Kevin G. Broadbelt

Late Development of the GABAergic System in the Human Cerebral Cortex and White Matter

Despite the key role of &ggr;-aminobutyric acid (GABA) neurons in the modulation of cerebral cortical output, little is known about their development in the human cortex. We analyzed several GABAergic parameters in standardized regions of the cerebral cortex and white matter in a total of 38 human fetuses and infants from 19 gestational weeks to 2.7 postnatal years using immunocytochemistry, Western blotting, tissue autoradiography, and computer-based cellular quantitation. At least 20% of GABAergic neurons in the white matter migrated toward the cortex over late gestation. After term, migration declined and ended within 6 postnatal months. In parallel, the GABAergic neuronal density increased in the cortex over late gestation, also with a peak at term. From midgestation to infancy, the pattern of GABAA receptor binding changed from uniformly low across all cortical layers to high levels concentrated in the middle laminae; glutamic acid decarboxylase (GAD65 and GAD67) levels differentially increased. Thus, the second half of gestation is a period of rapid development of the cortical GABAergic system that continues into early infancy. This period corresponds to the peak window of vulnerability to perinatal hypoxia-ischemia in which GABAergic neurons are potentially developmentally susceptible, including in the preterm infant.

Spatially Heterogeneous Choroid Plexus Transcriptomes Encode Positional Identity and Contribute to Regional CSF Production

A sheet of choroid plexus epithelial cells extends into each cerebral ventricle and secretes signaling factors into the CSF. To evaluate whether differences in the CSF proteome across ventricles arise, in part, from regional differences in choroid plexus gene expression, we defined the transcriptome of lateral ventricle (telencephalic) versus fourth ventricle (hindbrain) choroid plexus. We find that positional identities of mouse, macaque, and human choroid plexi derive from gene expression domains that parallel their axial tissues of origin. We then show that molecular heterogeneity between telencephalic and hindbrain choroid plexi contributes to region-specific, age-dependent protein secretion in vitro. Transcriptome analysis of FACS-purified choroid plexus epithelial cells also predicts their cell-type-specific secretome. Spatial domains with distinct protein expression profiles were observed within each choroid plexus. We propose that regional differences between choroid plexi contribute to dynamic signaling gradients across the mammalian cerebroventricular system.

Brainstem Deficiency of the 14-3-3 Regulator of Serotonin Synthesis: A Proteomics Analysis in the Sudden Infant Death Syndrome

Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42–75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MSE-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT1A receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process.

Decreased GABAA Receptor Binding in the Medullary Serotonergic System In the Sudden Infant Death Syndrome

&ggr;-Aminobutyric acid (GABA) neurons in the medulla oblongata help regulate homeostasis, in part through interactions with the medullary serotonergic (5-HT) system. Previously, we reported abnormalities in multiple 5-HT markers in the medullary 5-HT system of infants dying from sudden infant death syndrome (SIDS), suggesting that 5-HT dysfunction is involved in its pathogenesis. Here, we tested the hypothesis that markers of GABAA receptors are decreased in the medullary 5-HT system in SIDS cases compared with controls. Using tissue receptor autoradiography with the radioligand 3H-GABA, we found 25% to 52% reductions in GABAA receptor binding density in 7 of 10 key nuclei sampled of the medullary 5-HT system in the SIDS cases (postconceptional age [PCA] = 51.7 ± 8.3, n = 28) versus age-adjusted controls (PCA = 55.3 ± 13.5, n = 8) (p ≤ 0.04). By Western blotting, there was 46.2% reduction in GABAA&agr;3 subunit levels in the gigantocellularis (component of the medullary 5-HT system) of SIDS cases (PCA = 53.9 ± 8.4, n = 24) versus controls (PCA = 55.3 ± 8.3, n = 8) (56.8% standard in SIDS cases vs 99.35% in controls; p = 0.026). These data suggest that medullary GABAA receptors are abnormal in SIDS infants and that SIDS is a complex disorder of a homeostatic network in the medulla that involves deficits of the GABAergic and 5-HT systems.

