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Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes- Fgf3 , Fgf4 , and Fgf15 -that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes-for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.

Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gβ5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Introduction: Enhancer of zeste homolog 2 (Ezh2) is responsible for trimethylation of histone 3 at lysine 27 (H3K27me3), resulting in repression of gene expression. Here, we explore the role of Ezh2 in forebrain GABAergic interneuron development., Methods: We removed Ezh2 in the MGE by generating Nkx2-1Cre;Ezh2 conditional knockout mice. We then characterized changes in MGE-derived interneuron fate and electrophysiological properties in juvenile mice, as well as alterations in gene expression, chromatin accessibility and histone modifications in the MGE., Results: Loss of Ezh2 increases somatostatin-expressing (SST+) and decreases parvalbumin-expressing (PV+) interneurons in the forebrain. We observe fewer MGE-derived interneurons in the first postnatal week, indicating reduced interneuron production. Intrinsic electrophysiological properties in SST+ and PV+ interneurons are normal, but PV+ interneurons display increased axonal complexity in Ezh2 mutant mice. Single nuclei multiome analysis revealed differential gene expression patterns in the embryonic MGE that are predictive of these cell fate changes. Lastly, CUT&Tag analysis revealed that some genomic loci are particularly resistant or susceptible to shifts in H3K27me3 levels in the absence of Ezh2 , indicating differential selectivity to epigenetic perturbation., Discussion: Thus, loss of Ezh2 in the MGE alters interneuron fate, morphology, and gene expression and regulation. These findings have important implications for both normal development and potentially in disease etiologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Rhodes, Asokumar, Sohn, Naskar, Elisha, Stevenson, Lee, Zhang, Rocha, Dale, Lee and Petros.)

Efficient protocols to generate single-cell and single-nuclei suspensions are critical for the burgeoning field of single-cell/single-nuclei sequencing. Here we describe procedures to generate single-cell and single-nuclei suspensions from embryonic and adult mouse brains. This protocol can be modified for any brain region and/or neural cell type. For complete details on the use and execution of this protocol, please refer to Lee et al. (2022), 1 Rhodes et al. (2022), 2 Mahadevan et al. (2021), 3 Ekins et al. (2020), 4 and Wester et al. (2019). 5 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

How enhancers activate their distal target promoters remains incompletely understood. Here we dissect how CTCF-mediated loops facilitate and restrict such regulatory interactions. Using an allelic series of mouse mutants, we show that CTCF is neither required for the interaction of the Sox2 gene with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs, between Sox2 and its distal enhancers, generated boundaries with varying degrees of insulation that directly correlated with reduced transcriptional output. However, in both epiblast and neural tissues, enhancer contacts and transcriptional induction could not be fully abolished, and insertions failed to disrupt implantation and neurogenesis. In contrast, Sox2 expression was undetectable in the anterior foregut of mutants carrying the strongest boundaries, and these animals fully phenocopied loss of SOX2 in this tissue. We propose that enhancer clusters with a high density of regulatory activity can better overcome physical barriers to maintain faithful gene expression and phenotypic robustness., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

A comprehensive characterization of epigenomic organization in the embryonic mouse forebrain will enhance our understanding of neurodevelopment and provide insight into mechanisms of neurological disease. Here we collected single-cell chromatin accessibility profiles from four distinct neurogenic regions of the embryonic mouse forebrain using single nuclei ATAC-Seq (snATAC-Seq). We identified thousands of differentially accessible peaks, many restricted to distinct progenitor cell types or brain regions. We integrated snATAC-Seq and single cell transcriptome data to characterize changes of chromatin accessibility at enhancers and promoters with associated transcript abundance. Multi-modal integration of histone modifications (CUT&Tag and CUT&RUN), promoter-enhancer interactions (Capture-C) and high-order chromatin structure (Hi-C) extended these initial observations. This dataset reveals a diverse chromatin landscape with region-specific regulatory mechanisms and genomic interactions in distinct neurogenic regions of the embryonic mouse brain and represents an extensive public resource of a 'ground truth' epigenomic landscape at this critical stage of neurogenesis., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression, but a detailed characterization of transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the ganglionic eminences (GEs), the source of all forebrain GABAergic neurons. By using a transgenic mouse line to enrich for VZ cells, we characterize significant transcriptional heterogeneity, both between GEs and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of VZ cells in the ventral forebrain that will aid our understanding of initial fate decisions in the forebrain., Competing Interests: DL, CR, AM, YZ, DM, RD, TP No competing interests declared

Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical, and physiological properties. However, the molecular mechanisms regulating their maturation remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates transcriptional regulation of gene expression in specific subtypes of MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific early developmental Grin1 loss results in a broad downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs in the juvenile brain. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mahadevan, Mitra, Zhang, Yuan, Peltekian, Chittajallu, Esnault, Maric, Rhodes, Pelkey, Dale, Petros and McBain.)

In vertebrates, motor control relies on cholinergic neurons in the spinal cord that have been extensively studied over the past hundred years, yet the full heterogeneity of these neurons and their different functional roles in the adult remain to be defined. Here, we develop a targeted single nuclear RNA sequencing approach and use it to identify an array of cholinergic interneurons, visceral and skeletal motor neurons. Our data expose markers for distinguishing these classes of cholinergic neurons and their rich diversity. Specifically, visceral motor neurons, which provide autonomic control, can be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons is also reflected in our analysis with alpha, gamma, and a third subtype, possibly corresponding to the elusive beta motor neurons, clearly distinguished. In combination, our data provide a comprehensive transcriptomic description of this important population of neurons that control many aspects of physiology and movement and encompass the cellular substrates for debilitating degenerative disorders.

GluN3A subunits endow N-Methyl-D-Aspartate receptors (NMDARs) with unique biophysical, trafficking, and signaling properties. GluN3A-NMDARs are typically expressed during postnatal development, when they are thought to gate the refinement of neural circuits by inhibiting synapse maturation, and stabilization. Recent work suggests that GluN3A also operates in adult brains to control a variety of behaviors, yet a full spatiotemporal characterization of GluN3A expression is lacking. Here, we conducted a systematic analysis of Grin3a (gene encoding mouse GluN3A) mRNA expression in the mouse brain by combining high-sensitivity colorimetric and fluorescence in situ hybridization with labeling for neuronal subtypes. We find that, while Grin3a mRNA expression peaks postnatally, significant levels are retained into adulthood in specific brain regions such as the amygdala, medial habenula, association cortices, and high-order thalamic nuclei. The time-course of emergence and down-regulation of Grin3a expression varies across brain region, cortical layer of residence, and sensory modality, in a pattern that correlates with previously reported hierarchical gradients of brain maturation and functional specialization. Grin3a is expressed in both excitatory and inhibitory neurons, with strong mRNA levels being a distinguishing feature of somatostatin interneurons. Our study provides a comprehensive map of Grin3a distribution across the murine lifespan and paves the way for dissecting the diverse functions of GluN3A in health and disease., (© The Author(s) 2020. Published by Oxford University Press.)

Type I lissencephaly is a neuronal migration disorder caused by haploinsuffiency of the PAFAH1B1 (mouse: Pafah1b1 ) gene and is characterized by brain malformation, developmental delays, and epilepsy. Here, we investigate the impact of Pafah1b1 mutation on the cellular migration, morphophysiology, microcircuitry, and transcriptomics of mouse hippocampal CA1 parvalbumin-containing inhibitory interneurons (PV+INTs). We find that WT PV+INTs consist of two physiological subtypes (80% fast-spiking (FS), 20% non-fast-spiking (NFS)) and four morphological subtypes. We find that cell-autonomous mutations within interneurons disrupts morphophysiological development of PV+INTs and results in the emergence of a non-canonical 'intermediate spiking (IS)' subset of PV+INTs. We also find that now dominant IS/NFS cells are prone to entering depolarization block, causing them to temporarily lose the ability to initiate action potentials and control network excitation, potentially promoting seizures. Finally, single-cell nuclear RNAsequencing of PV+INTs revealed several misregulated genes related to morphogenesis, cellular excitability, and synapse formation., Competing Interests: TE, VM, YZ, JD, GA, TP, CM No competing interests declared

