Abstracts

Rationale: γ-Aminobutyric acid (GABA) type A receptors (GABAR) are heteropentameric chloride ionotropic receptors that mediate majority of CNS inhibition and play vital roles in many neurological and neuropsychiatric conditions, especially epilepsy. However, during development, GABAergic signaling can act in excitatory capacity, with important roles in developmental processes like neuronal migration and circuit formation. Progressive changes in GABAR composition and expression of K-Cl transporter KCC2 alter GABAergic signals during development to fit unique developmental functions. To date, nineteen main GABAR subunits have been characterized, allowing a large number of possible GABAR combinations, which determine GABAR subcellular localization, GABA potency, activation and deactivation rates, and other properties. On the other hand, transport activity of KCC2 is a significant determinant of GABAR reversal potential. In this study, we investigate these adaptations in developing somatosensory cortex by characterizing expression of nine major GABAR subunits and KCC2 at different developmental stages in mice.

Methods: Antibodies against GABAR subunits α1-5, β2-3, γ2, and δ were first validated using HEK cells transfected to express GABAR subunit protein, and then analyzed by immunocytochemistry or Western blot (WB) of extracted proteins. GABAR subunit expression was measured by WB in cortical lysates, and GABAR subunit and KCC2 expression was visualized by immunohistochemistry in cortical sections (Figure). Tissue samples progressively staged from embryonic day 13.5 to postnatal day 25 (P25) were used.

Results: General patterns of cortical expression are marked by very early expression of α3 and α5, predominantly in layer (L) 5/6 and subplate. On the other hand, α1, α2, and then α4 and δ were primarily expressed at later developmental stages, often most strongly in L4 and superficial layers. Interestingly, α2 and α3 were seen in the intermediate zone. α2 expression overlapped with the radial glia marker RC2, while subcortical α3 expression seemed to follow the internal capsule. Expression of β2, β3, and γ2 was more ubiquitous than α subunits, but still had laminar-specific expression. β3 expression came online early and generally preceded expression of β2. Especially interesting is an abrupt increase in α1, α2, α4, β2 and δ expression in L4 of the barrel fields around P5, in addition to the β2/3 and γ2 proteins that are already there. These changes coincide with 4 barrels formation. Similar to later-expressed subunits, KCC2 expression was low until P5 with exception of some KCC2-expressing interneurons. At P4, KCC2 began to be expressed in L4, and then increased in expression, with stronger expression in superficial layers.

Conclusions: Similarly to previous studies, we found that embryonic cortex expresses mainly GABAR subunits ɑ3, ɑ5, β3, and γ2, while expression of ɑ1, ɑ2, ɑ4, β2, δ, and KCC2 begins at later points in development. However, we also found unique patterns of temporal, regional, and laminar expression of each subunit and KCC2 that have previously not been known.

Funding: This work was funded by Veteran Affairs Merit Grant I01 BX001189.