Electrophysiological phenotyping of left ventricular noncompaction cardiomyopathy in pediatric populations: A systematic review.

Left ventricular noncompaction cardiomyopathy (LVNC) is a structural heart defect that has been associated with generation of arrhythmias in the population and is a cause of sudden cardiac death with severe systolic dysfunction and fatal arrhythmias. LVNC has gained increasing acknowledgment with increased prevalence. We conducted a systematic review of reported electrocardiogram (ECG) results for pediatric LVNC patients. EMBASE database query was performed, yielding 4531 articles related to LVNC between 1990 and December 2023. Patient age ranged from prenatal to 18 years of age. Qualitative analyses were performed to characterize individual arrhythmias, and summative interpretation of ECG evaluations was gathered for the entire cohort. Systematic review of 57 LVNC cases and ECG presentation revealed many waveform consistencies, including abnormal left ventricular, atrioventricular node, and interventricular septal patterns, and specifically a high incidence of Mobitz type II and Wolff-Parkinson-White waveforms. This review of ECG analysis reinforces the clinical and etiologic significance of pediatric LVNC. While LVNC in pediatric populations may not always present as acute clinical cases, further investigation into the electrophysiology of the disease supports the need for further evaluation and risk stratification for patients with suspected LVNC and/or ventricular arrhythmia.

The Primary Cilium and its Hedgehog Signaling in Nociceptors Contribute to Inflammatory and Neuropathic Pain.

The primary cilium, a 1-3 µm long hair-like structure protruding from the surface of almost all cells in the vertebrate body, is critical for neuronal development and also functions in the adult. As the migratory neural crest settles into dorsal root ganglia (DRG) sensory neurons elaborate a single primary cilium at their soma that is maintained into adult stages. While it is not known if primary cilia are expressed in nociceptors, or their potential function in the mature DRG neuron, recent studies have shown a role for Hedgehog, whose signaling demonstrates a dependence on primary cilia, in nociceptor sensitization. Here we report the expression of primary cilia in rat and mouse nociceptors, where they modulate mechanical nociceptive threshold, and contribute to inflammatory and neuropathic pain. When siRNA targeting Ift88, a primary cilium-specific intraflagellar transport (IFT) protein required for ciliary integrity, was administered by intrathecal injection, in the rat, it resulted in loss of Ift88 mRNA in DRG, and primary cilia in neuronal cell bodies, which was associated with an increase in mechanical nociceptive threshold, and abrogation of hyperalgesia induced by the pronociceptive inflammatory mediator, prostaglandin E2, and painful peripheral neuropathy induced by a neurotoxic chemotherapy drug, paclitaxel. To provide further support for the role of the primary cilium in nociceptor function we also administered siRNA for another IFT protein, Ift52. Ift52 siRNA results in loss of Ift52 in DRG and abrogates paclitaxel-induced painful peripheral neuropathy. Attenuation of Hedgehog-induced hyperalgesia by Ift88 knockdown supports a role for the primary cilium in the hyperalgesia induced by Hedgehog, and attenuation of paclitaxel chemotherapy-induced neuropathy (CIPN) by cyclopamine, which attenuates Hedgehog signaling, suggests a role of Hedgehog in CIPN. Our findings support a role of nociceptor primary cilia in the control of mechanical nociceptive threshold and in inflammatory and neuropathic pain, the latter, at least in part, Hedgehog dependent.

Primary cilia are critical for tracheoesophageal septation.

INTRODUCTION: Primary cilia play pivotal roles in the patterning and morphogenesis of a wide variety of organs during mammalian development. Here we examined murine foregut septation in the cobblestone mutant, a hypomorphic allele of the gene encoding the intraflagellar transport protein IFT88, a protein essential for normal cilia function. RESULTS: We reveal a crucial role for primary cilia in foregut division, since their dramatic decrease in cilia in both the foregut endoderm and mesenchyme of mutant embryos resulted in a proximal tracheoesophageal septation defects and in the formation of distal tracheo(broncho)esophageal fistulae similar to the most common congenital tracheoesophageal malformations in humans. Interestingly, the dorsoventral patterning determining the dorsal digestive and the ventral respiratory endoderm remained intact, whereas Hedgehog signaling was aberrantly activated. CONCLUSIONS: Our results demonstrate the cobblestone mutant to represent one of the very few mouse models that display both correct endodermal dorsoventral specification but defective compartmentalization of the proximal foregut. It stands exemplary for a tracheoesophageal ciliopathy, offering the possibility to elucidate the molecular mechanisms how primary cilia orchestrate the septation process. The plethora of malformations observed in the cobblestone embryo allow for a deeper insight into a putative link between primary cilia and human VATER/VACTERL syndromes.