GABAergic interneurons have many important functions in cortical circuitry, a reflection of their cell diversity. The developmental origins of this diversity are poorly understood. Here, we identify rostral-caudal regionality in Wnt exposure within the interneuron progenitor zone delineating the specification of the two main interneuron subclasses. Caudally situated medial ganglionic eminence (MGE) progenitors receive high levels of Wnt signaling and give rise to somatostatin (SST)-expressing cortical interneurons. By contrast, parvalbumin (PV)-expressing basket cells originate mostly from the rostral MGE, where Wnt signaling is attenuated. Interestingly, rather than canonical signaling through β-catenin, signaling via the non-canonical Wnt receptor Ryk regulates interneuron cell-fate specification in vivo and in vitro. Indeed, gain of function of Ryk intracellular domain signaling regulates SST and PV fate in a dose-dependent manner, suggesting that Ryk signaling acts in a graded fashion. These data reveal an important role for non-canonical Wnt-Ryk signaling in establishing the correct ratios of cortical interneuron subtypes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Neocortical circuits consist of stereotypical motifs that must self-assemble during development. Recent evidence suggests that the subtype identity of both excitatory projection neurons (PNs) and inhibitory interneurons (INs) is important for this process. We knocked out the transcription factor Satb2 in PNs to induce those of the intratelencephalic (IT) type to adopt a pyramidal tract (PT)-type identity. Loss of IT-type PNs selectively disrupted the lamination and circuit integration of INs derived from the caudal ganglionic eminence (CGE). Strikingly, reprogrammed PNs demonstrated reduced synaptic targeting of CGE-derived INs relative to controls. In control mice, IT-type PNs targeted neighboring CGE INs, while PT-type PNs did not in deep layers, confirming this lineage-dependent motif. Finally, single-cell RNA sequencing revealed that major CGE IN subtypes were conserved after loss of IT PNs, but with differential transcription of synaptic proteins and signaling molecules. Thus, IT-type PNs influence CGE-derived INs in a non-cell-autonomous manner during cortical development., (Published by Elsevier Inc.)

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

The mammalian brain develops from a simple sheet of neuroepithelial cells into an incredibly complex structure containing billions of neurons with trillions of synapses. Understanding how intrinsic genetic programs interact with environmental cues to generate neuronal diversity and proper connectivity is one of the most daunting challenges in developmental biology. We recently explored this issue in forebrain GABAergic inhibitory interneurons, an extremely diverse population of neurons that are classified into distinct subtypes based on morphology, neurochemical markers, and electrophysiological properties. Immature interneurons were harvested from one brain region and transplanted into a different region, allowing us to assess how challenging cells in a new environment affected their fate. Do these grafted cells adopt characteristics of the host environment or retain features from the donor environment? We found that the proportion of interneuron subgroups is determined by the host region, but some interneuron subtypes maintain features attributable to the donor environment. In this commentary, I expound on potential mechanisms that could underlie these observations and explore the implications of these findings in a greater context of developmental neuroscience., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

During embryogenesis, neural progenitors in the ganglionic eminences give rise to diverse GABAergic interneuron subtypes that populate all forebrain regions. The extent to which these cells are genetically predefined or determined by postmigratory environmental cues remains unknown. To address this question, we performed homo- and heterotopic transplantation of early postnatal MGE-derived cortical and hippocampal interneurons. Grafted cells migrated, and displayed neurochemical, electrophysiological, morphological, and neurochemical profiles similar to endogenous interneurons. Our results indicate that the host environment regulates the proportion of interneuron classes in the brain region. However, some specific interneuron subtypes retain characteristics representative of their donor brain regions., (Published by Elsevier Inc.)

