Li WC and Soffe SR (2019), Stimulation of Single, Possible CHX10 Hindbrain...

XB-ART-55783

Front Cell Neurosci 2019 Jan 01;13:47. doi: 10.3389/fncel.2019.00047.

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Stimulation of Single, Possible CHX10 Hindbrain Neurons Turns Swimming On and Off in Young Xenopus Tadpoles.

Li WC , Soffe SR .

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Vertebrate central pattern generators (CPGs) controlling locomotion contain neurons which provide the excitation that drives and maintains network rhythms. In a simple vertebrate, the developing Xenopus tadpole, we study the role of excitatory descending neurons with ipsilateral projecting axons (descending interneurons, dINs) in the control of swimming rhythms. In tadpoles with both intact central nervous system (CNS) and transections in the hindbrain, exciting some individual dINs in the caudal hindbrain region could start swimming repeatedly. Analyses indicated the recruitment of additional dINs immediately after such evoked dIN spiking and prior to swimming. Excitation of dINs can therefore be sufficient for the initiation of swimming. These "powerful" dINs all possessed both ascending and descending axons. However, their axon projection lengths were not different from those of other excitatory dINs at similar locations. The dorsoventral position of dINs, as a population, significantly better matched that of cells marked by immunocytochemistry for the transcription factor CHX10 than other known neuron types in the ventral hindbrain and spinal cord. The comparison suggests that the excitatory interneurons including dINs are CHX10-positive, in agreement with CHX10 as a marker for excitatory neurons with ipsilateral projections in the spinal cord and brainstem of other vertebrates. Overall, our results further demonstrate the key importance of dINs in driving tadpole swimming rhythms.

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Species referenced: Xenopus
Genes referenced: shox2 vsx2
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	Figure 1. Fictive swimming in a stage 37/38 tadpole and the neural circuit controlling swimming. (A) A side-view scale diagram of a developing tadpole at stage 37/38 with a top view of its central nervous system (CNS; gray) showing: m swimming myotomes; mb, midbrain; hb, hindbrain; sc, spinal cord; oc, otic capsule. Obex is where the hindbrain ventricle closes and the landmark separating hb and sc. (B) Diagram showing most critical neuron populations and their synaptic connections in tadpole swimming central pattern generator (CPG). Each circle represents a neuron population. dIN, descending interneuron; cIN, commissural interneuron; MN, motoneuron. l.m.n. and r.m.n.: left and right motor nerve. (C) Typical activity of a right side dIN (r.dIN) and simultaneous left motor nerve (l.m.n) activity at the beginning of an episode of fictive swimming started by electrical stimulation to the head skin (arrow). *Indicates cIN inhibition.
	Figure 2. Stimulating single dINs in tadpoles with hindbrain transections starts fictive swimming. (A1) Step current injection into a right side dIN (r.dIN1) initiates swimming-like rhythms in a tadpole with the left side spinal cord and caudal hindbrain removed (black trace shows a failed trial). (A2) Nine superimposed trials as in (A1) but on a faster time scale and with different injected current levels. (B1) Injecting depolarizing currents into a dIN in a tadpole with its right side hindbrain transected starts swimming. (B2) Seven successful trials are overlapped on a faster time scale. (C1) The rebound spiking of a right side dIN (r.dIN1) following the withdrawal of a hyperpolarizing current injection, which has stopped swimming for 1 s, re-starts swimming in a dual whole-cell recording. Dotted line indicates the resting membrane potential of l.dIN. (C2) Eight superimposed trials. Traces are lined up to the rising phase of the first dIN spike in (B2,C2). Diagrams on the left show CNS as in Figure 1A with location of lesions and electrodes (same color-coded as the recording traces). Arrowheads indicate secondary EPSPs and spiking following the initial dIN spiking. Inset shows sequence of activity in the CPG (arrows) with resistor sign representing electrical coupling among ipsilateral dINs.
	Figure 3. The features of swimming evoked by powerful dINs. (A) The beginning of a swimming episode started by tail skin stimulation (arrow). m.n. trace is rectified and threshold set to trigger burst events. Box area is stretched in the inset to show burst duration (t1), swimming frequency (1/t2) and duty cycle (t1/t2). (B) Swimming burst durations, frequencies and duty cycles for the first 50 cycles are lower in the episodes evoked by powerful dINs (red) than those started by skin stimulation (black). (C) The beginning of two swimming episodes started by a powerful dIN (traces before the initial spiking is off scale). Numbers in red and black mark dIN spikes and m.n. bursts used for calculating spiking/cycle periods, respectively. Both dIN and m.n. are recorded from the left side (diagram, l.dIN, l.m.n.). Arrowhead points at secondary dIN spiking. (D) The first, but not the second dIN spiking period is longer than swimming cycle period measured using m.n. bursts. Gray lines link measurements from the same recordings in (B,D). *Indicates p < 0.05.
	Figure 4. Single spiking in a right side dIN (r.dIN1) in a tadpole with intact CNS reliably evokes fictive swimming (right inset drawing with arrows showing activity sequence). (A) In nine trials (two 10 ms and seven 1,000 ms current pulses), r.dIN1 spiking initiates swimming. (B) The distribution of r.dIN1 spike and EPSP time relative to the first spike after the first l.m.n. bursts (time 0, 94 trials for the histogram). The timing of three events (see the trace below for examples) are measured: initial r.dIN1 spike evoked by current injections (“a,” unfilled histogram); onset of the first EPSP (“b,” arrowhead, gray); 2nd r.dIN1 spike after m.n. bursts (“c,” filled). (C) Comparison between the time of the three events in (B). ***Indicates p < 0.001.
	Figure 5. The location of powerful dIN somata and longitudinal axonal projections. (A) A diagram of the tadpole hindbrain and rostral spinal cord outline (dashed) showing the location of seven somata of the 10 powerful dINs in Figures 2, 3, 4 (red dots) relative to the mid/hindbrain border (0). The red lines above the sketch show the trajectories of their ascending and descending axons (dotted line indicates a broken axon). Arrowhead points at the obex. (B) Drawing of the axon branches and their trajectories in the CNS for dIN B marked in (A; also recording in Figure 2B). (C) Drawing of the axons and their trajectories for dIN C in (A; also recording in Figure 4). The area within the dashed box is photographed (inset). (D) Axon lengths of the seven powerful dINs are similar to those of other 12 dINs within the same region.
	Figure 6. Comparing the location of CHX10 immuno-positive cells and neurons identified by their anatomy and physiology. (A) Photograph of CHX10 immuno-positive cells in the right side CNS of a stage 37/38 tadpole (image flipped). The hindbrain and rostral spinal cord is outlined by the white dashed line. Arrowhead marks obex. (B) Comparing the dorsoventral soma positions of identified neuron groups [red: dINs, dINrs; blue: ascending interneurons (aINs), cINs; green: MNs, number of neurons given next to names] to the CHX10 immuno-positive cells (brown). The bottom of the hindbrain/spinal cord is set as 0 μm in the dorsoventral dimension and the mid/hindbrain border is set as 0 μm in the longitudinal dimension. Indicates significance at p < 0.05 and * at p < 0.01.

References [+] :

Borodinsky, Activity-dependent homeostatic specification of transmitter expression in embryonic neurons. 2004, Pubmed, Xenbase