Circuitry of structures implicated in VOR and its calibration. Integration of vestibular and visual inputs in the floccular complex modulates PC outputs. Floccular target neurons in the MVN are partitioned depending on the density of innervations received from the flocculi (thin or thick lines; density of synaptic contacts), on their neurotransmitter content, and projection sites. 1°VN and 2°VN, first- and second-order vestibular neuron; MN, motoneuron; CF, MF, and PF, climbing, mossy, and parallel fiber; GC, granule cell; Abd and Omn, abducens and oculomotor nucleus.
VVM protocol and open-field experiments. A, Pictures of a mouse during VVM (top) or sham (bottom) protocols. B, Mean body weight of sham (n = 24, gray line) and VVM mice (n = 57, red) during the 2 weeks of the protocol. C, Locomotion of mice before VVM (top) or after 5 d of VVM protocol (bottom) recorded while the animal explores the open field. Left panels, examples of 3D reconstruction of the path of the same animal. Right panels, distribution of velocities for the population of mice (n = 4) before VVM (blue) or after 5 d of VVM (red). D, Plots of the mean velocity (Vmean, in cm/s), total covered distance (in cm), and vertical explorations (number of rearings) of mice (n = 4) before and after 5 d of VVM. Error bars represent ± SEM.
VOR reduction. A, Illustration of the setup used to test the VOR. IR, infrared light. B, Example raw traces of the VOR in the dark recorded before (blue line) and after 2 weeks of VVM (red line) from the same animal. Gray trace, sham mouse tested after 2 weeks of wearing the helmet. White arrows indicate example of quick phases observed after VVM. Head and eye traces show rightward movements in the upward and downward directions, respectively. C, Mean VOR gain (left) and corresponding phase (right) plotted as a function of tested velocity (n = 13 mice; fixed frequency of 0.5 Hz) measured before (blue lines) and after 2 weeks of VVM (red lines). Insets: sham (n = 6 mice) before (filled squares) and after (empty squares) the protocol. D, Occurrence (left) and amplitude (right) of quick phases (n = 10 mice). E, Quick-phase amplitude ratio (after/before values) is significantly correlated to slow-phase gain ratio (***p < 0.001). F, Example raw traces of the VOR reduction at 0.2 or 2Hz stimulation. G, Mean percentage of gain decrease (left, n = 12) or phase change (right, n = 12) depending on stimulating frequencies. The gray triangles represent the individual values, and the black lines represent the mean values. Error bars represent ± SEM.
Flocculi shutdown experiments. A, Left, coronal section of the brainstem and cerebellum illustrating the lidocaine injections in flocculi complex. PF, paraflocculus; Fl, flocculus; PLF, posterolateral fissure; 4V, 4th ventricle; BS, brainstem. Right, example of a lidocaine injection coupled to fluorescein isothiocyanate. Dors, dorsal; Vent, ventral; Med, medial; Lat, lateral. B, Left, example raw traces of VOR in the dark recorded before (blue line), after 2 weeks of VVM (red line), and after flocculi shutdown (black line). All traces are from the same animal. Right, Bode plots of VOR gain and phase (n = 5 mice). C, Left, example raw traces of eye movements recorded during optokinetic stimulation (60s-long full-field stimulation at 7.5°/s constant velocity). All traces are from the same animal. Right, mean OKR gain recorded before, after VVM, after lidocaine injection, or on sham animals. Error bars represent ± SEM.
Stimulation of afferents: vestibular synapses efficacy. A, Left, illustration of in vitro patch-clamp recordings of MVN neurons on coronal brainstem slice. The stimulating electrode is placed on the central vestibular fiber bundles. PF, parafloccular region; 4V, 4th ventricle. Right, example raw traces of superimposed eEPSC recorded from a MVN neuron of a control mouse (blue line) and from a mouse after VVM (red line). B, Top, evoked EPSCs AUC (in fC), time constant (τ, in ms), and amplitude (in pA) recorded from control neurons (n = 17) or after VVM (n = 31). Bottom, distribution of eEPSC amplitude for control (blue bars) and VVM (red bars) conditions. Arrows and dashed lines indicate the medians (∼135 pA for control and ∼85 pA for VVM). C, Plasticity of vestibular nerve synapses onto MVN neurons. Left, eEPSC amplitude recorded on control (n = 8) and VVM (n = 14) neurons before (filled bars) and after (empty bars) LTD protocol. Right, mean eEPSC peak amplitude before and after LTD protocol for control (blue line) and VVM (red line) neurons. The eEPSC values are normalized to the mean baseline value before LTD protocol (filled circles). Empty circles represent measures following LTD protocol. Error bars represent ± SEM.
Excitability of second-order vestibular neurons in response to step-like currents. A, Example raw traces of MVN neurons recorded in control (top) type A (left) and type B (right), or VVM (bottom) conditions. B, Relation between the injected current and the frequency of discharge (I/F curves) for all MVN neurons (left, n = 38 control and n = 24 VVM), type A (middle, n = 23 control and n = 7 VVM) and type B (right, n = 15 control and n = 17 VVM) neurons. (***p < 0.001).
Parameters of the spontaneous activity of MVN neurons recorded on control and VVM slices. Static parameters are based on the analysis of the pacemaker discharge of the neurons and the quantification of the AHP and interspike interval. Neurons are pooled (all) or segmented in type A and type B. Overall, the intrinsic membrane properties recorded in controls and VVM mice show limited differences between type A and type B neurons. AHPR, dAHP, and AHP are parameters representing kinetics of the AHP and interspike interval (see Materials and methods). Statistically significant differences are highlighted in red. NS, not significant.