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A 'tool box' for deciphering neuronal circuits in the developing chick spinal cord.

Hadas Y, Etlin A, Falk H, Avraham O, Kobiler O, Panet A, Lev-Tov A, Klar A - Nucleic Acids Res. (2014)

Bottom Line: Here we present a circuit-deciphering 'tool box' for fast, reliable and cheap genetic targeting of neuronal circuits in the developing spinal cord of the chick.We demonstrate targeting of motoneurons and spinal interneurons, mapping of axonal trajectories and synaptic targeting in both single and populations of spinal interneurons, and viral vector-mediated labeling of pre-motoneurons.We also demonstrate fluorescent imaging of the activity pattern of defined spinal neurons during rhythmic motor behavior, and assess the role of channel rhodopsin-targeted population of interneurons in rhythmic behavior using specific photoactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Neurobiology, IMRIC, Hebrew University Medical School, Jerusalem, Israel.

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Imaging of flexor and extensor motoneurons targeted with GCaMP3 during rhythmic activity produced by electrical stimulation of the sacral segments. (A) GCaMP3 was cloned into a Cre-dependent PiggyBac vector. This plasmid was electroporated into E3 chick neural tube along with ubiquitously-expressed PBase and Cre under the control of the chick TAG1/Axonin1 that drives expression in motor neurons. The embryos were incubated for 9 days. At E12, the embryos were sacrificed and their spinal cords were isolated. (B) Confocal image of a 100-μm cross-section through the LS6 segment in E13 chick embryo spinal cord. The section is stained for GFP (green) and ChAT (blue). The GFP-expressing cell is localized (cyan) within ChAT-positive motoneuron pool at the ventrolateral aspect of the cord. (C) The isolated spinal cord preparation is illustrated mounted with ventral side up in an in vitro chamber. The most caudal part of the spinal cord is inserted into a stimulation suction electrode. Suction electrode recordings of the motor output are obtained from the sartorius and femorotibialis nerves and the activity of GCaMP3-targeted motoneurons is viewed using water immersion objective on an upright epifluorescent microscope equipped with a digital CCD. Combined calcium imaging of the activity of motoneurons targeted with GCaMP3 and the motor output produced by stimulation of the caudal segments of the spinal cord. Stimulation of the sacral part of the cord (3 pulse 10 Hz train at 50 μA) elicited an alternating flexor and extensor rhythm, recorded from the satrorius and femorotibialis nerves, respectively. The activity pattern of two different motoneurons targeted with GCaMP3 was imaged from the ventral surface of the LS1 segment (see methods) concurrently with the recorded motor output. The fluorescence changes (ΔF/F) of one of these neurons were in phase with the rhythmic bursting recorded from the sartorius nerve (imaging of flexor motoneuron), while the ΔF/F of the other motoneuron was in phase with femorotibialis bursting (imaging of extensor motoneuron).
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Figure 6: Imaging of flexor and extensor motoneurons targeted with GCaMP3 during rhythmic activity produced by electrical stimulation of the sacral segments. (A) GCaMP3 was cloned into a Cre-dependent PiggyBac vector. This plasmid was electroporated into E3 chick neural tube along with ubiquitously-expressed PBase and Cre under the control of the chick TAG1/Axonin1 that drives expression in motor neurons. The embryos were incubated for 9 days. At E12, the embryos were sacrificed and their spinal cords were isolated. (B) Confocal image of a 100-μm cross-section through the LS6 segment in E13 chick embryo spinal cord. The section is stained for GFP (green) and ChAT (blue). The GFP-expressing cell is localized (cyan) within ChAT-positive motoneuron pool at the ventrolateral aspect of the cord. (C) The isolated spinal cord preparation is illustrated mounted with ventral side up in an in vitro chamber. The most caudal part of the spinal cord is inserted into a stimulation suction electrode. Suction electrode recordings of the motor output are obtained from the sartorius and femorotibialis nerves and the activity of GCaMP3-targeted motoneurons is viewed using water immersion objective on an upright epifluorescent microscope equipped with a digital CCD. Combined calcium imaging of the activity of motoneurons targeted with GCaMP3 and the motor output produced by stimulation of the caudal segments of the spinal cord. Stimulation of the sacral part of the cord (3 pulse 10 Hz train at 50 μA) elicited an alternating flexor and extensor rhythm, recorded from the satrorius and femorotibialis nerves, respectively. The activity pattern of two different motoneurons targeted with GCaMP3 was imaged from the ventral surface of the LS1 segment (see methods) concurrently with the recorded motor output. The fluorescence changes (ΔF/F) of one of these neurons were in phase with the rhythmic bursting recorded from the sartorius nerve (imaging of flexor motoneuron), while the ΔF/F of the other motoneuron was in phase with femorotibialis bursting (imaging of extensor motoneuron).

