11912 • The Journal of Neuroscience, December 6, 2017 • 37(49):11912–11929 Development/Plasticity/Repair Transient Hypoxemia Chronically Disrupts Maturation of Preterm Fetal Ovine Subplate Neuron Arborization and Activity XEvelyn McClendon,1 Daniel C. Shaver,1 Kiera Degener-O’Brien,1 Xi Gong,1 Thuan Nguyen,2 Anna Hoerder-Suabedissen,3 Zolta´n Molna´r,3 Claudia Mohr,4 XBen D. Richardson,4 David J. Rossi,4 and Stephen A. Back1,5 1Department of Pediatrics and 2Public Health and Preventive Medicine, Oregon Health & Science University, Portland, Oregon 97239, 3Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, United Kingdom OX1 3QX, 4Department of Integrative Physiology and Neuroscience, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164, and 5Department of Neurology, Oregon Health & Science University, Portland, Oregon 97239 Preterm infants are at risk for a broad spectrum of neurobehavioral disabilities associated with diffuse disturbances in cortical growth and development. During brain development, subplate neurons (SPNs) are a largely transient population that serves a critical role to establish functional cortical circuits. By dynamically integrating into developing cortical circuits, they assist in consolidation of intra- cortical and extracortical circuits. Although SPNs reside in close proximity to cerebral white matter, which is particularly vulnerable to oxidative stress, the susceptibility of SPNs remains controversial. We determined SPN responses to two common insults to the preterm brain: hypoxia-ischemia and hypoxia. We used a preterm fetal sheep model using both sexes that reproduces the spectrum of human cerebral injury and abnormal cortical growth. Unlike oligodendrocyte progenitors, SPNs displayed pronounced resistance to early or delayed cell death from hypoxia or hypoxia-ischemia. We thus explored an alternative hypothesis that these insults alter the maturational trajectory of SPNs. We used DiOlistic labeling to visualize the dendrites of SPNs selectively labeled for complexin-3. SPNs displayed reduced basal dendritic arbor complexity that was accompanied by chronic disturbances in SPN excitability and synaptic activity. SPN dysmaturation was significantly associated with the level of fetal hypoxemia and metabolic stress. Hence, despite the resistance of SPNs to insults that trigger white matter injury, transient hypoxemia disrupted SPN arborization and functional maturation during a critical window in cortical development. Strategies directed at limiting the duration or severity of hypoxemia during brain development may mitigate disturbances in cerebral growth and maturation related to SPN dysmaturation. Key words: anatomy and physiology; dendrite; hypoxia; ischemia; preterm brain injury; subplate neurons Significance Statement The human preterm brain commonly sustains blood flow and oxygenation disturbances that impair cerebral cortex growth and cause life-long cognitive and learning disabilities. We investigated the fate of subplate neurons (SPNs), which are a master regulator of brain development that plays critical roles in establishing cortical connections to other brain regions. We used a preterm fetal sheep model that reproduces key features of brain injury in human preterm survivors. We analyzed the responses of fetal SPNs to transient disturbances in fetal oxygenation. We discovered that SPNs are surprisingly resistant to cell death from low oxygen states but acquire chronic structural and functional changes that suggest new strategies to prevent learning problems in children and adults that survive preterm birth. Introduction birth, these insults contribute to a broad spectrum of unexplained During human development, the preterm brain commonly sus- neurological disabilities in motor control, vision, language, cogni- tains antenatal, perinatal, or postnatal disturbances in cerebral tion, attention, learning, and social development (Vohr, 2014). blood flow and metabolism. In 25%–50% of survivors of preterm These behavioral abnormalities coincide with significant impair- ment in cortical growth and connectivity defined by volumetric Received Aug. 24, 2017; revised Oct. 18, 2017; accepted Oct. 25, 2017. Authorcontributions:E.M.,Z.M.,D.J.R.,andS.A.B.designedresearch;E.M.,D.C.S.,K.D.-O.,X.G.,A.H.