Known pathogenic gene variants and new candidates detected in Sudden Unexpected Infant Death using Whole Genome Sequencing.

Purpose: To gain insights into potential genetic factors contributing to the infant's vulnerability to Sudden Unexpected Infant Death (SUID). Methods: Whole Genome Sequencing (WGS) was performed on 145 infants that succumbed to SUID, and 576 healthy adults. Variants were filtered by gnomAD allele frequencies and predictions of functional consequences. Results: Variants of interest were identified in 86 genes, 63.4% of our cohort. Seventy-one of these have been previously associated with SIDS/SUID/SUDP. Forty-three can be characterized as cardiac genes and are related to cardiomyopathies, arrhythmias, and other conditions. Variants in 22 genes were associated with neurologic functions. Variants were also found in 13 genes reported to be pathogenic for various systemic disorders. Variants in eight genes are implicated in the response to hypoxia and the regulation of reactive oxygen species (ROS) and have not been previously described in SIDS/SUID/SUDP. Seventy-two infants met the triple risk hypothesis criteria (Figure 1). Conclusion: Our study confirms and further expands the list of genetic variants associated with SUID. The abundance of genes associated with heart disease and the discovery of variants associated with the redox metabolism have important mechanistic implications for the pathophysiology of SUID.

Purinergic signaling mediates neuroglial interactions to modulate sighs.

Sighs prevent the collapse of alveoli in the lungs, initiate arousal under hypoxic conditions, and are an expression of sadness and relief. Sighs are periodically superimposed on normal breaths, known as eupnea. Implicated in the generation of these rhythmic behaviors is the preBötzinger complex (preBötC). Our experimental evidence suggests that purinergic signaling is necessary to generate spontaneous and hypoxia-induced sighs in a mouse model. Our results demonstrate that driving calcium increases in astrocytes through pharmacological methods robustly increases sigh, but not eupnea, frequency. Calcium imaging of preBötC slices corroborates this finding with an increase in astrocytic calcium upon application of sigh modulators, increasing intracellular calcium through g-protein signaling. Moreover, photo-activation of preBötC astrocytes is sufficient to elicit sigh activity, and this response is blocked with purinergic antagonists. We conclude that sighs are modulated through neuron-glia coupling in the preBötC network, where the distinct modulatory responses of neurons and glia allow for both rhythms to be independently regulated.

Genetically Targeted Connectivity Tracing Excludes Dopaminergic Inputs to the Interpeduncular Nucleus from the Ventral Tegmentum and Substantia Nigra.

The "habenulopeduncular system" consists of the medial habenula (MHb) and its principal target of innervation, the interpeduncular nucleus (IP). Neurons in the ventral MHb (MHbV) express acetylcholine along with glutamate, and both the MHb and IP are rich in nicotinic acetylcholine receptors. Much of the work on this system has focused on nicotinic mechanisms and their clinical implications for nicotine use, particularly because the IP expresses the α5 nicotinic receptor subunit, encoded by the CHRNA5 gene, which is genetically linked to smoking risk. A working model has emerged in which nicotine use may be determined by the balance of reinforcement mediated in part by nicotine effects on dopamine reward pathways, and an aversive "brake" on nicotine consumption encoded in the MHb-IP pathway. However, recent work has proposed that the IP also receives direct dopaminergic input from the ventral tegmental area (VTA). If correct, this would significantly impact the prevailing model of IP function. Here, we have used Chrna5Cre mice to perform rabies virus-mediated retrograde tracing of global inputs to the IP. We have also used Cre-dependent adeno-associated virus (AAV) anterograde tracing using Slc6a3Cre (DATCre ) mice to map VTA dopaminergic efferents, and we have examined tract-tracing data using other transgenic models for dopaminergic neurons available in a public database. Consistent with the existing literature using non-genetic tracing methods, none of these experiments show a significant anatomic connection from the VTA or substantia nigra (SN) to the IP, and thus do not support a model of direct dopaminergic input to the habenulopeduncular system.

Mapping Cell Types and Efferent Pathways in the Ascending Relaxin-3 System of the Nucleus Incertus.

