Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: ( A ) Kv3.1 in the cochlear nucleus (outlined) of WT mouse at 1 month of age. ( B ) Kv3.3 in the same section of cochlear nucleus (outlined). ( C ) Overlay of Kv3.1 (green) and Kv3.3 (magenta) with DAPI (blue). ( D–F ) magnified section of cochlear nucleus showing Kv3.1 ( D ) and Kv3.3 ( E ) immunoreactivity. Images are overlayed in F. All scale bars = 100 µm. Antibodies and conditions have been independently validated previously using Kv3.1KO and Kv3.3KO tissues .

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques:

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: ( A–B ) Kv3.1 immunoreactivity spiral ganglion neurons in the WT mouse at 1 month of age. In addition to the spiral ganglion cell membranes, Kv3.1 was also present at the nodes of Ranvier (B, arrows). ( C ) No Kv3.1 staining was observed in the spiral ganglion cell bodies of Kv3.1KO mice. Non-specific signal was associated with the bony tissues of the modiolus, and assumed to be autofluorescence from the collagen fibers. ( D–E ) Kv3.3 immunoreactivity spiral ganglion neurons in the WT mouse at 1 month of age. Strong immunoreactivity was observed in the cell bodies. ( F ) Faint non-specific signal was observed in Kv3.3KO tissues, but did not appear to be associated with the spiral ganglion neurons. All scale bars = 100 µm.

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Staining

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: ( A ) Current-Voltage (I–V) relationship for potassium currents in the calyx of Held terminal of WT mice (P10–P12) in control (black; n=7 terminals, 4 mice) and TEA, 1 mM (red, n=6 terminals, 5 mice; HP = –70 mV). Inset (top): Example current traces in response to voltage command of +10 mV step (grey box in IV) in WT (black) and WT +1 mM TEA (red). Scale bars = 5 nA and 20ms. Inset (lower): Bar graph of mean currents ± SD, measured on step depolarisation to +10 mV (from HP –70 mV) ±1 mM TEA. Outward Currents are significantly reduced by TEA (student’s t-test, unpaired, P =0.0386). ( B ) WT calyx AP (black trace) evoked by 100 pA step current injection; inset - diagram of recording configuration. ( C ) WT calyx AP in the presence of TEA (1 mM, red trace); inset – overlaid WT APs ±TEA (red) as indicated by dotted box (grey) around APs in B and C. ( D ) Representative AP traces from calyx terminals of WT (black), Kv3.3KO (blue), and Kv3.1KO (orange); double arrows indicate the half-width of WT AP. AP threshold is indicated by the grey dashed line. ( E ) AP half-width measured as time difference between rise and decay phases at 50% maximal amplitude. Half-width is significantly increased in TEA and in Kv3.3KO; N is individual terminals: WT N=9 from 6 animals; TEA = 7 from 5 mice; Kv3.3KO = 6 from 3 mice and Kv3.1KO = 5 from 3 mice. ( F ) AP amplitude, ( G ) AP Decay slope, ( H ) AP rise slope (10–90%) and ( I ) membrane resistance for calyceal recordings. Average data presented as mean ± SD. Statistical test (parts E-I) were one-way ANOVAs and Tukey’s post hoc for multiple comparisons, with significant Ps indicated on the graph. Figure 1—source data 1. Relates to .

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Injection

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: Individual images are identified in rows 1–6 and columns A-F, as indicated by the central labels. Four quadrants of 9 images are shown, each 3 × 3 matrix is from the named genotype and stained as specified in the title of each quadrant. The top row of each quadrant (rows 1 and 4) are single optical sections from 3 different MNTB neurons, in which their calyceal synaptic profiles are labelled with bassoon (purple) and co-labelled with either Kv3.1 or Kv3.3 antibodies (yellow): from Kv3.1KO and stained for Kv3.3 ( A1-C1 ); the Kv3.3KO stained for Kv3.1 ( D1-F1 ); WT stained for Kv3.3 ( A4-C4 ); WT stained for Kv3.1 ( D4-F4 ). In each MNTB neuron (rows 1 & 4) two synaptic regions of interest (ROI) containing bassoon are indicated by the red and green arrowheads. These magnified ROIs are displayed below (in rows 2+3 or 5+6) bordered by the same colour, respectively. The neuronal compartments are labelled: ‘post’ – postsynaptic; ‘pre’ – presynaptic; ‘term’ – synaptic terminal. In each image, the dark grey arrows point to presynaptic Kv3 labelling, and the white arrows point to postsynaptic Kv3 labelling. Scale bars are indicated for each row in column A (5 µm in rows 1 and 4: 1 µm in rows 2,3,5, and 6). Tissue was used from mice aged P28-P30.

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Staining

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: ( A ) Superimposed calyceal EPSCs generated from each genotype (age P21-P25): wildtype (WT; black), Kv3.3KO (blue), and Kv3.1KO mice (orange). Thin lines show traces from individual neurons (each is mean of 5 EPSCs) with thick line showing the population mean for each genotype. Grey dashed line indicates the average WT amplitude; N=WT, 22 neurons (11 mice); Kv3.3KO, 22 neurons (10 mice); Kv3.1KO, 17 neurons (8 mice). Inset shows recording and stimulation configuration. ( B ) EPSC amplitude increased in the Kv3.3KO. ( C ) EPSC rise time (10–90%) no difference was found between groups (one-way ANOVA, p=0.1576). ( D ) EPSC decay tau and ( E ) EPSC total charge were increased in the Kv3.3KO relative to WT. ( F ) EPSC traces from WT, Kv3.3KO, and Kv3.1KO mice, before and after the addition of 1 mM TEA. ( Centre): EPSC amplitudes recorded before and after perfusion of TEA (1 mM); n=WT, 9 neurons (7 mice); Kv3.3KO, 6 neurons (3 mice); Kv3.1KO, 5 neurons (3 mice). ( G ) Increase in EPSC amplitude by 1 mM TEA. ( H ). The amplitude increase induced by TEA was significantly reduced in Kv3.3KO mice compared to WT. Average data presented as mean ± SD; statistics was using one-way ANOVA with Tukey’s post hoc for multiple comparisons. Kruskal-Wallis ANOVA with Dunn’s multiple corrections was used to compare change to EPSC amplitude in TEA due to a non-gaussian distribution in WT. Figure 3—source data 1. Relates to .

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Generated

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: Short-term depression was accelerated and enhanced in mice lacking Kv3.3. Values shown are for parameters measured from data presented in Figure 3 for WT, Kv3.3KO and Kv3.1KO genotypes at 100 Hz to 600 Hz range. Paired pulse depression of EPSC responses recorded in MNTB neurons (EPSC 2 /EPSC 1 ) was increased in Kv3.3 KO animals during high frequency stimulation of the calyx. The increased depression was maintained throughout the stimulation train (EPSC 80 /EPSC 1 ) across all frequencies. The rate of short term-depression in EPSC amplitudes during EPSC trains (duration 800ms), measured as short-term depression (STD) decay tau was significantly increased in Kv3.3 KOs at 100 and 200 Hz compared to WT. This STD was more severe in mice lacking Kv3.3, as shown by decreased normalized steady-state EPSC amplitudes compared to WT. STD tau and steady state amplitudes were measured using a single exponential fit to normalized EPSC amplitudes throughout the 800ms stimulation trains. n=number of neurons. Values in bold are significantly different to WT (see statistics table for more detail). Data represented as mean ± SD.

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques:

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet:

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Knock-Out, Software