The Primary Cilium and its Hedgehog Signaling in Nociceptors Contribute to Inflammatory and Neuropathic Pain.

The primary cilium, a 1-3 µm long hair-like structure protruding from the surface of almost all cells in the vertebrate body, is critical for neuronal development and also functions in the adult. As the migratory neural crest settles into dorsal root ganglia (DRG) sensory neurons elaborate a single primary cilium at their soma that is maintained into adult stages. While it is not known if primary cilia are expressed in nociceptors, or their potential function in the mature DRG neuron, recent studies have shown a role for Hedgehog, whose signaling demonstrates a dependence on primary cilia, in nociceptor sensitization. Here we report the expression of primary cilia in rat and mouse nociceptors, where they modulate mechanical nociceptive threshold, and contribute to inflammatory and neuropathic pain. When siRNA targeting Ift88 , a primary cilium-specific intra-flagellar transport (IFT) protein required for ciliary integrity, was administered by intrathecal injection, in the rat, it resulted in loss of Ift88 mRNA in DRG, and primary cilia in neuronal cell bodies, which was associated with an increase in mechanical nociceptive threshold, and abrogation of hyperalgesia induced by the pronociceptive inflammatory mediator, prostaglandin E 2 , and painful peripheral neuropathy induced by a neurotoxic chemotherapy drug, paclitaxel. To provide further support for the role of the primary cilium in nociceptor function we also administered siRNA for another IFT protein, Ift 52. Ift 52 siRNA results in loss of Ift 52 in DRG and abrogates paclitaxel-induced painful peripheral neuropathy. Attenuation of Hedgehog-induced hyperalgesia by Ift88 knockdown supports a role for the primary cilium in the hyperalgesia induced by Hedgehog, and attenuation of paclitaxel chemotherapy-induced neuropathy (CIPN) by cyclopamine, which attenuates Hedgehog signaling, suggests a role of Hedgehog in CIPN. Our findings support a role of nociceptor primary cilia in the control of mechanical nociceptive threshold and in inflammatory and neuropathic pain, the latter, at least in part, Hedgehog dependent.

Hedgehog Morphogens Act as Growth Factors Critical to Pre- and Postnatal Cardiac Development and Maturation: How Primary Cilia Mediate Their Signal Transduction.

Primary cilia are crucial for normal cardiac organogenesis via the formation of cyto-architectural, anatomical, and physiological boundaries in the developing heart and outflow tract. These tiny, plasma membrane-bound organelles function in a sensory-integrative capacity, interpreting both the intra- and extra-cellular environments and directing changes in gene expression responses to promote, prevent, and modify cellular proliferation and differentiation. One distinct feature of this organelle is its involvement in the propagation of a variety of signaling cascades, most notably, the Hedgehog cascade. Three ligands, Sonic, Indian, and Desert hedgehog, function as growth factors that are most commonly dependent on the presence of intact primary cilia, where the Hedgehog receptors Patched-1 and Smoothened localize directly within or at the base of the ciliary axoneme. Hedgehog signaling functions to mediate many cell behaviors that are critical for normal embryonic tissue/organ development. However, inappropriate activation and/or upregulation of Hedgehog signaling in postnatal and adult tissue is known to initiate oncogenesis, as well as the pathogenesis of other diseases. The focus of this review is to provide an overview describing the role of Hedgehog signaling and its dependence upon the primary cilium in the cell types that are most essential for mammalian heart development. We outline the breadth of developmental defects and the consequential pathologies resulting from inappropriate changes to Hedgehog signaling, as it pertains to congenital heart disease and general cardiac pathophysiology.

