Article Figures & Data
Figures
- Figure 1.
Pathway-selective optogenetic activation of SC output neurons. A, Viral vector constructions. B, Experimental protocols. C, Schematic diagram for the double injection of the viral vectors into the brainstem and SC, and the interaction of NeuRet-MSCV-Cre and AAV-EF1α-DIO-hChR2(E123T/T159C)-EYFP in the double infected SC neurons. Upper insets in left and right panels, Photomicrographs of the somata of the crossed (orienting) and uncrossed (defense) SC-brainstem pathway neurons, respectively. Lower insets in left and right panels, Injection sites of the NeuRet-MSCV-Cre, indicated by a mock injection of Fluoro-Ruby into the brainstem reticular formation at medial PMRF and CnF, respectively. Scale bar in the upper insets = 100 μm. Scale bar in the lower insets = 1 mm. D, Responses of a mouse SC output neuron to blue laser stimulation (blue line) with a 500-μm diameter fiber. (1) Responses of the crossed (orienting) pathway neurons. (2) Responses of uncrossed (defense) pathway neurons. Figure Contributions: Thongchai Sooksawate, Kenta Kobayashi, and Kaoru Isa performed the experiments. Thongchai Sooksawate analyzed the data and prepared the figure.
- Figure 2.
Categorization of behavioral responses to selective optogenetic activation of each SC output pathway. A–D, Sequential photographs illustrating each of typical behavioral response patterns of the mice following optogenetic activation of SC neurons. E, Categorization and percentage of responses in 10 trials of behavioral responses induced by the crossed (orienting) pathway with various stimulus intensities (50, 200, and 50 ms × 20). Left panel for the responses in the closed box and right panel for the responses on the open platform (*p < 0.05, **p < 0.01, ***p < 0.001, Bonferroni’s multiple comparison test, eight tested animals). F, Categorization and percentage of responses in 10 trials of behavioral responses induced by the uncrossed SC-brainstem pathway with various stimulus intensities. Left panel for the responses in the closed box and right panel for the responses on the open platform (*p < 0.05, **p < 0.01, ***p < 0.001, Bonferroni’s multiple comparison test, seven tested animals). Frequencies of retreat and flight responses at 200-ms pulse were significantly different between the closed box and open platform environment (dotted green and blue lines with asterisks; **p < 0.01, ***p < 0.001, Bonferroni’s multiple comparison test, n = 7). G, upper panel, Examples of the trajectory of the orienting responses of the mouse during stimulation of the crossed (orienting) pathway (20 trains of 50-ms pulses at 10 Hz) in the closed box and on the open platform. Lower panel, Histogram of the velocity of body movements before, during and after stimulation. H, upper panel, Examples of the trajectory of the defense-like responses of the mouse during stimulation of the uncrossed (defense) pathway (20 trains of 50-ms pulses at 10 Hz) in the closed box and the open platform. Lower panel, Histogram of the velocity of body movements before, during and after stimulation. Figure Contributions: Thongchai Sooksawate, Kenta Kobayashi, and Kaoru Isa performed the experiments. Thongchai Sooksawate and Kaoru Isa analyzed the data and prepared the figure.
- Figure 3.
Axonal trajectories of the crossed (orienting) pathway neurons visualized by anti-GFP immunohistochemistry with Alexa Fluor 594. A, Crossed (orienting) pathway. 1–6, Photomicrographs of the frontal sections of the diencephalon, brainstem. Scale bars = 1 mm. 7, Spinal cord (C1 level). Upper panel, Low-magnification bright field view (scale bar = 1 mm). Lower panel, High-magnification fluorescent view of the square area in the upper panel (scale bar = 100 μm). B, A diagram showing the location of the somata of crossed (orienting) pathway neurons. C, Targets of the crossed (orienting) pathway neurons. The numerals with arrows indicate the rostrocaudal levels of photomicrographs in A1–A7. An asterisk indicates the injection site of viral vector in PMRF. CM/PC/CL: rostral intralaminar thalamic nuclei, such as central medial thalamic nucleus (CM)/paracentral thalamic nucleus (PC)/centrolateral thalamic nucleus (CL), coSCd; contralateral SC deeper layers, IO: inferior olive, MDL: mediodorsal thalamic nucleus lateral part, mRt: mesencephalic reticular formation, NRTP: nucleus reticularis tegmenti pontis, PDB: predorsal bundle, PF: parafascicular thalamic nucleus, PMRF: medial PMRF, PPN: pedunculopontine nucleus, SAI: intermediate white layer, SC: superior colliculus, SCd: SC deeper layers, SGP: deep gray layer, SGS: superficial gray layer, SNc: substantia nigra pars compacta, SO: optic layer, Sp.c.: spinal cord, VM: ventral mediothalamic nucleus, ZI: zona incerta. Figure Contributions: Thongchai Sooksawate, Kenta Kobayashi, and Kaoru Isa performed the experiments. Kaoru Isa, Peter Redgrave, and Tadashi Isa analyzed the data.
- Figure 4.
