Journal: eLife

Article Title: Neuropeptide Y-expressing dorsal horn inhibitory interneurons gate spinal pain and itch signalling

doi: 10.7554/eLife.86633

Figure Lengend Snippet:

Article Snippet: Strain, strain background ( Adeno-associated virus ) , AAV1 pCAG-FLEX-tdTomato-WPRE (AAV.flex.tdTomato; 1.76 × 10 9 GC) , Penn Vector Core, PA, USA; available from Addgene, MA, USA (deposited by Hongkui Zeng) , Cat#: 51503 RRID: Addgene_51503 , .

Techniques: Virus, Plasmid Preparation, Sequencing, RNAscope, Recombinant, Adjuvant, Software

Journal: bioRxiv

Article Title: Inositol Polyphosphate-5-phosphatase K ( Inpp5k ) enhances sprouting of corticospinal tract axons after CNS trauma

doi: 10.1101/2021.04.27.441184

Figure Lengend Snippet: Schematic ( A ) shows approach to identify intact CSNs undergoing functional plasticity after uPyX. Adult ngr1 +/+ crym -GFP ngr1 -/- crym -GFP transgenic mice received a uPyX, 14 days post lesion mice received a contralateral infusion of the retrograde tracer fast blue, 28 days post lesion mice were prepared for laser capture microdissection of intact quiescent CSNs (GFP+FB-) and intact sprouting CSNs (GFP+/FB+) . Differential gene expression analysis calculated using a Wilcoxon rank sum test, showed that 2738 genes were significantly upregulated and 506 down regulated in sprouting vs. quiescent CSNs. Volcano plot ( B )shows every gene profiled as log2 fold change versus –log10 of the false discovery rate (FDR) corrected p value. Non-SDE genes (grey dots) are separated from SDE genes (black dots) by p < 0.05 cut off (stippled) line. Inositol phosphate genes (dark blue) and genes associated with the cytoskeleton (magenta dots) are primarily enriched in sprouting CSNs. Five 5-phosphatases ( Inpp5k , INPP5J, SYNJ1, OCRL, ) and Cofilin are highlighted. Ingenuity Pathway Analysis (IPA) was conducted on genes enriched in CSNs undergoing functional plasticity. Table ( C ) shows the IPA output of significantly upregulated ‘Canonical Pathways’ and ‘Diseases and Biofunctions’ across the whole dataset of differentially expressed genes (with P values corrected for multiple comparisons), canonical pathways associated with inositol phosphate signaling are highlighted in blue and cytoskeletal dynamics in magenta. E17.5 neurons cultured transduced with Inpp5k -V5 ( E ), showed significant longer neurites compared to GFP ( D, F , average total axon length of GFP (n = 45, grey dots) and Inpp5k -V5 (n = 45, light blue dots) neurons from n=3 independent experiments ( t 4 = 4.066, * p = 0.015, unpaired two-tailed t test). Data shown are the mean length of axon (µm) (biological n, darker dots) ± SEM. To assess if Inpp5k mediated enhanced neurite growth was mTOR dependent we transduced E17.5 cortical neurons with AAV-mCherry or AAV- Inpp5k +/-300 nM Rapamycin in DMSO ( G-J ). Neurons expressing mCherry showed minimal regeneration into the scrapped zone, while Inpp5k expressing neurons showed significant increase in regeneration compared to control. There was no significant difference in the neurite regeneration upon addition of rapamycin to mCherry or Inpp5k treated neurons (one-way ANOVA, **** p < 0.0001 [F(5, 44) = 18.24], using post hoc Tukey’s HSD test). Scale bars, D = 100 µm, H = 200 µm.

Article Snippet: To generate AAV-CAG-mCherry, Dr. Kim modified two AAV vectors (Addgene plasmid #18917 and #38044).

Techniques: Functional Assay, Transgenic Assay, Laser Capture Microdissection, Gene Expression, Cell Culture, Transduction, Two Tailed Test, Expressing, Control

Journal: bioRxiv

Article Title: Inositol Polyphosphate-5-phosphatase K ( Inpp5k ) enhances sprouting of corticospinal tract axons after CNS trauma