Targeted Ablation of Primary Cilia in Differentiated Dopaminergic Neurons Reduces Striatal Dopamine and Responsiveness to Metabolic Stress.

Primary cilia (PC) are microtubule-based protrusions of the cell membrane transducing molecular signals during brain development. Here, we report that PC are required for maintenance of Substantia nigra (SN) dopaminergic (DA) neurons highly vulnerable in Parkinson's disease (PD). Targeted blockage of ciliogenesis in differentiated DA neurons impaired striato-nigral integrity in adult mice. The relative number of SN DA neurons displaying a typical auto-inhibition of spontaneous activity in response to dopamine was elevated under control metabolic conditions, but not under metabolic stress. Strikingly, in the absence of PC, the remaining SN DA neurons were less vulnerable to the PD neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP). Our data indicate conserved PC-dependent neuroadaptive responses to DA lesions in the striatum. Moreover, PC control the integrity and dopamine response of a subtype of SN DA neurons. These results reinforce the critical role of PC as sensors of metabolic stress in PD and other disorders of the dopamine system.

Report on 7 Years' Experience Implementing an Undergraduate Medical Curriculum for Osteopathic Medical Students Using Entrustable Professional Activities.

Calls for changes in undergraduate medical education and the advent of the single graduate medical education accreditation system have challenged the osteopathic medical profession to maintain its identity and distinctiveness while adapting to innovations. For the osteopathic medical profession to thrive, its colleges must provide students with an educational framework that solidifies their osteopathic identity. The authors developed an integrated anatomy-clinical skills course at the University of New England College of Osteopathic Medicine, Osteopathic Clinical Skills, that used the performance benchmarks of the Entrustable Professional Activities and the Osteopathic Core Competencies for Medical Students from the American Association of Colleges of Osteopathic Medicine. A primary tenet of osteopathic medicine is the relationship of structure and function; Osteopathic Clinical Skills fuses anatomical sciences with clinical skills and underscores this tenet in clinical diagnosis and treatment. This article describes the development and implementation of an educational framework that integrates anatomy, physical examination, history taking, and other clinical skills with osteopathic medicine principles and practice and osteopathic manipulative treatment.

Noninvasive Electrocardiography in the Perinatal Mouse.

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

RgIA4 Accelerates Recovery from Paclitaxel-Induced Neuropathic Pain in Rats.

Chemotherapeutic drugs are widely utilized in the treatment of human cancers. Painful chemotherapy-induced neuropathy is a common, debilitating, and dose-limiting side effect for which there is currently no effective treatment. Previous studies have demonstrated the potential utility of peptides from the marine snail from the genus Conus for the treatment of neuropathic pain. α-Conotoxin RgIA and a potent analog, RgIA4, have previously been shown to prevent the development of neuropathy resulting from the administration of oxaliplatin, a platinum-based antineoplastic drug. Here, we have examined its efficacy against paclitaxel, a chemotherapeutic drug that works by a mechanism of action distinct from that of oxaliplatin. Paclitaxel was administered at 2 mg/kg (intraperitoneally (IP)) every other day for a total of 8 mg/kg. Sprague Dawley rats that were co-administered RgIA4 at 80 µg/kg (subcutaneously (SC)) once daily, five times per week, for three weeks showed significant recovery from mechanical allodynia by day 31. Notably, the therapeutic effects reached significance 12 days after the last administration of RgIA4, which is suggestive of a rescue mechanism. These findings support the effects of RgIA4 in multiple chemotherapeutic models and the investigation of α9α10 nicotinic acetylcholine receptors (nAChRs) as a non-opioid target in the treatment of chronic pain.

Targeted Depletion of Primary Cilia in Dopaminoceptive Neurons in a Preclinical Mouse Model of Huntington's Disease.