Axonal trajectories of the uncrossed (defense) pathway neurons visualized by anti-GFP immunohistochemistry with Alexa Fluor 594. A, Uncrossed (defense) pathway. 1–6, Photomicrographs of the frontal sections of the diencephalon and brainstem. Scale bars = 1 mm. B, A diagram showing location of the somata of uncrossed (defense) pathway neurons. C, Targets of the uncrossed (defense) pathway neurons. The numerals with arrows indicate the rostrocaudal levels of photomicrographs in A1–A6. An asterisk indicates the injection site of viral vector in the right MRF around CnF. The same abbreviations as Figure 3 and additional abbreviations: dPAG: dorsal periaqueductal gray matter, IC: inferior colliculus, LP: lateral posterior thalamic nucleus, PIL: posterior intralaminar nucleus, PoT: posterior thalamic nucleus, triangular, Re: reuniens thalamic nucleus. Figure Contributions: Thongchai Sooksawate, Kenta Kobayashi, and Kaoru Isa performed the experiments. Kaoru Isa, Peter Redgrave, and Tadashi Isa analyzed the data.
- Figure 5.
EMG responses of dorsal neck muscles to optogenetic stimulation of the SC neurons. A, Schematic diagram showing the experimental protocol. B, EMG response to the activation of the tectal orienting pathway, which was inhibited by a muscimol at 2 and 5 min after injection into the brainstem. C, EMG responses to the activation of the tectal defense pathway, which were also abolished by unilateral microinjection of muscimol at 2 and 5 min after injection into the brainstem. The horizontal blue lines above individual traces indicate the period of laser irradiation. Figure Contributions: Thongchai Sooksawate, Kenta Kobayashi, and Kaoru Isa performed the experiments. Thongchai Sooksawate analyzed the data. Thongchai Sooksawate and Kaoru Isa prepared the figure.
- Figure 6.
Nonselective optogenetic activation of the SC neurons. A, Fluorescent photomicrographs of the SC that expressed ChR2 and tdTomato. Scale bar = 1 mm. B, Site of laser stimulation on the topographic map of the mouse SC. C, Responses of mouse SCs neuron to blue laser stimulation; raw traces (upper row), peristimulus time histogram (PSTH; middle row), and raster plots (lower row). D, Responses of mouse SCd neuron to blue laser stimulation; raw traces (upper row), PSTH (middle row), and raster plots (lower row). The horizontal blue lines above individual traces indicate the period of laser irradiation; single, long pulse (100 ms) on the left (horizontal blue line), and repetitive stimulation with short pulses on the right (10 trains of 50-ms duration pulses with 50-ms interval (10 Hz), horizontal blue broken line). PAG: periaqueductal gray matter, SAI: intermediate white layer, SC: superior colliculus, SCd: SC deeper layers, SCs: SC superficial layers, SGI: intermediate gray layer, SGP: deep gray layer, SGS: superficial gray layer, SO: optic layer. Figure Contributions: Thongchai Sooksawate, Kenta Kobayashi, and Kaoru Isa performed the experiments. Thongchai Sooksawate analyzed the data. Thongchai Sooksawate and Kaoru Isa prepared the figure.
- Figure 7.
Categorization of behavioral responses to nonselective optogenetic activation of the SC neurons in the closed box or on the open platform environment. Behavioral response categories (body turn, head-only turn, retreat, flight, and freezing responses) are plotted on the horizontal axis and their percentage of occurrence in 10 trials to stimulation with each parameter on the vertical axis. A, Stimulation of the caudo-lateral SC by a 250-μm diameter fiber with various stimulation parameters (50, 200, and 50 ms × 20). Left panel for the responses in the closed box and right panel for the responses on the open platform (*p < 0.05, **p < 0.01, ***p < 0.001, Bonferroni’s multiple comparison test, six tested animals). B, Stimulation of the rostro-medial SC by a 250-μm diameter fiber with various stimulation parameters. The same arrangement as A (six tested animals) C, Stimulation of the central SC by a 500-μm diameter fiber with various stimulation parameters. The same arrangement as A, B (six tested animals). Figure Contributions: Thongchai Sooksawate, Kenta Kobayashi, and Kaoru Isa performed the experiments. Thongchai Sooksawate and Kaoru Isa analyzed the data and prepared the figure.
Movies
- Movie 1.
Head-only turn response. Pathway selective optogenetic activation-induced head-only turn of mouse in the crossed (orienting) pathway group to blue laser (50-ms duration) in the closed box.
- Movie 2.
Body turn in closed box. Pathway selective optogenetic activation-induced body turn response of mouse in the crossed (orienting) pathway group to blue laser (50-ms duration, 10 Hz, 20 trains) in the closed box.
- Movie 3.
Body turn response in open platform. Pathway selective optogenetic activation-induced body turn response of mouse in the crossed (orienting) pathway group to blue laser (50-ms duration, 10 Hz, 20 trains) on the open platform. This mouse was the same one as in Movie 2.
- Movie 4.
Flight response in closed box. Pathway selective optogenetic activation-induced flight response of mouse in the uncrossed (defense) pathway group to blue laser (50-ms duration, 10 Hz, 20 trains) in the closed box.
- Movie 5.
Retreat response in open platform. Pathway selective optogenetic activation-induced retreat response of mouse in the uncrossed (defense) pathway group to blue laser (50-ms duration, 10 Hz, 20 trains) on the open platform.
- Movie 6.
Freezing response in closed box. Pathway-nonselective stimulation (100-ms duration) of central SC induced freezing responses which last during the stimulation and for a few seconds after the stimulus offset.
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