The electrophysiological recording of the probability of release from

in the CA1 region of the hippocampus after 2 weeks of EE.

Paired pulse (PP) facilitation or depression is a form of short term synaptic plasticity reflecting the probability of release of neurotransmitter from the presynaptic terminal (Andersen, 2007). Experimentally this is measured by delivering two stimuli in close succession and the slope of the first and second synaptic responses are compared. The PP ratio is calculated by dividing the second fEPSP slope by the first fEPSP slope and is a reflection of the increase in the probability of transmitter release. The mechanism behind this phenomenon is due to the influx of calcium into the presynaptic terminal after the first stimuli which will still be present if the second stimulus is delivered in close succession. Therefore the second postsynaptic response will be larger as the calcium influx of the second stimuli will add to the residual calcium concentration from the first evoked fEPSP. Thus an enhanced calcium concentration will increase the probability of release as calcium is required for the fusion of the vesicle to the presynaptic membrane releasing neurotransmitter by exocytosis (Zucker and Regehr, 2002). Therefore differences in PP facilitation between treatments/genotypes will indicate that the probability of release will be different.

A PP ratio was calculated for all four groups in this study, WT SH, WT EE, MSK1 KD SH and MSK1 KD EE. The PP ratio was also calculated over an interpulse interval (IPI) of (units; ms) 50, 100, 150, 200 and 250 using fEPSP of ~50% of the maximal fEPSP slope. In all groups PP facilitation was observed as the ratio was above 1 at all IPI. However there were no significant shifts in the PP relationships between any of the conditions or genotypes (p>0.05, Two way ANOVA). Therefore there were no changes in the probability of release.

166 Figure 5.8; There were no changes in the general trend of the paired pulse (PP) ratios in CA1 region of the hippocampus after EE in either WT or MSK1 KD. Parasagittal hippocampal slices (400 µm) were prepared from 6 week old male mice that had been exposed to SH or EE conditions for 2 weeks. Extracellular fEPSP were recorded from the stratum radiatum in the area CA1. The linear part of the fEPSP slope was measured throughout the recording which usually measured over a 1 ms range. The PP ratio was also calculated over an interpulse interval (IPI) of (units; ms) 50, 100, 150, 200 and 250 using fEPSPs of ~50% of the maximal fEPSP slope. The graphs represent the average PP ratio for each ISI ±SEM for WT SH versus WT EE (A), MSK1 KD SH and EE (B) WT SH and KD SH (C) and WT EE and MSK1 KD EE (D). A Two Way ANOVA was carried out for each coupled comparison in each graph which rendered all groups not significantly different to each other (p>0.05). (WT SH; n=33 slices from 10 mice, WT EE; n=37 slices from 10 mice, KD SH; 17 slices from 8 mice and KD EE; n=24 slices from 7 mice).

167

5.2.3.3 Tetanus-induced LTP in WT and MSK1 KD CA1 region of the hippocampus after 2 weeks of EE.

Long-term potentiation (LTP) is a form of synaptic plasticity that can be initiated in hippocampal synapses through the delivery of a high-frequency stimulation (HFS) consisting of 100 pulses at 100 Hz, also known as a tetanus (T- LTP). The question that was addressed was whether EE of WT or MSK1 KD mice have changes in LTP expression in response to EE, and whether this response was MSK1-dependent.

T-LTP was successfully induced in all four groups, WT SH and EE as well as MSK1 KD SH and EE (Figure 5.9 A-D). This was confirmed after carrying out a paired t test on the average values prior to T-LTP (2 minutes prior to T-LTP) and at the end LTP (58th-60th minute post T-LTP) (Figure 5.9 E, ** p<0.01 and *** p<0.001 in the orange boxes). When the WT SH and WT EE LTP traces were compared after ~60 minutes of LTP there was no significant difference in LTP between the two groups (Figure 5.9 A and E; unpaired t test). Using the same analysis the MSK1 KD SH LTP showed no significant difference in LTP from the KD EE LTP trace (Figure 5.9 B and E). The SH condition in both WT and MSK1 KD showed no changes in LTP expression between 58th -60th minutes post T-LTP (Figure 5.9 C; unpaired t test shown in E). This was also the case for the EE condition in both WT and MSK1 KD (Figure 5.9 D, unpaired t test shown in E).

169 Figure 5.9; There were no changes in tetanus induced LTP in CA1 region of the hippocampus after EE in either WT or MSK1 KD. Parasagittal hippocampal slices (400 µm) were prepared from 6 week old male mice that had been exposed to SH or EE conditions for 2 weeks. Extracellular fEPSP were recorded from the stratum radiatum in area CA1 where the linear part of the fEPSP slope was measured. Tetanus LTP (T-LTP; 100 pulses at 100 Hz) was induced at the time indicated by the arrow. The stimulation was applied after 10 minutes of stable baseline was collected at 0.067 Hz (1 pulse per 15 sec). The recordings continued for at least an hour after the induction of LTP. Each graph represents the average fEPSP slope normalised to the baseline ± SEM for WT SH versus WT EE (A), KD SH and KD EE (B), WT SH and KD SH (C) and WT EE and KD EE (D). A representative fEPSP is shown above each of the graphs with a letter (a-d) which is indicative of the time at which the fEPSP was recorded shown on the graph. The fEPSP represented as a dotted line represents (a) and (c) pre-tetanus induction as oppose to (b) and (d) which are after LTP induction. (E) The statistical tests used to identify if LTP was successfully induced, compared 8 time points before LTP induction and the last 8 time points. The other test was to see if there were any differences in the traces between the groups which compared the last 8 time points (n numbers; WT SH= 11 slices, 5 mice; WT EE= 9 slices, 6 mice; KD SH=4 slices, 4 mice; KD EE=8 slices, 5 mice)

5.2.3.4 Theta burst-induced LTP in WT and MSK1 KD CA1 region of the