The firing patterns were recorded from a single cell

The firing patterns were recorded from a single cell over a prolonged period of time. PC activity was identified by the presence of a complex spike (CS) in the discharge pattern, as well as by an inhibitory pause after a complex discharge. CSs emerge due to synaptic activation of PCs by climbing fiber afferents, which leads to the generation of calcium-dependent action potentials in the dendrites, whereas SSs are synaptically activated by parallel fibers, also known as granule-cell phospholipase inhibitor [15]. All the experiments on the extracellular recording of PC activity were performed over a time period of not more than five hours after the last injection of the anesthetic. The detected firings were amplified by a differential amplifier (AC/DC Differential Amplifier, A-M Systems Inc., USA), processed with filters for high (10kHz) and low (100Hz) frequencies, digitized by an NI PCI-6221 analog-digital converter (National Instruments, USA), and stored for further computer analysis. The Bioactivity Recorder v. 5.9 software was used for recording the electrophysiological activity. The characteristics of the firing activity were assessed using the Clampfit v10.3.1.5 software. Subsequent statistical analysis was performed in Origin and MS Excel. The test substances were intravenously injected into the tail vein of the test animal. The solution concentration used was 1mM of СуРРА. The activity was recorded continuously for 5min before the injection, and for 30min after. Short (30s) recordings were also performed 60, 90, 120, 150 and 180min after the intravenous injection. The effect of the modulator was assessed for each cell 30, 60, 90, 120, 150 and 180min after the intravenous injection by a change in SS frequency. A total of 33 cases of PC activity was recorded in the experiments, of which 12 cells were statistically processed, as the recorded firing activity of the rest of the neurons did not persist for three hours after the intravenous injection. Since our experiments were aimed at studying the effect of the SK2/SK3-channel modulator CyPPA, we did not analyze the CS frequency, as it is known that the SK channels are involved in SS generation [8]. In order to analyze the data, we determined the average values of the SS frequency. The data was presented as relative frequencies allowing for the root-mean-square deviation, i.e., in the form (/F0) ± σ, where F0 is the SS frequency value 5min before the injection of the tested substance, is the SS frequency value after the intravenous injection (after 30, 60, 90, 120, 150 and 180min for each cell), and σ is the standard deviation. Next we tested the statistical hypothesis by Pearson\'s χ-squared test (that a series of experimental data has the form of a normal distribution at each moment of time). Statistical processing of the obtained data was performed by one-way analysis of variance (ANOVA) with the subsequent application of the Bonferroni test. We analyzed the effect of the tested substances on the relative frequency of the recorded firing activity of PCs. At any given moment of time we assessed the effect of a 1mM CyPPA injection on the relative SS frequency to determine the effect of a DMSO solution injection on the same factor in normal saline.
Key findings and discussion This study included a series of experiments on the extracellular recording of PC activity of the intact cerebellar cortex carried out in vivo on six-month-old male outbred laboratory mice. The frequency of spontaneous SSs ranged from 15 to 50Hz in most experiments; however, signals with frequencies of 4–90Hz were also registered. Experiments in which pure saline was intravenously injected into the tail vein of the mouse were conducted as controls. A typical example of the recorded pattern of the PC discharge five minutes before the intravenous injection of saline and three hours after it is shown in Fig. 2a (curve 1). The lower part of Fig. 2a shows the time dependence for the moving average of the SS firing frequency of PCs (curve 2). The temporal behavior of the firing frequency was obtained in increments of 60s. Fig. 2b shows the fragments corresponding to the signal recording in the control experiment after 1, 2 and 3h after the intravenous injection of saline. Both simple and complex spikes (indicated by a dot above) were detected in each of the fragments. The moving average curve for the PC firing frequency (curve 2 in Fig. 2a), as well as the enlarged fragments of the activity recordings (Fig. 2b) illustrate a slight decrease in the PC firing frequency. The presence of both simple and complex spikes in the activity recording, as well as of an inhibitory pause after a complex discharge clearly identifies the neuron as a PC.