The Journal of Neuroscience, November 15, 2006, ():
Massive and Specific Dysregulation of Direct Cortical Input to the Hippocampus in Temporal Lobe Epilepsy
J. Neurosci. Ang et al.
26: 11850
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1, Subthreshold membrane potential changes in the apical dendrites of CA1 pyramidal cell in stratum radiatum are closely correlated with the local voltage sensitive dye signal. Schematic in A shows the horizontal hippocampal slice with stimulating electrodes positioned to activate the hippocampal afferents: DG stim, Perforant path stimulation; SC stim, Schaffer collateral stimulation; TA stim, temporoammonic pathway stimulation; and AB stim, angular bundle stimulation. (B) A schematic illustration of the optical set-up used in the study. The voltage-sensitive-dye signal is recorded with a CCD camera simultaneously with dendritic whole cell recordings. (C) In control, the I-clamp dendritic recording in response to a single stimulus applied in stratum radiatum is superimposed onto the local voltage sensitive dye signal (in red) quantified from a region of interest near the recording electrode in stratum radiatum. Changes in the membrane potential of the area CA1 apical dendrite appear to be closely correlated with the voltage-sensitive-dye signal which results from dendritic activity of many area CA1 pyramidal neurons. Inset, changes in the local voltage sensitive dye fluorescence are plotted as a function of the changes in membrane potential of the area CA apical dendrite. The two measurements are correlated linearly with a linear correlation coefficient of r = 0.95. Residuals of the linear fit indicate that there is no systematic variability in the voltage-sensitive-dye signal not accounted for by the change in dendritic membrane potential. (D) In epileptic animals, the I-clamp dendritic recording in response to a burst stimulation (4 stimuli at 100 Hz) applied in the temporoammonic pathway is superimposed onto the local voltage-sensitive-dye signal. Summary data of all I-clamp recording in epileptic animals (n=5, black ) and in controls (n=6, grey) where each current clamp recording having a 10 mV change in membrane voltage from baseline is plotted as a function of the local voltage-sensitive-dye response.
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Supplemental Figure 2, Temporoammonic pathway stimulation in epileptic animals demonstrates persistent throughput to stratum radiatum in individual voltage sensitive dye imaging trials. Because averaging in trials where a network bursting is generated can generate prolonged activity we analyzed individual trials from slices derived from epileptic animals to assess whether bursting had occurred (A; Epileptic). A snapshot of activation at 30 ms of the temporoammonic evoked EPSPs (denoted by asterisk) in response to a burst stimulus (4 stimuli at 100 Hz) in stratum lacunosum moleculare (left image) and the activation profile (right image) generated from the raster line scan along the path of interest (green line). VSD SR and SLM are the local voltage sensitive dye signals quantified from the regions of interest in stratum oriens (blue box), stratum radiatum (green box) and stratum lacunosum moleculare (black box) respectively. The voltage sensitive dye signals were averaged over 12 trials in (B). Note the propagation of temporoammonic activity to stratum radiatum in every trial, demonstrating that the averaged responses as shown in B are faithful to the individual trials.
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Supplemental Figure 3, Sections of ventral hippocampus and medial entorhinal cortex from epileptic rats illustrating the typical pattern neuronal loss in animals used in this study. A number of rats were video monitored for at least 48 hours, and three were chosen with seizure frequencies close to the mean frequency of rats used for the current imaging experiments (#1528, 2/day; #1535, 1.5/day; and #1564, 2/day). These rats were perfused with fixative, and sections of the forebrain were cut at the same angle used for physiology. This primarily horizontal angle includes the entorhinal cortex and hippocampus and is designed to maintain the connections between those two areas. Sections were cut on a vibrotome and stained with cresyl violet to identify cell bodies and their stratification within the dentate, hippocampus proper and entorhinal cortex. The most apparent differences between sections from the Naïve animal (top) and the epileptic animals below are the reduction of hilar neurons in the dentate (DG; hilus) and the narrowing and focal disruptions of the area CA1 cell layer (dark band under stratum radiatum; SR, see arrows), particularly in animals #1535 and #1564. Except for animal #1582 showing some reduction in layer III, in the remaining animals, the entorhinal cortex appears intact.
