Article Figures & Data
Figures
- Figure 1.
Layer-specific profiling of theta coordination in the dorsal hippocampus. A, Representative 16-channel recordings of LFPs during exploratory theta activity in a normal rat. B, Representative LFP recordings from an epileptic rat engaged in active exploration. Recordings were obtained in periods between seizures, in the absence of interictal spikes and other types of epileptiform events. C, A combination of electrophysiological and immunostaining tools was used to identify recording sites and hippocampal layers. Averaged LFP (black traces) and CSD signals (color map: sinks are blue, sources are red) during theta, SPW, and DS1 and DS2 were used to identify specific layers. Data from the control rat shown in A. Numbers in parenthesis indicate the number of events of each type used. D, Postmortem immunostaining against netrinG1 and G2 together with the neuronal marker NeuN aided to assign recording channels to specific hippocampal lamina. Images from the control rat shown in A and C. E, F, Same as in C and D for the epileptic rat shown in B. G, Laminar profile of theta coherence (cohe; 4–12 Hz) between all channels and a reference channel at the SLM (blue) or at the MLm (green) for the normal rat shown in A. G′, Layer-based theta coherence matrix depicting pairwise estimation of theta coherence between (nondiagonal elements) and within layers (diagonal elements) for the control rat shown in A. Arrows from G to G′ indicate how coherence values represented in the matrix were obtained from pairwise estimations between recording sites. H, H′, Same as in G and G′ for the epileptic rat shown in B. Arrows from H to H′ indicate symmetric coherence values between SLM and MLm. GCL, granule cell layer.
- Figure 2.
Group layer-based theta coherence (cohe) and statistical contrast matrices. A, Mean group layer-based group coherence matrices for LFP signals recorded during exploratory theta activity, together with the contrast matrix (Welch's test) showing p values as −log10. Only significant p values (<0.05) are depicted. The level for Bonferroni correction is at 2.75 = −log10(0.05/28). Data from n = 10 control and n = 9 epileptic rats. B, Same as in A for CSD signals. GCL, granule cell layer.
- Figure 3.
Proximodistal distribution of theta coherence in normal and epileptic rats. A, Schematic representation of the entorhinal input pathways converging into the dorsal hippocampus. The temporoammonic pathway originates in layer III of the entorhinal cortex to target the SLM of CA1 region. Layer III neurons from the LEC project to distal CA1, i.e., close to the subiculum. Layer III neurons from the MEC project to the proximal CA1, i.e., close to CA3. The perforant pathway originates in layer II with LEC cells projecting to the MLo and MEC cells projecting to the MLm of the DG. LEC and MEC presumably process different aspects of information. B, Proximodistal distribution of theta coherence among SLM–MLo, SLM–MLm, and MLo–MLm for both LFP (left column) and CSD signals (right column). Individual data obtained from rats implanted at different mediolateral and anteroposterior locations are represented by dots scaled according to coherence (cohe) values (scale at right). Data from n = 10 control (black), n = 9 epileptic rats (blue). The approximate limits between proximal and distal CA1 are marked by discontinuous lines. One epileptic rat recorded at proximal coordinates had a defective site at MLo and could not be included in the SLM–MLo and MLm–MLo analysis. SUB, subiculum. C, Mean LFP coherence values per group at distal (n = 5 control, n = 4 epileptic) and proximal coordinates (n = 5 control, n = 5 epileptic). Data are represented as mean ± 95% confidence interval. Asterisks indicate significant differences between groups (one-tailed Welch's test); *p < 0.05, **p < 0.005. C′, Same for CSD coherence values. D, Mean group layer-based coherence matrices for LFP and CSD signals recorded at distal locations together with the statistical contrast matrix (Welch's tests) for comparisons among SLM, MLo, MLm, and MLi layers as in matrices shown in Fig. 2. The level for Bonferroni correction is at 2.25 = −log10(0.05/9). D′, Same as in D for rats recorded at proximal coordinates. E, Within-group comparisons (control) of distal (n = 5 rats) versus proximal (n = 5 rats) CSD coherence values. The contrast matrix shows only significant values (Welch's test). The level for Bonferroni correction is at 2.25 = −log10(0.05/9). E′, Same as in E for proximal (n = 5 rats) and distal (n = 4 rats) comparisons within the epileptic group. GCL, granule cell layer.