Fate determination in the mammalian telencephalon, with its diversity of neuronal subtypes and relevance to neuropsychiatric disease, remains a critical area of study in neuroscience. Most studies investigating this topic focus on the diversity of neural progenitors within spatial and temporal domains along the lateral ventricles. Often overlooked is whether the location of neurogenesis within a fate-restricted domain is associated with, or instructive for, distinct neuronal fates. Here, we use in vivo fate mapping and the manipulation of neurogenic location to demonstrate that apical versus basal neurogenesis influences the fate determination of major subgroups of cortical interneurons derived from the subcortical telencephalon. Somatostatin-expressing interneurons arise mainly from apical divisions along the ventricular surface, whereas parvalbumin-expressing interneurons originate predominantly from basal divisions in the subventricular zone. As manipulations that shift neurogenic location alter interneuron subclass fate, these results add an additional dimension to the spatial-temporal determinants of neuronal fate determination., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Medial ganglionic eminence (MGE)-derived GABAergic cortical interneurons (cINs) consist of multiple subtypes that are involved in many cortical functions. They also have a remarkable capacity to migrate, survive and integrate into cortical circuitry after transplantation into postnatal cortex. These features have engendered considerable interest in generating distinct subgroups of interneurons from pluripotent stem cells (PSCs) for the study of interneuron fate and function, and for the development of cell-based therapies. Although advances have been made, the capacity to generate highly enriched pools of subgroup fate-committed interneuron progenitors from PSCs has remained elusive. Previous studies have suggested that the two main MGE-derived interneuron subgroups--those expressing somatostatin (SST) and those expressing parvalbumin (PV)--are specified in the MGE from Nkx2.1-expressing progenitors at higher or lower levels of sonic hedgehog (Shh) signaling, respectively. To further explore the role of Shh and other factors in cIN fate determination, we generated a reporter line such that Nkx2.1-expressing progenitors express mCherry and postmitotic Lhx6-expressing MGE-derived interneurons express GFP. Manipulations of Shh exposure and time in culture influenced the subgroup fates of ESC-derived interneurons. Exposure to higher Shh levels, and collecting GFP-expressing precursors at 12 days in culture, resulted in the strongest enrichment for SST interneurons over those expressing PV, whereas the strongest enrichment for PV interneurons was produced by lower Shh and by collecting mCherry-expressing cells after 17 days in culture. These findings confirm that fate determination of cIN subgroups is crucially influenced by Shh signaling, and provide a system for the further study of interneuron fate and function., (© 2015. Published by The Company of Biologists Ltd.)

Forebrain GABAergic interneurons are divided into subgroups based on their neurochemical markers, connectivity and physiological properties. Abnormal interneuron function is implicated in the pathobiology of neurological disorders such as schizophrenia, autism, and epilepsy. Studies on interneuron development and their role in disease would benefit from an efficient mechanism for the production and selection of specific interneuron subgroups. In this study, we engineered a mouse embryonic stem cell (mESC) line for doxycycline-inducible expression of Nkx2.1, a required transcription factor for cortical interneurons derived from the medial ganglionic eminence (MGE). This mESC line was modified to express GFP in Lhx6(+) cells, a marker of newly postmitotic and mature MGE-derived cortical interneurons. The addition of doxycycline to differentiating ESCs efficiently induced Nkx2.1 protein and increased the production of GFP(+) cells. Transplantation of GFP(+) putative interneuron precursors resulted in migratory, morphological, and neurochemical features consistent with cortical interneuron fates. To test the hypothesis that Sonic hedgehog (Shh) primarily influences cortical interneuron fate determination through the induction of Nkx2.1, ESCs were grown with doxycycline and the Shh antagonist cyclopamine. We found induced Nkx2.1 renders Shh signaling dispensable for the generation of MGE-derived interneurons. These results demonstrate that inducible expression of fate determining genes in embryonic stem cells can be used to study fate determination of the developing forebrain., (Copyright © 2013. Published by Elsevier B.V.)

