In vivo analysis of the spontaneous firing of cerebellar Purkinje cells in awake transgenic mice that model spinocerebellar ataxia type 2

Abstract

Cerebellar Purkinje cells (PCs) fire spontaneously in a tonic mode, although the precision of this pacemaking activity is disturbed in many abnormal conditions involving cerebellar atrophy, such as many spinocerebellar ataxias (SCAs). In our previous studies we used the single-unit extracellular recording method to analyze spontaneous PC firing in vivo in the anesthetized SCA2-58Q transgenic mice. We realized that PCs from aging SCA2-58Q mice fire much less regularly compared to PCs from their wild type (WT) littermates and this abnormal activity can be reversed with an intraperitoneal (i. p.) injection of SK channel-positive modulator chlorzoxazone (CHZ). Here we used the same single-unit extracellular recording method to analyze the spontaneous firing in vivo in awake SCA2-58Q transgenic mice. For this purpose, we used the Mobile HomeCage (Neurotar, Finland) floating platform to immobilize the experimental animal's head during the recording sessions. We discovered that generally PCs from awake animals fired much more frequently and much less regularly than previously observed PCs from anesthetized animals. In vivo recordings from awake SCA2/WT mice revealed that complex spikes, which are generated by PCs in reply to the excitation coming by climbing fibers, as well as simple spikes, were much less frequent in SCA2 mice compared to their WT littermates. To test the effect of the SK channel positive modulation on the PCs firing activity in awake SCA2 mice and also the effect on their motor coordination, we started the CHZ trial in these mice. We discovered that the long-term i. p. injections of CHZ did not affect the spike generation in SCA2-58Q mice, however, they did recover the precision of this spontaneous pacemaking activity. Furthermore, we also showed that treatment with CHZ alleviated the age-dependent motor impairment in SCA2-58Q mice. We propose that the lack of precision in PC spike generation might be a key cause for the progression of ataxic symptoms in different SCAs and that the activation of calcium-activated potassium channels, including SK channels, can be used as a potential way to treat SCAs on the physiological level of the disease.

Keywords: Cerebellum; Chlorzoxazone; In vivo electrophysiology; SK channels; Spinocerebellar ataxia; Transgenic mice.

Figure 1.. Spontaneous activity of cerebellar PCs in 6- and 9-month-old WT and SCA2-58Q mice.

(A, B) Examples of PC firing activity in 9-month-old WT (A) and SCA2 (B) mice. Recording traces are 3 s in duration with enlarged 100-ms fragment containing CS is shown. (C) The average SS firing frequency of PCs for 6- and 9-month-old WT (n = 7 and 11 cells; m = 4 and 3 mice) and SCA2 mice (n = 6 and 11 cells; m = 3 and 4 mice) is shown as mean ± SE. * p < 0.05. (D) The average CS firing frequency of PCs for 6- and 9-month-old WT (n = 7 and 11 cells; m = 4 and 3 mice) and SCA2 mice (n = 6 and 11 cells; m = 3 and 4 mice) is shown as mean ± SE. * p < 0.05. (E) The average post-CS pause in PC discharge for 6- and 9-month-old WT (n = 7 and 11 cells; m = 4 and 3 mice) and SCA2 mice (n = 6 and 11 cells; m = 3 and 4 mice) is shown as mean ± SE. * p < 0.05. (F) The CV ISI of cerebellar PCs was analyzed. Spontaneously firing PCs in 9-month-old SCA2 mice fired more irregularly. * p < 0.05.

Figure 2.. Long-term injections of 30 mg/kg CHZ alleviate the regularity in the firing activity of 12-month-old SCA2-58Q PCs.

(A) Examples of typical PC activity in 12-month-old WT and SCA2 mice in control and CHZ groups are given. Recording traces 2 s in duration are shown. Complex spikes are marked by filled circles. (B) The average SS firing frequency was significantly less in 12-month-old SCA2-58Q mice compared to WT mice and did not recover after treatment with CHZ. * p < 0.05. (C) The average CS firing frequency was significantly reduced in 12-month-old SCA2-58Q mice compared to WT mice and was recovered after treatment with CHZ. * p < 0.05. (D) The average post-CS pause in PC discharge was significantly higher in 12-month-old SCA2-58Q mice compared to WT mice and was recovered after treatment with CHZ. ** p < 0.01. (E) CHZ injections recover the precision of the PC pacemaker activity in 12-month-old SCA2-58Q mice. At 12 months of age, the systematic CHZ injections decreased the firing variability (E) in SCA2-58Q mice. * p < 0.05. For all the measured properties number of recorded cells (n) and number of animals (m) were following: n = 12 cells for WT mice in the control group (WT Ctrl), m = 4 WT Ctrl mice; n = 12 cells for WT mice injected with CHZ, m = 4 WT CHZ mice; n = 8 cells for SCA2-58Q mice in the control group (SCA2 Ctrl), m = 3 SCA2 Ctrl mice; n = 7 cells for SCA2-58Q mice injected with CHZ, m = 3 SCA2 CHZ mice.

Figure 3.. CHZ treatment improves performance of SCA2-58Q mice in the beam-walk test.

The average mean latency as the mice traverse the 1 meter length of the 18 mm (A), 12 mm (C), and 8 mm (E) beam is plotted for the 2, 4, 6, 8, 10, and 12-month-old WT and SCA2-58Q mice in control and CHZ treatment groups as mean ± SE. Average number of foot slips as the mice traverse the 1 meter length of the 18 mm (B), 12 mm (D), and 8 mm (F) beam is plotted for the 2, 4, 6, 8, 10, and 12-month-old WT and SCA2-58Q mice as mean ± SE. * p < 0.05; ** p < 0.01; *** p < 0.001 significant differences between control WT group and control SCA2-58Q group.