doi: 10.1101/2021.04.27.441184

Figure Lengend Snippet: Schematics ( A ) show relative locations of PyX and cortical injection of AAV1 mCherry and AAV1- Inpp5k -V5. Lower right schematic of transverse cervical sections with mCherry (red) labeling in four quadrants that were analyzed and right shows experimental timeline. Photomicrographs B-E show V5 ( B, D ) and mCherry ( C, E ) staining in M1 cortex ( B, C ) and C4 spinal cord 42 days after AAV infusion, confirming expression of Inpp5k and the reporter in CSNs and CST axons. Photomicrographs F-I show mCherry+ CST axon staining in transverse sections of C6 spinal cord from mice that received AAV1-FLEX-GFP after sham lesion (F) and PyX (G) and Inpp5k treatment after sham lesion (H) and PyX (I). Insets ( F’, H’ ) show PKC γ immunoreactivity both dorsal column projections in sham lesioned mice and intact contralateral dorsal columns after PyX ( G’, I’ ). There was no significant difference in the number of CST axons traced between groups ( J , two-way ANOVA with Bonferroni post hoc comparisons, p > 0.05, data are shown as average number of labeled CST axons ± SEM). Densitometric analysis of mCherry+ CST axons on the intact side of the spinal cord showed that there was a main effect of treatment, with Inpp5k treated mice having significantly more CST axon terminal density compared to controls ( F (3, 50) = 18.97, p < 0.0001, two-way ANOVA with Bonferroni post hoc comparisons, n = 5 control sham, n = 11 control PyX, n = 6 Inpp5k sham, and n = 7 Inpp5k PyX). Specifically, Inpp5k treatment increased density of mCherry+ CST axons in the DI quadrant regardless of surgery condition ( F (1, 50) = 34.69, p < 0.0001, two-way ANOVA with Bonferroni post hoc comparisons, data shown are average mCherry signal density ± SEM. (L) For the denervated side of the cervical cord Inpp5k treatment increased the density of mCherry+ CST axons regardless of surgical condition ( F (3, 50) = 47.79, p < 0.0001, two-way ANOVA with Bonferroni post hoc comparisons), and this was true for both the DC and VC quadrants ( F (1, 50) = 199.6, p < 0.0001, two-way ANOVA with Bonferroni post hoc comparisons, data shown are average mCherry+ signal density ± SEM. CST function was assessed using the grid-walking apparatus. No significant differences were found between Inpp5k PyX and control PyX treated subjects for the forelimbs ( M , two-way ANOVA with repeated measures with Bonferroni post hoc comparisons, p > 0.05, data shown are average percent missteps ± SEM), or the hind limbs ( N , two-way ANOVA with repeated measures with Bonferroni post hoc comparisons, p > 0.05, data shown are average percent missteps ± SEM. Scale bars, B = 1 mm; D = 100 µm; I = 500 µm; I’ = 100 µm.

Article Snippet: To generate AAV-CAG-mCherry, Dr. Kim modified two AAV vectors (Addgene plasmid #18917 and #38044).

Techniques: Injection, Labeling, Staining, Expressing, Control

Journal: bioRxiv

Article Title: Subcortical source and modulation of the narrowband gamma oscillation in mouse visual cortex

doi: 10.1101/050245

Figure Lengend Snippet: We made whole-cell voltage-clamp recordings of EPSCs in neurons in layer 4 of V1 while simultaneously recording single-unit activity in the LGN of VGAT-ChR2 mice under light urethane anesthesia. V1 could be silenced by shining a blue LED over the region. B. Spike time autocorrelation of an LGN single unit when V1 activity was intact (black) or silenced (cyan) C. Average EPSCs recorded by voltage-clamping a V1 layer 4 neuron at −70mV, triggered on spikes of an LGN neuron (same neuron as in B, D), when V1 activity was intact (black) or silenced (cyan). Excitatory current negative. D. Mean (± s.e.m) spectral power across LGN neurons (n = 47 neurons). E. Comparison of the peak power of the narrow-band gamma when cortical activity is intact or silenced (n = 47 neurons). F. Mean (± s.e.m) EPSC spectral power across neurons without and without cortical silencing (n = 14 neurons). G. Comparison of residual narrowband gamma power when the cortical activity is intact or silenced (n = 14 neurons). H. The cross-coherence between EPSCs recorded in V1 and LGN spiking activity shows a peak at the narrow-band gamma frequency, which does not change with cortical silencing (n = 47 pairs). I. Comparison of maximum LGN-V1 cross-coherence when cortical activity is intact or silenced (n = 14 neurons).

Article Snippet: For experiments inactivating the LGN, V1 extracellular (Buzsaki16 probe, Neuronexus) recordings were performed in awake Gad2-Cre mice (Jackson Labs stock number: 010802 ) expressing Cre-dependent ChR2 (AAV2/1.CAGGS.flex.ChR2.tdTomato.SV40, Addgene 18917 , from the University of Pennsylvania viral vector core) stereotactically confined to the thalamic reticular nucleus (TRN).

Techniques: Activity Assay, Comparison

Journal: eLife

Article Title: Extended field-of-view ultrathin microendoscopes for high-resolution two-photon imaging with minimal invasiveness

doi: 10.7554/eLife.58882

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , AAV pCAG- FLEX-EGFP-WPRE , Penn Vector Core , RRID: Addgene_51502 ; Addgene viral prep # 51502-AAV1 , .

Techniques: Recombinant, Plasmid Preparation, Adhesive, Software