Figure 2. Oscillatory burst firing varies across TRN sectors and depends on Ca_V3.3 Ca²⁺ channels.

(A) Representative traces of paired EPSCs in whole-cell voltage-clamped TRN neurons at −60 mV of WT and Ca_V3.3 KO mice upon light activation of S1 (top), AC (middle) and MD (bottom) afferents. Afferent-specific forms of short-term plasticity are preserved across genotypes (see Results for averaged data). (B) Box-and-whisker-plots of the mean capacitance (C_m) and the resting membrane potential (V_RMP) of the recorded TRN neurons (WT: n = 12 for S1, n = 13 for AC, n = 14 for MD; Ca_V3.3 KO: n = 12 for S1, n = 13 for AC, n = 11 for MD). It can be noted that the C_m of S1-innervated TRN neurons in Ca_V3.3 KO showed a reduction compared to WT, suggesting smaller cell size (Mann-Whitney test, p=0.007). (C) Representative current-clamp recordings of oscillatory bursting responses of TRN neurons across sectors, induced through hyperpolarizing current injections (−50 to −300 pA for 500 ms). Horizontal lines denote −60 mV. Note the strong repetitive burst firing in sensory sectors that is impaired in the Ca_V3.3 KO cells, whereas MD-innervated cells mostly discharge a single burst. (D) Graph of the number of repetitive bursts as a function of the membrane potential prior to the hyperpolarizing pulse (Cueni et al., 2008). This yields a U-shaped curve reaching a peak at −65 and −60 mV in all sectors of WT mice (D1) that was abolished in Ca_V3.3 KO mice (D2). Dashed horizontal lines at ordinate value one indicate the border between repetitive and not-repetitive bursting conditions. (E) Mean number of repetitive bursts of TRN neurons (between −60 and −65 mV) across sectors and genotype. Mann-Whitney tests were used for comparison between genotypes, and p-values are given above the bars. (F) Histogram of the proportion of repetitive (colored rectangles) and non-repetitive bursting (grey rectangles with color surroundings) TRN neurons in the different sectors. Chi-square test followed by pairwise proportion test with Holm’s p-value adjustment was used for statistical evaluation, with significant value given above the bars.

Figure 2—figure supplement 1. Oscillatory burst firing varies across the anteroposterior extent of TRN and depends on Ca_V3.3 Ca²⁺ channels.

(A) Epifluorescent micrograph of horizontal brain sections (A1, WT; A2), Ca_V3.3 KO mouse) showing the TRN’s anteroposterior extent (magenta, PV-staining, PV+) and two biocytin-filled neurons (red, biocytin+) recorded through in vitro whole-cell patch-clamp in anterior and posterior locations. Squares defined by dashed-lines on the left micrograph (5x) are shown expanded on the right (10x). (B) Left: Representative traces of oscillatory burst firing of TRN neurons in response to hyperpolarizing current injections (left, WT; right Ca_V3.3 KO) in the posterior/sensory sector of the TRN (blue) and in the anterior/limbic sector (red). (C) Inverted U-shaped voltage-dependence of burst firing for posterior (WT, n = 4; Ca_V3.3 KO, n = 6) and anterior (WT, n = 4; Ca_V3.3 KO, n = 6) cells. Neurons in the posterior location of WT cells showed more repetitive burst discharge between −65 and −60 mV than anterior neurons (posterior: 4.4 ± 0.8, anterior: 1.7 ± 0.3, Mann-Whitney test, p=0.042). Ca_V3.3 KO mice showed an abolition of repetitive bursting in both locations in comparison to WT (posterior: 1.0 ± 0.0, anterior: 0.3 ± 0.2, Mann-Whitney tests, p=5.7×10⁻⁴ for posterior, p=0.019 for anterior).