Article Figures & Data
Figures
- Figure 1.
The distribution of Kv2 channel α and auxiliary subunit immunolabeling in s.p. changes at the CA1-CA2 boundary, and again within region CA3 of mouse hippocampus. A, Representative low-magnification (10×, wide-field mosaic) images of C57BL/6J mouse coronal brain sections immunelabeled for combinations of Kv2.1, Kv2.2, AMIGO-1, and the CA2 marker RGS14. Arrowheads indicate the boundaries of region CA2 based on RGS14 immunolabeling. Scale bar, 500 µm. B, Quantification of mean fluorescence intensity from linescans across s.p. of regions CA1, CA2, and CA3. Values are normalized to the maximum average intensity (n = 8 mice).
- Figure 2.
Individual CA2 PNs have reduced levels of Kv2 channel α and auxiliary subunit immunolabeling compared to CA1 neurons. A, High-magnification (63×) representative images of C57BL/6J mouse coronal brain sections immunolabeled for Kv2.1, Kv2.2, AMIGO-1, and RGS14. Single optical z-section images (ApoTome Zeiss). Scale bar, 35 µm. B, Quantification of mean fluorescence intensity from ROIs corresponding to individual RGS14 + and RGS14 – PNs. Values are normalized to average values in CA1 PNs. (n = 4 mice). Error bars show SEM. Asterisks denote samples exhibiting significant differences (p < 0.001, unpaired t test).
- Figure 3.
Amigo-2/Cre GFP-positive CA2 PNs have reduced levels of Kv2 channel α and auxiliary subunit immunolabeling. A, Representative low-magnification (10×, wide-field mosaic) images of Amigo-2/Cre mice expressing GFP in CA2 PNs. Coronal brain sections from five mice were immunolabeled for Kv2.1, Kv2.2, AMIGO-1, and RGS14. Scale bar, 500 μm. B, High-magnification (63×) representative images of Amigo-2/Cre mice expressing GFP in CA2 PNs. Single optical z-sections images (ApoTome Zeiss) were obtained from five Amigo-2/Cre mice immunolabeled for Kv2.1, Kv2.2, AMIGO-1, and RGS14. Scale bar, 35 μm.
- Figure 4.
GxTX effects on AP characteristics in CA1 PNs. Bath application of 100 µM GxTX altered the AP shape and AHP of CA1 PNs. A, Example traces of an AP in a CA1 PN as recorded in whole-cell current clamp configuration in response to a current injection of 260 pA before (black) and after (red) the application of 100 nM GxTX. B, Summary graph showing the measured AP widths for the 1st and 2nd AP before and after the application of GxTX. Gray symbols are individual cells, black is the mean. C, Summary graph showing the minimum AHP trough potential following the 1st and 2nd AP before and after GxTX application. Gray, individual points, black, mean. Error bars show SEM.
- Figure 5.
GxTX has different effects on CA1 and CA2 PN AP properties. A, Example traces from CA1 and CA2 PNs recorded in whole-cell current clamp mode in response to current injections of different duration and amplitude before (black) and after (gray) the application of 100 nM GxTX. B, Summary graph showing the change in AP width following GxTX application as a function of current injection over threshold for CA1 (red) and CA2 (black) PNs. Data are presented for both the 1st and 2nd AP. C, Summary graph of the change in minimal potential of the AHP trough following GxTX application as a function of current injection for the 1st and 2nd AP.
- Figure 6.
GxTX has different effects on repetitive firing in CA1 versus CA2 PNs. A, Example traces from CA1 and CA2 PNs recorded in whole-cell current clamp mode in response to a 1-s long current injection of 460 pA before (black) and after (orange) the application of 100 nM GxTX. Note how GxTX altered several properties of CA1 PN AP firing but had little to no effect on CA2 PNs. B, Summary graph showing the change in AP width with GxTX application as a function of AP number. Note the consistent change in AP width in CA1 whereas no increase was observed with CA2. C, Summary graph of the change in minimal potential of the AHP trough following GxTX application as a function of AP number. D, The instantaneous frequency of AP firing in CA1 PNs before (open circles) and after (closed circles) the application of GxTX. The block of Kv2 channels significantly increased the instantaneous frequency of the first two APs, but had no significant effect on the subsequent instantaneous firing frequencies. E, The instantaneous frequency of AP firing in CA2 PNs before (open circles) and after (closed circles) the application of GxTX. F, The instantaneous frequency normalized to the first AP, to illustrate the changes in adaptive AP firing for CA1 PNs before (open circles) and after (closed circles) the application of GxTX. Current injection step was around 300 pA over AP threshold. G, Summary graph of the normalized instantaneous frequency for CA2 PNs before (open circles) and after application of GxTX (filled circles) as a function of AP number. Current injection step was 500 pA over AP threshold. Error bars show SEM.
Tables
Antibody name Species/isotype/immunogen Manufacturer information Concentration used AMIGO-1, anti-AMIGO-1 rabbit pAb Raised against aa 394–492 of mouse AMIGO-1 (cytoplasmic C-terminus). Trimmer Lab. Rabbit 28330 RRID:AB_2571515 1:400 dilution of affinity purified pAb, concentration unknown L98/12, anti-AMIGO-1 mouse IgG1 mAb Raised against aa 28–370 of mouse AMIGO-1 (extracellular N-terminus). Trimmer lab.
RRID:AB_25715161:3 dilution of tissue culture supernatant, concentration unknown K89/34, anti-Kv2.1 mouse IgG1 mAb Raised against aa 837–853 of rat Kv2.1. Trimmer lab. NeuroMab catalog 73-014
RRID:AB_106722535 µg/ml purified mAb L61C/30, anti-Kv2.1 mouse IgG1 mAb Raised against aa 595–616 of rat Kv2.1. Trimmer lab. RRID:AB_2532100 1:5 dilution of tissue culture supernatant, concentration unknown N372B/1, anti-Kv2.2 mouse IgG1 mAb Raised against aa 717–907 of rat Kv2.2. Binds within aa 764–907. Species reactivity with mouse, rat, ferret, macaque and human NeuroMab catalog 73–369, RRID:AB_2315869 1:3 dilution of tissue culture supernatant, concentration unknown N372B/60, anti-Kv2.2 mouse IgG2b mAb Raised against aa 717–907 of rat Kv2.2. Binds within aa 764–907. Species reactivity with mouse and rat NeuroMab catalog 73–360, RRID:AB_2315867 1:10 dilution of tissue culture supernatant, concentration unknown N372C/51, anti-Kv2.2 mouse IgG1 mAb Raised against aa 717–907 of rat Kv2.2. Binds within aa 717–763 Species reactivity with mouse and rat NeuroMab catalog 73–358, RRID:AB_2315865 1:2 dilution of tissue culture supernatant, concentration unknown N133/21, anti-RGS14 mouse IgG2a mAb Raised against aa 1–544 of rat RGS14. NeuroMab catalog 73–170, RRID:AB_10698026 1:10 dilution of tissue culture supernatant, concentration unknown Details of the polyclonal (pAb) and monoclonal (mAb) Abs used in this study.
PN type RMP (mV) RM (MΩ) CM (pF) CA1 (N = 6) −70.2 ± 1.3 84.8 ± 13.8 151.4 ± 38.6 CA2 (N =5) −73.1 ± 1.6 49.1 ± 3.7 316.3 ± 56.3
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