Characterization Of The Facial Stimulation-Evoked Field Potential Responses In Cerebellar Granule And Molecular Layer In Vivo In Mice

Sensory information transferred into cerebellar cortex via climbing and mossy fibers, which regulates the cerebellar neuronal activities, involves in motor regulation and motor learning. However, the synaptic properties of sensory information processed in the cerebellar cortex are not so clear. Therefore, we studied the characterization of cerebellar molecular layer and granule cell layer in response to sensory stimulation of trigeminal nerve. We aimed to understand the dynamic properties and the pharmacological mechanisms of the facial stimulation evoked local field potential in cerebellar granule cell layer and molecular layer in vivo in mice.Following6-8-week old Kunming mice were anesthetized by urathane (1.3g/kg body weight) intraperitoneal injection, a craniotomy of1-1.5mm in diameter were opened to expose the surface of cerebellar cortex lobule Crus II. After carefully removed the dura, and the surface of cerebellum was perfuse using oxygen bubbled ACSF artificial cerebrospinal fluid. Facial stimulation was carried by air-puff (5-500ms,50-60psi) of ipsilateral whisker pad. The air-puff stimulation was controlled by a personal computer and was also synchrinized with the electrophysiologial recording. Local field potentials of both granule cell and molecular layers were induced by the air-puff stimulation on ipsilateral whisker pad. The electrodes for recording were filled with ACSF (20-30μl). The resistance of recording electrode was4-6MΩ.. Facial stimuli evoked the responses of local field potential in cerebellar granule cell and molecular layer were recorded using an amplifier (Axopatch-200B, Molecular devices) and acquired through the software of clampex (version,10.3). The data of electrophysiological recording were analyzed by software of clampfit. All of the data were expressed by Mean±S.E.M. The field potential recordings’s number was indicated by n. The difference of the mean values between the control and experimental conditions were examined by Student’s paired t-test using the SPSS software (version16.0). When P less than0.05, which was considered as significant differences.The data of the present study is showed that,(1) Under conditions of urathane anesthetizzing, facial stimuli of ipsilateral whisker-pad induced responses of local field potential in lobule Crus II molecular layer of cerebellar cortex, which exhibited a N1, negative component, followed by a P1, big positive component. The mean amplitude of N1and P1are0.13±0.06mV and0.36±0.08mV, respectively. The mean delay value is13.21±0.32ms. The stimulation off could evoke field potential responses (P2) in molecular layer, but required the duration of the air-puff stimulation over than60ms.(2) GABAA receptor antagonist not only blocked completely P1, but also reversed P1to another negative wave (N2), indicated that Nl was the parallel fiber excitatory input and P1was the inhibitory responses of PC. Blockade of GABAA receptor activity, the sensory stimulation evoked excitation of PC which expressed N2.(3) Air-puff stimulation of ipsilateral whisker-pad evoked field potential responses in granule cell layer. When the stimulation duration was5-10ms, the evoked responses expressed single peak. When the stimulation duration>30ms, the stimulation evoked double-peak responses. The first peak is stimulation-on response, which expressed "all or none" and the mean amplitude of N1was0.82±0.06mV. The second peak is stimulation-off response, which became stronger with the increase in stimulation duration.(4) Cerebellar cortical granule cell layer expressed high frequency properties in response to sensory stimulation. Sensory stimulation train at1-33Hz can evoke granule cell layer responses. However, with the increase of the stimulation frequency, the responses expressed different degree of inhibition after the first stimulation. When the stimulation frequency was>4Hz, the peak of second response was significant decrease.(5) The sensory stimulation evoked responses in granule cell layer was completely blocked by application of synaptic transmission blocker, TTX. The amplitude of N1was decreased by4.6±1.31%of control.The results of the present study are indicated that,The granule cell layer expresses high frequency and fidelity during the sensory information transmission, which can exactly present the information of stimulation-on and stimulation off, but expresses high frequency response inhibition. The molecular layer is insensitive to high frequency stimulation and expresses poor fidelity than granule cell layer, indicated that he molecular layer plays important roles in information integration and delay of cerebellar cortex.