Mentions: The calcium indicator GCaMP3 was cloned into a Cre-dependent PiggyBac vector. Targeting to motoneurons was attained using electroporation of the conditional GCaMP3 plasmid (Figure 6A) into E3 chick neural tube along with ubiquitously-expressed PBase and Cre under the control of the chick motoneuron-specific enhancer of the TAG1/Axonin1 gene (19) (Table 1, Figure 6B). Electroporated embryos were sacrificed at E12; the spinal cords were isolated and mounted in an in vitro chamber equipped for combined optical imaging of the targeted neurons and electrophysiological recording of the motor output. Fluorophores were excited at the appropriate wavelength, and images were acquired from the ventral or lateral surface of the cord using epifluorescence microscopy and a cooled 14bit CCD camera, before and during motor rhythm produced by electrical stimulation (Figure 6C). Figure 6C shows that the stimulation elicited an alternating flexor-extensor rhythm recorded from the satrorius and femorotibialis nerves, respectively, and that the simultaneous calcium imaging and electrophysiological recordings could be used to resolve the activity patterns of flexor and extensor motoneurons during the motor rhythm (Figure 6C and Supplementary Movie 1).


A 'tool box' for deciphering neuronal circuits in the developing chick spinal cord.

Hadas Y, Etlin A, Falk H, Avraham O, Kobiler O, Panet A, Lev-Tov A, Klar A - Nucleic Acids Res. (2014)

