Computational protocol: Network Deficiency Exacerbates Impairment in a Mouse Model of Retinal Degeneration

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Protocol publication

[…] Each GC was filled with sulforhodamine B, included in patch pipette solution. At the end of each recording session, contrast and fluorescent images of the cell were documented with a modified Nikon D5000 DSLR attached to a Nikon FN1 microscope. The preparation was immediately placed in glass bottom culture dish (Matek, Ashland, MA, USA) and transferred to a stage of a Nikon C1 confocal microscope. A z-stack of 160 images was acquired at 0.5 μm steps at a resolution of 1024 × 1024 pixels. A nuclear stain stock solution, 2 μL of an equal mixture of 12 mM ethidium bromide and 100 μM To-Pro-3 (Invitrogen, Carlsbad, CA, USA) was added for determining the borders of the inner plexiform layer (IPL, Figure ). GCs were distinguished from displaced ACs by the presence of an axon. As previously described (Sun et al., ), for dendritic field (DF) size, a polygon was drawn by linking the tips of dendrites, and the area calculated. The area was converted back to diameter by assuming a circular DF. Cell body size was measured similarly. The level at which the GC dendritic processes stratified in the IPL was measured as the distance of its processes from the proximal (0%) to distal margin (100%) of the IPL. In general, ON GCs were defined as those whose dendrites stratified <60% of the IPL depth, and OFF GCs stratified >60% of the IPL depth. Measurement of cell properties was performed with ImageJ and Nikon EZ-C1 software. Cells were classified under two different methods. First, cell body size, DF diameter, and depth of dendritic stratification were used to classify cells in adherence to the groups described by Sun et al. (). This was done to verify a broad sampling of previously identified classes in both wild type (Sun et al., ) and RD (Mazzoni et al., ) GCs, and to establish a baseline for cluster analysis (Kong et al., ). Second, a cluster analysis was performed using SPSS (SPSS Inc., Chicago, IL, USA), with stratification depth and DF diameter as parameters (Badea and Nathans, ; Kong et al., ). This followed the method described by Badea and Nathans: Ward’s joining method was used to determine the number of clusters by a separation threshold of 25% of the greatest distance between nodes, followed by a k-means analysis to determine cluster membership. Monostratified and bistratified cells were analyzed separately. For bistratified cells, the dendritic depth and the area were obtained for both branches. [...] Currents were analyzed using Signal. The term “bursting” refers to periodic spiking activity measured with extracellular GC recordings, while “oscillations” refer to periodic current activity measured in voltage-clamp mode. To quantify the strength of synaptic oscillations, the power spectra of traces were obtained using a Hanning window, with 0.076 Hz bins. Frequency of oscillations was determined by finding the peak power within the range of 0.1 and 30 Hz. Cells with a peak greater than 2 SD above the mean of this range were considered to be oscillating at that frequency. Otherwise, cells were considered to be non-oscillating. Changes in oscillatory activity across a given experiment were illustrated in heat maps, where a series of power spectra for a given cell was obtained (using 20 s segments) and plotted on a time scale. Excitatory-to-inhibitory ratios were calculated by dividing the oscillatory power measured from EPSC traces by that measured from IPSC traces (Margolis et al., ). To measure efficiency of synaptic transmission of evoked response, signal-to-noise ratio (SNR) was calculated from DC-adjusted traces by dividing the charge transfer of the response (signal) by the charge transfer of unstimulated activity (noise) over equivalent epoch (250 ms). Spectrograms were generated for consecutive recording frames using custom scripts written for Matlab (Mathworks, Natick, MA, USA). Statistical analyses were performed using SigmaPlot (Systat Software Inc. Richmond, CA, USA) and SPSS. All data are reported as means ± SEM. Student’s t-test or paired t-tests were used for group comparisons or ANOVA for multiple comparisons. Two-way ANOVA was used where multiple factors were considered; one-way ANOVA was used unless otherwise specified. Multivariate pair-wise comparisons across treatment conditions were made with repeated-measures MANOVA. […]

Pipeline specifications

Software tools ImageJ, SPSS, SigmaPlot
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Deficiency Diseases, Epilepsy, Muscular Dystrophies, Parkinson Disease, Retinal Degeneration