GABAergic interneurons of the cerebral cortex (cINs) play crucial roles in many aspects of cortical function. The diverse types of cINs are classified into subgroups according to their morphology, intrinsic physiology, neurochemical markers and synaptic targeting. Recent advances in mouse genetics, imaging and electrophysiology techniques have greatly advanced our efforts to understand the role of normal cIN function and its dysfunction in neuropsychiatric disorders. In schizophrenia (SCZ), a wealth of data suggests that cIN function is perturbed, and that interneuron dysfunction may underlie key symptoms of the disease. In this review, we discuss the link between cINs and SCZ, focusing on the evidence for GABAergic signaling deficits from both SCZ patients and mouse models., (Copyright © 2013 Elsevier Inc. All rights reserved.)

At the optic chiasm, retinal ganglion cells (RGCs) project ipsi- or contralaterally to establish the circuitry for binocular vision. Ipsilateral guidance programs have been characterized, but contralateral guidance programs are not well understood. Here, we identify a tripartite molecular system for contralateral RGC projections: Semaphorin6D (Sema6D) and Nr-CAM are expressed on midline radial glia and Plexin-A1 on chiasm neurons, and Plexin-A1 and Nr-CAM are also expressed on contralateral RGCs. Sema6D is repulsive to contralateral RGCs, but Sema6D in combination with Nr-CAM and Plexin-A1 converts repulsion to growth promotion. Nr-CAM functions as a receptor for Sema6D. Sema6D, Plexin-A1, and Nr-CAM are all required for efficient RGC decussation at the optic chiasm. These findings suggest a mechanism by which a complex of Sema6D, Nr-CAM, and Plexin-A1 at the chiasm midline alters the sign of Sema6D and signals Nr-CAM/Plexin-A1 receptors on RGCs to implement the contralateral RGC projection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The mammalian central nervous system is a complex neuronal network consisting of a diverse array of cellular subtypes generated in a precise spatial and temporal pattern throughout development. Achieving a greater understanding of the molecular and genetic mechanisms that direct a relatively uniform population of neuroepithelial progenitors into diverse neuronal subtypes remains a significant challenge. The advent of pluripotent stem cell (PSC) technology allows researchers to generate diverse neural populations in vitro. Although the primary focus of PSC-derived neural cells has been their therapeutic potential, utilizing PSCs to study neurodevelopment is another frequently overlooked and equally important application. In this review, we explore the potential for utilizing PSCs to study neural development. We introduce the types of neurodevelopmental questions that PSCs can help to address, and we discuss the different strategies and technologies that researchers use to generate diverse subtypes of PSC-derived neurons. Additionally, we highlight the derivation of several thoroughly characterized neural subtypes; spinal motoneurons, midbrain dopaminergic neurons and cortical neurons. We hope that this review encourages researchers to develop innovative strategies for using PSCs for the study of mammalian, and specifically human, neurodevelopment.

Rural communities face an impending surgical workforce crisis. The purpose of this study is to describe perceptions of rural Tennessee hospital administrators regarding the importance of surgical services to their hospitals. In collaboration with the Tennessee Hospital Association, we developed and administered a 13-item survey based on a recently published national survey to 80 rural Tennessee hospitals in August 2008. A total of 29 responses were received for an overall 36.3 per cent response rate. Over 44 per cent of rural surgeons were older than 50 years of age, and 27.6 per cent of hospitals reported they would lose at least one surgeon in the next 2 years. The responding hospitals reported losing 10.4 per cent of their surgical workforce in the preceding 2 years. Over 53 per cent were actively recruiting a general surgeon with an average time to recruit a surgeon of 11.8 months. Ninety-seven per cent stated that having a surgical program was very important to their financial viability with the mean and median reported revenue generated by a single general surgeon being $1.8 million and $1.4 million, respectively. Almost 11 per cent of the hospitals stated they would have to close if they lost surgical services. Although rural Tennessee hospitals face similar difficulties to national rural hospitals with regard to retaining and hiring surgeons, slightly more Tennessee hospitals (54 vs 36%) were actively attempting to recruit a general surgeon. The shortage of general surgeons is a threat to the accessibility of comprehensive hospital-based care for rural Tennesseans.

