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. 2021 Nov 1;10:e64695. doi: 10.7554/eLife.64695

Figure 4. The biophysical properties of PNs are significantly perturbed in AtmR35X/R35X; Aptx−/− mice.

(A) Schematic diagram of intracellular recording from a single Purkinje neuron (PN) in an acute cerebellar tissue slice preparation used to examine their physiological properties. (B) Left: Voltage-clamp measurements of PN membrane properties made from a 1 s, –5 mV step pulse as illustrated. Right: The membrane input resistance (Rm), time constant (τ), and capacitance (Cm) were perturbed in AtmR35X/R35X; Aptx−/− compared to Atm+/+; Aptx+/+ mice. (C) Current-clamp recordings of PN action potentials (APs) after 2 nA step pulses from a –70 mV holding potential. PN APs recorded from AtmR35X/R35X; Aptx−/− fail to maintain constant firing and summary plots show that they have lower 1st AP amplitudes, firing threshold, and area under the curve. (D) Top: Example sEPSC traces taken from a PN under voltage clamp at a –80 mV holding potential. Bottom: Median frequency and amplitude data, along with the overall probability distribution function are plotted for both Atm+/+; Aptx+/+ (n=11) and AtmR35X/R35X; Aptx−/− (n=11) mice. The frequency but not amplitude of PNs recorded in AtmR35X/R35X; Aptx−/− mice was found to be perturbed. (E, F) Left: Example traces of evoked EPSCs recorded from PNs as a result of a two-pulse stimulation (50 ms interval) of either parallel (E) or climbing (F) fiber axons. Traces illustrate the first (A1) and second (A2) amplitude (normalized) and time course of decay (blue fitted line) of each synaptic response. Right: Summary plots of the paired-pulse ratio. While parallel fiber paired-pulse facilitation was normal in AtmR35X/R35X; Aptx−/− mice, climbing fiber paired-pulse depression and halfwidth was significantly perturbed compared to Atm+/+; Aptx+/+ mice. (G) Schematic diagram of extracellular recording from a single PN in an acute cerebellar tissue slice preparation. Example electrophysiological traces for Atm+/+; Aptx+/+ (purple, top) and AtmR35X/R35X; Aptx−/− (orange, bottom) PNs in the medial area (i.e., vermis) of the cerebellum. (H) AtmR35X/R35X; Aptx−/− PN AP firing frequency progressively decreased with age and was significantly slower in comparison to all control genotypes expressing at least one copy of the Atm or Aptx gene [Atm+/+; Aptx+/+ (n=52–59), Atm+/+; Aptx−/− (n=51–64), AtmR35X/R35X; Aptx+/+ (n=39–52), AtmR35X/R35X; Aptx−/− (n=24–71), AtmR35X/+; Aptx−/− (n=69)]. Data in (B) were compared using an ANOVA (Kruskal-Wallis) followed by Dunn’s multiple comparisons test, data in (D–F) were compared via Welch’s t-test, and data in (H) using a two-way ANOVA followed by Holm-Šídák’s multiple comparisons test. Symbol/color key: Atm+/+; Aptx+/+ (purple circle), Atm+/+; Aptx−/− (blue diamond), AtmR35X/R35X; Aptx+/+ (green triangle), AtmR35X/R35X; Aptx−/− (orange square), AtmR35X/+; Aptx−/− (red inverted triangle). sEPSC, spontaneous excitatory postsynaptic current.

Figure 4—source data 1. Numerical data of electrophysiological recordings for each panel.

Figure 4.

Figure 4—figure supplement 1. Current versus voltage responses significantly differ between Atm+/+; Aptx+/+ and AtmR35X/R35X; Aptx−/− mice.

Figure 4—figure supplement 1.

(A) PN voltage responses to various current steps between –500 and 2250 pA (250 pA steps) from a –70 mV holding current in Atm+/+; Aptx+/+ (top, purple) and AtmR35X/R35X; Aptx−/− (bottom, orange) mice. (B) I‒V curves calculated from either max deflection (Vm max) or steady state (Vm end) for Atm+/+; Aptx+/+ (purple) and AtmR35X/R35X; Aptx−/− (orange) mice. (C) Various measurements of the voltage response to –500 pA step pulse (blue box in B) in Atm+/+; Aptx+/+ (purple) and AtmR35X/R35X; Aptx−/− (orange) mice. Significance was tested using a non-parametric Mann-Whitney test. PN, Purkinje neuron.
Figure 4—figure supplement 2. Mean PN firing frequency across the cerebellum.

Figure 4—figure supplement 2.

Average PN firing frequency (Hz) is plotted across the indicated locations at P45, P120, P210, and P400. Significance tested via two-way ANOVA with age and genotype as factors. PN, Purkinje neuron.
Figure 4—figure supplement 3. Mean PN firing frequency across genotype and sex.

Figure 4—figure supplement 3.

Average PN firing frequency (Hz) for all cells recorded from male and female mice is plotted for the indicated genotype. No significant differences were observed between sex. Two-way ANOVA with age and sex as factors, Atm+/+; Aptx+/+ (F(1, 751)=1.15, p=0.3), Atm+/+; Aptx−/− (F(1, 797)=1.10, p=0.3), AtmR35X/R35X; Aptx+/+ (F(1, 630)=0.17, p=0.7), AtmR35X/R35X; Aptx−/− (F(1, 666)=1.10, p=0.4), t-test for P400 AtmR35X/+; Aptx−/− (p=0.9). PN, Purkinje neuron.
Figure 4—figure supplement 4. Coefficient of variation (CV) of PN firing frequency across the cerebellum.

Figure 4—figure supplement 4.

Average CV of PN firing frequency is plotted across the indicated locations at P45, P120, P210, and P400. No significant differences (p<0.5) were detected across all areas using two-way ANOVA with age and genotype as factors. PN, Purkinje neuron.
Figure 4—figure supplement 5. Mean variation between PN firing intervals across the cerebellum.

Figure 4—figure supplement 5.

Average CV2 of PN firing frequency is plotted across the indicated locations at P45, P120, P210, and P400. No significant differences (p<0.5) were detected across all areas using two-way ANOVA with age and genotype as factors. PN, Purkinje neuron.