Relaxin-3 (Rln3) is an insulin-family peptide neurotransmitter expressed primarily in neurons of the nucleus incertus (NI) of the pontine tegmentum, with smaller populations located in the deep mesencephalon (DpMe) and periaqueductal gray (PAG). Here, we have used targeted recombination at the Rln3 gene locus to generate an Rln3Cre transgenic mouse line, and characterize the molecular identity and axonal projections of Rln3-expressing neurons. Expression of Cre recombinase in Rln3Cre mice, and the expression of Cre-mediated reporters, accurately reflect the expression of Rln3 mRNA in all brain regions. In the NI, Rln3 mRNA is expressed in a subset of a larger population of tegmental neurons that express the neuropeptide neuromedin-b (NMB). These Rln3-expressing and NMB-expressing neurons also express the GABAergic marker GAD2 but not the glutamatergic marker Slc17a6 (VGluT2). Cre-mediated anterograde tracing with adeno-associated viruses (AAVs) shows that the efferents of the Rln3-expressing neurons in the DpMe and PAG are largely confined to the brain regions in which they originate, while the NI-Rln3 neurons form an extensive ascending system innervating the limbic cortex, septum, hippocampus, and hypothalamus. Viral anterograde tracing also reveals the potential synaptic targets of NI-Rln3 neurons in several brain regions, and the distinct projections of Rln3-expressing and non-expressing neurons in the pontine tegmentum. Rabies virus (RV)-mediated transsynaptic retrograde tracing demonstrates a probable synaptic link between NI-Rln3 neurons and GABAergic neurons in the septum, with implications for the modulation of neural activity in the septo-hippocampal system. Together, these results form the basis for functional studies of the NI-Rln3 system.

GAD2 Expression Defines a Class of Excitatory Lateral Habenula Neurons in Mice that Project to the Raphe and Pontine Tegmentum.

The lateral habenula (LHb) sends complex projections to several areas of the mesopontine tegmentum, the raphe, and the hypothalamus. However, few markers have been available to distinguish subsets of LHb neurons that may serve these pathways. In order to address this complexity, we examined the mouse and rat LHb for neurons that express the GABA biosynthesis enzymes glutamate decarboxylase 1 (GAD1) and GAD2, and the vesicular GABA transporter (VGAT). The mouse LHb contains a population of neurons that express GAD2, while the rat LHb contains discrete populations of neurons that express GAD1 and VGAT. However, we could not detect single neurons in either species that co-express a GABA synthetic enzyme and VGAT, suggesting that these LHb neurons do not use GABA for conventional synaptic transmission. Instead, all of the neuronal types expressing a GABAergic marker in both species showed co-expression of the glutamate transporter VGluT2. Anterograde tract-tracing of the projections of GAD2-expressing LHb neurons in Gad2Cre mice, combined with retrograde tracing from selected downstream nuclei, show that LHb-GAD2 neurons project selectively to the midline structures in the mesopontine tegmentum, including the median raphe (MnR) and nucleus incertus (NI), and only sparsely innervate the hypothalamus, rostromedial tegmental nucleus (RMTg), and ventral tegmental area (VTA). Postsynaptic recording of LHb-GAD2 neuronal input to tegmental neurons confirms that glutamate, not GABA, is the fast neurotransmitter in this circuit. Thus, GAD2 expression can serve as a marker for functional studies of excitatory neurons serving specific LHb output pathways in mice.

Dual recombinase fate mapping reveals a transient cholinergic phenotype in multiple populations of developing glutamatergic neurons.

Cholinergic transmission shapes the maturation of glutamatergic circuits, yet the developmental sources of acetylcholine have not been systematically explored. Here, we have used Cre-recombinase-mediated genetic labeling to identify and map both mature and developing CNS neurons that express choline acetyltransferase (ChAT). Correction of a significant problem with a widely used ChatCre transgenic line ensures that this map does not contain expression artifacts. ChatCre marks all known cholinergic systems in the adult brain, but also identifies several brain areas not usually regarded as cholinergic, including specific thalamic and hypothalamic neurons, the subiculum, the lateral parabrachial nucleus, the cuneate/gracilis nuclei, and the pontocerebellar system. This ChatCre fate map suggests transient developmental expression of a cholinergic phenotype in areas important for cognition, motor control, and respiration. We therefore examined expression of ChAT and the vesicular acetylcholine transporter in the embryonic and early postnatal brain to determine the developmental timing of this transient cholinergic phenotype, and found that it mirrored the establishment of relevant glutamatergic projection pathways. We then used an intersectional genetic strategy combining ChatCre with Vglut2Flp to show that these neurons adopt a glutamatergic fate in the adult brain. The transient cholinergic phenotype of these glutamatergic neurons suggests a homosynaptic source of acetylcholine for the maturation of developing glutamatergic synapses. These findings thus define critical windows during which specific glutamatergic circuits may be vulnerable to disruption by nicotine in utero, and suggest new mechanisms for pediatric disorders associated with maternal smoking, such as sudden infant death syndrome.

Specific connections of the interpeduncular subnuclei reveal distinct components of the habenulopeduncular pathway.

