avidin biotin immunoperoxidase complex technique  (Alomone Labs)

Journal: eLife

Article Title: TRPV3 channel activity helps cortical neurons stay active during fever

doi: 10.7554/eLife.102412

Figure Lengend Snippet: ( A ) TRPV3 immunostaining (green) in mouse primary somatosensory (S1) cortex, striatum, thalamus, and hippocampus, with DAPI counterstain (blue). Top row left: Postnatal day (P)7; bottom row left: P14; bottom row right: P21. Top row right: Sections incubated with TRPV3 antibody plus TRPV3 blocking peptide in S1 cortex, striatum, thalamus, and hippocampus (green) with DAPI counterstain (blue). Remaining TRPV3 staining is shown in green with DAPI in blue. ( B ) TRPV3 and TRPV4 immunostaining in S1 cortex, striatum, thalamus, and hippocampus at P14. Top row left: TRPV3 (green) and TRPV4 (brick red). Top row right: TRPV3 (green) and TRPV4 (brick red) with DAPI counterstain (blue). Bottom row left: TRPV4 immunostaining in S1BF (inset from top row left) at 10x. Bottom row right: TRPV3 immunostaining in S1BF (inset from top row left) at 10x.

Article Snippet: Slides were blocked in 10% normal goat serum for 1 hr at room temperature, then incubated overnight at 4°C with anti-TRPV3-Biotin antibody (#ACC-033-B), TRPV3 blocking peptide (#BLP-CC033), and/or anti-TRPV4 antibody (#ACC-034) (Alomone Labs, Israel).

Techniques: Immunostaining, Incubation, Blocking Assay, Staining

Journal: eLife

Article Title: TRPV3 channel activity helps cortical neurons stay active during fever

doi: 10.7554/eLife.102412

Figure Lengend Snippet: ( A ) Setup for recording whole-cell TRPV3 currents at 30°C (black), 36°C (gray), and 39°C (red) in cortical excitatory pyramidal neurons (PNs) with bath application of camphor (5 mM), a TPRV3 agonist. ( B ) Current density-voltage (I–V) relationship of TRPV3 currents at 30°C (black), 36°C (gray) and 39°C (red) in wild-type (WT) mice: 11 cells from four mice. ( C ) Scatter dot plots of the current density-voltage measurements. ( D ) Current density-voltage (I–V) relationship of TRPV3 currents at 30°C (black) in the presence of camphor (5 mM), a TPRV3 agonist, or camphor (5 mM) + TRPV3 blocker (Forsythoside B, 50 µM) (blue). ( E ) Same as ( D ) but for 36°C. ( F ) Same as ( D ) but for 39°C. ( G ) Current density-voltage (I–V) plot showing the net TRPV3 current (opener – (opener+blocker) condition). In B - F , statistical significance was assessed using a two-way repeated-measures ANOVA with Tukey’s or Sidak post-hoc test ( α =0.05).

Article Snippet: Slides were blocked in 10% normal goat serum for 1 hr at room temperature, then incubated overnight at 4°C with anti-TRPV3-Biotin antibody (#ACC-033-B), TRPV3 blocking peptide (#BLP-CC033), and/or anti-TRPV4 antibody (#ACC-034) (Alomone Labs, Israel).

Techniques:

Journal: eLife

Article Title: TRPV3 channel activity helps cortical neurons stay active during fever

doi: 10.7554/eLife.102412

Figure Lengend Snippet: ( A ) Setup for recording L4-evoked post-synaptic potential and spiking in an excitatory cortical pyramidal neuron (PN) with an intracellular blocker of TRPV3 channels (Forsythoside B, 50 µM) (left) or TRPV4 channels (RN1734, 10 µM) (right) at just-subthreshold V m at 30°C, 36°C, and 39°C in mouse S1 cortex. ( B ) Percentages of cell types obtained from experiment in A . ( C ) Evoked spikes in L2/3 cortical PNs during temperature elevations to 30°C, 36°C, and 39°C under three conditions: no blockers, TRPV3 blocker (Forsythoside B, 50 µM), or TRPV4 blocker (RN1734, 10 µM). ( D ) Correlation between post-synaptic potential (PSP) peak and spike threshold (ST). r =Pearson correlation coefficient with Deming linear regression. ( E ) Same as ( C ) for the L4-evoked late PSP peak. ( F ) Same as ( C ) for input resistance (R in ). Each data point in C – F represents an individual cell. Data were collected from 26 cells in 7 animals for the TRPV3 blocker, 24 cells in 6 animals for the TRPV4 blocker, and 37 cells in 14 animals for the no-block condition. Mean ± SEM is shown in C , E , and F . Statistical significance was assessed using one- or two-way repeated-measures ANOVA with Tukey’s or Sidak post-hoc test ( α =0.05). In D , correlations were evaluated using Pearson’s r with Deming linear regression.

