Experiments on the intravenous administration

Experiments on the intravenous administration of 1mM of CyPPA showed that this SK channel activator is capable of modulating the electrophysiological properties of PCs in vivo. A typical example of recording the PC discharge pattern before and after the intravenous injection of 1mM of CyPPA into the tail vein of a six-month-old mouse is shown in Fig. 3. Curve 1 in Fig. 3a illustrates the pattern we observed for five minutes before and three hours after the intravenous injection of 1mM of CyPPA. The lower part of Fig. 3 presents the moving average frequency of SS firing by the PC (curve 2), the plot corresponds to the respective entry for PC activity. The values ​​of the firing frequency were calculated in increments of 60s. Fig. 3b shows the fragments corresponding to the signal recording in the control experiment 1, 2 and 3h after the intravenous injection of 1mM of CyPPA. The graphical representation of the moving average of the firing frequency (curve 2 in Fig. 3a), as well as the enlarged fragments of the activity recording (Fig. 3b) illustrate the significant decrease in the frequency of SS firing by the Purkinje cell. For example, three hours after the intravenous injection of 1mM of CyPPA, SS firing was observed to decrease by about 7 times (curve 2 in Fig. 3a). Fig. 4 presents the generalized data obtained in the above-described experiments. For example, it was shown that a slight increase in the SS firing frequency (about 10–11%) was observed during the first hour after the intravenous injection of saline (n=3), a slight decrease in the firing frequency relative to the initial value (by about 2–7%) was observed during the second hour after injection, while during the third hour after the intravenous injection of 0.9% NaCl, a further decrease in the firing frequency was observed, amounting on average to 25%. Our experiments also investigated the effect of the selective SK3/SK2 cAMPS-Rp, triethylammonium salt modulator СуРРА in a concentration of 1mM. These experiments revealed two types of PC reactions in response to the injection of the activator. In the first case, there was a progressive decrease in the frequency of SS firing by the PC, while in the second one there were no significant changes in the electrophysiological properties of the PC. According to our assumptions, the lack of reaction to the CyPPA injection was because the syringe needle missed the tail vein (the number of failed experiments n=3). In connection with this, the analysis was performed only for the first case, i.e., for successful intravenous injections of 1mM of CyPPA (see Fig. 4 for n=6). Statistical analysis of the obtained experimental data revealed that there was no change in the SS frequency during the first 30min after the intravenous injection of 1mM of CyPPA; however, as early as 60min after the injection of the activator, a decrease by 16% relative to the initial value was observed in the firing frequency (see Fig. 4). After that there was a progressive decrease in the SS firing frequency. For example, an average decrease by 32% relative to the initial value was observed in the SS frequency 90min after the CyPPA injection, by 49% after 120min, by 50% after 150min, and by 61% after 180min (see Fig. 4). Statistical analysis of all data has demonstrated (see Fig. 4) that the effect of CyPPA on the PC activity is reliably different from the effect of normal saline as early as 90min after the time of injection (the reliability was 95%) and continues to differ with the same reliability up to the end of the analyzed three-hour period. After three hours the relative SS frequency following the intravenous injection of saline averages 0.75, whereas the value of this parameter after the same time interval after the injection of CyPPA is 0.39 relative units (see Fig. 4).
Conclusion This study presented the experimental results for the intravenous delivery of a positive SK channel modulator СуРРА. SK channel activation is a potential method for therapeutic treatment of cerebellar ataxias [6, 10–12]. The chosen delivery method has demonstrated that СуРРА molecules are capable of controlling PC activity in vivo, directly or indirectly, through interacting with other metabolic intermediaries. These results are incredibly important because injecting a potential therapeutic agent intravenously is more acceptable for clinical trials than the methods previously used in similar experiments assessing the effect of SK channel modulators on PC activity in vivo [13].