Neocortical post-traumatic epileptogenesis is associated with loss of GABAergic neurons

Abstract

The subtle mechanisms of post-traumatic epileptogenesis remain unknown, although the incidence of chronic epilepsy after penetrating cortical wounds is high. Here, we investigated whether the increased frequency of seizures occurring within 6 weeks following partial deafferentation of the suprasylvian gyrus in cats is accompanied with a change in the ratio between the number of excitatory and inhibitory neurons. Immuno-histochemical labeling of all neurons with neuronal-specific nuclear protein (NeuN) antibody, and of the GABAergic inhibitory neurons with either gamma-aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD 65&67) antibodies, was performed on sections obtained from control and epileptic animals with chronically deafferented suprasylvian gyrus. Quantification of the labeled neurons was performed in control animals and at 2, 4, and 6 weeks following cortical deafferentation, in the suprasylvian and marginal gyri, both ipsi- and contra-lateral to the cortical trauma. In all epileptic animals, the neuronal loss was circumscribed to the deafferented suprasylvian gyrus. Inhibitory GABAergic neurons were particularly more sensitive to cortical deafferentation than excitatory ones, leading to a progressively increasing ratio between excitation and inhibition towards excitation, potentially explaining the increased propensity to seizures in chronic undercut cortex.

FIG. 1.

Increased propensity to seizures after chronic cortical deafferentation. (A) Frontal section (schema in the left panel, Nissl staining in the right panel) of cat brain. The extent of the damage caused by the knife is expanded in the inset. Scale bar = 4 mm (for the complete brain section), 2 mm (for the inset). (B) Electroencephalography (EEG) field potentials during slow oscillation (left) and seizures (right) at 2 weeks (2W), 4 weeks (4W), and 6 weeks (6W) respectively, after brain injury. The EEG was recorded in the undercut gyrus (SS ip), in the marginal gyrus ipsi-lateral (MA ip) and contra-lateral (MA ct) to the undercut and in the contra-lateral suprasylvian gyrus (SS ct). (C) Fast Fourier transformation (FFT) of EEG activity during cortical slow-oscillation (left) and seizures (right) at different time intervals following cortical trauma. With progression in time, 3–4-Hz spike wave (SW) and poli-spike-wave (PSW) activities (PSW) emerged from the slow-oscillation and seizures progressively developed from SW complexes to SW/PSW activities intermingled with fast-runs (10–20 Hz).

FIG. 2.

Penetrating brain wounds cause a reduction of gray matter's thickness and the disorganization of cortical architecture. (A) Nissl staining of the suprasylvian gyrus in control (left) and 6 weeks following cortical undercut (right). (B) Cortical depth profile of the suprasylvian gyrus with Nissl staining, showing the normal hexalaminar (I–VI) distribution in control (CTRL), and the progressive disorganization and shrinkage of the gray matter at 2 weeks (2W), 4 weeks (4W), and 6 weeks (6W), respectively, after deafferentation. (C) Illustration of neuronal morphology in the cortical layer 2 (upper panel) and layer 5 (lower panel) in control (left) and undercut cortex (right). Note in chronic undercut cortex the reduced staining of neuronal cytoplasm, the variability of shape of the nuclei, and the condensation of genetic material and the lack of the nucleolus. (D) Quantification of cortical width in control (CTRL) and at 2 weeks (2W), 4 weeks (4W), and 6 weeks (6W) after undercut (average, n = 10). **p < 0.01, Student's t-test. Scale bar (at the bottom right corner of each panel) = 1 mm (A), 200 μm (B), 25 μm (C).

FIG. 3.

Immunohistochemical labeling of cortical neurons. (A) Staining for glutamic acid decarboxylase (GAD 65&67; upper panel), gamma-aminobutyric acid (GABA; middle panel) and neuronal nuclear protein (NeuN; lower panel) in control (left) and undercut cortex (right). Insets show the simple GAD, GABA, and NeuN labeling of one neuron. (B) Double staining GAD and NeuN (left panel), and GABA and NeuN (right panel). Insets depict the double labeling of GABAergic neurons. Note the nucleus labeled in gray-black (DAB-Ni, Cr enhancement) and the cytoplasm in brown (DAB). Scale bar = 100 μm (50 μm in insets) for A; 20 μm (10 μm in insets) for B.

FIG. 4.

Examples of cortical depth profiles showing the distribution of excitatory and inhibitory neurons in control and undercut cortex. (A) Individual examples of neuronal profiles double stained with GAD and NeuN in control (CTRL) and after deafferentation at 2 weeks (2W), 4 weeks (4W), and 6 weeks (6W). (B) Examples similar to those in A for neurons double stained with GABA and NeuN. Graphs on the right side of the profiles depict the number of labeled cells (x-axis), red for inhibitory GABAergic neurons and black for non-GABAergic excitatory neurons, for each 100 μm of depth (y-axis) on a total area of 1.8 mm² per section. Note the reduction of neurons in layers II–III and V–VI in early stages of the undercut and the generalized decrease in neuronal density at 6 weeks following cortical trauma.

FIG. 5.

Depth profile distribution of neuronal densities in control and after cortical trauma. (A) Average number of excitatory neurons (NeuN minus GAD [left panel] and NeuN minus GABA [right panel]) on 0.03 mm² areas (x-axis) at each 100 μm of cortical depth (y-axis) from five different animals in control (black circles) and at 2 weeks (black squares), 4 weeks (black triangles), and 6 weeks (black rhombi). (B) Average distribution of inhibitory neurons (GAD [left panel] and GABA [right panel]) on 0.03 mm² areas (x-axis) at each 100 μm of cortical depth (y-axis) from five different animals in control (black circle) and at 2 weeks (black square), 4 weeks (black triangle), and 6 weeks (black rhombus). Note the progressive loss of excitatory neurons mostly in the deep layers, and the loss of inhibitory neurons both in the deep layers and in the more superficial ones. The diagram on the right side of each graph represents the statistical significant difference (control - 2 weeks left; control - 4 weeks middle; control - 6 weeks right) in neuronal densities for every 100 μm of cortical depth. *p < 0.05, Student's t-test.

FIG. 6.

Changes in the balance between excitation and inhibition towards excitation in chronically deafferented cortex. (A) Stack images were computed from pictures acquired at every 5 μm through the thickness of the slice. Note different neurons stained at different levels, demonstrating antibody penetration through the entire thickness of the slice. (B) Images depict the selection process, used for automatic quantification with the Image-Pro software, of all neurons (labeled with NeuN) and of specifically labeled inhibitory neurons (double-labeled with either GAD or GABA) based on predefined ranges of image analysis parameters equally applied to all acquired z-stack average images. (C) Distribution of the total number of neurons (NeuN distribution) and of inhibitory neurons (GAD and GABA distribution) in control (black bars), and after undercut at 2 weeks (white bars), 4 weeks (light gray bars) and 6 weeks (dark gray bars). Neuronal densities are expressed as number of neurons per each analyzed area of 0.07 mm². (D) Relative distribution of total neuronal loss in the undercut gyrus, together with progressive loss of excitatory neurons and inhibitory neurons. Note the more significant reduction of inhibitory GABAergic neurons. (E) The ratio of excitatory/inhibitory neurons in control (CTRL) and at 2 weeks (2W), 4 weeks (4W), and 6 weeks (6W) after deafferentation. Note the increased shift in the excitation-inhibition balance toward excitation following cortical trauma which might account for the increased propensity to seizures. *p < 0.05, **p < 0.01, ***p < 0.001, analysis of variance (ANOVA) with post hoc Tukey test.