cav2 flp  (Addgene inc)

Journal: The Journal of Neuroscience

Article Title: A Discrete Glycinergic Neuronal Population in the Ventromedial Medulla That Induces Muscle Atonia during REM Sleep and Cataplexy in Mice

doi: 10.1523/JNEUROSCI.0688-20.2020

Figure Lengend Snippet: Axonal projections of GlyVMM neurons. A, Schematic of AAV injection. AAV8-hSyn-FLEX(loxP)-hM3Dq-mCherry was injected unilaterally into the VMM of GlyT2-iCre mice. B, Representative image of mCherry-positive neurons. Scale bar: 1 mm. C, Representative images show mCherry-positive axonal fibers (magenta) in brain regions and spinal cord. ChAT-positive neurons are stained (green). TPH2 and TH are stained to characterize serotonergic neurons in the dorsal raphe and noradrenergic neurons in the locus coeruleus (yellow). Scale bars: 100 μm.

Article Snippet: CAV2-FLEX(loxP)-Flp virus was obtained from Biocampus Montpellier.

Techniques: Injection, Staining

Journal: The Journal of Neuroscience

Article Title: A Discrete Glycinergic Neuronal Population in the Ventromedial Medulla That Induces Muscle Atonia during REM Sleep and Cataplexy in Mice

doi: 10.1523/JNEUROSCI.0688-20.2020

Figure Lengend Snippet: Axonal projections of GlyVMM→AH neurons. A, Experimental procedure of cTRIO study. We injected CAV2-FLEX(loxP)-Flp into the AH of the spinal cord (L1) and AAV-FLEX(Frt)-TVA and AAV-FLEX(Frt)-RG into the VMM (day 1). SADΔG-GFP(EnvA) was injected into the VMM (day 15). B, Representative images of starter cells in VMM. Upper panel, Low-power image. Lower panel, High-power image. Nissl is stained blue, TVA-mCherry is stained magenta, and GFP is stained green. Scale bar: 1 mm (upper), 100 μm (lower). C. Schematic drawings showing location of starter neurons in VMM. D, TVA-mCherry-positive axons in the brainstem and spinal cord. ChAT is stained green, and TPH2 or TH is stained green, while TVA-mCherry is stained magenta. E, Percentage of total mCherry axon arborization of GlyVMM neurons (upper) and GlyVMM→AH neurons (below) across brain and spinal cord regions (n = 4 in each group). F, Comparison of mCherry fraction in motoneurons between direct GlyVMM labeling and projection-specific GlyVMM→AH labeling; *p < 0.05. Unpaired t test. APT, anterior pretectal nucleus; CL, centrolateral thalamic nucleus; ECu, external cuneate nucleus; InCo, intercollicular nucleus; IRT, intermediate reticular nucleus; LPO, lateral preoptic nucleus; MDL, mediodorsal thalamic nucleus; MPO, medial preoptic nucleus; MS, medial septum; PB, parabrachial nucleus; PCRT, parvicellular reticular nucleus; RtTg, reticulotegmental nucleus of the pons; Ve, vestibular nucleus; ZI, zona incerta.

Article Snippet: CAV2-FLEX(loxP)-Flp virus was obtained from Biocampus Montpellier.

Techniques: Injection, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: A Discrete Glycinergic Neuronal Population in the Ventromedial Medulla That Induces Muscle Atonia during REM Sleep and Cataplexy in Mice

doi: 10.1523/JNEUROSCI.0688-20.2020

Figure Lengend Snippet: Silencing of GlyVMM neurons and GluSLD→VMM pathways. A, Schematic drawing of procedure. AAV2-hSyn-FLEX(loxP)-TeNTLC-P2A-EYFP or AAV10-EF1a-FLEX (loxP)-EYFP was injected into the VMM of GlyT2-iCre mice. B, Representative EYFP-positive neurons are shown. Scale bar: 1 mm. C, Schematic depicting EYFP expression areas in the brain; n = 7 in each group. D, Representative EEG trace (blue) and EMG trace (black) during REM sleep in control group (top) and TeNTLC group (bottom). E–G, EMG integral during wakefulness (E), NREM (F), and REM (G). H, REM/NREM ratio of EMG integral. I, Schematics of AAV injection and representative image of expression. AAV-CAG-FLEX(Frt)-TeNTLC-P2A-EYFP or AAV-CAG-FLEX (Frt)-EYFP was injected into the SLD and CAV2-FLEX (loxP)-Flp was injected into the VMM of vGlut2-iresCre mice. J, Representative EYFP-positive neurons in SLD. ChAT is stained red whereas EYFP is stained green. Nissl is stained blue. Left lower shows a magnified image. Scale bar: 100 µm. K, EYFP-positive axons in VMM. Scale bar: 1 mm (upper), 100 µm (lower). L, Outlines of EYFP expression sites in SLD; n = 5 in each group. M, Representative EEG trace (blue) and EMG trace (black) during REM sleep in control group (top) and TeNTLC group (bottom). N–P, EMG integral during wakefulness (N), NREM (O), and REM (P). Q, REM/NREM ratio of EMG integral. All values are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired t test. n.s., not significant.

