Mechanisms of Heterosynaptic Metaplasticity. Hulme Et Al

Mechanisms of Heterosynaptic Metaplasticity. Hulme et al.

Mechanisms of Heterosynaptic Metaplasticity.
Sarah R. Hulme, Owen D. Jones, Clarke R. Raymond, Pankaj Sah, Wickliffe C. Abraham

doi: 10.1098/rstb.2013.0148

SupplementaryInformation

SUPPLEMENTARY METHODS

Calcium imaging in astrocytes

Hippocampal slices were cut from 3-4 week old ketamine/xylazine(10 µL/gram body weight: 10% ketamine, 5% xylazine in saline) anesthetised Sprague-Dawley rats which had been given ~1.5-3 hours previously an in vivointrahippocampal~2-3 µL injection (targeting the CA1/subiculum border) of the astrocytic-specific fluorphoresulforhodamine-101 (1 µM) and the calcium indicator Fluo-4 (10 µM), in modified ACSF vehicle. Astrocytes were visualised using a BX50 microscope (Olympus) and images sampled at 10 Hzwith whole-field epifluorescence imaging were recorded for off-line analysis with Image J and Axograph software. Fluorescence across time was measured in regions of interest exclusively containing astrocytic somata, as identified by SR-101 labelling, and paired adjacent background regions. Background fluorescence was subtracted from that of the astrocyte somata and responses were normalised (ΔF/F) to the fluorescence fromthe initial 2.5 s of recording for each burst of tetani. Data were then measured for the responses to each tetanus. Data were analysed using 2-way repeated measure ANOVA. The amplitude of the “no drug” astrocytic calcium signals were not normally distributed (P < 0.001, n = 40, D'angostini omnibus test). However, after being subjected to a logarithmic transformation, the data became gaussian (P = 0.2, n=40). Thus, all calcium signals were log-transformed before parametric statistical analysis. As there was no significant difference in the amplitude data across the two bursts of tetani (F (1,156)=0.4, P = 0.5),the data werecollapsed across this factor by averaging.

Figure 4D group numbers: No drug: tet 1 n = 40, tet 2 n = 39, tet 3 = 39, tet 4 = 40, tet 5 = 39, tet 6 = 36; CBX: tet 1 n = 10, tet 2 n = 5, tet 3 n = 5, tet 4 n = 10, tet 5 n = 4, tet 6 n = 5.