Early Appearance and Spread of Fast Ripples in the Hippocampus in a Model of Cortical Traumatic Brain Injury

Abstract

Fast ripples (FRs; activity of >250 Hz) have been considered as a biomarker of epileptic activity in the hippocampus and entorhinal cortex; it is thought that they signal the focus of seizure generation. Similar high-frequency network activity has been produced in vitro by changing extracellular medium composition, by using pro-epileptic substances, or by electrical stimulation. Here we study the propagation of these events between different subregions of the male rat hippocampus in a recently introduced experimental model of FRs in entorhinal cortex-hippocampal slices in vitro By using a matrix of 4096 microelectrodes, the sites of initiation, propagation pathways, and spatiotemporal characteristics of activity patterns could be studied with unprecedented high resolution. To this end, we developed an analytic tool based on bidimensional current source density estimation, which delimits sinks and sources with a high precision and evaluates their trajectories using the concept of center of mass. With this methodology, we found that FRs can arise almost simultaneously at noncontiguous sites in the CA3-to-CA1 direction, underlying the spatial heterogeneity of epileptogenic foci, while continuous somatodendritic waves of activity develop. An unexpected, yet important propagation route is the propagation of activity from CA3 into the hilus and dentate gyrus. This pathway may cause reverberating activation of both regions, supporting sustained pathological network events and altered information processing in hippocampal networks.SIGNIFICANCE STATEMENT Fast ripples (FRs) have been considered as a biomarker of epileptic activity and may signal the focus of seizure generation. In a model of traumatic brain injury in the rat, FRs appear in the hippocampus within a couple of hours after an extrahippocampal, cortical lesion. We analyzed the origin and dynamics of the FRs in the hippocampus using massive electrophysiological recordings, allowing an unprecedented high spatiotemporal resolution. We show that FRs originate in distinct and noncontiguous locations within the CA3 region and uncover, with high precision, the extent and dynamics of their current density. This activity propagates toward CA1 but also backpropagates to the hilus and the dentate gyrus, suggesting activation of defined microcircuits that can sustain recurrent excitation.

Keywords: ca3; dentate gyrus; epilepsy; fast ripples; hippocampus; traumatic brain injury.

Figure 1.

Fast ripple activity in the hippocampus. A, Recurrent fast ripple events in different regions of the DG and hippocampus proper. B, Numbers to the left of the traces correspond to the positions depicted on the photograph of the hippocampus. C, Expanded raw and filtered traces of the event in the rectangle in A. Numbers indicate the recording position, as shown in B. Filtered traces (250–600 Hz) uncover the presence of fast ripples along the DG, hilus, and CA regions. These events with very high frequencies presented high amplitude in the CA subregions (n = 9) but could also be recorded in the DG (n = 4), albeit with low amplitude. The vertical calibration bar in microvolts applies traces 1 to 9, both raw and filtered; the calibration in mV applies to traces 10 to 21. D, Power spectral analysis of the fast ripples recorded in the DG, hilus, stratum pyramidale (sp.) and stratum lucidum (sl.) of CA3, and strata radiatum (sr.) and pyramidale of CA1. Notice a distinctive peak at around 340 Hz in all the structures.

Figure 2.

Simultaneous voltage recording in the entire hippocampus. Traces of 85 ms were taken from a 16 × 16 array out of the 64 × 64 microelectrode matrix for better depiction. Notice the strong presence of FRs in the strata radiatum and pyramidale of CA3 through CA1.

Figure 3.

Statistical parameters of the fast ripples. A, Topographic maps of the different parameters measured in CA (n = 9) and DG/hilus (n = 4). Values in the color scale are superimposed to a photograph of the hippocampus to signal incidence (A1), amplitude (A2), duration (A3), and frequency (A4) of the fast ripples. Note that the middle portion of the DG is not invaded by the events. The green square on the photograph denotes the MEA recording area. B, Whereas frequency (B4) and duration (B3) of the events are similar in all structures, the incidence (B1) and amplitude (B2) of the events are significantly higher in CA with respect to DG and hilus. Moreover, these data do not follow a normal but a log-normal distribution.

Figure 4.

Propagation of the fast ripples. A, Propagation sequence of a representative event. Fast ripples normally initiated in area CA3 and spread to CA1 and to the hilus/DG. B, An analysis of the incidence of the initiation site showed that fast ripples initiated mainly in medial and distal CA3, with no statistically significant differences between these areas. C, The incidence distribution through the different hippocampal areas. D, Depiction of the progression of the event, whereby the color indicates the time at which the electrodes detected the event (time–color coding on the right scale). Note that the invasion of electrodes by fast ripples presented a time gradient from CA3 to CA1 and DG, although isolated regions could present them in an apparent spatiotemporally disorganized manner.

Figure 5.

Current source density analysis. A1, A fast ripple event at an expanded time scale, whereby high frequencies appear on a slow-developing component. A2, A color map (in voltage) of the traces in A1. The locations where the selected traces in A1 were acquired are signaled with the letters on the color map. B, Time sequence of the CSD dynamics throughout a fast ripple at a high spatiotemporal resolution. Events initiated in CA3, often in different sites with a sink in the distal dendritic zone and alternate sink/source regions up to stratum oriens (frames at t = 12.1 and at t = 16.23). This slow component was followed by high-frequency activity that showed discontinuities along the CA3 that spread to CA1, after which patches of sink/source signals appeared in CA3 with concurrent signals in the DG (t = 26.77–29.76 ms). The patchy nature of the signals suggests activation of discrete zones of CA. The later phase of the slow component was characterized by a source along CA stratum pyramidale and sinks in strata radiatum and oriens. The size of the figure corresponds to the whole active zone of the recording matrix (see Materials and Methods).

Figure 6.

Fine topographic evolution of current source density in time. A, Snapshots of the sinks and sources obtained every millisecond, during the first 25 ms of a fast ripple event. Patches in red are sources, and patches in blue are sinks. The boundaries of the components were obtained by setting a threshold of the current density above the noise level, yielding disjoint components that can appear, disappear, or move. Contrary to local field potentials, which obscure the micrometer resolution of activity, current densities can be measured with a resolution <20 μm and followed in time. Trajectories of the disjoint components were traced by obtaining the center of mass at high frequency. Thus, instant velocities could be obtained from this analysis. Interestingly, displacements of sinks and sources can be observed within the DG–hilar region, close to the proximal part of CA3. The dynamics of the wave-like activity can be better observed in supplemental Movie 3. B, Histogram depicting the distribution of instantaneous velocity measurements. Ordinates are the number of displacement segments for each instantaneous velocity range (bin, 0.025 m/s). The resolution of the center-of-mass computation was of ∼10 μm because it is very sensitive to the differences of value of the CSD in the neighboring electrodes.

Figure 7.

Granger causal interactions and calcium dependence. A, Bidirectional interaction of CA3 and DG and interactions from CA3 to CA1 in a representative experiment. Fast ripples were recorded in 833 electrodes, of which 581 were in CA3, 230 were in CA1, and 22 were from the dentate gyrus and hilus. Related electrodes/sites were joined by lines. B, Mean Granger causal interactions (p < 0.05) were computed per fast ripple event. Note that most Granger causality interactions happen between CA3 electrodes; however, CA3 causes fast ripples in the DG, and the DG causes fast ripples in CA3. C, Voltage map showing the remaining multiunitary activity in the pyramidal and granular cell layers in a low-calcium (0.2 mm) recording solution. FR activity ceased, indicating its dependence on chemical synaptic transmission. D, Representative multiunitary activity recordings from the DG granule cell layer and CA3 and CA1 pyramidal layers.