The habenulopeduncular pathway consists of the medial habenula (MHb), its output tract, the fasciculus retroflexus, and its principal target, the interpeduncular nucleus (IP). Several IP subnuclei have been described, but their specific projections and relationship to habenula inputs are not well understood. Here we have used viral, transgenic, and conventional anterograde and retrograde tract-tracing methods to better define the relationship between the dorsal and ventral MHb, the IP, and the secondary efferent targets of this system. Although prior studies have reported that the IP has ascending projections to ventral forebrain structures, we find that these projections originate almost entirely in the apical subnucleus, which may be more appropriately described as part of the median raphe system. The laterodorsal tegmental nucleus receives inhibitory inputs from the contralateral dorsolateral IP, and mainly excitatory inputs from the ipsilateral rostrolateral IP subnucleus. The midline central gray of the pons and nucleus incertus receive input from the rostral IP, which contains a mix of inhibitory and excitatory neurons, and the dorsomedial IP, which is exclusively inhibitory. The lateral central gray of the pons receives bilateral input from the lateral IP, which in turn receives bilateral input from the dorsal MHb. Taken together with prior studies of IP projections to the raphe, these results form an emerging map of the habenulopeduncular system that has significant implications for the proposed function of the IP in a variety of behaviors, including models of mood disorders and behavioral responses to nicotine.

Efferent pathways of the mouse lateral habenula.

The lateral habenula (LHb) is part of the habenula complex of the dorsal thalamus. Recent studies of the LHb have focused on its projections to the ventral tegmental area (VTA) and rostromedial tegmental nucleus (RMTg), which contain γ-aminobutyric acid (GABA)ergic neurons that mediate reward prediction error via inhibition of dopaminergic activity. However, older studies in the rat have also identified LHb outputs to the lateral and posterior hypothalamus, median raphe, dorsal raphe, and dorsal tegmentum. Although these studies have shown that the medial and lateral divisions of the LHb have somewhat distinct projections, the topographic specificity of LHb efferents is not completely understood, and the relative extent of these projections to brainstem targets is unknown. Here we have used anterograde tracing with adeno-associated virus-mediated expression of green fluorescent protein, combined with serial two-photon tomography, to map the efferents of the LHb on a standard coordinate system for the entire mouse brain, and reconstruct the efferent pathways of the LHb in three dimensions. Using automated quantitation of fiber density, we show that in addition to the RMTg, the median raphe, caudal dorsal raphe, and pontine central gray are major recipients of LHb efferents. By using retrograde tract tracing with cholera toxin subunit B, we show that LHb neurons projecting to the hypothalamus, VTA, median raphe, caudal dorsal raphe, and pontine central gray reside in characteristic, but sometimes overlapping regions of the LHb. Together these results provide the anatomical basis for systematic studies of LHb function in neural circuits and behavior in mice. J. Comp. Neurol. 523:32-60, 2015. © 2014 Wiley Periodicals, Inc.

Medial habenula output circuit mediated by α5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus.

The Chrna5 gene encodes the α5 nicotinic acetylcholine receptor subunit, an "accessory" subunit of pentameric nicotinic receptors, that has been shown to play a role in nicotine-related behaviors in rodents and is genetically linked to smoking behavior in humans. Here we have used a BAC transgenic mouse line, α5(GFP), to examine the cellular phenotype, connectivity, and function of α5-expressing neurons. Although the medial habenula (MHb) has been proposed as a site of α5 function, α5(GFP) is not detectable in the MHb, and α5 mRNA is expressed there only at very low levels. However, α5(GFP) is strongly expressed in a subset of neurons in the interpeduncular nucleus (IP), median raphe/paramedian raphe (MnR/PMnR), and dorsal tegmental area (DTg). Double-label fluorescence in situ hybridization reveals that these neurons are exclusively GABAergic. Transgenic and conventional tract tracing show that α5(GFP) neurons in the IP project principally to the MnR/PMnR and DTg/interfascicular dorsal raphe, both areas rich in serotonergic neurons. The α5(GFP) neurons in the IP are located in a region that receives cholinergic fiber inputs from the ventral MHb, and optogenetically assisted circuit mapping demonstrates a monosynaptic connection between these cholinergic neurons and α5(GFP) IP neurons. Selective inhibitors of both α4ß2- and α3ß4-containing nicotinic receptors were able to reduce nicotine-evoked inward currents in α5(GFP) neurons in the IP, suggesting a mixed nicotinic receptor profile in these cells. Together, these findings show that the α5-GABAergic interneurons form a link from the MHb to serotonergic brain centers, which is likely to mediate some of the behavioral effects of nicotine.