Imaging of flexor and extensor motoneurons targeted with GCaMP3 during rhythmic activity produced by electrical stimulation of the sacral segments. (A) GCaMP3 was cloned into a Cre-dependent PiggyBac vector. This plasmid was electroporated into E3 chick neural tube along with ubiquitously-expressed PBase and Cre under the control of the chick TAG1/Axonin1 that drives expression in motor neurons. The embryos were incubated for 9 days. At E12, the embryos were sacrificed and their spinal cords were isolated. (B) Confocal image of a 100-μm cross-section through the LS6 segment in E13 chick embryo spinal cord. The section is stained for GFP (green) and ChAT (blue). The GFP-expressing cell is localized (cyan) within ChAT-positive motoneuron pool at the ventrolateral aspect of the cord. (C) The isolated spinal cord preparation is illustrated mounted with ventral side up in an in vitro chamber. The most caudal part of the spinal cord is inserted into a stimulation suction electrode. Suction electrode recordings of the motor output are obtained from the sartorius and femorotibialis nerves and the activity of GCaMP3-targeted motoneurons is viewed using water immersion objective on an upright epifluorescent microscope equipped with a digital CCD. Combined calcium imaging of the activity of motoneurons targeted with GCaMP3 and the motor output produced by stimulation of the caudal segments of the spinal cord. Stimulation of the sacral part of the cord (3 pulse 10 Hz train at 50 μA) elicited an alternating flexor and extensor rhythm, recorded from the satrorius and femorotibialis nerves, respectively. The activity pattern of two different motoneurons targeted with GCaMP3 was imaged from the ventral surface of the LS1 segment (see methods) concurrently with the recorded motor output. The fluorescence changes (ΔF/F) of one of these neurons were in phase with the rhythmic bursting recorded from the sartorius nerve (imaging of flexor motoneuron), while the ΔF/F of the other motoneuron was in phase with femorotibialis bursting (imaging of extensor motoneuron).
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Figure 6: Imaging of flexor and extensor motoneurons targeted with GCaMP3 during rhythmic activity produced by electrical stimulation of the sacral segments. (A) GCaMP3 was cloned into a Cre-dependent PiggyBac vector. This plasmid was electroporated into E3 chick neural tube along with ubiquitously-expressed PBase and Cre under the control of the chick TAG1/Axonin1 that drives expression in motor neurons. The embryos were incubated for 9 days. At E12, the embryos were sacrificed and their spinal cords were isolated. (B) Confocal image of a 100-μm cross-section through the LS6 segment in E13 chick embryo spinal cord. The section is stained for GFP (green) and ChAT (blue). The GFP-expressing cell is localized (cyan) within ChAT-positive motoneuron pool at the ventrolateral aspect of the cord. (C) The isolated spinal cord preparation is illustrated mounted with ventral side up in an in vitro chamber. The most caudal part of the spinal cord is inserted into a stimulation suction electrode. Suction electrode recordings of the motor output are obtained from the sartorius and femorotibialis nerves and the activity of GCaMP3-targeted motoneurons is viewed using water immersion objective on an upright epifluorescent microscope equipped with a digital CCD. Combined calcium imaging of the activity of motoneurons targeted with GCaMP3 and the motor output produced by stimulation of the caudal segments of the spinal cord. Stimulation of the sacral part of the cord (3 pulse 10 Hz train at 50 μA) elicited an alternating flexor and extensor rhythm, recorded from the satrorius and femorotibialis nerves, respectively. The activity pattern of two different motoneurons targeted with GCaMP3 was imaged from the ventral surface of the LS1 segment (see methods) concurrently with the recorded motor output. The fluorescence changes (ΔF/F) of one of these neurons were in phase with the rhythmic bursting recorded from the sartorius nerve (imaging of flexor motoneuron), while the ΔF/F of the other motoneuron was in phase with femorotibialis bursting (imaging of extensor motoneuron).
Mentions: The calcium indicator GCaMP3 was cloned into a Cre-dependent PiggyBac vector. Targeting to motoneurons was attained using electroporation of the conditional GCaMP3 plasmid (Figure 6A) into E3 chick neural tube along with ubiquitously-expressed PBase and Cre under the control of the chick motoneuron-specific enhancer of the TAG1/Axonin1 gene (19) (Table 1, Figure 6B). Electroporated embryos were sacrificed at E12; the spinal cords were isolated and mounted in an in vitro chamber equipped for combined optical imaging of the targeted neurons and electrophysiological recording of the motor output. Fluorophores were excited at the appropriate wavelength, and images were acquired from the ventral or lateral surface of the cord using epifluorescence microscopy and a cooled 14bit CCD camera, before and during motor rhythm produced by electrical stimulation (Figure 6C). Figure 6C shows that the stimulation elicited an alternating flexor-extensor rhythm recorded from the satrorius and femorotibialis nerves, respectively, and that the simultaneous calcium imaging and electrophysiological recordings could be used to resolve the activity patterns of flexor and extensor motoneurons during the motor rhythm (Figure 6C and Supplementary Movie 1).

Bottom Line: Here we present a circuit-deciphering 'tool box' for fast, reliable and cheap genetic targeting of neuronal circuits in the developing spinal cord of the chick.We demonstrate targeting of motoneurons and spinal interneurons, mapping of axonal trajectories and synaptic targeting in both single and populations of spinal interneurons, and viral vector-mediated labeling of pre-motoneurons.We also demonstrate fluorescent imaging of the activity pattern of defined spinal neurons during rhythmic motor behavior, and assess the role of channel rhodopsin-targeted population of interneurons in rhythmic behavior using specific photoactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Neurobiology, IMRIC, Hebrew University Medical School, Jerusalem, Israel.

Show MeSH
Related in: MedlinePlus