The circuit for binocular vision and stereopsis is established at the optic chiasm, where retinal ganglion cell (RGC) axons diverge into the ipsilateral and contralateral optic tracts. In the mouse retina, ventrotemporal (VT) RGCs express the guidance receptor EphB1, which interacts with the repulsive guidance cue ephrin-B2 on radial glia at the optic chiasm to direct VT RGC axons ipsilaterally. RGCs in the ventral retina also express EphB2, which interacts with ephrin-B2, whereas dorsal RGCs express low levels of EphB receptors. To investigate how growth cones of RGCs from different retinal regions respond upon initial contact with ephrin-B2, we utilized time-lapse imaging to characterize the effects of ephrin-B2 on growth cone collapse and axon retraction in real time. We demonstrate that bath application of ephrin-B2 induces rapid and sustained growth cone collapse and axon retraction in VT RGC axons, whereas contralaterally-projecting dorsotemporal RGCs display moderate growth cone collapse and little axon retraction. Dose response curves reveal that contralaterally-projecting ventronasal axons are less sensitive to ephrin-B2 treatment compared to VT axons. Additionally, we uncovered a specific role for Rho kinase signaling in the retraction of VT RGC axons but not in growth cone collapse. The detailed characterization of growth cone behavior in this study comprises an assay for the study of Eph signaling in RGCs, and provides insight into the phenomena of growth cone collapse and axon retraction in general.

Partial decussation of sensory pathways allows neural inputs from both sides of the body to project to the same target region where these signals will be integrated. Here, to better understand mechanisms of eye-specific targeting, we studied how retinal ganglion cell (RGC) axons terminate in their thalamic target, the dorsal lateral geniculate nucleus (dLGN), when crossing at the optic chiasm midline is altered. In models with gain- and loss-of-function of EphB1, the receptor that directs the ipsilateral projection at the optic chiasm, misrouted RGCs target the appropriate retinotopic zone in the opposite dLGN. However, in EphB1(-/-) mice, the misrouted axons do not intermingle with normally projecting RGC axons and segregate instead into a distinct patch. We also revisited the role of retinal activity on eye-specific targeting by blocking correlated waves of activity with epibatidine into both eyes. We show that, in wild-type mice, retinal waves are necessary during the first postnatal week for both proper distribution and eye-specific segregation of ipsilateral axons in the mature dLGN. Moreover, in EphB1(-/-) mice, refinement of ipsilateral axons is perturbed in control conditions and is further impaired after epibatidine treatment. Finally, retinal waves are required for the formation of the segregated patch of misrouted axons in EphB1(-/-) mice. These findings implicate molecular determinants for targeting of eye-specific zones that are independent of midline guidance cues and that function in concert with correlated retinal activity to sculpt retinogeniculate projections.

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups, adult rats, and embryonic murine retinae in vitro, none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs. With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

At the optic chiasm, retinal ganglion cell (RGC) axons make the decision to either avoid or traverse the midline, a maneuver that establishes the binocular pathways. In mice, the ipsilateral retinal projection arises from RGCs in the peripheral ventrotemporal (VT) crescent of the retina. These RGCs express the guidance receptor EphB1, which interacts with ephrin-B2 on radial glia cells at the optic chiasm to repulse VT axons away from the midline and into the ipsilateral optic tract. However, because VT RGCs express more than one EphB receptor, the sufficiency and specificity of the EphB1 receptor in directing the ipsilateral projection is unclear. In this study, we use in utero retinal electroporation to demonstrate that ectopic EphB1 expression can redirect RGCs with a normally crossed projection to an ipsilateral trajectory. Moreover, EphB1 is specifically required for rerouting RGC projections ipsilaterally, because introduction of the highly similar EphB2 receptor is much less efficient in redirecting RGC fibers, even when expressed at higher surface levels. Introduction of EphB1-EphB2 chimeric receptors into RGCs reveals that both extracellular and juxtamembrane domains of EphB1 are required to efficiently convert RGC projections ipsilaterally. Together, these data describe for the first time functional differences between two highly similar Eph receptors at a decision point in vivo, with EphB1 displaying unique properties that efficiently drives the uncrossed retinal projection.