Deletion of a conserved regulatory element required for Hmx1 expression in craniofacial mesenchyme in the dumbo rat: a newly identified cause of congenital ear malformation.

Hmx1 is a homeodomain transcription factor expressed in the developing eye, peripheral ganglia, and branchial arches of avian and mammalian embryos. Recent studies have identified a loss-of-function allele at the HMX1 locus as the causative mutation in the oculo-auricular syndrome (OAS) in humans, characterized by ear and eye malformations. The mouse dumbo (dmbo) mutation, with similar effects on ear and eye development, also results from a loss-of-function mutation in the Hmx1 gene. A recessive dmbo mutation causing ear malformation in rats has been mapped to the chromosomal region containing the Hmx1 gene, but the nature of the causative allele is unknown. Here we show that dumbo rats and mice exhibit similar neonatal ear and eye phenotypes. In midgestation embryos, dumbo rats show a specific loss of Hmx1 expression in neural-crest-derived craniofacial mesenchyme (CM), whereas Hmx1 is expressed normally in retinal progenitors, sensory ganglia and in CM, which is derived from mesoderm. High-throughput resequencing of 1 Mb of rat chromosome 14 from dmbo/dmbo rats, encompassing the Hmx1 locus, reveals numerous divergences from the rat genomic reference sequence, but no coding changes in Hmx1. Fine genetic mapping narrows the dmbo critical region to an interval of ∼410 kb immediately downstream of the Hmx1 transcription unit. Further sequence analysis of this region reveals a 5777-bp deletion located ∼80 kb downstream in dmbo/dmbo rats that is not apparent in 137 other rat strains. The dmbo deletion region contains a highly conserved domain of ∼500 bp, which is a candidate distal enhancer and which exhibits a similar relationship to Hmx genes in all vertebrate species for which data are available. We conclude that the rat dumbo phenotype is likely to result from loss of function of an ultraconserved enhancer specifically regulating Hmx1 expression in neural-crest-derived CM. Dysregulation of Hmx1 expression is thus a candidate mechanism for congenital ear malformation, most cases of which remain unexplained.

Hmx1 is required for the normal development of somatosensory neurons in the geniculate ganglion.

Hmx1 is a variant homeodomain transcription factor expressed in the developing sensory nervous system, retina, and craniofacial mesenchyme. Recently, mutations at the Hmx1 locus have been linked to craniofacial defects in humans, rats, and mice, but its role in nervous system development is largely unknown. Here we show that Hmx1 is expressed in a subset of sensory neurons in the cranial and dorsal root ganglia which does not correspond to any specific sensory modality. Sensory neurons in the dorsal root and trigeminal ganglia of Hmx1dm/dm mouse embryos have no detectable Hmx1 protein, yet they undergo neurogenesis and express sensory subtype markers normally, demonstrating that Hmx1 is not globally required for the specification of sensory neurons from neural crest precursors. Loss of Hmx1 expression has no obvious effect on the early development of the trigeminal (V), superior (IX/X), or dorsal root ganglia neurons in which it is expressed, but results in marked defects in the geniculate (VII) ganglion. Hmx1dm/dm mouse embryos possess only a vestigial posterior auricular nerve, and general somatosensory neurons in the geniculate ganglion are greatly reduced by mid-gestation. Although Hmx1 is expressed in geniculate neurons prior to cell cycle exit, it does not appear to be required for neurogenesis, and the loss of geniculate neurons is likely to be the result of increased cell death. Fate mapping of neural crest-derived tissues indicates that Hmx1-expressing somatosensory neurons at different axial levels may be derived from either the neural crest or the neurogenic placodes.

Brn3a and Nurr1 mediate a gene regulatory pathway for habenula development.

The habenula is a dorsal diencephalic structure consisting of medial and lateral subnuclei and a principal output tract, the fasciculus retroflexus, which together form a link between the limbic forebrain and ventral midbrain. Here, we have used microarray and bioinformatic approaches in the mouse to show that the habenula is a distinctive molecular territory of the CNS, with a unique profile of neurotransmitter, ion channel, and regulatory factor expression. Neurons of the medial habenula and part of the lateral habenula express the transcription factor Brn3a/Pou4f1, and Brn3a-expressing habenular neurons project exclusively to the interpeduncular nucleus in the ventral midbrain. In Brn3a mutant embryos, the fasciculus retroflexus is directed appropriately, but habenular neurons fail to innervate their targets. Microarray analysis of Brn3a null embryos shows that this factor regulates an extensive program of habenula-enriched genes, but not generic neural properties. The orphan nuclear receptor Nurr1/Nr4a2 is coexpressed with Brn3a in the developing habenula, is downstream of Brn3a, and mediates expression of a subset of Brn3a-regulated transcripts. Together, these findings begin to define a gene regulatory pathway for habenula development in mammals.

