Article Figures & Data
Figures
- Figure 1.
AAV-TeLC induces selective expression of TeLC in PV-containing interneurons in the subiculum of PV-cre mice. A, Distribution of TeLC (immunoreactivity for the GFP tag) after unilateral injection of AAV-TeLC into the subiculum of PV-cre mice and after EEG monitoring for up to 8 weeks. B, C, The overall distribution of PV-containing interneurons and fibers in the subiculum of C57BL/6N WT mice (B) is similar to the distribution of TeLC-expressing neurons in AAV-TeLC-injected mice (C). A–C, Representative images for WT mice (B; n = 5) and AAV-TeLC-injected mice (A, C; n = 25). See Table 1 for regional distribution of TeLC expression in individual animals. D–L, TeLC is specifically expressed in PV-containing interneurons. D–F, Only a subpopulation of GABA neurons (blue) expressed TeLC (green; white arrows in F indicate double-labeled neurons). The majority of GABA-containing neurons did not express TeLC (red arrows in F). G–L, The majority of PV-containing neurons were positive for TeLC/GFP at the site of AAV-TeLC injection, shown at high magnification (G–I) and low magnification (J–L; white arrows indicate some of the double-labeled cells). M, N, Cell counts of double-labeled cells (in total 2724 GFP-positive neurons were evaluated in 25 mice). M, More than 95% of TeLC-expressing cells contain GABA, 80% PV and ∼20% also somatostatin (interneurons containing somatostatin and PV; Jinno and Kosaka, 2000). N, In reverse, ∼60% of PV neurons express TeLC at the injection site, whereas <20% of GABA-expressing neurons and ∼15% of SOM-expressing interneurons are TeLC/GFP positive. O, Scheme of PV-containing interneurons in the subiculum. The major population comprises PV-containing basket cells forming perisomatic synapses on pyramidal cells (PCs). Minor populations are axo-axonic cells forming synapses on PC axon initial segments and subpopulations of somatostatin-containing O-LM cells and bistratified cells mediating feedback inhibition through PC dendrites. DG, Dentate gyrus; PaS, parasubiculum; PrS, presubiculum; Sub, subiculum; oml, outer molecular layer; iml, inner molecular layer; pcl, pyramidal cell layer. The dashed line in B indicates the border between the subiculum and sector CA1. Scale bars: A, 600 μm; C (for B, C), 300 μm; I (for D–I), 25 μm; L (for J–L), 100 μm.
- Figure 2.
TeLC expression in PV-containing interneurons attenuates inhibitory input at synapses on pyramidal neurons of the subiculum. To quantify the loss of inhibition, 2–3 weeks after AAV-TeLC or AAV-GFP injection (time when C-SWDs had become manifest), whole-cell voltage-clamp recordings from pyramidal neurons of the subiculum were obtained and eIPSCs were recorded in the presence of glutamate receptor antagonists (DNQX, 10 μm; dl-AP5, 100 μm). In the subiculum, eIPSCs originate from various types of local interneurons, including CCK- and PV-containing neurons. To partially isolate the eIPSC component from PV-containing neurons, the CB1 agonist WIN 55,212–2 (1 μm) was bath applied to selectively suppress GABA release from CCK-containing neurons (known to express the CB1 receptor), as previously described for pyramidal neurons of sector CA1 (Glickfeld et al., 2008; Murray et al., 2011). A, B, The baseline maximal amplitude of eIPSCs and the stimulation intensity required to evoke maximal responses was