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, 3 (6), e2473

Epilepsy in Dcx Knockout Mice Associated With Discrete Lamination Defects and Enhanced Excitability in the Hippocampus

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Epilepsy in Dcx Knockout Mice Associated With Discrete Lamination Defects and Enhanced Excitability in the Hippocampus

Marika Nosten-Bertrand et al. PLoS One.

Abstract

Patients with Doublecortin (DCX) mutations have severe cortical malformations associated with mental retardation and epilepsy. Dcx knockout (KO) mice show no major isocortical abnormalities, but have discrete hippocampal defects. We questioned the functional consequences of these defects and report here that Dcx KO mice are hyperactive and exhibit spontaneous convulsive seizures. Changes in neuropeptide Y and calbindin expression, consistent with seizure occurrence, were detected in a large proportion of KO animals, and convulsants, including kainate and pentylenetetrazole, also induced seizures more readily in KO mice. We show that the dysplastic CA3 region in KO hippocampal slices generates sharp wave-like activities and possesses a lower threshold for epileptiform events. Video-EEG monitoring also demonstrated that spontaneous seizures were initiated in the hippocampus. Similarly, seizures in human patients mutated for DCX can show a primary involvement of the temporal lobe. In conclusion, seizures in Dcx KO mice are likely to be due to abnormal synaptic transmission involving heterotopic cells in the hippocampus and these mice may therefore provide a useful model to further study how lamination defects underlie the genesis of epileptiform activities.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Morphological Abnormalities in the Dcx KO Hippocampus.
(A–D) Cresyl violet staining showed that dentate gyri were indistinguishable between the genotypes, however, abnormally organized CA3 pyramidal cells in the KO hippocampus were observed (red arrows in D). (E,F) Calbindin-labeling shows less well organized mossy fibers in the KO section (F) compared to an equivalent WT section (E). Red arrowheads indicate a more fragmented appearance of fibers in the KO. Only strongly CB-labeled KO sections were used in this analysis. (G–K) Golgi-Cox labeling shows the association of interneuron-like cells (red arrows) with CA3 pyramidal cells, even those displaced in a separate upper pyramidal layer in the KO (H). Pyramidal cells (blue, stained with Nissl) were present in 3 approximate layers in this KO animal (2 are observed here labeled 1, 2 in H). (I–K) The three cells shown in H are shown in better focus individually in I–K. These cells show the typical morphology of CA3 basket cell or axo-axonic-like interneurons , with fusiform cell bodies, and one or two dendrites originating from the apical pole, which then branch proximally to give radially oriented dendrites in the stratum radiatum. In addition, such cells have several basal, spine-free dendrites, branched close to the cell body and extended toward the alveus. Scale bars: A (for A, B), 500 µm; and C (for C, D); E (for E, F); G (for G, H), 50 µm; I (for I, J, K), 44 µm. dg, dentate gyrus; fi, fimbria; so, stratum oriens.
Figure 2
Figure 2. Dcx KO Mice are Hyperactive.
Time-course of mean±SEM spontaneous horizontal (locomotion) and vertical (rearings) behavioural activity in WT (n = 31) and KO (n = 30) mice. Adult mice (aged 5–6 months) were introduced individually in activity boxes and automatically recorded over a 2 h period. An ANOVA test revealed significance between the 2 strains for horizontal activity, (genotype, F 1,1416 = 14.48, P<0.0001; time, F 23,1416 = 80.82, P<0.0001 and genotype×time interaction, F 23,1416 = 2.78, P<0.01); and for vertical activity, (genotype, F 1,1416 = 102.71, P<0.0001; time, F 23,1416 = 76.41, P<0.0001 and genotype×time interaction, F 23,1416 = 1.785, P<0.01).
Figure 3
Figure 3. Untreated KO Mice Show Changes in Seizure Sensitive Marker Expression.