Multiple pathomechanisms triggered by mutant Huntingtin (mHTT) underlie progressive degeneration of dopaminoceptive striatal neurons in Huntington's disease (HD). The primary cilium is a membrane compartment that functions as a hub for various pathways that are dysregulated in HD, for example, dopamine (DA) receptor transmission and the mechanistic target of rapamycin (mTOR) pathway. The roles of primary cilia (PC) for the maintenance of striatal neurons and in HD progression remain unknown. Here, we investigated PC defects in vulnerable striatal neurons in a progressive model of HD, the mHTT-expressing knock-in zQ175 mice. We found that PC length is affected in striatal but not in cortical neurons, in association with the accumulation of mHTT. To explore the role of PC, we generated conditional mutant mice lacking IFT88, a component of the anterograde intraflagellar transport-B complex lacking PC in dopaminoceptive neurons. This mutation preserved the expression of the dopamine 1 receptor (D1R), and the survival of striatal neurons, but resulted in a mild increase of DA metabolites in the striatum, suggesting an imbalance of ciliary DA receptor transmission. Conditional loss of PC in zQ175 mice did not trigger astrogliosis, however, mTOR signaling was more active and resulted in a more pronounced accumulation of nuclear inclusions containing mHTT. Further studies will be required of aged mice to determine the role of aberrant ciliary function in more advanced stages of HD.

Definition of a critical spatiotemporal window within which primary cilia control midbrain dopaminergic neurogenesis.

Midbrain dopaminergic (mDA) neurons are generated in the ventral midbrain floor plate depending on Sonic Hedgehog (SHH) signaling for induction. Primary cilia transduce canonical SHH signals. Loss of intraflagellar transport protein IFT88, essential for ciliary function, disrupts SHH signaling in the ventral midbrain and results in the reduction in mDA progenitors and neurons. We investigate whether conditional inactivation of the kinesin motor protein KIF3A recapitulates phenotypes observed in conditional Ift88 mutants. Conditional Kif3a inactivation reduced the mDA progenitor domain size, but did not result in mDA neuron reduction, most likely because of a delayed loss of cilia and delayed inactivation of SHH signaling. We thereby define a precise spatiotemporal window within which primary cilia-dependent SHH signaling determines mDA fate.

Primary cilia are critical for Sonic hedgehog-mediated dopaminergic neurogenesis in the embryonic midbrain.

Midbrain dopaminergic (mDA) neurons modulate various motor and cognitive functions, and their dysfunction or degeneration has been implicated in several psychiatric diseases. Both Sonic Hedgehog (Shh) and Wnt signaling pathways have been shown to be essential for normal development of mDA neurons. Primary cilia are critical for the development of a number of structures in the brain by serving as a hub for essential developmental signaling cascades, but their role in the generation of mDA neurons has not been examined. We analyzed mutant mouse lines deficient in the intraflagellar transport protein IFT88, which is critical for primary cilia function. Conditional inactivation of Ift88 in the midbrain after E9.0 results in progressive loss of primary cilia, a decreased size of the mDA progenitor domain, and a reduction in mDA neurons. We identified Shh signaling as the primary cause of these defects, since conditional inactivation of the Shh signaling pathway after E9.0, through genetic ablation of Gli2 and Gli3 in the midbrain, results in a phenotype basically identical to the one seen in Ift88 conditional mutants. Moreover, the expansion of the mDA progenitor domain observed when Shh signaling is constitutively activated does not occur in absence of Ift88. In contrast, clusters of Shh-responding progenitors are maintained in the ventral midbrain of the hypomorphic Ift88 mouse mutant, cobblestone. Despite the residual Shh signaling, the integrity of the mDA progenitor domain is severely disturbed, and consequently very few mDA neurons are generated in cobblestone mutants. Our results identify for the first time a crucial role of primary cilia in the induction of mDA progenitors, define a narrow time window in which Shh-mediated signaling is dependent upon normal primary cilia function for this purpose, and suggest that later Wnt signaling-dependent events act independently of primary cilia.

Regulation of proliferation and histone acetylation in embryonic neural precursors by CREB/CREM signaling.

