Progressive Brain Damage, Synaptic Reorganization and NMDA Activation in a Model of Epileptogenic Cortical Dysplasia

<div><p>Whether severe epilepsy could be a progressive disorder remains as yet unresolved. We previously demonstrated in a rat model of acquired focal cortical dysplasia, the methylazoxymethanol/pilocarpine - MAM/pilocarpine - rats, that the occurrence of status epilepticus (SE) and subsequent seizures fostered a pathologic process capable of modifying the morphology of cortical pyramidal neurons and NMDA receptor expression/localization. We have here extended our analysis by evaluating neocortical and hippocampal changes in MAM/pilocarpine rats at different epilepsy stages, from few days after onset up to six months of chronic epilepsy. Our findings indicate that the process triggered by SE and subsequent seizures in the malformed brain <b>i</b>) is steadily progressive, deeply altering neocortical and hippocampal morphology, with atrophy of neocortex and CA regions and progressive increase of granule cell layer dispersion; <b>ii</b>) changes dramatically the fine morphology of neurons in neocortex and hippocampus, by increasing cell size and decreasing both dendrite arborization and spine density; <b>iii</b>) induces reorganization of glutamatergic and GABAergic networks in both neocortex and hippocampus, favoring excitatory <i>vs</i> inhibitory input; <b>iv</b>) activates NMDA regulatory subunits. Taken together, our data indicate that, at least in experimental models of brain malformations, severe seizure activity, i.e., SE plus recurrent seizures, may lead to a widespread, steadily progressive architectural, neuronal and synaptic reorganization in the brain. They also suggest the mechanistic relevance of glutamate/NMDA hyper-activation in the seizure-related brain pathologic plasticity.</p></div

Altered synaptic input on hippocampal neurons of epileptic MP rats.

<p><b>A–H</b>) VGAT⁺ (green) and VGLUT1⁺ (red) synaptic terminals on granule cells (A–B and E–F) and CA3 pyramidal neurons (C–D and G–H) from a representative non-epileptic MDP (A–D) and MP-6m rats (E–H). Sections were counterstained with Neurotrace™ (blue) to reveal neuronal nuclei. Note in MDP rats the intense VGAT⁺ staining outlining cell bodies (A and C, green) and the virtual absence of VGLUT1⁺ staining in both GC and CA3 layers (B and D, red). In MP-6m rats, by contrast, perisomatic VGAT⁺ staining was reduced (compare E <i>vs</i> A, G <i>vs</i> C) but VGLUT1⁺ synaptic terminals were more evident in both GC and CA3 layers (compare F <i>vs</i> B, H <i>vs</i> D), sometimes outlining neuronal profiles (arrowheads in F, arrows in H). <b>I–J</b>) Quantification of VGAT/VGLUT1 IF ratio at peri-somatic and -dendritic terminals on granule cells (I) and CA3 pyramidal neurons (J). Note the significant reduction VGAT/VGLUT1 ratio in both layers in MP rats compared to aged-matched MDP controls (<b>I</b>: MP-6m <i>vs</i> MDP-3m ***p<0.001; MP-6m <i>vs</i> MDP-6m **p<0.01; MP-3m <i>vs</i> MDP3m/MDP-6m *p<0.05; <b>J</b>: MP-3m/MP-6m <i>vs</i> MDP-3m/MDP-6m rats, *p<0.05). No differences were found in MDP-3m <i>vs</i> MDP-6m and MP-3m <i>vs</i> MP-6m. Scale bar: 20 µm.</p

Progressive hippocampal changes in epileptic MP rats.

