Comparison of wholemount dissection methods for neuronal subtype marker expression in the mouse myenteric plexus.

BACKGROUND: Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study, we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression. METHODS: We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation and a third rod-mounted peeling technique. We also tested a reversed orientation variation of flat-sheet peeling, two step-by-step variations of the rod peeling technique, and whole-gut fixation as a tube. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis. KEY RESULTS AND CONCLUSIONS: We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin or neuronal nitric oxide synthase (nNOS) as a proportion of total neurons in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased proportion of neurons labeled for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in distal colon and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling. The step-by-step variations of this method point to mechanical manipulation of the tissue as the likely cause of decreased labeling. Our study thereby demonstrates a critical variability in wholemount muscularis dissection methods.

Spiral NeuroString: High-Density Soft Bioelectronic Fibers for Multimodal Sensing and Stimulation.

Bioelectronic fibers hold promise for both research and clinical applications due to their compactness, ease of implantation, and ability to incorporate various functionalities such as sensing and stimulation. However, existing devices suffer from bulkiness, rigidity, limited functionality, and low density of active components. These limitations stem from the difficulty to incorporate many components on one-dimensional (1D) fiber devices due to the incompatibility of conventional microfabrication methods (e.g., photolithography) with curved, thin and long fiber structures. Herein, we introduce a fabrication approach, ‶spiral transformation″, to convert two-dimensional (2D) films containing microfabricated devices into 1D soft fibers. This approach allows for the creation of high density multimodal soft bioelectronic fibers, termed Spiral NeuroString (S-NeuroString), while enabling precise control over the longitudinal, angular, and radial positioning and distribution of the functional components. We show the utility of S-NeuroString for motility mapping, serotonin sensing, and tissue stimulation within the dynamic and soft gastrointestinal (GI) system, as well as for single-unit recordings in the brain. The described bioelectronic fibers hold great promises for next-generation multifunctional implantable electronics.

Comparison of wholemount dissection methods for neuronal subtype marker expression in the mouse myenteric plexus.

Background: Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI tract function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression. Methods: We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation as well as a rod-mounted peeling technique. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis. Key Results and Conclusions: We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin, neuronal nitric oxide synthase (nNOS), or somatostatin (SST) in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased marker labeling for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in ileum and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling, demonstrating a critical variability in wholemount muscularis dissection methods.

Regional cytoarchitecture of the adult and developing mouse enteric nervous system.

The organization and cellular composition of tissues are key determinants of their biological function. In the mammalian gastrointestinal (GI) tract, the enteric nervous system (ENS) intercalates between muscular and epithelial layers of the gut wall and can control GI function independent of central nervous system (CNS) input.1 As in the CNS, distinct regions of the GI tract are highly specialized and support diverse functions, yet the regional and spatial organization of the ENS remains poorly characterized.2 Cellular arrangements,3,4 circuit connectivity patterns,5,6 and diverse cell types7-9 are known to underpin ENS functional complexity and GI function, but enteric neurons are most typically described only as a uniform meshwork of interconnected ganglia. Here, we present a bird's eye view of the mouse ENS, describing its previously underappreciated cytoarchitecture and regional variation. We visually and computationally demonstrate that enteric neurons are organized in circumferential neuronal stripes. This organization emerges gradually during the perinatal period, with neuronal stripe formation in the small intestine (SI) preceding that in the colon. The width of neuronal stripes varies throughout the length of the GI tract, and distinct neuronal subtypes differentially populate specific regions of the GI tract, with stark contrasts between SI and colon as well as within subregions of each. This characterization provides a blueprint for future understanding of region-specific GI function and identifying ENS structural correlates of diverse GI disorders.

The Cell-Autonomous Clock of VIP Receptor VPAC2 Cells Regulates Period and Coherence of Circadian Behavior.

Circadian (approximately daily) rhythms pervade mammalian behavior. They are generated by cell-autonomous, transcriptional/translational feedback loops (TTFLs), active in all tissues. This distributed clock network is coordinated by the principal circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). Its robust and accurate time-keeping arises from circuit-level interactions that bind its individual cellular clocks into a coherent time-keeper. Cells that express the neuropeptide vasoactive intestinal peptide (VIP) mediate retinal entrainment of the SCN; and in the absence of VIP, or its cognate receptor VPAC2, circadian behavior is compromised because SCN cells cannot synchronize. The contributions to pace-making of other cell types, including VPAC2-expressing target cells of VIP, are, however, not understood. We therefore used intersectional genetics to manipulate the cell-autonomous TTFLs of VPAC2-expressing cells. Measuring circadian behavioral and SCN rhythmicity in these temporally chimeric male mice thus enabled us to determine the contribution of VPAC2-expressing cells (∼35% of SCN cells) to SCN time-keeping. Lengthening of the intrinsic TTFL period of VPAC2 cells by deletion of the CK1εTau allele concomitantly lengthened the period of circadian behavioral rhythms. It also increased the variability of the circadian period of bioluminescent TTFL rhythms in SCN slices recorded ex vivo Abrogation of circadian competence in VPAC2 cells by deletion of Bmal1 severely disrupted circadian behavioral rhythms and compromised TTFL time-keeping in the corresponding SCN slices. Thus, VPAC2-expressing cells are a distinct, functionally powerful subset of the SCN circuit, contributing to computation of ensemble period and maintenance of circadian robustness. These findings extend our understanding of SCN circuit topology.