- Figure 4.
Structural alterations of the temporoammonic and perforant pathways in TLE. A, Immunostaining against netrinG1 and G2 was used to quantify changes of specific lamina in TLE. The thickness of different sublayers was measured at proximal and distal locations (squares) in sections adjacent to the recording probe. Images taken at 3.2× magnification. Images arranged vertically share scale bar. A′, For quantification purposes, images were taken at higher magnification (10×) at the proximal locations. c, control; e, epileptic. A″, Higher magnification images taken at distal locations. B, Proximal and distal group differences of CA1 and netrinG1-positive SLM thickness (mean ± SD). Data from n = 10 control (black) and n = 9 epileptic rats (blue) as estimated from images at 10× magnification. *p < 0.05, **p < 0.01. C, Proximal and distal group differences of the thickness of molecular strata as measured from netrinG1 (MLo) and G2 (MLm) immunostaining. MLi was measured from the ventral limit of netrinG2 band to the granule cell layer. Same dataset as in B. D, Schematic representation of horizontal sections used to evaluate neuronal loss at LEC and MEC at different dorsoventral levels (A–C; see Materials and Methods). E, Quantification of neuronal density from different layers of the entorhinal cortex at dorsal levels. Data (mean ± SD) from n = 3 control (black), n = 4 epileptic rats (blue). *p < 0.05, **p < 0.01, and ***p < 0.001 for Bonferroni corrected t tests. E′, Same as in E for intermediate horizontal levels. E″, At ventral levels we differentiated between layer II fan cells and pyramidal cells. Same dataset as in E and E′. F, Representative dorsal sections from a control and an epileptic rat. G, Representative ventral sections from a control and epileptic rat.
- Figure 5.
Temporal precision between entorhinal current sinks during exploratory theta. A, Theta current sinks at SLM and MLm are systematically out of phase in control rats (left), but exhibited large fluctuations in epileptic animals (right). Three individual events from a representative control and epileptic rat are shown. Average traces were estimated from all events in the given session. B, Timing variability (jitter; mean ± 95% confidence interval) of intersink interval estimated in windows of 2 s is larger in epileptic (blue) than control rats (black). *p < 0.016, Bonferroni corrected. C, P values for between-group statistical differences of intersink jitter estimated over a different number of cycles (time windows). Data show significant p values (expressed as −log10) resulting from a Welch's test. The level for Bonferroni correction is at 2.85 = −log10(0.05/36). D, Group data (epileptic, blue; control, black) of CSD theta cycle jitter between SLM–MLm (left) and MLo–MLm (right). Data are given as mean ± 95% confidence interval. E, Group data (mean ± 95% confidence interval) on variability of CSD waveform did not reach significance. F, Proximodistal distribution of intersink jitter between different layers (SLM–MLm, SLM–MLm, and MLo–MLm) for control (black) and epileptic (blue) rats. Dot size reflects CSD jitter values (in milliseconds) according to the scale shown at top. One epileptic rat recorded at proximal coordinates had a defective site at MLo and could not be included in the SLM–MLo and MLm–MLo analysis. G, Mean jitter values per group for CSD (left) and LFP (right) signals recorded at distal (n = 5 control, n = 4 epileptic) and proximal locations (n = 5 control, n = 5 epileptic) from animals shown in F. *p < 0.05, **p < 0.01, and ***p < 0.005, Bonferroni corrected.
- Figure 6.
