• ADHD
• audiogenic seizure
• Grin2A(N596S)

The voltage dependent Mg2+ block of GluN2A-containing NMDARs is essential
for activity-induced neuroplasticity. The GRIN2A gene, encoding the NMDAR
subunits GluN2A, was found to be disrupted or mutated in individuals with
mental retardation and/or epilepsy.
In my thesis work I performed behavioral test, genotyping, western-blot analysis
and many different histological technics to analyze protein expression and
anatomical structures of neurons and glia of mice carrying a gene targeted point
mutation, Grin2A(N596S), in the gene encoding NMDAR subunit GluN2A. This
mutation causes the loss of the Mg2+ block and a decrease in Ca2+ permeability
and it is homologous to a human de novo point mutation Grin2A(N615K), found
in a young patient with severe mental retardation and epileptic seizures (Endele S.
et al., 2010).
As in humans, mice homozygous for the mutation (Grin2aS/S) have profound
cognitive impairments and, analyzing their performance in a battery of behavioral
test such as the Burrowing test, the Crawley’s sociability test, the Cliff avoidance
reaction (CAR) test and the passive avoidance (PA) test, emerged that Grin2aS/S
mice express also features related to ADHD, which are hyperactivity, inattention
and impulsivity. On the other hand, in heterozygous Grin2aS/N mice the mutation
had a lower penetrance, resulting in an intermediate behavior and ambiguous
ADHD-like phenotype compared to the other two groups of mice, Grin2aS/S and
Grin2aN/N courts phenotype.
Grin2aS/S mice exhibited a 100% penetrance of generalized convulsive seizure
induced by an external stimulus (11 kHz tone), namely Audiogenic Seizure (AGS).
The AGS ended always with respiratory arrest and the death, resembling the same
human process called SUDEP (sudden unexpected death in epilepsy). The
epileptic phenotype could be genetically rescued in a different genetic
background of FVB mice or pharmacologically by low doses of memantine.
Western blot analysis reveled no differences in NMDAR subunits protein
expression and other kinds of scaffolding proteins enriched in the PSD in forebrain
membrane fraction of P28 Grin2aS/S, Grin2aS/N and Grin2aN/N mice.
Moreover, c-fos and arc expression were verified by immunofluorescence after
tone induction, confirming published data about AGS-related brain circuit.
I also examined the presence of gliosis and neurodegeneration, 24 hours and 8
days after seizure induction, discovering a GFAP (glial fibrillary acidic protein)
reduction in some brain areas and gliosis-plaque in the cortex of Grin2aS/S mice;
furthermore, Silver and NeuN stainings highlighted signs of neurodegeneration in
the same brain areas related to AGS. Finally, using the Golgi impregnation
technique I compared the normal brain development and neuronal structure in
mutated and wild-type mice.
In summary, the results of my research revealed an ADHD-like phenotype in
Grin2aS/S mice, coupled with a strong epileptic phenotype, which caused the
death of the animal and some molecular adaptive responses in the brain circuit
related to the seizure, but that it could be prevented by memantine, a drug
commonly used in therapy.