vector suspensions  (World Precision Instruments)

Journal: Frontiers in Cellular Neuroscience

Article Title: Protein kinase Cγ negatively regulates the intrinsic excitability in zebrin-negative cerebellar Purkinje cells

doi: 10.3389/fncel.2024.1349878

Figure Lengend Snippet: Significant enhancement of intrinsic excitability in protein kinase C γ isoform (PKCγ)-conditional knock-out (cKO) Purkinje cells (PCs). (A) Schema depicting the experimental procedure. Three-week-old PKCγ fl/fl mice and wild-type (WT) mice were administered cerebellar injection of adeno-associated virus serotype 9 (AAV9) vectors expressing Cre recombinase (Cre) together with GFP (GFP-P2A-Cre) under the control of cerebellar PC-specific L7-6 promoter [1.0 × 10 9 viral genome (vg)/mouse]. ITR, inverted terminal repeat; L7-6 pr, L7-6 promoter with minimal cytomegalovirus sequence; P2A, porcine teschovirus-1 2A self-cleaving peptide; PolyA, polyadenylation signal; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. (B) Immunohistochemistry of the cerebellar section from the AAV-treated PKCγ-cKO mouse. The section was stained with antibodies for GFP (the upper image) and PKCγ (Red, the lower image). (C) Enhanced intrinsic excitability in PKCγ-cKO PCs. Intrinsic excitability was examined from PCs randomly chosen from vermal slices, which was assessed by the number of spikes evoked by injection of a somatic depolarizing current (100–500 pA, in 100 pA increments). The representative traces evoked with 300 pA pulses are shown above the graph. Scale bar = 500 μm for (B) , 200 ms, 10 mV for (C) . * p < 0.05 by two-way repeated measure ANOVA.

Article Snippet: AAV9 vector suspension (10 μl) was injected at a rate of 300 nl/min using a microprocessor-based controller (Micro4; World Precision Instrument).

Techniques: Knock-Out, Injection, Virus, Expressing, Sequencing, Immunohistochemistry, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Protein kinase Cγ negatively regulates the intrinsic excitability in zebrin-negative cerebellar Purkinje cells

doi: 10.3389/fncel.2024.1349878

Figure Lengend Snippet: Enhanced intrinsic excitability in zebrin-negative (Z–), but not in zebrin-positive (Z+), Purkinje cells (PCs) of protein kinase C γ isoform (PKCγ)-conditional knock-out (cKO) mice. (A) Schema depicting the experimental procedure. PKCγ fl/fl mice were crossed with RGS8-EGFP mice to obtain mice carrying both genotypes (PKCγ fl/fl × RGS8-EGFP mouse). Three-week-old PKCγ fl/fl mice and their wild-type (WT) littermates on RGS8-EGFP background were administered cerebellar injections of adeno-associated virus serotype 9 (AAV9) vectors expressing mCherry-P2A-Cre under the control of cerebellar Purkinje cell-specific L7-6 promoter (1.0 × 10 9 vg/mouse). Mice treated with AAVs were electrophysiologically analyzed 4 weeks after the viral injection. (B) (B1) Diagram depicting lobules IV–V (colored area) used for whole cell-recording of PCs. (B2) The fluorescent photo on the right shows lobule IV–V containing both PCs positive for GFP (Z–) and negative for GFP (Z+). The magnified images of the boxed areas in (B2) are shown in (B3) . (C,D) Graphs showing the change in spike number elicited by gradually increasing injecting currents to Z– (C) and Z+ (D) PCs from both AAV-treated WT mice and PKCγ-cKO mice. Traces above graphs show representatives evoked by injection of 300 pA current. Scale bar = 200 μm for (B2), 50 μm for (B3); 200 ms, 10 mV for (C,D) . ** p < 0.01 by two-way repeated measure ANOVA. N.S., not significant.

Article Snippet: AAV9 vector suspension (10 μl) was injected at a rate of 300 nl/min using a microprocessor-based controller (Micro4; World Precision Instrument).

Techniques: Knock-Out, Virus, Expressing, Injection