Dynamic interaction of local and transhemispheric networks is necessary for progressive intensification of hippocampal seizures

Abstract

The detailed mechanisms of progressive intensification of seizures often occurring in epilepsy are not well understood. Animal models of kindling, with progressive intensification of stimulation-induced seizures, have been previously used to investigate alterations in neuronal networks, but has been obscured by limited recording capabilities during electrical stimulations. Remote networks in kindling have been studied by physical deletions of the connected structures or pathways, inevitably leading to structural reorganisations and related adverse effects. We used optogenetics to circumvent the above-mentioned problems inherent to electrical kindling, and chemogenetics to temporarily inhibit rather than ablate the remote interconnected networks. Progressively intensifying afterdischarges (ADs) were induced by repetitive photoactivation of principal neurons in the hippocampus of anaesthetized transgenic mice expressing ChR2. This allowed, during the stimulation, to reveal dynamic increases in local field potentials (LFPs), which coincided with the start of AD intensification. Furthermore, chemogenetic functional inhibition of contralateral hippocampal neurons via hM4D(Gi) receptors abrogated AD progression. These findings demonstrate that, during repeated activation, local circuits undergo acute plastic changes with appearance of additional network discharges (LFPs), leading to transhemispheric recruitment of contralateral dentate gyrus, which seems to be necessary for progressive intensification of ADs.

Figure 1

Two main patterns of progressive or non-progressive afterdischarges (ADs) induced by repetitive pulse-train photostimulation in the Thy1-ChR2 mouse in vivo. (a,b) Isoflurane anaesthetized mice were subjected to 15 s blue light (463 nm) pulse trains. Representative in vivo local field recordings from the hippocampus of two mice are shown, number of stimulations indicated on the timeline arrow (left). (c) Top: illustration of unilateral stimulation & recording area in horizontal section, with optrode targeting CA3 at DV-2.75. Bottom: pulse train parameters. (d,e) Magnified views of ADs marked in (a & b) respectively. (f–h) Three AD parameters in the first 5 seconds after photostimulation were plotted, grouped by progressive (Progr, n = 4) or non-progressive (Non-Progr, n = 17) AD development. Comparing the mean of the last 5 trains, the two subsets were different for all three AD metrics (unpaired t-test). A control group (n = 3, grey) did not display any afterdischarge activity when subjected to orange (593 nm) light (see Methods), while blue light administered for the last 5 stimulations produced only limited non-progressive ADs. (i–k) Within-subject analysis using linear regression slopes of the data in (f–h) was performed. Increased slope was seen in the Progr AD group (unpaired t-tests). Note that two-segment axes with 10 times scale of bottom segment is used for coastline in (h & k) to be able to discern Non-Progr data points. Scale bars: (a,b) 3 mV, 5 s; (d,e) A1 & B1 3 mV, 200 ms; A2 & B2 1 mV, 200 ms. Error bars: ±SD. ***P < 0.001, ****P < 0.0001, see Results for exact P-values.

Figure 2

Increased post-light and spontaneous LFPs generated during the optogenetic pulse train is correlated with progressive AD occurrence. (a,b) Magnification of intra-train local field recordings from the 40th stimulation of two animals with progressive (a) and non-progressive (b) ADs. Blue traces: light pulses. Green traces: detected LFP events, categorized as induced (i-LFP) or spontaneous (s-LFP), while directly light-evoked LFPs, coinciding with the light pulse (L), were not included in analysis (see Results). (c,d) The number of i-LFPs and s-LFPs per light pulse was increased in the Progr AD group compared to the Non-Progr AD group (unpaired t-tests), especially s-LFPs. (e) In CaMKII-ChR2 mice with progressive light-induced ADs (n = 11, see Fig. 4), the amount of s-LFPs generated per light pulse was not different from the Thy1-ChR2 Progr group (n = 4, unpaired t-test). Thus groups were pooled for further analysis. (f,g) Aligning data by the stimulation producing the first progressive ipsilateral AD (see Results), a 20% increase in s-LFPs (f, repeated measures ANOVA with Dunnett’s post-hoc test) coincides in time with the start of progressive AD generation (g, Friedman test with Dunn’s post-hoc test). Binned data, three stimulations per bin; PreAD: consecutive bins preceding the first progressive AD; AD: consecutive bins from the first progressive AD. Asterisks: significance in post-hoc tests. (h) Boxplots of s-LFP characteristics for the pooled progressive AD group (1115 data points from 15 animals). Line: median, box: 25–75th percentile, bars: min/max. Scale bars: (a & b) 3 mV, 50 ms. Error bars: ±SD, except (h). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: non-significant; see Results for exact P-values.

