-mediated membrane potential depolarization followed by a delayed and slower excitation in current-clamp conditions. C, In voltage-clamp conditions, the same stimulation induced a ChR2-mediated inward current followed by a synaptic inward excitatory current. D, In voltage-clamp conditions, long-lasting optical stimulation induced a tonic inward current associated with episodes of synaptic currents in a V2a interneuron. E, The same interneuron recorded in current-clamp showed an episode of rhythmic synaptic oscillations superimposed on a tonic depolarization. F, Blocking ionotropic glutamate receptors abolished the synaptic oscillations, leaving only the tonic ChR2-mediated depolarization. G, A short optical stimulation induced a short bursting activity in the recorded V2a interneuron. H, Increasing the duration of the stimulation resulted in a long bout of bursting activity that outlasted the stimulus. I, Rhythmic bursting was abolished after blocking ionotropic receptors and only the direct ChR2-induced depolarization persisted.
Figure 3.
Optogenetic activation of V2a interneurons generates coordinated locomotor activity. A, Experimental setup with a spinalized larval zebrafish in which motor activity is monitored by recording motor nerves on the left (R-nerve-l) and right (R-nerve-r) side of the same rostral segment and on the left side of a caudal segment (C-nerve-l). B, Optical stimulation induced a bout of locomotor activity with the characteristic left–right alternation and rostrocaudal delay. C, Auto- and cross-correlation analysis showing the coordinated pattern of the swimming activity induced by V2a interneuron activation. D, E, Swimming activity induced by optical stimulation of V2a interneurons was not affected by blocking GABAA receptors with gabazine. F, Gabazine did not affect the frequency of the swimming activity. G, Bout duration of the swimming activity was not affected by gabazine.
Figure 4.
Optogenetic activation produces repetitive bursting in the absence of inhibition. A, B, Recording of motor nerves on the left (Nerve-l) and right (Nerve-r) side of the same segment. Optical stimulation of V2a interneurons elicited a swimming bout in a spinalized animal displaying left–right alternation. C–F, Left–right alternation was abolished after blocking glycinergic commissural synaptic transmission with strychnine. E, F, Long-lasting optical stimulation of V2a interneurons produced rhythmic burst activity that occurred synchronously in left and right motor nerves.
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