Article Snippet: Slides were blocked in 10% normal goat serum for 1 hr at room temperature, then incubated overnight at 4°C with anti-TRPV3-Biotin antibody (#ACC-033-B), TRPV3 blocking peptide (#BLP-CC033), and/or anti-TRPV4 antibody (#ACC-034) (Alomone Labs, Israel).

Techniques: Blocking Assay

Journal: eLife

Article Title: TRPV3 channel activity helps cortical neurons stay active during fever

doi: 10.7554/eLife.102412

Figure Lengend Snippet: ( A ) Setup for recording L4-evoked post-synaptic potentials and spiking in excitatory cortical pyramidal neurons (PNs) at just-subthreshold Vm at 30°C, 36°C, and 39°C in mouse S1 cortex of wild-type ( Trpv3 +/+ ) and Trpv3 knockout ( Trpv3 -/- ) mice. ( B ) Depolarization required to reach spike threshold (ST) in Trpv3 +/+ and Trpv3 -/- mice at 30°C, 36°C, and 39°C. ( C ) Same as ( B ) but for input resistance (R in ). ( D ) Same as ( B ) but for number of spikes. ( E ) Same as ( B ) but for post-synaptic potential (PSP). ( F ) Setup for recording mouse body temperature (T b ) at room temperature and during fever-range and higher, using an implanted transponder for non-invasive measurement, with exposure to infrared light. ( G ) Time to loss of postural control (LPC), defined as collapse and failure to maintain upright posture, in wild-type ( Trpv3 +/+ ), heterozygous ( Trpv3 +/- ), and Trpv3 knockout ( Trpv3 -/- ) mice. ( H ) Same as in ( G ) but showing T b at seizure onset. ( I ) Same as in ( G ) but for the time from LPC to seizure onset. Each data point in B – E represents an individual cell (three animals per genotype). Statistical significance was assessed using two-way repeated-measures ANOVA with Tukey’s or Sidak post-hoc test ( α =0.05). Each data point in G – I represents an individual animal. Statistical significance was assessed using one-way repeated-measures ANOVA with Tukey’s or Sidak post-hoc test ( α =0.05).

Article Snippet: Slides were blocked in 10% normal goat serum for 1 hr at room temperature, then incubated overnight at 4°C with anti-TRPV3-Biotin antibody (#ACC-033-B), TRPV3 blocking peptide (#BLP-CC033), and/or anti-TRPV4 antibody (#ACC-034) (Alomone Labs, Israel).

Techniques: Knock-Out, Control

Journal: eLife

Article Title: TRPV3 channel activity helps cortical neurons stay active during fever

doi: 10.7554/eLife.102412

Figure Lengend Snippet: ( A ) In cortical L2/3 pyramidal neurons (PNs) with synaptically evoked spiking, gradual increases in brain slice temperature from 30 °C to 36°C to 39°C result in four possible outcomes: neurons remain inactive, continue spiking (STAY), stop spiking, or initiate spiking. To spike, PNs must reach the spike threshold (ST), defined as the minimal V m that elicits an action potential, which requires sufficient depolarization via the post-synaptic potential (PSP). STAY neurons consistently reach ST and continue spiking. Neurons that stop spiking fall below the level of depolarization required to reach ST, while neurons that initiate spiking achieve sufficient depolarization to newly reach ST. ( B ) STAY neurons may contain unique ion channels, such as TRPV3. TRPV3 channels are highly permeable to Ca² + ions, and Ca² + influx contributes to PN depolarization. The presence of TRPV3 facilitates greater ion entry, enabling depolarization sufficient to reach ST and sustain spiking, whereas its absence reduces depolarization to levels insufficient for spiking.

Article Snippet: Slides were blocked in 10% normal goat serum for 1 hr at room temperature, then incubated overnight at 4°C with anti-TRPV3-Biotin antibody (#ACC-033-B), TRPV3 blocking peptide (#BLP-CC033), and/or anti-TRPV4 antibody (#ACC-034) (Alomone Labs, Israel).

Techniques: Slice Preparation