(A–D) CB immunoreactivity in an untreated WT mouse (A,C) and a KO mouse (B,D), sacrificed at 7 months of age. In the hippocampus of the KO mouse, the expression of CB is reduced in the dg cell layer and dendrites, and in mossy fibers (D). Although the corpus callosum appears thinner in the KO mouse section, this is not typical for Dcx KO mice on the C57BL/6 background (8). (E–H) NPY immunoreactivity in an untreated WT mouse (E,G) and a KO mouse (F,H), sacrificed at 7 months of age. High magnification of the hippocampus shows NPY neoexpression in the mossy fibers of the dg in the KO mouse (H). Ectopic NPY accumulates in the mossy fibers of dentate granule cells probably because it is transported through the fibers to the terminals, from which it can be released . No obvious differences in the expression of NPY and CB were observed in other brain structures, as previously reported by others . (I–L) Calretinin immunoreactivity in an untreated WT mouse (I,K) and a KO mouse (J,L). Note the disorganization and increase in expression and number of calretinin-labeled cells in the subgranular zone (SGZ) in KO sections. Analysis of 9 KO animals showed modifications in the SGZ in 7 of them (see also Figure S2), although 3 of these animals showed only a minor disorganization of the SGZ cells. The granular layer itself showed no obvious differences between the genotypes. Cx: cortex; dg: dentate gyrus; gc: granular cells; mf: mossy fibers; SGZ: subgranular zone. Scale bars: A (for A, B, E, F), 1 mm; C (for C, D, G, H), 200 µm; I (for I, J), 300 µm; K (for K, L), 100 µm.
Figure 4
Figure 4. Dcx KO Mice are More Susceptible to KA-induced Seizures.
(A) In the first 60 min after KA injection, a significant increase in the progression of seizure-related behavior was observed in KO mice (white circles), compared to WT (black squares). ANOVA: genotype, F 1,1565 = 37.65, P<0.0001; time, F 11,565 = 12.5, P<0.0001 and genotype×time interaction, F 11,565 = 1.4, P>0.05. KO mice were scored significantly higher at 15 min post-injection than WT mice and the increased severity of their reaction to KA continued throughout the initial 1 h monitoring period. *** differs from control at P<0.0001. (B) Bar histograms show that, during the three hour monitoring period, more KO mice show rearing and falling (score 4) or progression to severe tonic, clonic seizures (score 6) compared to WT mice. Inversely, more WT mice show less severe behavior (1, immobility). Thus, KO mice are significantly more susceptible to KA-induced seizures than WT mice. Seizures were rated according to a previously defined scale : 1: immobility; 2: forelimb and/or tail extension; 3: repetitive movements, head bobbing; 4: rearing and falling; 5: continuous rearing and falling: 6: severe tonic-clonic seizures.
Figure 5
Figure 5. Spontaneous Clonic Seizures in Dcx KO Mice.
(A) Typical example of hippocampal and cortical EEG recordings of a spontaneous seizure observed in a Dcx KO mouse associated with rearing, head nodding and forelimb clonus. (B) EEG recording at lower speed of the same seizure suggesting initiation in the hippocampus. Hipp: right hippocampus; Cx L: left cortex; Cx R: right cortex. (C) Averaged (n = 5 spontaneous seizures) time–frequency chart of signal power 5 sec before and 20 sec after the onset of the seizure in the hippocampus (Hipp) and the cortex (Cx). Hz, hertz. (D) Difference of GPDC (averaged over 5 seizures) between hippocampal and cortical recordings. A positive value indicates a direction of information transfer from hippocampus towards cortex. Red areas indicate a significant GPDC difference (p<0.005). Statistics were obtained using surrogate data in which phase relationships were destroyed by phase randomization of frequency spectra.
Figure 6
Figure 6. Enhanced Excitability and Lower Threshold for Epileptical Events in Dcx KO Hippocampal Slices.
(A) Position of the two tungsten electrodes placed in the stratum pyramidale of the CA3b and CA3c regions. (B) Bar graph shows a higher frequency of the synchronously generated sharp wave-like events in KO slices than in WT (1.91±0.34 Hz, KO, n = 10 vs. 0.48±0.15 Hz, WT, n = 11, Students's t-test, P<0.001). Error bars indicate SEM. (C) Extracellular recordings of synchronously generated sharp wave-like activities in the CA3b and CA3c regions from WT (left) and KO (center) animals. * indicates synchronous events, which are seen in both CA3b and c regions. Right panel shows 1 synchronous sharp wave-like activity in KO slices. (D) Interictal bursts initiated in the CA3 region on exposure to bicuculline at 4 µM in KO hippocampal slices. (E) Bar graph shows the percentage of slices that reached bursting threshold in bicuculline from 2 to 8 µM. KO mice are predisposed to epileptiform activity with moderate bicuculline application.

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