Research ArticleImpaired Organization of GABAergic Neurons Following Prenatal Hypoxia
Introduction
Several conditions related to the intrauterine environment have been proposed as risk factors for neurologic outcome, including pre- and perinatal hypoxia (Jones et al., 1998, Zornberg et al., 2000, Dalman et al., 2001, Cannon et al., 2002, Boog, 2004). These epidemiological findings are supported by a large number of animal studies that model the effects of early-life hypoxia on behavioral, histological and molecular variables relevant to learning disabilities, motor impairment and schizophrenia (Meyer et al., 1998, Boksa, 2004). In humans, approximately 1–2% of infants are exposed to perinatal hypoxia–ischemia or prolonged anoxic insult, and very low birth weight preterm infants are particularly vulnerable (Volpe, 2003). About 25–50% of these babies are at risk for significant cognitive, behavioral, attention and socialization impairments (Larroque et al., 2008, Marlow et al., 2005, Doyle and Anderson, 2010). The principal injury following hypoxia or hypoxia ischemia has been revealed through imaging methods, together with neurological and histological studies, to involve cerebral white and gray matter (Cannon et al., 2002, Boog, 2004, Volpe, 2003, Back et al., 2007, Jensen, 2006, Okereafor et al., 2008).
To increase our understanding of the mechanisms by which hypoxic insult affects the immature brain, animal models of pre- and neonatal hypoxia have been studied. Motor impairment and difficulties in learning and memory tasks were observed following prenatal hypoxia and hypoxia ischemia in mice and rats (McQuillen et al., 2003, Tashima et al., 2001, Zhuravin et al., 2004, Golan et al., 2004b, Golan et al., 2004a). In addition, deficits in tasks associated with schizophrenia, such as pre-pulse inhibition, were reported (Howell and Pillai, 2014). The search for the neuronal origin of the behavioral impairment revealed cell loss in various brain regions — among them, the cerebral cortex, hippocampus, striatum and cerebellum — in different animal models of prenatal and neonatal hypoxia (Golan and Huleihel, 2006, Van de Berg et al., 2002, Biran et al., 2010, Rees et al., 2011). Neuron populations that are particularly affected by hypoxic insult are the GABAergic interneurons. In response to perinatal anoxia, the density of GABA immunoreactive (IR) neurons in the cerebral cortex and hippocampus decreased seven days after insult, a condition that was maintained into adulthood (Dell'Anna et al., 1996). Reduced density of the sub-population of GABAergic neurons expressing the calcium-binding protein calbindin (CB) in the cortex was observed 20 days after hypoxic insult on embryonic day 17 (E17) (Louzoun-Kaplan et al., 2008). Similarly, hypoxic insult on E18 led to decreased densities both of cells expressing the GABA-synthesizing enzyme, GAD65, and of parvalbumin (PV) cells 12 days after insult (Mazur et al., 2010). Long-term decreases in the densities of PV and CB neurons in rat striatum were reported two months after perinatal asphyxia (Van de Berg et al., 2003) and in the cerebral cortex following prenatal hypoxia (Gerstein et al., 2005). Altered maturation of PV neurons following neonatal hypoxia ischemia has also been reported (Failor et al., 2010). In human infants, significant declines in GAD67-IR neurons in the sub-plate and white matter were observed following perinatal injury (Robinson et al., 2006). Functional analysis of inhibitory synaptic activity in newborn rats following hypoxia showed a transient effect on postsynaptic parameters and a persistent decrease in GABAergic neuron firing, which may reflect either changes in the internal properties of the neurons or a reduction in the population of interneurons (Sanchez et al., 2007). Collectively, these studies suggest that early-life hypoxia has long-lasting effects on the cortical interneuron population. In the search for the origin of the above-mentioned neuron loss, molecular pathways involved in angiogenesis and neuron migration in the developing hippocampus and cortical plate were studied. Enhancement of VEGF (24 h) and decreases in BDNF (24 h) and APP (2 h and 24 h), were reported (Golan et al., 2004b, Howell and Pillai, 2014, Golan et al., 2009). Cleaved reelin levels were sensitive to prenatal hypoxia in a region-specific manner, such that after hypoxia, they were elevated in the cortex (1 and 5 days) and reduced in the hippocampus (2 h and 24 h) (Golan et al., 2009). In the latter brain region, the suppression of their levels was shown to be long lasting (Howell and Pillai, 2014). In addition to the changes in these molecular cues, down regulation of the associated signaling molecules, BACE and Dab1, was also observed following prenatal hypoxia (Golan et al., 2009).