-S.,andC.M. Correspondence should be addressed to Dr. Stephen A. Back, Oregon Health and Science University, Department performed research; E.M., T.N., C.M., B.D.R., and D.J.R. analyzed data; E.M., Z.M., D.J.R., and S.A.B. wrote the paper. ofPediatrics,DivisionofPediatricNeuroscience,3181SWSamJacksonParkRoad,Portland,OR97239-3098.E-mail: This work was supported by National Institute of Neurological Disorders and Stroke Grants NS045737 and [email protected]. NS054044. DOI:10.1523/JNEUROSCI.2396-17.2017 The authors declare no competing financial interests. Copyright © 2017 the authors 0270-6474/17/3711912-18$15.00/0 McClendon et al. • Hypoxemia Triggers Subplate Neuron Dysmaturation J. Neurosci., December 6, 2017 • 37(49):11912–11929 • 11913 MRI studies (Ball et al., 2013; Vinall et al., 2013). With the excep- carotid and vertebral arteries. In the sheep, all collateral circulation to the tion of more severe cerebral injury (Ligam et al., 2009; Andiman brain, except the spinal arteries, is provided through branches of the et al., 2010; Kinney et al., 2012), most cortical volume loss appears brachiocephalic artery. An additional catheter was sewn to the fetal skin unrelated to significant cortical oxidative damage or neuronal to allow monitoring of the fetal amniotic fluid pressure. Following sur- degeneration (Back et al., 2005; Dean et al., 2013; Back and Miller, gical instrumentation, the fetus was returned to the uterus, the maternal incision was closed, and the ewe was allowed to recover from surgery for 2014). Given that subplate neurons (SPNs) mature when the pre- 3 d after which HI studies were conducted (see Fig. 1A). Fetuses of either term cerebrum displays peak vulnerability (Volpe, 2009), insults sex were included in the study (n ϭ 10 female, n ϭ 12 male). to SPNs were proposed to contribute to aberrant cortical growth Cerebral HI studies and physiological monitoring. Maternal Hx was in- and connectivity (Volpe, 1996). SPNs are a largely transient and duced by supplying the pregnant ewe with a mixture of 50% room air and unique neuronal population just below cortical layer 6 (Kostovic 50% nitrogen gas for an inhaled oxygen fraction of 10.5%. Following and Rakic, 1990; Hoerder-Suabedissen et al., 2013) that dynam- 5 min of maternal Hx, cerebral ischemia was initiated in one of the twin ically integrate into developing cortical circuits to promote fetuses (designated HI fetus) and maintained by inflating the brachioce- consolidation of functional intracortical and extracortical con- phalic occluder for 25 min. Four arterial blood gas (ABG) samples were nectivity. SPNs generate the first axons of the corticothalamic collected from each fetus throughout experimentation: pre-Hx (baseline tract (McConnell et al., 1989). These projections provide a scaf- or pre-ABG), at 5 min after initiation of maternal Hx (Hx ABG), 25 min fold for thalamic inputs to directly innervate cortical layer 4 after initiation of ischemia-induction (HI or HI ABG), and 10 min after cessation of ischemia (recovery or post-ABG). Blood samples were ana- (Kanold and Luhmann, 2010; Viswanathan et al., 2012). Most lyzed using an ABL800 flex blood gas analyzer (Radiometer Medical, A/S SPNs later undergo programmed cell death (Chun and Shatz, RRID:SCR_014772). If the fetal arterial oxygen concentration (ctO2) fell 1989; Torres-Reveron and Friedlander, 2007), which leaves a Ͻ5.0 vol% after a 5 min exposure to maternal Hx, the maternal inhaled monosynaptic thalamocortical projection to layer 4. oxygen fraction was enriched to 14% for the duration of the experiment. Because ablation of SPNs disrupts the entry or patterning of Fetuses exposed exclusively to maternal Hx were designated hypoxic corticothalamic projections (Ghosh et al., 1990; Kanold et al., controls (Hx), whereas fetuses exposed to maternal Hx in combination 2003), hypoxia-ischemia (HI) was proposed to similarly trigger with cerebral hypoperfusion were designated HI animals (see Fig. 1A). SPN degeneration to disrupt formation of thalamocortical affer- Each experimental twin pregnancy was comprised of a Hx and HI pair. ents. After HI, neonatal rat SPNs selectively degenerated unlike Tissue collection and processing. To ensure the viability of the fetal other neuronal populations (McQuillen et al., 2003). However, brains for concurrent electrophysiological studies, the ewe was placed under general anesthesia and ventilated. After fetal harvest, ewes were analysis of preterm human autopsy brains found no significant then killed with a 12 ml intravenous bolus of a pentobarbital sodium and loss of the total SPN population (Kinney et al., 2012). Recently, phenytoin sodium solution (Euthasol NAC# 10230760, Virbac). Fetal three different SPN-specific markers were used to quantify SPN brains were harvested either 24 h (Hx, n ϭ 5; HI, n ϭ 4) or 4 weeks (Hx, vulnerability to HI in the neonatal rat. SPN degeneration was n ϭ 8; HI, n ϭ 8) after exposure to maternal Hx and HI. Separate groups only observed in association with more severe cortical neuronal of appropriately age-matched normal twin fetuses were harvested to loss (Okusa et al., 2014). serve as true controls (TC: 4 weeks/120 dGA, n ϭ 7; 24 h/95 dGA, n ϭ 3) Because these