<p><b>A–C</b>) Low-power thionine-stained coronal sections from dorsal hippocampus revealing progressive morphologic changes in chronic epileptic rats. Note the progressive neuronal loss in CA (arrowheads in C <i>vs</i> B <i>vs</i> A), and the twisted, irregular shape of DG particularly evident in MP-6m rats (C). Asterisk in panel A marks a para-hippocampal heterotopic nodule. <b>D</b>) 3-D reconstruction and volumetric analysis of whole hippocampi showed a significant and progressive decrease of hippocampal volume during epilepsy course (MP-3m, 13.40±1.88 mm³, and MP-6m, 10.72±2.7 mm³, <i>vs</i> MDP-3m, 22.17±2.2 mm³: ***p<0.001; MP-6m <i>vs</i> MP-3m: *p<0.05). <b>E–G</b>) The CA1 region was severely affected during epilepsy, as indicated by progressive loss of pyramidal neurons and collapse of the stratum oriens (red arrows). <b>H–J</b>) Progressive loss of hilar neurons and increase of GCL thickness in epileptic MP (I–J) <i>vs</i> MDP-3m rats (H). <b>K</b>) Quantification of lower GCL thickness showed a progressive increase over time (MP-6m <i>vs</i> MDP-3m: *p<0.05). Scale bars: 200 µm in A–C; 100 µm in E–J.</p

Dysmorphic neocortical pyramidal neurons in epileptic MP rats.

<p><b>A–F</b>) Low- (A–C) and high-power (D–F) microphotographs of NF200⁺ enlarged pyramidal neurons in MDP (A, D) and epileptic MP-EC (B, E) and MP-6m (C, F) rats. Note the progressive perikaryal enlargement and dendritic simplification of dysplastic neurons in MP-6m rats <i>vs</i> both non-epileptic MDP and early-chronic epileptic MAM rats. <b>G–I</b>) Quantification and statistical analysis of somatic area (G), dendritic thickness (H) and cell numbers (I) of NF200⁺ enlarged pyramidal neurons (≥400 µm²) at different epilepsy stages in MAM rats (*p<0.05; **p<0.01; ***p<0.001; n.s: not significant). For somatic area and dendrite thickness at least 4 animals/each group were analyzed (25 neurons per animal). Scale bars: 100 µm in A–C; 10 µm in D–F.</p

Golgi-Cox analysis of neocortical and hippocampal neurons.

<p>A–H) Low- (A–D) and high-power (E–H) microphotographs of Golgiimpregnated neocortical (A–B, E–H) and hippocampal (C–D) neurons from MDP-3m (A, C, E, G) and epileptic MP-3m (B, D, F, H) rats. Note the dramatic and diffuse loss of dendritic arbors (B, D <i>vs</i> A, C), increase of both soma size and apical dendrite thickness (F <i>vs</i> E), and loss of spines (H <i>vs</i> G) in neurons from epileptic MP-3m <i>vs</i> non-epileptic MDP-3m rats. Dendrite fragmentations (black arrowhead in H), constrictions (white arrowhead in H) and varicosities (arrows in H, inset) are evident in higher magnification images. Dotted red and blue lines mark CA pyramidal and granule cell layers, respectively (C–D). Scale bars: A–B and C–D, 100 µm; E–H and inset, 10 µm.</p

Study design and number of rats used in the different experiments.

<p>MDP control rats were sacrificed 3 or 6–9 months after diazepam-pilocarpine administration (respectively MDP-3m and MDP-6m). Randomly chosen epileptic MP rats were sacrificed respectively 3–5 days (early chronic), 3 months and 6 months after epilepsy onset. Seven chronic epileptic MP rats were found dead (three rats, ∼1 month post-SE; two rats, ∼4 months post-SE and two rats, ∼6 months post-SE). MDP, MAM/diazepam/pilocarpine treated rats; MP, MAM/pilocarpine treated rats; WB, western blot; SE, status epilepticus. No SE: rats not experiencing SE.</p

Quantification of dendritic branching and spine density in epileptic MP rats.

<p><b>A–B</b>) Neurolucida tracings of representative Golgi-impregnated cortical pyramidal neurons from chronic epileptic MP-3m (A) and non-epileptic MDP-3m (B) rats. Note the clear reduction of dendritic tree complexity and spine density in the MP neuron in panel A. Scale bar: 20 µm. <b>C–D</b>) Quantitative analysis of total dendrite length (C) and spine density (D) of basal and apical dendrites from fully reconstructed cortical pyramidal neurons in MDP-3m <i>vs</i> MP-3m (*p<0.05; **p<0.01). <b>E–F</b>) Sholl dendrite analysis, obtained by placing a series of concentric circles spaced at 10 µm intervals centered on the soma, demonstrated a significant reduction in dendritic arborization of both basal (**p<0.01) and apical (*p<0.05) dendrites in MP-3m <i>vs</i> MDP-3m rats.</p

Progressive neocortical atrophy in epileptic MP rats.