The VIP-VPAC2 neuropeptidergic axis is a cellular pacemaking hub of the suprachiasmatic nucleus circadian circuit.

The hypothalamic suprachiasmatic nuclei (SCN) are the principal mammalian circadian timekeeper, co-ordinating organism-wide daily and seasonal rhythms. To achieve this, cell-autonomous circadian timing by the ~20,000 SCN cells is welded into a tight circuit-wide ensemble oscillation. This creates essential, network-level emergent properties of precise, high-amplitude oscillation with tightly defined ensemble period and phase. Although synchronised, regional cell groups exhibit differentially phased activity, creating stereotypical spatiotemporal circadian waves of cellular activation across the circuit. The cellular circuit pacemaking components that generate these critical emergent properties are unknown. Using intersectional genetics and real-time imaging, we show that SCN cells expressing vasoactive intestinal polypeptide (VIP) or its cognate receptor, VPAC2, are neurochemically and electrophysiologically distinct, but together they control de novo rhythmicity, setting ensemble period and phase with circuit-level spatiotemporal complexity. The VIP/VPAC2 cellular axis is therefore a neurochemically and topologically specific pacemaker hub that determines the emergent properties of the SCN timekeeper.

Insulin/IGF-1 Drives PERIOD Synthesis to Entrain Circadian Rhythms with Feeding Time.

In mammals, endogenous circadian clocks sense and respond to daily feeding and lighting cues, adjusting internal ∼24 h rhythms to resonate with, and anticipate, external cycles of day and night. The mechanism underlying circadian entrainment to feeding time is critical for understanding why mistimed feeding, as occurs during shift work, disrupts circadian physiology, a state that is associated with increased incidence of chronic diseases such as type 2 (T2) diabetes. We show that feeding-regulated hormones insulin and insulin-like growth factor 1 (IGF-1) reset circadian clocks in vivo and in vitro by induction of PERIOD proteins, and mistimed insulin signaling disrupts circadian organization of mouse behavior and clock gene expression. Insulin and IGF-1 receptor signaling is sufficient to determine essential circadian parameters, principally via increased PERIOD protein synthesis. This requires coincident mechanistic target of rapamycin (mTOR) activation, increased phosphoinositide signaling, and microRNA downregulation. Besides its well-known homeostatic functions, we propose insulin and IGF-1 are primary signals of feeding time to cellular clocks throughout the body.

Chandelier Cells Swipe Right for L1CAM.

Establishing a functional neuronal circuit requires not only synapsing with the right cell type, but also targeting the right subcellular compartment. In this issue of Neuron, Tai et al. (2019) identify the cell adhesion molecule L1CAM as integral to the mechanism by which chandelier cells establish subcellular compartment-specific innervation of pyramidal neurons in the mammalian cerebral cortex.

Vasoactive intestinal peptide controls the suprachiasmatic circadian clock network via ERK1/2 and DUSP4 signalling.

The suprachiasmatic nucleus (SCN) co-ordinates circadian behaviour and physiology in mammals. Its cell-autonomous circadian oscillations pivot around a well characterised transcriptional/translational feedback loop (TTFL), whilst the SCN circuit as a whole is synchronised to solar time by its retinorecipient cells that express and release vasoactive intestinal peptide (VIP). The cell-autonomous and circuit-level mechanisms whereby VIP synchronises the SCN are poorly understood. We show that SCN slices in organotypic culture demonstrate rapid and sustained circuit-level circadian responses to VIP that are mediated at a cell-autonomous level. This is accompanied by changes across a broad transcriptional network and by significant VIP-directed plasticity in the internal phasing of the cell-autonomous TTFL. Signalling via ERK1/2 and tuning by its negative regulator DUSP4 are critical elements of the VIP-directed circadian re-programming. In summary, we provide detailed mechanistic insight into VIP signal transduction in the SCN at the level of genes, cells and neural circuit.

Temporally chimeric mice reveal flexibility of circadian period-setting in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non-Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells.