Gut enterochromaffin cells drive visceral pain and anxiety.

Gastrointestinal (GI) discomfort is a hallmark of most gut disorders and represents an important component of chronic visceral pain1. For the growing population afflicted by irritable bowel syndrome, GI hypersensitivity and pain persist long after tissue injury has resolved2. Irritable bowel syndrome also exhibits a strong sex bias, afflicting women three times more than men1. Here, we focus on enterochromaffin (EC) cells, which are rare excitable, serotonergic neuroendocrine cells in the gut epithelium3-5. EC cells detect and transduce noxious stimuli to nearby mucosal nerve endings3,6 but involvement of this signalling pathway in visceral pain and attendant sex differences has not been assessed. By enhancing or suppressing EC cell function in vivo, we show that these cells are sufficient to elicit hypersensitivity to gut distension and necessary for the sensitizing actions of isovalerate, a bacterial short-chain fatty acid associated with GI inflammation7,8. Remarkably, prolonged EC cell activation produced persistent visceral hypersensitivity, even in the absence of an instigating inflammatory episode. Furthermore, perturbing EC cell activity promoted anxiety-like behaviours which normalized after blockade of serotonergic signalling. Sex differences were noted across a range of paradigms, indicating that the EC cell-mucosal afferent circuit is tonically engaged in females. Our findings validate a critical role for EC cell-mucosal afferent signalling in acute and persistent GI pain, in addition to highlighting genetic models for studying visceral hypersensitivity and the sex bias of gut pain.

Differential Expression Analysis Identifies Candidate Synaptogenic Molecules for Wiring Direction-Selective Circuits in the Retina.

An organizational feature of neural circuits is the specificity of synaptic connections. A striking example is the direction-selective (DS) circuit of the retina. There are multiple subtypes of DS retinal ganglion cells (DSGCs) that prefer motion along one of four preferred directions. This computation is mediated by selective wiring of a single inhibitory interneuron, the starburst amacrine cell (SAC), with each DSGC subtype preferentially receiving input from a subset of SAC processes. We hypothesize that the molecular basis of this wiring is mediated in part by unique expression profiles of DSGC subtypes. To test this, we first performed paired recordings from isolated mouse retinas of both sexes to determine that postnatal day 10 (P10) represents the age at which asymmetric synapses form. Second, we performed RNA sequencing and differential expression analysis on isolated P10 ON-OFF DSGCs tuned for either nasal or ventral motion and identified candidates which may promote direction-specific wiring. We then used a conditional knock-out strategy to test the role of one candidate, the secreted synaptic organizer cerebellin-4 (Cbln4), in the development of DS tuning. Using two-photon calcium imaging, we observed a small deficit in directional tuning among ventral-preferring DSGCs lacking Cbln4, though whole-cell voltage-clamp recordings did not identify a significant change in inhibitory inputs. This suggests that Cbln4 does not function primarily via a cell-autonomous mechanism to instruct wiring of DS circuits. Nevertheless, our transcriptomic analysis identified unique candidate factors for gaining insights into the molecular mechanisms that instruct wiring specificity in the DS circuit.

Contributions of Rod and Cone Pathways to Retinal Direction Selectivity Through Development.

UNLABELLED: Direction selectivity is a robust computation across a broad stimulus space that is mediated by activity of both rod and cone photoreceptors through the ON and OFF pathways. However, rods, S-cones, and M-cones activate the ON and OFF circuits via distinct pathways and the relative contribution of each to direction selectivity is unknown. Using a variety of stimulation paradigms, pharmacological agents, and knockout mice that lack rod transduction, we found that inputs from the ON pathway were critical for strong direction-selective (DS) tuning in the OFF pathway. For UV light stimulation, the ON pathway inputs to the OFF pathway originated with rod signaling, whereas for visible stimulation, the ON pathway inputs to the OFF pathway originated with both rod and M-cone signaling. Whole-cell voltage-clamp recordings revealed that blocking the ON pathway reduced directional tuning in the OFF pathway via a reduction in null-side inhibition, which is provided by OFF starburst amacrine cells (SACs). Consistent with this, our recordings from OFF SACs confirmed that signals originating in the ON pathway contribute to their excitation. Finally, we observed that, for UV stimulation, ON contributions to OFF DS tuning matured earlier than direct signaling via the OFF pathway. These data indicate that the retina uses multiple strategies for computing DS responses across different colors and stages of development. SIGNIFICANCE STATEMENT: The retina uses parallel pathways to encode different features of the visual scene. In some cases, these distinct pathways converge on circuits that mediate a distinct computation. For example, rod and cone pathways enable direction-selective (DS) ganglion cells to encode motion over a wide range of light intensities. Here, we show that although direction selectivity is robust across light intensities, motion discrimination for OFF signals is dependent upon ON signaling. At eye opening, ON directional tuning is mature, whereas OFF DS tuning is significantly reduced due to a delayed maturation of S-cone to OFF cone bipolar signaling. These results provide evidence that the retina uses multiple strategies for computing DS responses across different stimulus conditions.