not different between groups (p = 0.286 and p = 0.651, respectively; t test). C, Two-way repeated-measures ANOVA revealed that the amplitude of eIPSCs in the presence of WIN 55,212–2 was significantly reduced in AAV-TeLC-injected mice compared with AAV-GFP-injected mice (treatment: F(1,20) = 9.83, p = 0.0052; time: F(8,160) = 5.97, p < 0.0001). D, Importantly, the WIN 55,212-2-insensitive component of the eIPSC was significantly larger in mice injected with AAV-GFP compared with those injected with AAV-TeLC (treatment: F(1,20) = 4.72, p = 0.0419; time: F(8,160) = 7.74, p < 0.0001). E, Specifically, WIN 55,212–2 reduced the amplitude of eIPSCs by 33% in AAV-GFP-injected mice, which is consistent with previous reports (Glickfeld et al., 2008), while mice injected with AAV-TeLC exhibited a 59% reduction in the amplitude of eIPSCs. Thus, TeLC expression in PV-containing neurons results in a significant reduction of inhibitory input to subicular pyramidal neurons (n = 10 cells from 6 AAV-TeLC-injected mice; n = 12 cells from 5 AAV-GFP-injected mice). Values are given as the mean ± SEM. F, The representative traces of the mIPSC recordings of subiculum pyramidal neurons from AAV-GFP (black) and AAV-TeLC-injected (gray) mice. G, Visualization of subiculum pyramidal neurons (magenta color) after patch clamping (arrows) with a solution containing biocytin. PV-positive neurons in the subiculum are visible in green because of stereotaxic injection of a Cre-driven AAV-GFP construct. H–I, Analysis of mEPSC (H) and mIPSC (I) amplitudes (H, I, left) and frequencies (H, I, right) in subiculum pyramidal neurons from Pvalbtm1(cre)Arbr mice injected with AAV-GFP (black bars, n = 10 cells for mEPSC recordings; n = 9 cells for mIPSC recordings) and AAV-TeLC (red bars, n = 11 cells for mEPSC recordings; n = 10 cells for mIPSC recordings). Data are not significantly different between the two experimental groups.
- Figure 3.
Permanent silencing of PV neurons by unilateral AAV-TeLC injection into the ventral subiculum of PV-cre mice results in SRSs. A, Scheme of electrode positions and anatomical site of vector injection. B, Cumulative presentation of C-SWDs and of SRSs. Eighty-eight percent of mice developed C-SWDs by day 16, and 64% of mice presented at least one SRS by day 28. C–E, Representative 8 min EEG traces obtained in mice 18 d after AAV-GFP (C) or AAV-TeLC (D, E) injection. Whereas AAV-GFP-injected mice showed an unchanged EEG, AAV-TeLC-injected mice presented either C-SWDs only (D) or C-SWDs and SRSs (16 of 25 mice; E). The intermittent C-SWDs lasted ∼5 min and were detected in 22 of 25 AAV-TeLC-injected mice (D). E, Approximately 90% of SRSs occurred immediately after preictal C-SWDs. F, Magnitude spectrum (0–80 Hz, logarithmic power scale) of the EEG recording in E. Note the profound and long-lasting post-seizure depression of EEG amplitude in all frequency bands. G, H, High-resolution EEG traces of a preictal single SWD (G, duration, ∼300 ms; marked as “G” in E) and of a spontaneous tonic-clonic seizure (H; marked as “H” in E). E, F, H, EEG seizures lasted ∼25 s and were always accompanied by generalized tonic–clonic motor seizures with loss of posture (stage 3 to 4). I, J, Mean C-SWDs (±SEM; I) and mean SRSs (±SEM) presented per week (J). Note the reduction in C-SWDs and seizure frequencies after 5–6 weeks.
- Figure 4.
AAV-TeLC injection or related seizures did not induce signs of neurodegeneration. A, B, AAV-TeLC did not affect cell numbers of PV neurons at the injection site. Numbers (mean ± SEM) of PV-positive and GABA-positive neurons were reduced neither in the subiculum of mice that had experienced C-SWD only (n = 6) nor in mice presenting SRSs after AAV-TeLC injection (n = 16). Cell counts were performed 6–8 weeks after AAV-TeLC injection (and monitoring). Numbers are shown for the injected subiculum; they did not differ from those in the contralateral subiculum (data not shown). Controls: uninjected mice (n = 6); data were not different from those for AAV-GFP-injected mice (n = 9; data not shown). C, D, Representative Nissl stains in brain slices at the level of the ventral hippocampus of mice with SRS after AAV-TeLC injection (n = 16; C), and after AAV-GFP injection (seizure free; n = 9; D). E, To test for possible mossy fiber sprouting as a consequence of loss in highly vulnerable mossy cells, we labeled hippocampal mossy fibers by immunohistochemistry for dynorphin. Although mossy fibers were strongly positive for dynorphin, no immunoreactivity was observed in the inner molecular layer of the dentate gyrus, which would be indicative for sprouted mossy fibers (red arrow in E) in AAV-TeLC-injected mice that had experienced SRSs for 6–8 weeks. The black arrow (E) indicates the area of the granule cell layer that is also devoid of dynorphin immunoreactivity (representative for 16 mice). F, Double labeling for the apoptosis marker caspase 3 (red cells, white arrows) and for TeLC/GFP (green cells, blue arrows) at the injection site of AAV-TeLC 10 d after injection. Labeling was performed in three horizontal sections obtained at different dorsoventral levels (∼240 μm apart from each other) from four mice killed 3 or 10 d after AAV-TeLC injection. Zero to maximally five apoptotic cells per section were detected close to the needle track. Note the intact GFP-positive cells expressing TeLC but not caspase 3 in close vicinity to presumably apoptotic cells. As positive controls, we investigated caspase 3 expression in the dorsal hippocampus of mice injected locally with kainic acid 2 and 10 d before (Jagirdar et al., 2015) and observed caspase 3-positive cells at both intervals (data not shown). Scale bars: D (for C, D), 500 μm; E, 200 μm; F, 20 μm.
- Figure 5.
Expression of ΔFosB in the hippocampal formation after spontaneous seizures induced by AAV-TeLC injection. To identify neurons stimulated during spontaneous seizures, sections of the ventral hippocampus were obtained after EEG monitoring (up to 8 weeks after AAV-TeLC or AAV-GFP injection) and labeled for ΔFosB-immunoreactivity, a cellular marker accumulating in activated neurons (Morris et al., 2000). A, D, Only very faint ΔFosB labeling was detected in some neurons of the hippocampal formation (A), including the subiculum (D; higher magnification) in AAV-GFP-injected mice (no EEG alterations) and in AAV-TeLC-injected mice with C-SWDs but no SRSs (data not shown). B, C, E, F, ΔFosB was clearly expressed in the neurons of mice that experienced one to six SRSs (B) and high numbers of ΔFosB-positive neurons were present throughout the hippocampal formation and the deep and superficial layers of the entorhinal and perirhinal cortices of mice that had experienced between 9 and 19 SRSs after AAV-TeLC (C, E, F). E, Higher magnification of the subiculum of these mice. C, F, Note the extremely strong expression of ΔFosB in the granule cell layer of these mice (C), sometimes particularly concentrated in the inner and outer portions of the granule cell layer (F). G–J, Semiquantitative estimates of ΔFosB-positive cells using the ImageJ program were done in blinded fashion and are depicted in sector CA3 (G), granule cells of the dentate gyrus (H), the subiculum (I), and layers V to VI of the perirhinal cortex after injection of AAV-GFP (GFP; n = 10) or AAV-TeLC (TeLC; J). AAV-TeLC-injected mice had experienced at most C-SWDs (TeLC sw; n = 9) or 1–6 (TeLC rs+; n = 10) or more frequent (9–19) SRSs (TeLC rs++, n = 5). Scale bars: C (for A–C), 500 μm; F (for D–F), 100 μm. Similar increases in numbers of ΔFosB-positive neurons were also obtained for sector CA1, the superficial layers of the perirhinal cortex (PRC), and the deep and superficial entorhinal cortices (data not shown). Statistical analyses were conducted using the Kruskal–Wallis test with Dunn's multiple-comparison post hoc test (all groups compared with controls; *p < 0.05; **p < 0.01; ***p < 0.001 vs AAV-GFP group). Values are given as the mean ± SEM. CA1 and CA3, Hippocampal sectors CA1 and CA3; DG, dentate gyrus; EC, entorhinal cortex; PRC, perirhinal cortex; Sub, subiculum.