Lack of Association of the Serotonin Transporter Polymorphism With the Sudden Infant Death Syndrome in the San Diego Dataset

Dysfunction of medullary serotonin (5-HT)-mediated respiratory and autonomic function is postulated to underlie the pathogenesis of the majority of sudden infant death syndrome (SIDS) cases. Several studies have reported an increased frequency of the LL genotype and L allele of the 5-HT transporter (5-HTT) gene promoter polymorphism (5-HTTLPR), which is associated with increased transcriptional activity and 5-HT transport in vitro, in SIDS cases compared with controls. These findings raise the possibility that this polymorphism contributes to or exacerbates existing medullary 5-HT dysfunction in SIDS. In this study, we tested the hypothesis that the frequency of LL genotype and L allele are higher in 179 SIDS cases compared with 139 controls of multiple ethnicities in the San Diego SIDS Dataset. We observed no significant association of genotype or allele with SIDS cases either in the total cohort or on stratification for ethnicity. These observations do not support previous findings that the L allele and/or LL genotype of the 5-HTTLPR are associated with SIDS.

Neuroanatomic relationships between the GABAergic and serotonergic systems in the developing human medulla

gamma-Amino butyric (GABA) critically influences serotonergic (5-HT) neurons in the raphé and extra-raphé of the medulla oblongata. In this study we hypothesize that there are marked changes in the developmental profile of markers of the human medullary GABAergic system relative to the 5-HT system in early life. We used single- and double-label immunocytochemistry and tissue receptor autoradiography in 15 human medullae from fetal and infant cases ranging from 15 gestational weeks to 10 postnatal months, and compared our findings with an extensive 5-HT-related database in our laboratory. In the raphé obscurus, we identified two subsets of GABAergic neurons using glutamic acid decarboxylase (GAD65/67) immunostaining: one comprised of small, round neurons; the other, medium, spindle-shaped neurons. In three term medullae cases, positive immunofluorescent neurons for both tryptophan hydroxylase and GAD65/67 were counted within the raphé obscurus. This revealed that approximately 6% of the total neurons counted in this nucleus expressed both GAD65/67 and TPOH suggesting co-production of GABA by a subset of 5-HT neurons. The distribution of GABA(A) binding was ubiquitous across medullary nuclei, with highest binding in the raphé obscurus. GABA(A) receptor subtypes alpha1 and alpha3 were expressed by 5-HT neurons, indicating the site of interaction of GABA with 5-HT neurons. These receptor subtypes and KCC2, a major chloride transporter, were differentially expressed across early development, from midgestation (20 weeks) and thereafter. The developmental profile of GABAergic markers changed dramatically relative to the 5-HT markers. These data provide baseline information for medullary studies of human pediatric disorders, such as sudden infant death syndrome.

Serotonin-Related FEV Gene Variant in the Sudden Infant Death Syndrome Is a Common Polymorphism in the African-American Population

An important subset of the sudden infant death syndrome (SIDS) is associated with multiple serotonergic (5-HT) abnormalities in regions of the medulla oblongata. The mouse ortholog of the fifth Ewing variant gene (FEV) is critical for 5-HT neuronal development. A putatively rare intronic variant [IVS2-191_190insA, here referred to as c.128-(191_192)dupA] has been reported as a SIDS-associated mutation in an African-American population. We tested this association in an independent dataset: 137 autopsied cases (78 SIDS, 59 controls) and an additional 296 control DNA samples from Coriell Cell Repositories. In addition to the c.128-(191_192)dupA variant, we observed an associated single-base deletion [c.128-(301-306)delG] in a subset of the samples. Neither of the two FEV variants showed significant association with SIDS in either the African-American subgroup or the overall cohort. Although we found a significant association of c.128-(191_192)dupA with SIDS when San Diego Hispanic SIDS cases were compared with San Diego Hispanic controls plus Mexican controls (p = 0.04), this became nonsignificant after multiple testing correction. Among Coriell controls, 33 of 99 (33%) African-American and 0 of 197 (0%) of the remaining controls carry the polymorphism (c.128-(191_192)dupA). The polymorphism seems to be a common, likely nonpathogenic, variant in the African-American population.

