Capítulo 1: Análisis de la expresión génica relativa de genes codificantes de lacasas de
1.2. Optimización de la qPCR:
1.3.2 Cuantificación de la expresión por el método 2 ∆ Ct
The first strategy employed to block T-type Ca2+ channels in the nRT involved increasing the concentration of TTA-P2 administered by microdialysis to the centre of the VB from 300μM to 1mM. This increased the distance from the microdialysis membrane at which a 90% block of burst firing was achieved at 1 hour post-drug from 600μm to 750μm. This change resulted in the theoretical volume of effective channel block at 1 hour including the majority of the nRT as well as the VB (see Fig. 2.5).
3
Post-drug is used in this chapter, for the sake of brevity, to refer to the hour during which dialysis was affecting the target area.
Figure 4.1 300μM TTA-P2 in the central VB doesn’t suppress absence seizures A Total time spent in seizure, B mean seizure duration, C number of seizures and D ictal powear at 6-8 Hz were unchanged by microdialysis of 300 μM TTA-P2 (red) relative to during microdialysis of aCSF (blue). In this and Figures 4.2 & 4.3, parameters are plotted in 20-min bins during pre-drug control and 2 hours of drug administration. At time zero dialysis solution was changed from aCSF to test solution (either aCSF or 300 μM TTA-P2). Black horizontal bars indicate the hour used for effect quantification. E All the parameters of interest during the target hour only, for both TTA-P2 and aCSF.
In 11 animals the administration of 1mM TTA-P2 throughout the VB and nRT resulted in a significant decrease (by 56.16% of pre-drug control4) in time spent in seizure (as a percentage of the pre-drug control hour) compared to aCSF control (P = 0.0063, 19.82 to 92.51) [Fig. 4.2A]. This decrease involved both shorter (by 31.1%) seizures (P = 0.0129, 8.17 to 54.03) [Fig. 4.2B], and a lower (by 33.52%) incidence of seizure (P = 0.0258, 4.97 to 62.06) [Fig. 4.2C]. Despite this decrease in time spent in seizure there was no detectable change in ictal power at 6-8Hz (0.695, −0.18 to 0.26) [Fig. 4.2D].
Two way repeated measures ANOVA using time (pre vs post drug) and treatment (aCSF vs TTA-P2) found that interaction between factors was a significant source of variation of time spent in seizure (P = 0.0013) and of number of seizures (P = 0.0068). Bonferroni post-hoc tests showed that 1mM TTA-P2 treatment involved a significant difference between pre- and post- drug time spent in seizure (P < 0.001) and number of seizures (P < 0.001) while aCSF treatment did not in either case (P > 0.05). Time/treatment interaction was not found to be a significant source of variation of average seizure duration or power at peak seizure frequency. Treatment was not a significant source of variation of any parameter.
These results suggest that blocking T-type Ca2+ channels throughout the VB and nRT decreases the time spent in ASs by GAERS. This effect could be attributed either to a cumulative effect of VB and nRT T-type Ca2+ channel inhibition, an nRT-only effect (given the lack of change when the VB only was reached), or even an effect that required T-type Ca2+ channels throughout the entirety of the VB to be blocked before emerging, i.e. the 600μm block distance achieved by 300μM TTA-P2 (see Fig. 2.5) may not have been sufficient for this. In order to clarify this situation, a complementary strategy to differentiate nRT and VB effects was employed.
Moving the microdialysis probe positioning laterally and reverting to a 300μM TTA-P2 concentration (see Fig. 2.2) was estimated to achieve a 90% (or higher) block of bursts throughout a similar or greater proportion of the nRT than the central VB 1mM protocol, while simultaneously achieving a block throughout a significantly reduced proportion of the VB. This method resulted in a significant decrease (46.74%) in time spent in seizure (as a percentage of the control hour) for 300μM TTA-P2- compared to aCSF-treated animals (n = 6,
4
Differences between groups are given in percentages (of pre-drug control) as both are measured in those units, these do not refer to a ratio of the TTA-P2 and aCSF groups.
Figure 4.2 1mM TTA-P2 in the VB and nRT suppresses absence seizures
A Total time spent in seizure was decreased by 56% during microdialysis of 1mM TTA-P2 relative to during microdialysis of aCSF control. B There was a 31% reduction in mean duration of seizure and (C) a 34% reduction in number of seizures. D There was no detectable change in ictal power at 6-8Hz. At time zero dialysis solution was changed from aCSF to test solution (either aCSF or 1 mM TTA-P2). Black horizontal bars indicate the hour used for effect quantification. E All the parameters of interest during the target hour only, for both TTA-P2 and aCSF.
Figure 4.3 300μM TTA-P2 adjacent to the nRT suppresses absence seizures
A Total time spent in seizure was decreased by 46% during microdialysis of 300μM TTA-P2, compared to during microdialysis of aCSF control. B The decrease was not accompanied by a detectable change in mean length of seizure but rather (C) solely by a 47% decrease in number of seizures. D Ictal power at 6-8Hz was not detectably changed. At time zero dialysis solution was changed from aCSF to test solution (either aCSF or 300 μM TTA-P2). Black horizontal bars indicate the hour used for effect quantification. E All the parameters of interest during the target hour only, for both TTA-P2 and aCSF.
P = 0.0326, 95% CI = 5.68 to 87.15) [Fig. 4.3A]. This decrease was of a similar order to that observed in the 1mM VB group, but unlike that group the decreased time spent in seizure did not involve shorter seizures (P = 0.832, 95% CI = −27.26 to 32.44) [Fig. 4.3B]. Rather, there was a mean 46.74% decrease in number of seizures (P = 0.0148, 95% CI = 13.8 to 79.68) [Fig. 4.3C]. Again, power at 6-8Hz showed no detectable difference between groups (P = 0.275, 95% CI = −0.31 to 0.11) [Fig. 4.3D].
Again the result was confirmed by 2 way repeated measures ANOVA using time (pre vs. post drug) and treatment (aCSF vs. TTA-P2) as factors. Interaction between the factors was found to be a significant source of variation in time spent in seizure (P = 0.0265) and in number of seizures (P = 0.0118). Bonferroni post-hoc tests showed that 300μM TTA-P2 treatment involved a significant difference between pre- and post-drug time spent in seizure (P < 0.01) and number of seizures (P < 0.01) while aCSF treatment did not in either case (P > 0.05). Time/treatment interaction was not found to be a significant source of variation of average seizure duration or power at peak seizure frequency. Treatment was not a significant source of variation of any parameter.
The observation of a similar level of seizure suppression when nRT T-type Ca2+ channel inhibition was maintained and VB T-type Ca2+ channel inhibition was decreased suggested that seizure suppression could be attributed to nRT, rather than TC or combined TC and nRT, block of these channels.