The impact of prenatal insult on cortical GABAergic interneurons, which originate in the ganglionic eminences, may vary depending on the stage of neurogenesis (Super et al., 1998, Soriano and Del Rio, 2005). From the ganglionic eminences, these cells migrate via a long, tangential course into the developing cortex where they complete their final differentiation to generate the various classes of inhibitory interneurons (Xu et al., 2004, Cobos et al., 2005). There is substantial evidence that the specific germinal origin controls the final fate of these cells and that broad subtypes of interneurons originate from each of the three divisions of the ganglionic eminences — the lateral, medial and caudal ganglionic eminences (LGE, MGE and CGE, respectively) — and that they follow somewhat distinct migratory routes to the developing cortex (Yozu et al., 2005, Nery et al., 2002, Wonders and Anderson, 2005, Miyoshi et al., 2010). In addition, cells originating in the embryonic preoptic area (POA) contribute an additional population of cortical interneurons with distinct neurochemical and morphological fates (Metin and Pedraza, 2014, Gelman et al., 2009, Gelman and Marin, 2010).
The tangential migration of interneurons from the MGE to the cortex forms two streams, a superficial stream in the marginal zone (MZ) and a deep stream in the subventricular zone (SVZ) and intermediate zone (IZ), and both streams are dynamically organized during embryonic and early postnatal life. In mice at E13.5, migrating cells course through the SVZ of the LGE and along the LGE/cortex border, at which point the two prominent cortical migratory streams can be readily distinguished. From E13.5 until E17.5, the MZ stream accumulates MGE-derived neurons, and from E17.5 to postnatal day 2 (P2), the number of these young neurons in the MZ stream decreases as they migrate radially to populate the cortex in a laminar-specific manner. In the SVZ stream, the number of MGE-derived interneurons increases from E13.5 to E15.5 and then decreases thereafter. These dynamic changes in the distributions of MGE-derived neurons — as shown by different labeling methods (Tanaka et al., 2003, Yokota et al., 2007, Li et al., 2008, Ang et al., 2003) — provide an anatomical and temporal framework for assessing the effects of prenatal hypoxia during this period of interneuron development. In humans, the process of interneuron migration to and within the cortex takes place from mid-gestation to the age of two years (Xu et al., 2011). Cortical GABAergic interneuron migration during this time is also the peak window of vulnerability for hypoxia ischemia in term and preterm infants (Xu et al., 2011).
The overall aim of the current study was to search for a link between prenatal hypoxia and a defect in GABAergic interneuron migration, which may increase the risk for neurologic and psychiatric conditions such as schizophrenia and autism. We used an animal model of hypoxia, in which mice that undergo 2 h of prenatal hypoxia (9% O2, 3% CO2) present a clear defect in cortical GABAergic neuron numbers and distributions (Gerstein et al., 2005), a recapitulation of the major phenotype in human subjects. We hypothesize that this defect is due to the failure of the interneurons to populate their correct positions in the cortex. To assess this possibility and to understand the developmental mechanism that excludes a portion of GABAergic interneurons from the developing cortex, we characterized the spatial and temporal patterns of the cortical distributions of inhibitory neurons induced by hypoxic insult.
Section snippets
Mice and hypoxia protocol
C57 black mice were ordered from Harlan, Israel. The mice were maintained in the animal facility of Ben-Gurion University of the Negev on a 12:12 h light/dark schedule with food and water provided ad libitum. Lhx6-GFP mice were obtained from the Gensat project and used as described in Li et al. (2008). All experiments were carried out in accordance with National Institutes of Health guidelines for the care and use of laboratory animals (NIH Publications No. 80-23, revised 1996) and with the
Effect of hypoxia on GABAergic cell numbers in the developing cortex
We began by investigating when interneuron loss was first evident in the developing cortex using two interneuron markers, GAD 65/67 (all interneurons) and Lhx6-GFP (MGE interneurons), in different sets of experiments.
Four days after hypoxic insult on E14.5, a significant drop in the total number of GAD+ cells in the developing cortex (22–26%) was first observed in lateral regions (F = 9.5; df 1,82; p < 0.001), particularly in the rostral sections (Fig. 1). Birth dating with BrdU indicated that
Discussion
The data collected during this study suggest that among the GABAergic cells, those that are the most vulnerable to maternal hypoxia comprise a population of interneurons that, under normoxic conditions, is already present in the cortical plate at E18.5. Considering that for the majority of the cortical neurons, peak proliferation took place around E12.5, our data suggest that interneurons that exited the ganglionic eminences before the hypoxic episode (before E14.5) were susceptible to the
Acknowledgments
This study was supported by a grant to H. Golan from the Israel Science Foundation (ISF) (grant number 309/13). The monoclonal antibody rat-401, deposited to the DSHB by Hockfield, S. from the Yale University School of Medicine (DSHB Hybridoma Product rat-401), was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.
Competing interests
The authors declare no competing financial interests.
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