- Figure 6.
The seizure threshold is reduced in mice exposing C-SWDs but not SRSs after AAV-TeLC. Seizure threshold was tested with a threshold dose of PTZ (30 mg/kg, i.p.) in PV-cre mice that did not develop SRSs after AAV-TeLC. A, PTZ induces acute (red bar) and then recurrent seizures and C-SWDs (data not shown) in mice that were seizure free for 6 weeks after AAV-TeLC. Bars represent the mean number of seizures (±SEM) per day. The red bar depicts acute PTZ-induced seizures, and the gray bars show SRSs. B, Representative EEG traces after PTZ in mice initially injected with AAV-GFP (top trace) and AAV-TeLC (bottom trace), respectively. C, After PTZ, seizures were observed in all AAV-TeLC-injected mice (n = 6) but in only 1 of 7 AAV-GFP-injected controls. D, Injection of a threshold dose of PTZ in still seizure-free mice (n = 6) 10 d after AAV-TeLC provoked one acute seizure (red bar) and subsequently SRSs (gray bars) and C-SWDs (data not shown), but not in AAV-GFP-injected controls (data not shown).
- Figure 7.
Only transient inhibition of PV neurons in the subiculum. A, Distribution of hM4Di/RFP expressed after AAV-hM4Di injection. B, hM4Di is found in neurons and dendrites at the injection site. C–E, At the injection site, the majority of PV neurons was also labeled for RFP (a tag for hM4Di). F, PV-cre mice injected with AAV-hM4Di (n = 7) were treated after 12 d with saline (not indicated) and after 15 d with CNO (10 mg/kg, i.p.). Neither treatment resulted in acute or spontaneous seizures (EEG recordings for 5 d after CNO). G, To investigate whether CNO treatment was efficient, we injected additional 12 mice with CNO or saline on day 15 after AAV-hM4Di and 45 min later with a threshold dose of PTZ (30 mg/kg, i.p.). EEGs were monitored for a further 24 h. All CNO- and PTZ-injected mice (n = 6) showed acute C-SWDs (4.2 ± 1.14 per 24 h) and two mice showed acute seizures (one and three seizures, respectively) during the initial 2 h, indicating that GABAergic transmission is reduced after CNO injection (right bars). Six AAV-hM4Di-injected mice (n = 6) treated concomitantly with saline (instead of CNO) and later with PTZ revealed neither C-SWDs nor seizures (left bars). CA1 and CA3, Hippocampal sectors CA1 and CA3; DG, dentate gyrus; EC, entorhinal cortex; Sub, subiculum. Scale bars: A, 500 μm; E (for B–E), 25 μm.
Tables
- Table 1.
