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.