Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase

Abstract

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms, GAD65 and GAD67. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that GAD65 may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding GAD65 and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to GAD67-/- animals, which are born with developmental abnormalities and die shortly after birth, GAD65-/- mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5' phosphate-inducible apo-enzyme reservoir is significantly decreased. GAD65-/- mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in GAD65-/- mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the GAD65-/- mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by GAD65 is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present GAD65-/- animals as a model of epilepsy involving GABA-ergic pathways.

Figure 1

Targeted disruption of the GAD65 gene in ES cells and mice. (a) Schematic of the wild-type locus, the targeting construct pmGAD65, and the expected targeted allele. Solid and open boxes represent coding and untranslated exons, respectively. Arrowed lines indicate the EcoRI restriction fragment sizes for the wild-type and targeted alleles. Restriction sites are: E, EcoRI; X, XbaI; Xh, XhoI; B, BamHI. (b) Genomic Southern blot analyses of ES cell DNA and tail biopsies. Genomic DNA from the parental and targeted ES cells along with genomic DNA from offspring obtained after breeding of heterozygote mice was digested with EcoRI and probed with a 5′ 500-bp genomic fragment (thick, black line). Wild-type and targeted restriction fragments are 6 kb and 4 kb, respectively. (c) Western blot analysis of GAD expression in GAD65+/+, GAD65+/−, and GAD65−/− brain extracts with an antibody recognizing GAD65 (1) or GAD65 and GAD67 (2).

Figure 2

Loss of GAD65 affects apo- but not holo-GAD enzyme activity. GAD enzyme activity was measured in brain extracts of GAD65+/+ (n = 4, solid bars) and GAD65−/− (n = 4, open bars) mice in the presence or absence of PLP. Data were converted to pmol CO₂ evolved per min/μg protein and expressed as percent of wild-type levels in the presence of PLP (2.69 ± 0.23 pmol per min/μg; mean ± SEM).

Figure 3

GAD65-deficient mice are prone to sudden death. Spontaneous deaths among homozygous mutant animals were observed as early as the fourth postnatal week. Solid, shaded, and open circles represent survival of wild-type, heterozygous, and homozygous mutant animals, respectively. Shown below each time point is the total number of animals represented for each genotype.

Figure 4

Decreased basal GABA levels in GAD65−/− {129 × NOD} but not in GAD65−/− {129 × B6} mice. GABA contents were measured in brain extracts of cerebral cortex, cerebellum, and hippocampus of GAD65+/+ (solid bars) and GAD65−/− (open bars) mice (n = 15 for each region for {129 × B6} animals and n = 24 for each region for {129 × NOD} animals) by using standard HPLC methods (21). GABA content values are significantly different between wild-type {129 × NOD} and homozygous mutant {129 × NOD} mice (∗∗, cortex and hippocampus, P < 0.001; ∗, cerebellum, P < 0.02; Student’s t test). GABA content values are also significantly different between wild-type {129 × B6} and wild-type {129 × NOD} mice (cortex, P < 0.01; cerebellum and hippocampus, P < 0.001; Student’s t test).

Figure 5

EEG recording and analysis of c-fos-like expression in GAD65−/− mouse brain during and after stress-induced seizures. (a) Monopolar EEG recording (left hemisphere) showing typical neocortical seizure activity in an adult GAD65−/− {129 × NOD} mouse after mild stress (mouse was placed on a small elevated platform). The seizure lasted approximately 40 sec, beginning with an abrupt transition from rhythmic background activity (1) to bilateral (right hemisphere not shown) continuous high-amplitude spiking during clonic forelimb activity (2), followed by a prolonged (>60 sec) postictal period of low-amplitude synchronous rhythms and behavioral depression (3, only initial stage is shown). Calibration: upper trace, 5 sec, 100 μV; lower traces, 2 sec, 300 μV. (b) Immunohistochemical staining of c-fos in brain sections of GAD65−/− {129 × B6} mice 2 hr after exposure to mild stress that either did (Left) or did not (Right) induce a seizure. c-fos expression (dark staining of neuronal nuclei) is observed in (1) the granule cell layer (GC) of the dentate gyrus and CA3 of the hippocampus, (2) amygdala (Am) and piriform cortex (Pir), and (3) lateral entorhinal cortex (L Ent) of GAD65−/− mice after a seizure, but not in nonseizure GAD65−/− controls. No significant staining was observed in GAD65+/+ controls (data not shown). H, hippocampus. (Bar = 500 μm).