Cortical Parvalbumin-Positive Interneuron Development and Function Are Altered in the APC Conditional Knockout Mouse Model of Infantile and Epileptic Spasms Syndrome.

Infantile and epileptic spasms syndrome (IESS) is a childhood epilepsy syndrome characterized by infantile or late-onset spasms, abnormal neonatal EEG, and epilepsy. Few treatments exist for IESS, clinical outcomes are poor, and the molecular and circuit-level etiologies of IESS are not well understood. Multiple human IESS risk genes are linked to Wnt/ß-catenin signaling, a pathway that controls developmental transcriptional programs and promotes glutamatergic excitation via ß-catenin's role as a synaptic scaffold. We previously showed that deleting adenomatous polyposis coli (APC), a component of the ß-catenin destruction complex, in excitatory neurons (APC cKO mice, APCfl/fl x CaMKIIαCre) increased ß-catenin levels in developing glutamatergic neurons and led to infantile behavioral spasms, abnormal neonatal EEG, and adult epilepsy. Here, we tested the hypothesis that the development of GABAergic interneurons (INs) is disrupted in APC cKO male and female mice. IN dysfunction is implicated in human IESS, is a feature of other rodent models of IESS, and may contribute to the manifestation of spasms and seizures. We found that parvalbumin-positive INs (PV+ INs), an important source of cortical inhibition, were decreased in number, underwent disproportionate developmental apoptosis, and had altered dendrite morphology at P9, the peak of behavioral spasms. PV+ INs received excessive excitatory input, and their intrinsic ability to fire action potentials was reduced at all time points examined (P9, P14, P60). Subsequently, GABAergic transmission onto pyramidal neurons was uniquely altered in the somatosensory cortex of APC cKO mice at all ages, with both decreased IPSC input at P14 and enhanced IPSC input at P9 and P60. These results indicate that inhibitory circuit dysfunction occurs in APC cKOs and, along with known changes in excitation, may contribute to IESS-related phenotypes.SIGNIFICANCE STATEMENT Infantile and epileptic spasms syndrome (IESS) is a devastating epilepsy with limited treatment options and poor clinical outcomes. The molecular, cellular, and circuit disruptions that cause infantile spasms and seizures are largely unknown, but inhibitory GABAergic interneuron dysfunction has been implicated in rodent models of IESS and may contribute to human IESS. Here, we use a rodent model of IESS, the APC cKO mouse, in which ß-catenin signaling is increased in excitatory neurons. This results in altered parvalbumin-positive GABAergic interneuron development and GABAergic synaptic dysfunction throughout life, showing that pathology arising in excitatory neurons can initiate long-term interneuron dysfunction. Our findings further implicate GABAergic dysfunction in IESS, even when pathology is initiated in other neuronal types.

Tonic Activation of GluN2C/GluN2D-Containing NMDA Receptors by Ambient Glutamate Facilitates Cortical Interneuron Maturation.

Developing cortical GABAergic interneurons rely on genetic programs, neuronal activity, and environmental cues to construct inhibitory circuits during early postnatal development. Disruption of these events can cause long-term changes in cortical inhibition and may be involved in neurological disorders associated with inhibitory circuit dysfunction. We hypothesized that tonic glutamate signaling in the neonatal cortex contributes to, and is necessary for, the maturation of cortical interneurons. To test this hypothesis, we used mice of both sexes to quantify extracellular glutamate concentrations in the cortex during development, measure ambient glutamate-mediated activation of developing cortical interneurons, and manipulate tonic glutamate signaling using subtype-specific NMDA receptor antagonists in vitro and in vivo We report that ambient glutamate levels are high (≈100 nm) in the neonatal cortex and decrease (to ≈50 nm) during the first weeks of life, coincident with increases in astrocytic glutamate uptake. Consistent with elevated ambient glutamate, putative parvalbumin-positive interneurons in the cortex (identified using G42:GAD1-eGFP reporter mice) exhibit a transient, tonic NMDA current at the end of the first postnatal week. GluN2C/GluN2D-containing NMDA receptors mediate the majority of this current and contribute to the resting membrane potential and intrinsic properties of developing putative parvalbumin interneurons. Pharmacological blockade of GluN2C/GluN2D-containing NMDA receptors in vivo during the period of tonic interneuron activation, but not later, leads to lasting decreases in interneuron morphological complexity and causes deficits in cortical inhibition later in life. These results demonstrate that dynamic ambient glutamate signaling contributes to cortical interneuron maturation via tonic activation of GluN2C/GluN2D-containing NMDA receptors.SIGNIFICANCE STATEMENT Inhibitory GABAergic interneurons make up 20% of cortical neurons and are critical to controlling cortical network activity. Dysfunction of cortical inhibition is associated with multiple neurological disorders, including epilepsy. Establishing inhibitory cortical networks requires in utero proliferation, differentiation, and migration of immature GABAergic interneurons, and subsequent postnatal morphological maturation and circuit integration. Here, we demonstrate that ambient glutamate provides tonic activation of immature, putative parvalbumin-positive GABAergic interneurons in the neonatal cortex via high-affinity NMDA receptors. When this activation is blocked, GABAergic interneuron maturation is disrupted, and cortical networks exhibit lasting abnormal hyperexcitability. We conclude that temporally precise activation of developing cortical interneurons by ambient glutamate is critically important for establishing normal cortical inhibition.