Prenatal Hypoxia-Ischemia Induces Abnormalities in CA3 Microstructure, Potassium Chloride Co-Transporter 2 Expression and Inhibitory Tone

Abstract

Infants who suffer perinatal brain injury, including those with encephalopathy of prematurity, are prone to chronic neurological deficits, including epilepsy, cognitive impairment, and behavioral problems, such as anxiety, inattention, and poor social interaction. These deficits, especially in combination, pose the greatest hindrance to these children becoming independent adults. Cerebral function depends on adequate development of essential inhibitory neural circuits and the appropriate amount of excitation and inhibition at specific stages of maturation. Early neuronal synaptic responses to γ-amino butyric acid (GABA) are initially excitatory. During the early postnatal period, GABAAR responses switch to inhibitory with the upregulation of potassium-chloride co-transporter KCC2. With extrusion of chloride by KCC2, the Cl(-) reversal potential shifts and GABA and glycine responses become inhibitory. We hypothesized that prenatal hypoxic-ischemic brain injury chronically impairs the developmental upregulation of KCC2 that is essential for cerebral circuit formation. Following late gestation hypoxia-ischemia (HI), diffusion tensor imaging in juvenile rats shows poor microstructural integrity in the hippocampal CA3 subfield, with reduced fractional anisotropy and elevated radial diffusivity. The loss of microstructure correlates with early reduced KCC2 expression on NeuN-positive pyramidal neurons, and decreased monomeric and oligomeric KCC2 protein expression in the CA3 subfield. Together with decreased inhibitory post-synaptic currents during a critical window of development, we document for the first time that prenatal transient systemic HI in rats impairs hippocampal CA3 inhibitory tone. Failure of timely development of inhibitory tone likely contributes to a lower seizure threshold and impaired cognitive function in children who suffer perinatal brain injury.

Keywords: KCC2; epilepsy; hippocampus; microstructure; prematurity; seizure.

Figure 1

Diffusion tensor imaging of young mature rats following prenatal TSHI shows microstructural abnormalities. (A) DTI directional diffusion color maps show differences in hippocampal structure in TSHI rats compared to shams. Red color indicates transverse tracts, blue color indicates anterior–posterior tracts, and green color indicates vertical tracts. (B) Fractional anisotropy (FA) is reduced in the CA3 subfield of P35–40 TSHI animals compared to shams. (C) Axial diffusivity (AD) is increased in CA3 of TSHI brains compared to shams, consistent with axonal injury. (D) Radial diffusivity is increased in CA3 of TSHI brains compared to shams in young mature CNS, consistent with impaired myelin integrity. *p < 0.05, ***p ≤ 0.001.

Figure 2

Prenatal TSHI diminishes KCC2 expression during postnatal development in the CA3. (A) NeuN-KCC2 double-labeling at P11 shows reduced KCC2 expression. Bar = 20 μm. (B) Western blot at P15 shows reduced monomeric KCC2 expression (140 kDa) in CA3 from TSHI brains compared to shams. (C) Similarly, oligomeric KCC2 (~270 kDa) expression is also reduced at P15 compared to shams (*p < 0.05, **p < 0.01).

Figure 3

Transient systemic hypoxia–ischemia (TSHI) decreases inhibitory post-synaptic currents (IPSCs). (A) Tracing showing decreased frequency of post-synaptic events in TSHI CA3 regions compared to sham; (B) biocytin-filled CA3 P10–P11 pyramidal neuron in hippocampal slice. Bar = 50 μm. (C) Frequency, charge transfer, and decay of IPSCs are significantly different in TSHI animals compared to shams (two-tailed t-test, p < 0.05, p = 0.006, and p = 0.019, respectively). (D) Consistent with the mean data, the cumulative distribution plots demonstrate a leftward shift in the population of TSHI IPSCs, and decreased inter-event intervals compared to shams. (E) Event distribution histograms confirm the leftward shift in decay and charge transfer following TSHI compared to shams.