Inactivation of calcium-binding protein genes induces 160 Hz oscillations in the cerebellar cortex of alert mice

Abstract

Oscillations in neuronal populations may either be imposed by intrinsically oscillating pacemakers neurons or emerge from specific attributes of a distributed network of connected neurons. Calretinin and calbindin are two calcium-binding proteins involved in the shaping of intraneuronal Ca2+ fluxes. However, although their physiological function has been studied extensively at the level of a single neuron, little is known about their role at the network level. Here we found that null mutations of genes encoding calretinin or calbindin induce 160 Hz local field potential oscillations in the cerebellar cortex of alert mice. These oscillations reached maximum amplitude just beneath the Purkinje cell bodies and are reinforced in the cerebellum of mice deficient in both calretinin and calbindin. Purkinje cells fired simple spikes phase locked to the oscillations and synchronized along the parallel fiber axis. The oscillations reversibly disappeared when gap junctions or either GABA(A) or NMDA receptors were blocked. Cutaneous stimulation of the whisker region transiently suppressed the oscillations. However, the intrinsic somatic excitability of Purkinje cells recorded in slice preparation was not significantly altered in mutant mice. Functionally, these results suggest that 160 Hz oscillation emerges from a network mechanism combining synchronization of Purkinje cell assemblies through parallel fiber excitation and the network of coupled interneurons of the molecular layer. These findings demonstrate that subtle genetically induced modifications of Ca2+ homeostasis in specific neuron types can alter the observed dynamics of the global network.

Figure 7.

Neuropharmacological regulation of the LFPOs in Cr^-/-Cb^-/- mice. A, Effect of SR95531 on LFPOs. Raw LFP recordings (top), WTA-resulting traces (bottom), and their power spectra (right insets) are shown before and 2 min after the microinjection. FFT, Fast Fourier transform. B, Maximal amplitude of the power of the WTA oscillation before and 2, 5, 10, and 60 min after microinjection of saline, bicuculline, APV, and carbenoxolone (carbenox) (values are expressed as a percentage of values before microinjection) (ANOVA; ^★p < 0.0001).

Figure 1.

Emergence and spatial coherence of high-frequency oscillations in the cerebellum of Cr^-/-Cb^-/- mice. A, Sample records of LFPOs from wild-type mice (wt; top trace) and Cr^-/-Cb^-/- mice (bottom trace). B, Spatial coherence of LFPOs was analyzed by recording simultaneously from electrodes aligned along the longitudinal [left, tracts 2, 1, 3 (0.5 mm apart)] or rostrocaudal (right, tracts 4, 1, 5) axis of a folium. Pair traces (left), using LFPO recording 2 as a trigger (Trig) for a WTA, show coherent oscillations of the same period and without any significant phase delay, whereas for pair traces (right), using channel 1 as a trigger, no oscillatory pattern was visible. C, Depth profile (Ca-Cf) of WTA traces throughout the last PC layer of lobule 10 indicates that the maximum amplitude was found just beneath the PC bodies. The arrow indicates an electrolytic lesion at the recording depth of the WTA trace (Cb). Scale bar, 0.2 mm. The dashed line represents the recording electrode tract.

Figure 2.

PC rhythmicity in alert WT and in Cb^-/-Cr^-/- mice. A, B, SS autocorrelograms for one representative WT (A) and one Cb^-/-Cr^-/- (B) PC demonstrating increased rhythmicity in the mutant. C, D, Cross-correlograms of SSs from PC pairs multirecorded along a PF beam (0.5 mm apart) in WT mice (C) and Cb^-/-Cr^-/- mice (D), demonstrating PC synchronization in the mutant.

Figure 3.

Temporal relationships between LFPOs and PC cell firing. Simultaneous recordings by the same electrode of an isolated PC and a 166 Hz LFPO were made. Shown is the superimposition of single traces (n = 6) (top). The WTA of the LFPO using PC SSs as a trigger (n = 1000) (middle) is displayed. An SS autocorrelogram, with a central peak truncated (bottom), has the same rhythmicity as the WTA trace. The dashed lines indicate the correspondence between the depth of the first two side waves and the first two side peaks of the SS autocorrelogram.

Figure 4.

Temporal relationships between LFPOs and PC cell firing. The same procedure as in Figure 3 was performed with the trigger adjusted on the CS of the same PC. Superimposition of unaveraged traces (n = 6) confirmed the recurrent occurrence of the CS in the ascending phase of the LFP oscillation (top). The WTA shows the presence of 166 Hz oscillation around the CS (middle). The SS cross-correlogram has the same rhythmicity as the WTA trace. The dashed lines indicate the correspondence between the depth of the first two side waves and the first two side peaks of the SS autocorrelogram.

Figure 5.

Temporal relationships between LFPOs and Golgi cell (Gc) firing. A, Single trace recordings of Gc spikes and LFPO. B, Quantitative relationship between LFPO amplitude and Gc interspike intervals demonstrates a Gc firing-associated suppression of LFPO (ANOVA; ^★p < 0.00001). Arrows indicate selected Golgi spike intervals.

Figure 6.

LFPO, PC firing, and EMG responses during cutaneous stimulation of the whisker region. A, B, An air puff is produced and evokes suppression of LFPO, PC firing response, and EMG burst (only in A). LFPO recording is filtered with a low-pass digital filter (200 Hz). Arrows indicate the extent of LFPO suppression. The dashed lines indicate stimulus onset. C, D, Peri-event histograms of SS frequency for two representative PCs excited (C) or inhibited (D) by air puff stimulation. Arrows indicate stimulus onset.