Article Snippet: CAV2-FLEX(loxP)-Flp virus was obtained from Biocampus Montpellier.

Techniques: Injection, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: A Discrete Glycinergic Neuronal Population in the Ventromedial Medulla That Induces Muscle Atonia during REM Sleep and Cataplexy in Mice

doi: 10.1523/JNEUROSCI.0688-20.2020

Figure Lengend Snippet: Silencing of GlyVMM neurons and GluSLD→VMM pathway in narcolepsy model mice. A, Experimental procedure. AAV2-hSyn-FLEX(loxP)-TeNTLC-P2A-EYFP or AAV10-EF1a-FLEX (loxP)-EYFP was injected into the VMM of GlyT2-iCre;orexin-ataxin3 mice. B, Area of EYFP expression site in VMM. EYFP, n = 4; TeNTLC, n =5.C, REM/NREM ratio of EMG integral. D, Representative EEG traces (blue) and EMG traces (black) of transitions from wakefulness to CLE (top left), from CLE to wakefulness (top middle), from wakefulness to NREM (top right), from NREM to REM (bottom left), from NREM to wakefulness (bottom middle), and from REM to wakefulness (bottom right). E, Time of CLEs in a day. F, Total amount of CLEs. G, Mean episode duration of CLEs. H, Episode number of CLEs. I, Experimental procedure. AAV2-CAG-FLEX(Frt)-TeNTLC-P2A-EYFP or AAV10-CAG-FLEX(Frt)-EYFP was injected into the SLD and CAV2-FLEX(loxP)-Flp was injected into the VMM of vGlut2-iresCre:orexin-ataxin3 mice. J, Areas containing EYFP expression in SLD; n = 5 in each group. K, REM/NREM ratio of EMG integral. L, Time of CLEs in a day. M, Total amount of CLEs. N, Mean episode duration of CLEs. O, Episode number of CLEs. All values are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired t test for single variables and two-way ANOVA followed by Bonferroni post hoc tests for multiple variables. n.s., not significant.

Article Snippet: CAV2-FLEX(loxP)-Flp virus was obtained from Biocampus Montpellier.

Techniques: Injection, Expressing

Journal: The Journal of Neuroscience

Article Title: Monoamines Inhibit GABAergic Neurons in Ventrolateral Preoptic Area That Make Direct Synaptic Connections to Hypothalamic Arousal Neurons

doi: 10.1523/JNEUROSCI.2835-17.2018

Figure Lengend Snippet: Visualization of GABAergic neurons in the POA, which make monosynaptic input to HDC and orexin neurons. A, Schematic representation of the procedure. We injected AAV10-FLEX-TVA-mCherry and AAV10-FLEX-RG stereotaxically into the TMN of Gad67-GFPΔNeo;Hdc-Cre or the LHA of Gad67-GFPΔNeo;Orexin-Cre mice. After 14 d, SADΔG-mCherry(EnvA) was injected into the same areas and the brains were analyzed 4–7 d after injection. B, Representative images of distribution of labeled cells in POA of Gad67-GFPΔNeo;Hdc-Cre mice. C, Representative image of distribution of labeled cells in POA of Gad67-GFPΔNeo;Orexin-Cre mice. Green, GFP. Red, SADΔG-mCherry. D, GABAergic input neurons to HDC neurons in the VLPO. Right, High-power view of rectangular region in left panel. E, GABAergic input neurons to orexin neurons in VLPO. Right, High-power view of rectangular region in the panel. F, Percentages of double-positive GABAergic neurons among input neurons to HDC neurons or orexin neurons in designated POA areas. G, Analysis of axonal arborization pattern of GABAVLPO→LHA neurons. CAV2-FLEX (loxP)Flp was injected into the LHA in vGAT-ires-Cre mice. AAV10-CAG-FLEX (Frt)-TVA-ChR2-eYFP was injected into the POA to express ChR2-eYFP in GABAPOA→LHA cells. H, YFP+ neurons observed in the POA (left). YFP+ fibers make close appositions to orexin and HDC neurons (middle and right). I, Specimen responses of GABAVLPO→HDC neurons (top traces) and GABAVLPO→orexin neurons (bottom traces) to 30 s bath application of 100 μm NA or 100 μm 5-HT. J, Summary of changes in membrane potential of GABAVLPO→HDC neurons and GABAVLPO→orexin neurons induced by drugs.

Article Snippet: CAV2-FLEX (loxP)-Flp virus was obtained from BioCampus Montpellier.

Techniques: Injection, Labeling