Figure 3

Differences in Fos immunoreactivity patterns in Thy1-ChR2 mice subjected to optogenetic stimulation in the hippocampus implies importance of contralateral DG. (a,b) Composite images of Fos immunoreactivity in horizontal sections of the hippocampus ipsilateral (a) and contralateral (b) to optogenetic stimulation from a mouse with progressive AD development. (c) Magnified areas from (a) of CA1, CA3 & DG, respectively. (d) Magnified area of DG from (b), indicating cell body localization of Fos by nuclear stain (Hoechst, blue). (e–g) Three general patterns of Fos immunoreactivity in the DGC (dashed outlines): (e) bilateral (BiL); (f) ipsilateral to optogenetic stimulation (IpL), (g) absence of Fos immunoreactivity bilaterally (non-activated, NA) (e & g are from the same animals as recordings in Fig. 1a & b, respectively). (h) Illustration of areas selected for quantification of Fos immunoreactivity. DGC marked in magenta. DM: dorsomedial, DL: dorsolateral, M: middle, V: ventral. (i) Ipsilateral and (j) contralateral mean Fos intensity in the BiL (n = 4), IpL (n = 10) and NA (n = 7) subsets (NA as control, Kruskal-Wallis test with Dunn’s post-test). Note that the BiL group completely matches the Progr AD group (Figs 1 & 2). (k) Control group (n = 3) exposed to trains of orange (593 nm) light. (l) Relative magnitude (ratio) of contra- to ipsilateral Fos intensity, comparing BiL and IpL (unpaired t-tests). Scale bars in a,b: 200 µm, c,d: 40 µm, e–g: 50 µm. Error bars: ±SD. *P < 0.05, **P < 0.01, ****P < 0.0001, ns: non-significant, see Table 1 and Results for exact P-values.

Figure 4

Inhibiting contralateral hippocampus via activation of hM4D blocks AD development induced by optogenetic train stimulation in CaMKIIa-ChR2 mice. (a) Illustration of unilateral blue light stimulation with bilateral field recording in horizontal section, targeting ventral CA3/DG at DV-2.1. hM4D(Gi)-mCherry was expressed by AAV vector contralateral to stimulation in the hM4D group. (b–d) Representative example of hM4D expression in composite images of horizontal sections from three levels of the hippocampus: dorsal, middle and ventral, respectively. hM4D is visible by mCherry autofluorescence (red; magenta when co-localized with nuclear counterstain (Hoechst, blue)) and ChR2 by YFP (green). (e) Representative section of hemisphere ipsilateral to optogenetic stimulation (opposite side of c), lacking hM4D expression. Post-experiment burn-in (electrode location) is visible in the hilus. (f,g) Traces of bilateral field recordings in control and hM4D mouse, respectively (I: ipsi, C: contra). Timeline arrow indicates number of stimulation trains from IP injection (dashed line) of vehicle or CNO. Bilateral progression of ADs is visibly abrogated after CNO injection in the hM4D-expressing mouse (g, bottom). (h,i) Counting contralateral intra-train s-LFPs and aligning bins of three stimulations according to the first ipsilateral progressive AD (see Fig. 2f–g), a 37% increase in s-LFPs (h, repeated measures ANOVA with Dunnett’s post-hoc test) is seen, coinciding in time with contralateral progressive AD onset (i, Friedman test with Dunn’s post-hoc test). (j) Quantification of AD durations, which were remarkably similar between ipsilateral (left graph) and contralateral sides (right graph). AD durations did not increase further in hM4D-expressing animals injected with CNO (red line) (repeated measures ANOVA with Dunnett’s post-hoc test, binned data, 5 stimulations per bin (see Results)). (k) AD durations of hM4D and control groups in three 5 stimulation bins (Post1-3) covering 75 minutes post-injection were directly compared. Durations were reduced after injection for the hM4D group both ipsi- and contralaterally (left and right graph, respectively) (One-way ANOVA with Sidak’s post-hoc test, see Table 2 for exact P values). Scale bars: (b–e) 100 µm; (f,g) 5 mV, 5 s. Error bars: ± SD. *P < 0.05, **P < 0.01, ns: non-significant.

Figure 5

Afterdischarge burst frequencies are not affected by inhibition of contralateral hippocampus. (a,b) 10 s post-stimulation periods immediately preceding CNO injection (a) and 10 stimulations post CNO injection (b), with details of ipsilateral bursts at the top (same animal as in Fig. 4g). Recording traces were Fast Fourier Transformed to examine burst frequencies (bottom). AD burst power was concentrated to frequency ranges of 15–30 Hz and 60–90 Hz in both groups. (c) Quantification of the burst frequencies on group level, taking the mean of the median burst frequency for each animal. There was no difference between groups (one way ANOVA), or from pre- to post-injection within groups (paired t-test), either ipsilateral or contralateral (see Results for P values). Scale bars (a & b): box: 5 mV, 50 ms; 5 mV, 1 s. Error bars: ± SD.

Figure 6

Schematic illustration of proposed mechanism for transhemispheric loop required for hippocampal progressive ADs. (a) Repetitive unilateral optogenetic stimulation leads to progressive increase in excitability of the local network, as evidenced by increased number of spontaneous LFPs during the stimulation train (ipsilateral s-LFPs, Fig. 2). These spontaneous LFPs spread to the contralateral hippocampus (red arrow), activating the contralateral dentate gyrus (contralateral s-LFPs, Fig. 4h), thus creating a transhemispheric feedback loop (black arrow) leading to progressive intensification of ADs bilaterally. Purple cells denotes increased activity indicated by Fos or LFP recordings. (b) If the contralateral hippocampus is inhibited, such as via hM4D-expressing neurons (located mainly in DG and CA3, orange) of CaMKIIa-ChR2 animals by CNO IP injection (green), feed-forward still exists (red arrow), while the feedback loop is selectively and temporarily disrupted (dashed arrow) and progressive intensification of ADs is halted.