Survivors of preterm birth are at high risk of pervasive cognitive and learning impairments, suggesting disrupted early brain development. The limits of viability for preterm birth encompass the third trimester of pregnancy, a "precritical period" of activity-dependent development characterized by the onset of spontaneous and evoked patterned electrical activity that drives neuronal maturation and formation of cortical circuits. Reduced background activity on electroencephalogram (EEG) is a sensitive marker of brain injury in human preterm infants that predicts poor neurodevelopmental outcome. We studied a rodent model of very early hypoxic-ischemic brain injury to investigate effects of injury on both general background and specific patterns of cortical activity measured with EEG. EEG background activity is depressed transiently after moderate hypoxia-ischemia with associated loss of spindle bursts. Depressed activity, in turn, is associated with delayed expression of glutamate receptor subunits and transporters. Cortical pyramidal neurons show reduced dendrite development and spine formation. Complementing previous observations in this model of impaired visual cortical plasticity, we find reduced somatosensory whisker barrel plasticity. Finally, EEG recordings from human premature newborns with brain injury demonstrate similar depressed background activity and loss of bursts in the spindle frequency band. Together, these findings suggest that abnormal development after early brain injury may result in part from disruption of specific forms of brain activity necessary for activity-dependent circuit development.

1Down syndrome (DS) is a devastating genetic disorder causing severe cognitive impairment. 2The staggering array of effects associated with an extra copy of human chromosome 21 3 (HSA21) complicates mechanistic understanding of DS pathophysiology. We developed an in 4 vitro system to examine the interplay of neurons and astrocytes in a fully recapitulated HSA21 5 trisomy model differentiated from DS patient-derived induced pluripotent stem cells (iPSCs). By 6 combining calcium imaging with genetic approaches, we utilized this system to investigate the 7 functional defects of DS astroglia and their effects on neuronal excitability. We found that, 8 compared with control isogenic astroglia, DS astroglia exhibited more-frequent spontaneous 9 calcium fluctuations, which reduced the excitability of co-cultured neurons. DS astrocytes 10 exerted this effect on both DS and healthy neurons. Neuronal activity could be rescued by 11 abolishing astrocytic spontaneous calcium activity either chemically by blocking adenosine-12 mediated astrocyte-neuron signaling or genetically by knockdown of inositol triphosphate (IP 3 ) 13 receptors or S100β, a calcium binding protein coded on HSA21. Our results suggest a novel 14 mechanism by which DS alters the function of astrocytes, which subsequently disturbs neuronal 15 excitability. Furthermore, our study establishes an all-optical neurophysiological platform for 16 studying human neuron-astrocyte interactions associated with neurological disorders.

Cytochrome c (Cytc) is a globular protein within the inner mitochondrial membrane that shuttles electrons from Complex III to Complex IV in the electron transport chain. Upon release from the mitochondria, Cytc participates in initiation of the caspase cascade leading to apoptosis. Cytc binds to cardiolipin in the inner mitochondrial membrane leading to loss of Met80-heme ligation, enabling Cytc to function as a peroxidase capable of oxidizing cardiolipin. The affinity of Cytc for oxidized cardiolipin is decreased and therefore Cytc can dissociate from the membrane facilitating release from mitochondria and propagation of apoptosis. In order for the heme coordination site to open for peroxidase activity, the heme crevice loop must undergo structural rearrangement similar to the alkaline transition. Unlike most residues in the heme crevice loop, positions 81 and 83 evolve to more sterically demanding residues in higher eukaryotes. Previous results suggest that although residues 81 and 83 are key residues for regulating the peroxidase activity of Cytc, there is evidence that substitutions in U-loop C can affect the stability of the heme crevice loop. We hypothesize that U loops C and D have evolved interactively to optimize the peroxidase activity of Cytc. To perform this test, we prepared a series of human-to-yeast substitutions in U-loop C of human Cytc. Using circular dichroism and UV-visible spectroscopies, we have investigated global stability of the protein and local stability of the heme crevice loop. Using stoppedflow methods, we investigated the peroxidase activity for both I57V and N54K human Cytc. We observe that the N54K substitution strongly enhances the peroxidase activity of human Cytc, whereas the I57V substitution has little effect.