Regulation of the development of tectal neurons and their projections by transcription factors Brn3a and Pax7.

The rostral part of the dorsal midbrain, known as the superior colliculus in mammals or the optic tectum in birds, receives a substantial retinal input and plays a diverse and important role in sensorimotor integration. However, little is known about the development of specific subtypes of neurons in the tectum, particularly those which contribute tectofugal projections to the thalamus, isthmic region, and hindbrain. Here we show that two homeodomain transcription factors, Brn3a and Pax7, are expressed in mutually exclusive patterns in the developing and mature avian midbrain. Neurons expressing these factors are generated at characteristic developmental times, and have specific laminar fates within the tectum. In mice expressing betagalactosidase targeted to the Pou4f1 (Brn3a) locus, Brn3a-expressing neurons contribute to the ipsilateral but not the contralateral tectofugal projections to the hindbrain. Using misexpression of Brn3a and Pax7 by electroporation in the chick tectum, combined with GFP reporters, we show that Brn3a determines the laminar fate of subsets of tectal neurons. Furthermore, Brn3a regulates the development of neurons contributing to specific ascending and descending tectofugal pathways, while Pax7 globally represses the development of tectofugal projections to nearly all brain structures.

Brn3a target gene recognition in embryonic sensory neurons.

Numerous transcription factors have been identified which have profound effects on developing neurons. A fundamental problem is to identify genes downstream of these factors and order them in developmental pathways. We have previously identified 85 genes with changed expression in the trigeminal ganglia of mice lacking Brn3a, a transcription factor encoded by the Pou4f1 gene. Here we use locus-wide chromatin immunoprecipitation in embryonic trigeminal neurons to show that Brn3a is a direct repressor of two of these downstream genes, NeuroD1 and NeuroD4, and also directly modulates its own expression. Comparison of Brn3a binding to the Pou4f1 locus in vitro and in vivo reveals that not all high affinity sites are occupied, and several Brn3a binding sites identified in the promoters of genes that are silent in sensory ganglia are also not occupied in vivo. Site occupancy by Brn3a can be correlated with evolutionary conservation of the genomic regions containing the recognition sites and also with histone modifications found in regions of chromatin active in transcription and gene regulation, suggesting that Brn3a binding is highly context dependent.

Brn3a-expressing retinal ganglion cells project specifically to thalamocortical and collicular visual pathways.

Retinal ganglion cells (RGCs) innervate several specific CNS targets serving cortical and subcortical visual pathways and the entrainment of circadian rhythms. Recent studies have shown that retinal ganglion cells express specific combinations of POU- and LIM-domain transcription factors, but how these factors relate to the subsequent development of the retinofugal pathways and the functional identity of RGCs is mostly unknown. Here, we use targeted expression of an genetic axonal tracer, tau/beta-galactosidase, to examine target innervation by retinal ganglion cells expressing the POU-domain factor Brn3a. Brn3a is expressed in RGCs innervating the principal retinothalamic/retinocollicular pathway mediating cortical vision but is not expressed in RGCs of the accessory optic, pretectal, and hypothalamic pathways serving subcortical visuomotor and circadian functions. In the thalamus, Brn3a ganglion cell fibers are primarily restricted to the outer shell of the dorsal lateral geniculate, providing new evidence for the regionalization of this nucleus in rodents. Brn3a RGC axons have a relative preference for the contralateral hemisphere, but known mediators of the laterality of RGC axons are not repatterned in the absence of Brn3a. Brn3a is coexpressed extensively with the closely related factor Brn3b in the embryonic retina, and the effects of the loss of Brn3a in retinal development are not severe, suggesting partial redundancy of function in this gene class.

Interleukin-6 produces neuronal loss in developing cerebellar granule neuron cultures.

CNS levels of the cytokine interleukin-6 (IL-6) are elevated during CNS injury and disease, but it is unclear if IL-6 contributes to the pathologic process. Our studies show that in a well-characterized CNS developmental model system, primary cultures of rodent cerebellar granule neurons, chronic exposure to IL-6 during neuronal development can result in cell damage and death in a subpopulation of developing granule neurons. Chronic exposure to IL-6 also increased the susceptibility of the granule neurons to a toxic insult produced by excessive activation of NMDA receptors. These results are consistent with a role for IL-6 in the neuropathology observed in the developing CNS during injury and disease.