The navigation of retinal axons to ipsilateral and contralateral targets in the brain depends on the decision to cross or avoid the midline at the optic chiasm, a critical guidance maneuver that establishes the binocular visual pathway. Previous work has identified a specific guidance receptor, EphB1, that mediates the repulsion of uncrossed axons away from its ligand, ephrinB2, at the optic chiasm midline (Williams et al., 2003), and a transcription factor Zic2, that, like EphB1, is required for formation of the ipsilateral retinal projection (Herrera et al., 2003). Although the reported similarities in localization implicated that Zic2 regulates EphB1 (Herrera et al., 2003; Williams et al., 2003; Pak et al., 2004), whether Zic2 drives expression of EphB1 protein has not been elucidated. Here we show that EphB1 protein is expressed in the growth cones of axons from ventrotemporal (VT) retina that project ipsilaterally and that repulsion by ephrinB2 is determined by the presence of this receptor on growth cones. Moreover, ectopic delivery of Zic2 into explants from non-VT retina induces expression of EphB1 mRNA and protein. The upregulated EphB1 receptor protein is localized to growth cones and is functional, because it is sufficient to change retinal ganglion cell axon behavior from extension onto, to avoidance of, ephrinB2 substrates. Our results demonstrate that Zic2 upregulates EphB1 expression and define a link between a transcription factor and expression of a guidance receptor protein essential for axon guidance at the vertebrate midline.

At the optic chiasm, retinal ganglion cell axons from each eye converge and segregate into crossed and uncrossed projections, a pattern critical for binocular vision. Here, we review recent findings on optic chiasm development, highlighting the specific transcription factors and guidance cues that implement retinal axon divergence into crossed and uncrossed pathways. Although mechanisms underlying the formation of the uncrossed projection have been identified, the means by which retinal axons are guided across the midline are still unclear. In addition to directives provided by transcription factors and receptors in the retina, gene expression in the ventral diencephalon influences chiasm formation. Throughout this review, we compare guidance mechanisms at the optic chiasm with those in other midline models and highlight unanswered questions both for retinal axon growth and axon guidance in general.

Eph receptors and their ephrin ligands play important roles in many aspects of visual system development. In this study, we characterized the spatial and temporal expression pattern of EphA4 in astrocyte precursor cell (APC) and astrocyte populations in the murine retina and optic nerve. EphA4 is expressed by immotile optic disc astrocyte precursor cells (ODAPS), but EphA4 is downregulated as these cells migrate into the retina. Surprisingly, mature astrocytes in the adult retina re-express EphA4. Within the optic nerve, EphA4 is expressed in specialized astrocytes that form a meshwork at the optic nerve head (ONH). Our in vitro and in vivo data indicate that EphA4 is dispensable for retinal ganglion cell (RGC) axon growth and projections through the chiasm. While optic stalk structure, APC proliferation and migration, retinal vascularization, and oligodendrocyte migration appear normal in EphA4 mutants, the expression of EphA4 in APCs and in the astrocyte meshwork at the ONH has implications for optic nerve pathologies.

The Krüppel-like transcription factors (KLFs) are important regulators of cell proliferation and differentiation in several different organ systems. The mouse Klf7 gene is strongly active in postmitotic neuroblasts of the developing nervous system, and the corresponding protein stimulates transcription of the cyclin-dependent kinase inhibitor p21waf/cip gene. Here we report that loss of KLF7 activity in mice leads to neonatal lethality and a complex phenotype which is associated with deficits in neurite outgrowth and axonal misprojection at selected anatomical locations of the nervous system. Affected axon pathways include those of the olfactory and visual systems, the cerebral cortex, and the hippocampus. In situ hybridizations and immunoblots correlated loss of KLF7 activity in the olfactory epithelium with significant downregulation of the p21waf/cip and p27kip1 genes. Cotransfection experiments extended the last finding by documenting KLF7's ability to transactivate a reporter gene construct driven by the proximal promoter of p27kip1. Consistent with emerging evidence for a role of Cip/Kip proteins in cytoskeletal dynamics, we also documented p21waf/cip and p27kip1 accumulation in the cytoplasm of differentiating olfactory sensory neurons. KLF7 activity might therefore control neuronal morphogenesis in part by optimizing the levels of molecules that promote axon outgrowth.