An Asymmetric Increase in Inhibitory Synapse Number Underlies the Development of a Direction Selective Circuit in the Retina.

Neural circuits rely upon a precise wiring of their component neurons to perform meaningful computations. To compute the direction of motion in the visual scene, the direction selective circuit in the mouse retina depends on an asymmetry in the inhibitory neurotransmission from starburst amacrine cells (SACs) to direction selective ganglion cells (DSGCs). Specifically, depolarization of a SAC on the null side of a DSGC causes a threefold greater unitary inhibitory conductance than depolarization of a SAC on the preferred side. This asymmetry emerges during the second postnatal week of development, but its basis remains unknown. To determine the source of this asymmetry in inhibitory conductance, we conducted paired recordings between SACs and DSGCs at the beginning and end of the second postnatal week. We replaced calcium with strontium to promote asynchronous neurotransmitter release and produce quantal events. During the second postnatal week the quantal frequency but not the quantal amplitude of synaptic events increased more than threefold for null-side SAC-DSGC pairs but remained constant for preferred-side pairs. In addition, paired-pulse depression did not differ between SACs located on the null and preferred sides of DSGCs, indicating that all inhibitory SAC synapses onto a DSGC exhibit the same probability of release. Thus, the higher quantal frequency seen in null-side pairs results from a greater number of inhibitory synapses, revealing that an asymmetry in synapse number between SACs and DSGCs underlies the development of an essential component in the retina's direction selective circuit.

V-ATPase-dependent ectodermal voltage and pH regionalization are required for craniofacial morphogenesis.

Using voltage and pH reporter dyes, we have discovered a never-before-seen regionalization of the Xenopus ectoderm, with cell subpopulations delimited by different membrane voltage and pH. We distinguished three courses of bioelectrical activity. Course I is a wave of hyperpolarization that travels across the gastrula. Course II comprises the appearance of patterns that match shape changes and gene expression domains of the developing face; hyperpolarization marks folding epithelium and both hyperpolarized and depolarized regions overlap domains of head patterning genes. In Course III, localized regions of hyperpolarization form at various positions, expand, and disappear. Inhibiting H(+) -transport by the H(+) -V-ATPase causes abnormalities in: (1) the morphology of craniofacial structures; (2) Course II voltage patterns; and (3) patterns of sox9, pax8, slug, mitf, xfz3, otx2, and pax6. We conclude that this bioelectric signal has a role in development of the face. Thus, it exemplifies an important, under-studied mechanism of developmental regulation.

A Dense Starburst Plexus Is Critical for Generating Direction Selectivity.

Starburst amacrine cell (SAC) morphology is considered central to retinal direction selectivity. In Sema6A-/- mice, SAC dendritic arbors are smaller and no longer radially symmetric, leading to a reduction in SAC dendritic plexus density. Sema6A-/- mice also have a dramatic reduction in the directional tuning of retinal direction-selective ganglion cells (DSGCs). Here we show that the loss of DSGC tuning in Sema6A-/- mice is due to reduced null direction inhibition, even though strong asymmetric SAC-DSGC connectivity and SAC dendritic direction selectivity are maintained. Hence, the reduced coverage factor of SAC dendrites leads specifically to a loss of null direction inhibition. Moreover, SAC dendrites are no longer strictly tuned to centrifugal motion, indicating that SAC morphology is critical in coordinating synaptic connectivity and dendritic integration to generate direction selectivity.

Motion Vision: Cortical Preferences Influenced by Retinal Direction Selectivity.