The transcription factor CREB (cAMP-response element binding protein) regulates differentiation, migration, survival and activity-dependent gene expression in the developing and mature nervous system. However, its specific role in the proliferation of embryonic neural progenitors is still not completely understood. Here we investigated how CREB regulates proliferation of mouse embryonic neural progenitors by a conditional mutant lacking Creb gene in neural progenitors. In parallel, we explored possible compensatory effects by the genetic ablation of another member of the same gene family, the cAMP-responsive element modulator (Crem). We show that CREB loss differentially impaired the proliferation, clonogenic potential and self-renewal of precursors derived from the ganglionic eminence (GE), in comparison to those derived from the cortex. This phenotype was associated with a specific reduction of histone acetylation in the GE of CREB mutant mice, and this reduction was rescued in vivo by inhibition of histone deacetylation. These observations indicate that the impaired proliferation could be caused by a reduced acetyltransferase activity in Creb conditional knock-out mice. These findings support a crucial role of CREB in controlling embryonic neurogenesis and propose a novel mechanism by which CREB regulates embryonic neural development.

Region-specific integration of embryonic stem cell-derived neuronal precursors into a pre-existing neuronal circuit.

Enduring reorganization is accepted as a fundamental process of adult neural plasticity. The most dramatic example of this reorganization is the birth and continuously occurring incorporation of new neurons into the pre-existing network of the adult mammalian hippocampus. Based on this phenomenon we transplanted murine embryonic stem (ES)-cell derived neuronal precursors (ESNPs) into murine organotypic hippocampal slice cultures (OHC) and examined their integration. Using a precise quantitative morphological analysis combined with a detailed electrophysiology, we show a region-specific morphological integration of transplanted ESNPs into different subfields of the hippocampal tissue, resulting in pyramidal neuron-like embryonic stem cell-derived neurons (ESNs) in the Cornu Ammonis (CA1 and CA3) and granule neuron-like ESNs in the dentate gyrus (DG), respectively. Subregion specific structural maturation was accompanied by the development of dendritic spines and the generation of excitatory postsynaptic currents (EPSCs). This cell type specific development does not depend upon NMDA-receptor-dependent synaptic transmission. The presented integration approach was further used to determine the cell-autonomous function of the pan-neurotrophin receptor p75 (P75(NTR)), as a possible negative regulator of ESN integration. By this means we used p75(NTR)-deficient ESNPs to study their integration into a WT organotypic environment. We show here that p75(NTR) is not necessary for integration per se but plays a suppressing role in dendritic development.

Primary cilia and forebrain development.

With a microtubule-based axoneme supporting its plasma membrane-ensheathed projection from the basal body of almost all cell types in the human body, and present in only one copy per cell, the primary cilium can be considered an organelle sui generis. Although it was first observed and recorded in histological studies from the late 19th century, the tiny structure was essentially forgotten for many decades. In the past ten years, however, scientists have turned their eyes once again upon primary cilia and realized that they are very important for the development of almost all organs in the mammalian body, especially those dependent upon the signaling from members Hedgehog family, such as Indian and Sonic hedgehog. In this review, we outline the roles that primary cilia play in forebrain development, not just in the crucial transduction of Sonic hedgehog signaling, but also new results showing that cilia are important for cell cycle progression in proliferating neural precursors. We will focus upon cerebral cortex development but will also discuss the importance of cilia for the embryonic hippocampus, olfactory bulb, and diencephalon.

Distinct and overlapping gene regulatory networks in BMP- and HDAC-controlled cell fate determination in the embryonic forebrain.

BACKGROUND: Both bone morphogenetic proteins (BMPs) and histone deacetylases (HDACs) have previously been established to play a role in the development of the three major cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. We have previously established a connection between these two protein families, showing that HDACs suppress BMP-promoted astrogliogenesis in the embryonic striatum. Since HDACs act in the nucleus to effect changes in transcription, an unbiased analysis of their transcriptional targets could shed light on their downstream effects on BMP-signaling. RESULTS: Using neurospheres from the embryonic striatum as an in vitro system to analyze this phenomenon, we have performed microarray expression profiling on BMP2- and TSA-treated cultures, followed by validation of the findings with quantitative RT-PCR and protein analysis. In BMP-treated cultures we first observed an upregulation of genes involved in cell-cell communication and developmental processes such as members of BMP and canonical Wnt signaling pathways. In contrast, in TSA-treated cultures we first observed an upregulation of genes involved in chromatin modification and transcription. Interestingly, we could not record direct changes in the protein levels of canonical members of BMP2 signaling, but we did observe an upregulation of both the transcription factor STAT3 and its active isoform phospho-STAT3 at the protein level. CONCLUSIONS: STAT3 and SMAD1/5/8 interact synergistically to promote astrogliogenesis, and thus we show for the first time that HDACs act to suppress BMP-promoted astrogliogenesis by suppression of the crucial partner STAT3.