<p><b>A–D</b>) Low-power thionine-stained coronal sections from rostral (A₁–D₁), somatosensory (A₂–D₂) and posterior (A₃–D₃) cortical areas in representative non-epileptic MDP and epileptic MP rats at different epilepsy stages (EC, early chronic; 3m, 3 months; 6m, 6 months. <b>E</b>) Statistical analysis of cortical thickness (at least n = 4 rats each group, except of MDP-6m, n = 3). Note the progressive epilepsy-dependent cortical thinning. Rostral cortex: MP-6m <i>vs</i> MDP-3m, MD6m and MP-EC **p<0.01; MP-6m <i>vs</i> MP-3m *p<0.05. Somatosensory cortex: MP-6m <i>vs</i> MDP-3m, MDP6m and MP-EC **p<0.01; MP-3m <i>vs</i> MDP-3m, MDP-6m and MP-EC *p<0.05; MP-6m <i>vs</i> MP-3m: *p<0.05. Posterior cortex: MP-6m <i>vs</i> MDP-3m, MDP-6m and MP-EC ***p<0.001; MP-3m <i>vs</i> MDP-3m, MDP-6m and MP-EC *p<0.05; MP-6m <i>vs</i> MP-3m: *p<0.05. Scale bar: 1 mm.</p

Altered synaptic input on neocortical pyramidal neurons of epileptic MP rats.

<p><b>A–F</b>) VGAT⁺ (green) and VGLUT1⁺ (red) synaptic terminals on pyramidal neurons from non-epileptic MDP (A–C) and epileptic MP-6m rats (D–F). Panels C and F are merged images counterstained with Neurotrace™ (blue) to reveal neuronal nuclei. <b>G</b>) Quantification of VGAT⁺ and VGLUT1⁺ peri-somatic and -dendritic terminals on neocortical pyramidal neurons expressed as VGAT/VGLUT1 IF ratio. VGAT/VGLUT1 ratio was significantly decreased in MP <i>vs</i> aged-matched MDP control (*p<0.05 MP-3m/6m <i>vs</i> MDP-3m/6m). No differences were found in MDP-3m <i>vs</i> MDP-6m and MP-3m <i>vs</i> MP-6m. Scale bar: 20 µm.</p

Molecular composition of the glutamatergic synapse in epileptic MP rats.

<p><b>A–B</b>) Representative WB analysis in neocortical (A) and hippocampal (B) homogenates from 4 MDP-3m and 4 epileptic MP-3m rats. Note the increased expression of the activated, phospho-tyr-1472-NR2B (pNR2B) subunit in both neocortex and hippocampus in MP-3m <i>vs</i> MDP-3m rats, which was associated in the neocortex with decreased NR2A/B subunits. <b>C–D</b>) Quantification of NMDA receptor subunits and associated proteins/actin ratio in epileptic MP-3m rats, expressed as percentage of values <i>vs</i> non-epileptic MDP rats. Significant increase of pNR2B in neocortex and hippocampus (*p<0.05) and decrease of NR2A/B (**p<0.01) in neocortex only were found in epileptic MP-3m <i>vs</i> MDP-3m rats (at least n = 4 rats each group). <b>E</b>) Representative WB of pNR2B and total NR2B expression from neocortical and hippocampal homogenates of MP rats at different epilepsy stages and non-epileptic MDP rats. Note the persistent NR2B phosphorylation in both neocortex and hippocampus of MP rats. <b>F</b>) Quantification of the pNR2B/total NR2B ratio. The ratio was significantly increased in the hippocampus in early chronic and chronic epilepsy stages (MP <i>vs</i> MDP rat *p<0.05) and it increased progressively in the neocortex during epilepsy course (MP <i>vs</i> MDP rat *p<0.05, **p<0.01; ***p<0.001; at least n = 4 rats each group).</p