Computational correction of intersink variability between layers. A, We implemented a procedure to evaluate the impact of correcting intersink variability between two layers, i.e., a reference layer (thick black trace) and the corrected layer (red trace), in LFP theta coherence. To correct waveform variability, each CSD-detected cycle in the corrected layer was substituted by the mean CSD waveform for the entire session while preserving variability of intersink interval and amplitude. LFP obtained from uncorrected (black traces) and corrected CSD signals (color map) were contrasted using z-scores and paired t tests. B, Jitter variability was corrected by shifting each detected CSD cycle to the mean intercycle interval between the reference and the corrected layers, thus neglecting temporal variability. C, Effect of correcting CSD waveform variability in LFP recordings from n = 3 epileptic rats. Data are represented as mean ± 95% confidence interval. C′, Individual changes of SLM–MLm theta coherence (cohe) before and after waveform corrections. Individual data are shown as mean (dots) and 95% confidence interval (bars) as calculated from several windows (2 s) in a given rat per session. C″, Same as in C′ for MLo–MLm. D, Effect of correcting CSD jitter variability in the same n = 3 epileptic rats corrected in C for the waveform (mean ± 95% confidence interval). D′, Individual changes of SLM–MLm theta coherence before and after jitter correction. D″, Same as in D′ for MLo–MLm. E, Statistical changes due to waveform correction obtained in the rat shown in A (z-score and −log10 of p value) as a function of the corrected and the reference layer. Nine different comparisons were performed using data from several windows (2 s) for each given animal. Only significant values (Bonferroni corrected) are represented. Note best results obtained when MLm is corrected using either SLM or MLo as reference layer. F, Same as in E for jitter correction as shown in B.
- Figure 7.
Pharmacological rescue of theta coherence. A, Mean layer-based coherence matrices for LFP (top) and CSD signals (bottom) 1 h before and after intraperitoneal injection of the type III mGluR antagonist CPPG (2 mg/kg) in n = 6 TLE rats. The contrast matrix (Welch's test) showing significant p values as −log10 for all pairs of layers is shown at right. The level for Bonferroni correction is at 2.75 = −log10(0.05/28). Note significant differences in LFPs, but poor statistical contrast for CSD signals. B, CPPG had strong effect in rescuing LFP theta coherence between SLM and ML layers in all epileptic rats. Group data before (blue) and after CPPG (red) are shown as mean ± 95% confidence interval. Note statistical differences concentrated at SLM–MLm. The arrow from A to B indicates data correspondence. B′, Individual data on LFP theta coherence between SLM and MLm before (blue) and after CPPG (red). P values correspond to results from paired t tests. Individual data are shown as mean (dot) and 95% confidence interval (bar) as estimated from several windows of 2 s from the whole recording session (300 s). C, CPPG injection had no effect on theta power along the CA1–DG axis. Data shown as mean ± 95% confidence interval. C′, No effect of vehicle (dark blue) on SLM–MLm LFP coherence was appreciated in n = 3 rats tested. D, Layer-based coherence matrices estimated from those epileptic rats shown in A recorded at distal locations (n = 3) before and after CPPG. D′, Layer-based coherence matrices estimated from epileptic rats shown in A recorded at proximal locations before and after treatment with CPPG (n = 3). E, Jitter between theta cycles in LFP recordings at SLM and MLm before and after CPPG. P value corresponds to results from paired t tests. Note opposite trends compared with LFP coherence (B′) indicating that CPPG restores theta coherence by reducing theta cycle jittering at the LFP. E′, CSD jitter between theta cycles in SLM–MLm before and after CPPG grouped according to proximal (n = 3) and distal locations (n = 3). P value corresponds to results from paired t tests. Note opposing trends at proximal and distal locations. F, Schematic representation summarizing differences of entorhinal input coordination (CSDs) between SLM and ML and within ML layers at distal and proximal locations and the effect on LFP theta coherence (denoted by arrows of different thicknesses) in the dorsal hippocampus of normal (black) and epileptic rats (blue). GCL, granule cell layer.