Potential Asphyxia and Brainstem Abnormalities in Sudden and Unexpected Death in Infants

OBJECTIVE: Sudden and unexplained death is a leading cause of infant mortality. Certain characteristics of the sleep environment increase the risk for sleep-related sudden and unexplained infant death. These characteristics have the potential to generate asphyxial conditions. We tested the hypothesis that infants may be exposed to differing degrees of asphyxia in sleep environments, such that vulnerable infants with a severe underlying brainstem deficiency in serotonergic, γ-aminobutyric acid-ergic, or 14-3-3 transduction proteins succumb even without asphyxial triggers (eg, supine), whereas infants with intermediate or borderline brainstem deficiencies require asphyxial stressors to precipitate death. METHODS: We classified cases of sudden infant death into categories relative to a “potential asphyxia” schema in a cohort autopsied at the San Diego County Medical Examiner’s Office. Controls were infants who died with known causes of death established at autopsy. Analysis of covariance tested for differences between groups. RESULTS: Medullary neurochemical abnormalities were present in both infants dying suddenly in circumstances consistent with asphyxia and infants dying suddenly without obvious asphyxia-generating circumstances. There were no differences in the mean neurochemical measures between these 2 groups, although mean measures were both significantly lower (P < .05) than those of controls dying of known causes. CONCLUSIONS: We found no direct relationship between the presence of potentially asphyxia conditions in the sleep environment and brainstem abnormalities in infants dying suddenly and unexpectedly. Brainstem abnormalities were associated with both asphyxia-generating and non–asphyxia generating conditions. Heeding safe sleep messages is essential for all infants, especially given our current inability to detect underlying vulnerabilities.

Progressive Differentiation and Instructive Capacities of Amniotic Fluid and Cerebrospinal Fluid Proteomes following Neural Tube Closure

After neural tube closure, amniotic fluid (AF) captured inside the neural tube forms the nascent cerebrospinal fluid (CSF). Neuroepithelial stem cells contact CSF-filled ventricles, proliferate, and differentiate to form the mammalian brain, while neurogenic placodes, which generate cranial sensory neurons, remain in contact with the AF. Using in vivo ultrasound imaging, we quantified the expansion of the embryonic ventricular-CSF space from its inception. We developed tools to obtain pure AF and nascent CSF, before and after neural tube closure, and to define how the AF and CSF proteomes diverge during mouse development. Using embryonic neural explants, we demonstrate that age-matched fluids promote Sox2-positive neurogenic identity in developing forebrain and olfactory epithelia. Nascent CSF also stimulates SOX2-positive self-renewal of forebrain progenitor cells, some of which is attributable to LIFR signaling. Our Resource should facilitate the investigation of fluid-tissue interactions during this highly vulnerable stage of early brain development.

Serotonin metabolites in the cerebrospinal fluid in sudden infant death syndrome.

Forensic biomarkers are needed in sudden infant death syndrome (SIDS) to help identify this group among other sudden unexpected deaths in infancy. Previously, we reported multiple serotonergic (5-HT) abnormalities in nuclei of the medulla oblongata that help mediate protective responses to homeostatic stressors. As a first step toward their assessment as forensic biomarkers of medullary pathology, here we test the hypothesis that 5-HT-related measures are abnormal in the cerebrospinal fluid (CSF) of SIDS infants compared with those of autopsy controls. Levels of CSF 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA), the degradative products of 5-HT and dopamine, respectively, were measured by high-performance liquid chromatography in 52 SIDS and 29 non-SIDS autopsy cases. Tryptophan (Trp) and tyrosine (Tyr), the substrates of 5-HT and dopamine, respectively, were also measured. There were no significant differences in 5-HIAA, Trp, HVA, or Tyr levels between the SIDS and non-SIDS groups. These data preclude the use of 5-HIAA, HVA, Trp, or Tyr measurements as CSF autopsy biomarkers of 5-HT medullary pathology in infants who have died suddenly and unexpectedly. They do, however, provide important information about monoaminergic measurements in human CSF at autopsy and their developmental profile in infancy that is applicable to multiple pediatric disorders beyond SIDS.