Multiple essential roles for primary cilia in heart development.

BACKGROUND: The primary cilium is a microtubule-based, plasma membrane-ensheathed protrusion projecting from the basal bodies of almost all cell types in the mammalian body. In the past several years a plethora of papers has indicated a crucial role for primary cilia in the development of a wide variety of organs. We have investigated heart development in cobblestone, a hypomorphic allele of the gene encoding the intraflagellar transport protein Ift88, and uncovered a number of the most common congenital heart defects seen in newborn humans. METHODS: We generated serial sections of mutant cobblestone and wild type embryos in the region encompassing the heart and the cardiac outflow tract. The sections were further processed to generate three-dimensional reconstructions of these structures, and immunofluorescence confocal microscopy, transmission electron microscopy, and in situ hybridization were used to examine signal transduction pathways in the relevant areas. Whole mount in situ hybridization was also employed for certain developmental markers. RESULTS: In addition to an enlarged pericardium and failure of both ventricular and atrial septum formation, the cobblestone mutants displayed manifold defects in outflow tract formation, including persistent truncus arteriosus, an overriding aorta, and abnormal transformation of the aortic arches. To discern the basis of these anomalies we examined both the maintenance of primary cilia as well as endogenous and migratory embryonic cell populations that contribute to the outflow tract and atrioventricular septa. The colonization of the embryonic heart by cardiac neural crest occurred normally in the cobblestone mutant, as did the expression of Sonic hedgehog. However, with the loss of primary cilia in the mutant hearts, there was a loss of both downstream Sonic hedgehog signaling and of Islet 1 expression in the second heart field, a derivative of the pharyngeal mesoderm. In addition, defects were recorded in development of atrial laterality and ventricular myocardiogenesis. Finally, we observed a reduction in expression of Bmp4 in the outflow tract, and complete loss of expression of both Bmp2 and Bmp4 in the atrioventricular endocardial cushions. Loss of BMP2/4 signaling may result in the observed proliferative defect in the endocardial cushions, which give rise to both the atrioventricular septa as well as to the septation of the outflow tract. CONCLUSIONS: Taken together, our results potentially identify a novel link between Sonic hedgehog signaling at the primary cilium and BMP-dependent effects upon cardiogenesis. Our data further point to a potential linkage of atrioventricular septal defects, the most common congenital heart defects, to genes of the transport machinery or basal body of the cilia.

Primary cilia and organogenesis: is Hedgehog the only sculptor?

The primary cilium is a small microtubule-based organelle projecting from the plasma membrane of practically all cells in the mammalian body. In the past 8 years, a flurry of papers has indicated a crucial role of this long-neglected organelle in the development of a wide variety of organs, including derivatives of all three germ layers. A common theme of these studies is the critical dependency of signal transduction of the Hedgehog pathway upon functionally intact cilia to regulate organogenesis. Another common theme is the role that the cilium plays, not necessarily in the determination of the embryonic anlagen of these organs, although this too occurs but rather in the proliferation and morphogenesis of the previously determined organ. We outline the various organ systems that are dependent upon primary cilia for their proper development and we discuss the cilia-dependent roles that Sonic and Indian Hedgehog play in these processes. In addition and most importantly for the field, we discuss the controversial involvement of another major developmental pathway, Wnt signaling, in cilia-dependent organogenesis.

Organotypic slice culture of GFP-expressing mouse embryos for real-time imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system(1), allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques(2). The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period(2). Representative results using the tauGFP mouse line are demonstrated.