Speed and segmentation control mechanisms characterized in rhythmically-active circuits created from spinal neurons produced from genetically-tagged embryonic stem cells
- Howard Hughes Medical Institute, Salk Institute for Biological Studies, United States
- University of California, San Diego, United States
- University of Illinois at Chicago, United States
- Salk Institute for Biological Studies, United States
Figures
Figure 1 with 3 supplements
Spontaneous activity emerges from networks created from ES cell-derived spinal neurons.
(A) Mouse ES cell lines were derived from embryos with genetic tags for defined spinal neuron subclasses. The number of individual ES cell lines generated for each genotype is shown in parentheses. …
Figure 1—figure supplement 1
Neurosphere differentiation and composition.
(A and B) 10 days-post ES cell differentiation neurospheres were dissociated and cell composition was quantified using FACS. Plots of V3, V2a, and V1 interneurons and motor neurons from Sim1:Cre;R26/…
Figure 1—figure supplement 2
ES-cell-derived neurons express neuronal subtype markers.
(A–B) Motor neurons and V2a interneurons were differentiated from Hb9:GFP and Chx10:Cre;R26/C:LSL:Tomato ES cell lines, respectively. FACS-purified cells were gene-expression profiled using next …
Figure 1—figure supplement 3
Circuitoid activity and synaptic structures.
(A) Plated ES-cell-derived V3 interneurons labeled with calcium indicator dye (OGB-1, green) and gradient contrast transmitted light (Dodt). Cells simultaneously recorded with electrode and calcium …
Figure 2
Physiological properties of circuitoids.
Circuitoids were generated with 1000 nm SAG (CirH) for analysis using calcium imaging with a ubiquitously expressed GCaMP3 ES cell line. (A) Spontaneous bursting frequency increased from week 2 to 5.…
Figure 3
Cellular composition influences circuitoid rhythmicity and burst speed.
(A–F) Circuitoids generated with 1000 nM SAG (CirH) were recorded in drugs that evoke CPG activity (evoked, NMA +5 HT, green) 16–17 days post ES cell differentiation. Activity was classified as …
Figure 4 with 3 supplements
Rhythmic activity in networks with purified neuron subclasses.
(A) Circuitoids of defined cellular composition were created by re-aggregating FACS purified ES cell-derived neurons (red) with astrocytes (blue) following the outlined scheme. (B–Q) Bursting …
Figure 4—figure supplement 1
Cell purifications to generate synthetic neural networks.
ES cells were differentiated, dissociated, and neuronal subtypes were FACS purified to evaluate differentiation efficiency and purity. (A) Wild-type non-fluorescent cell line, negative control. (B) …
Figure 4—figure supplement 2
Frequency and rhythmicity of V3 interneuron networks.
(A) Sim1:Cre;R26/C:LSL:Tomato ES cell lines were differentiated and cell sorted. 50,000 V3 interneurons were aggregated and calcium imaged at weekly intervals. Spontaneous and evoked bursting …
Figure 4—figure supplement 3
Cholinergic antagonists do not affect motor neuron networks.
(A) Hb9:GFP ES cell lines were differentiated and purified with FACS to create pure motor neuron networks. Three weeks after reaggregation spontaneous and evoked bursts were detected with calcium …
Figure 5 with 1 supplement
V3 network burst speed is tuned by inhibitory V1 interneurons.
(A) ES cell lines with fluorescent reporters for neuronal subtypes were differentiated, and tomato +V3 and V1 interneurons were purified by FACS. Monolayer circuitoids were created by plating …
Figure 5—figure supplement 1
Generation of synthetic networks comprised of defined neuronal subtypes.
Hb9:GFP ES cell lines were differentiated and GFP+ motor neurons purified using FACS to generate base networks of 100,000 motor neurons. In parallel, En1:Cre;R26/C:LSL:Tomato ES cell lines were …
Figure 6 with 1 supplement
V1 interneurons control the segmentation of motor neuron network activity.
Circuitoids were established with either 100,000 motor neurons or 100,000 V3 interneurons combined with 0–40,000 V1 interneurons, plated on astrocytes. Networks were imaged 14 days after sorting …
Figure 6—figure supplement 1
Activity across different network configurations.
Motor neuron networks, V3 interneuron networks, and V3:V1 mixed networks were created by differentiating and sorting 100,000 motor neurons (using Hb9:GFP ES cell lines), 100,000 V3 interneurons …
Figure 7
Motor neuron burst frequency is set by V3-V1 network activity.
(A–D) Tripartite networks of 50,000 motor neurons, 50,000 V3 interneurons, and 0–40,000 V1 interneurons were formed, and burst activity monitored using calcium dyes (see Figure 5A). (A) Spontaneous …
Figure 8
Model of neuronal interactions in an oscillatory circuit.
(A–C) V3 interneurons form strong synaptic interactions, whereas motor neurons have weaker connections. (A) Strongly connected neurons in V3 networks become rhythmically active. (B) Weakly connected …
Videos
Video 1
Mature circuitoids display spontaneous activity.
Heterogeneous circuitoids display spontaneous bursts of network activity that appear to be synchronous throughout each sphere. Here, an En1:Cre;R26/C:LSL:Tomato ES cell line was differentiated with …
Video 2
V1 interneurons generate subnetwork activity in motor neuron-V1 networks.
A network of 100,000 motor neurons and 10,000 V1 interneurons displays segmented activity. Calcium intensity change (dF/F) was pseudocolored (scale from black to white), showing different active …
Video 3
Inhibitory antagonists synchronize motor neuron-V1 networks.
The same network (see Video 2) displays synchronous network activity after the application of inhibitory antagonists (strychnine+picrotoxin), suggesting that synaptic activity from V1 inhibitory …