Summary of mice injected with AAV-TeLC or with AAV-GFP, the anatomical distribution of TeLC expression, and the total number of seizures observed
No. Injection: AAV-FLEX- Sub/CA1 CA3 PrS PaS DG EC C-SWD Number of seizures Days recorded Group 1: AAV-FLEX-TeLC injected, no seizures 1 TeLC ++ + ++ ++ + ++ − 0 28 2 TeLC ++ + − 0 32 3 TeLC +++ ++ − 0 48 4 TeLC +++ + ++ +++ ++ ++ + 0 55 5 TeLC +++ +++ + ++ +++ + 0 56 6 TeLC ++ + 0 48 7 TeLC ++ + 0 55 8 TeLC +++ ++ ++ +++ +++ ++ + 0 56 9 TeLC ++ + ++ + + 0 55 Group 2: AAV-FLEX-TeLC injected, with seizures 10 TeLC +++ + + + 1 35 11 TeLC ++ + ++ ++ ++ + 3 56 12 TeLC +++ + +++ + + + 19 47 13 TeLC ++ ++ +++ +++ ++ + 5 30 14 TeLC +++ + + 2 47 15 TeLC +++ + ++ + 5 47 16 TeLC +++ + + 2 18 17 TeLC +++ + + + + 6 32 18 TeLC +++ + ++ +++ +++ + 12 56 19 TeLC ++ ++ + 9 30 20 TeLC +++ + + + 9 40 21 TeLC ++ + + 6 30 22 TeLC +++ + ++ + 9 25 23 TeLC ++ + 2 38 24 TeLC ++ + 5 28 25 TeLC +++ + ++ + 7 34 Group 3: AAV-FLEX-GFP injected 26 GFP ++ + +++ − 0 56 27 GFP ++ + + +++ +++ +++ − 0 55 28 GFP +++ ++ +++ + + − 0 47 29 GFP n.e. − 0 55 30 GFP n.e. − 0 43 31 GFP +++ ++ +++ +++ ++ ++ − 0 57 32 GFP ++ − 0 54 33 GFP ++ + − 0 52 34 GFP ++ ++ ++ +++ ++ + − 0 52 Group 4: AAV-FLEX-TeLC injected and 10 d later with PTZ 35 TeLC + PTZ ++ + + 14 28 36 TeLC + PTZ ++ + 6 53 37 TeLC + PTZ ++ + + + 43 se 34 38 TeLC + PTZ ++ + + 9 53 39 TeLC + PTZ ++ + 9 52 40 TeLC + PTZ ++ + + 7 52 Injections were performed unilaterally into the ventral subiculum, and mice were subjected to telemetric EEG and video monitoring. Group 1 (1–9), Mice that revealed no spontaneous seizures, but (except 1–3) C-SWDs. Six of these mice (4–9) that had experienced C-SWDs but were seizure free at day 42 (Fig. 3) were then injected with a threshold dose of PTZ resulting in acute and then recurrent spontaneous seizures (Fig. 6). Group 2 (10–25), 16 of 25 AAV-TeLC-injected mice (10–25) revealed spontaneous recurrent seizures and C-SWDs. Group 3 (26–34), none of nine AAV-GFP-injected mice exhibited recurrent seizures and were considered as controls. The fact that these mice also did not present C-SWDs argues against an unspecific effect of vector injection. Group 4 (35–40), these mice were injected with AAV-TeLC and after 10 d with PTZ. They were seizure free after AAV-TeLC injection but exhibited acute and then spontaneous recurrent C-SWDs and seizures after PTZ injection. Mouse 37 died from a status epilepticus on day 34. Expression of GFP (tag for AAV-TeLC) was most prominent in the subiculum/sector CA1, often extending to the presubiculum and parasubiculum, and, in some instances to the entorhinal cortex or to the dentate gyrus. se, Status epilepticus; PrS, presubiculum; PaS, parasubiculum; EC, entorhinal cortex; DG, dentate gyrus; n.e., not evaluated.
Spontaneous recurrent seizures Incidence: 64% of mice presented at least 1 spontaneous seizure during the first 6 weeks after virus injection
Onset of spontaneous seizures: 9.5 ± 1.86 d after AAV injection
50% of mice with seizures presented first seizure before day 6
Frequency of seizures: 5.3 ± 0.83 seizures/month
Mean seizure duration: 24.6 ± 0.62 s
Preictal C-SWD: 89.5% of spontaneous seizures were preceded by C-SWDs
C-SWDs Incidence: 88% of mice
50% of mice with seizures presented first C-SWD before day 5
Number of SWDs per C-SWD: 42.1 ± 3.14
Frequency of SWDs within C-SWDs: 0.14 ± 0.005 Hz
Number of C-SWDs: 40.0 ± 9.07 per month/mouse
Mean duration of C-SWDs: 294 ± 13.7 s
Data are presented as the mean ± SEM.