Background: Endocannabinoids may participate in the homeostasis of arterial pressure. Recently, anandamide, the most extensively studied endocannabinoid, has been proposed as a key mediator in the peripheral arterial vasodilation of cirrhosis., Objectives: To determine if circulating levels of anandamide are related to the extent of the peripheral arterial vasodilation, the severity of portal hypertension and the degree of liver and renal dysfunction of patients with cirrhosis., Methods: Plasma levels of anandamide and several systemic, portal and renal hemodynamic parameters were determined in 18 patients with cirrhosis and eight healthy subjects (control group)., Results: Plasma levels of anandamide were elevated in patients compared to the control group (P<0.05), nevertheless, no differences between patients with ascites and well-compensated patients were found. There was no correlation between anandamide concentration and arterial pressure, cardiac output and systemic vascular resistance, Child-Pugh's score, portal pressure, renal vascular resistance, plasma renin activity or plasma aldosterone concentration., Conclusions: Circulating levels of anandamide are increased in cirrhotic patients. However, this elevation was unrelated to the extent of arterial vasodilation, the severity of portal hypertension or the degree of hepatic and renal dysfunction. Although a local hormonal action cannot be excluded, our results do not support a relevant contribution of this system in the hemodynamic disturbance of cirrhosis.

N-arachidonylglycine (NAGly), the carboxylic analog of the endocannabinoid anandamide, occurs in rat and bovine brain as well as in peripheral sites and shows activity against tonic, formalin-induced pain. It was also observed, using cell membrane preparations, that it inhibits the hydrolytic activity of fatty acid amide hydrolase (FAAH) on anandamide (N-arachidonylethanolamide). These data suggested that it may serve as an endogenous regulator of tissue anandamide concentrations. In this report, we show findings derived from mass spectrometric analyses, indicating that blood levels of anandamide in rats given 10 mg/kg p.o. of NAGly were increased significantly by more than 9-fold when compared with vehicle-treated controls. In vitro evidence in RAW 264.7 cells using a deuterium-labeled NAGly demonstrated that it was not a precursor or source of arachidonic acid for the observed 50% rise in anandamide levels, suggesting that the increase was due to some effect other than increased biosynthesis of anandamide. Moreover, the findings presented here suggest that NAGly can serve as a model for the design of agents to provide pharmacological control of tissue anandamide concentrations.

The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and the pungent ingredient of "hot" chili peppers, capsaicin. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. Here we examined the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous "capsaicin-like" substance in mammalian nervous tissues. We found that NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. We also gained evidence for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency (EC(50) approximately 50 nM) and efficacy similar to those of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia (EC(50) = 1.5 +/- 0.3 microg). Our data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors.

In mammals, specific lipids and amino acids serve as crucial signaling molecules. In bacteria, conjugates of lipids and amino acids (referred to as lipoamino acids) have been identified and found to possess biological activity. Here, we report that mammals also produce lipoamino acids, specifically the arachidonyl amino acids. We show that the conjugate of arachidonic acid and glycine (N-arachidonylglycine (NAGly)) is present in bovine and rat brain as well as other tissues and that it suppresses tonic inflammatory pain. The biosynthesis of NAGly and its degradation by the enzyme fatty acid amide hydrolase can be observed in rat brain tissue. In addition to NAGly, bovine brain produces at least two other arachidonyl amino acids: N-arachidonyl gamma-aminobutyric acid (NAGABA) and N-arachidonylalanine. Like NAGly, NAGABA inhibits pain. These findings open the door to the identification of other members of this new class of biomolecules, which may be integral to pain regulation and a variety of functions in mammals.