A recent study shows that retinal direction selectivity influences a subset of cells in primary visual cortex which respond to the optic flow associated with forward motion, while other cortical direction selective cells perform this computation independently.

Development of synaptic connectivity in the retinal direction selective circuit.

Direction selectivity is a classic neuronal computation that has been described in many different sensory systems. The circuit basis of this computation is perhaps best understood in the retina, where direction selectivity is the result of asymmetric connectivity patterns between excitatory and inhibitory circuit components. Retinal direction selective circuits emerge before eye-opening, though components of the circuit undergo refinement after vision begins. These features make the direction selective circuit a rich model in which to investigate neuronal circuit assembly. In this Opinion, we highlight recent experiments investigating the contribution of various molecular cues, as well as neuronal activity, to the development of the retinal direction selective circuit.

A Role for Synaptic Input Distribution in a Dendritic Computation of Motion Direction in the Retina.

The starburst amacrine cell in the mouse retina presents an opportunity to examine the precise role of sensory input location on neuronal computations. Using visual receptive field mapping, glutamate uncaging, two-photon Ca(2+) imaging, and genetic labeling of putative synapses, we identify a unique arrangement of excitatory inputs and neurotransmitter release sites on starburst amacrine cell dendrites: the excitatory input distribution is skewed away from the release sites. By comparing computational simulations with Ca(2+) transients recorded near release sites, we show that this anatomical arrangement of inputs and outputs supports a dendritic mechanism for computing motion direction. Direction-selective Ca(2+) transients persist in the presence of a GABA-A receptor antagonist, though the directional tuning is reduced. These results indicate a synergistic interaction between dendritic and circuit mechanisms for generating direction selectivity in the starburst amacrine cell.

Visual stimulation switches the polarity of excitatory input to starburst amacrine cells.

Direction-selective ganglion cells (DSGCs) are tuned to motion in one direction. Starburst amacrine cells (SACs) are thought to mediate this direction selectivity through precise anatomical wiring to DSGCs. Nevertheless, we previously found that visual adaptation can reverse DSGCs's directional tuning, overcoming the circuit anatomy. Here we explore the role of SACs in the generation and adaptation of direction selectivity. First, using pharmacogenetics and two-photon calcium imaging, we validate that SACs are necessary for direction selectivity. Next, we demonstrate that exposure to an adaptive stimulus dramatically alters SACs' synaptic inputs. Specifically, after visual adaptation, On-SACs lose their excitatory input during light onset but gain an excitatory input during light offset. Our data suggest that visual stimulation alters the interactions between rod- and cone-mediated inputs that converge on the terminals of On-cone BCs. These results demonstrate how the sensory environment can modify computations performed by anatomically defined neuronal circuits.

Rab GTPases are required for early orientation of the left-right axis in Xenopus.

The earliest steps of left-right (LR) patterning in Xenopus embryos are driven by biased intracellular transport that ensures a consistently asymmetric localization of maternal ion channels and pumps in the first 2-4 blastomeres. The subsequent differential net efflux of ions by these transporters generates a bioelectrical asymmetry; this LR voltage gradient redistributes small signaling molecules along the LR axis that later regulate transcription of the normally left-sided Nodal. This system thus amplifies single cell chirality into a true left-right asymmetry across multi-cellular fields. Studies using molecular-genetic gain- and loss-of-function reagents have characterized many of the steps involved in this early pathway in Xenopus. Yet one key question remains: how is the chiral cytoskeletal architecture interpreted to localize ion transporters to the left or right side? Because Rab GTPases regulate nearly all aspects of membrane trafficking, we hypothesized that one or more Rab proteins were responsible for the directed, asymmetric shuttling of maternal ion channel or pump proteins. After performing a screen using dominant negative and wildtype (overexpressing) mRNAs for four different Rabs, we found that alterations in Rab11 expression randomize both asymmetric gene expression and organ situs. We also demonstrated that the asymmetric localization of two ion transporter subunits requires Rab11 function, and that Rab11 is closely associated with at least one of these subunits. Yet, importantly, we found that endogenous Rab11 mRNA and protein are expressed symmetrically in the early embryo. We conclude that Rab11-mediated transport is responsible for the movement of cargo within early blastomeres, and that Rab11 expression is required throughout the early embryo for proper LR patterning.