Differential adhesion regulates neurite placement via a retrograde zippering mechanism

During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the C. elegans brain neuropil, and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers. Differential adhesion is orchestrated via developmentally-regulated expression of the IgCAM SYG-1, and its partner ligand SYG-2. Changes in SYG-1 expression across neuropil layers result in changes in adhesive forces, which sort SYG-2-expressing neurons. Sorting to layers occurs, not via outgrowth from the neurite tip, but via an alternate mechanism of retrograde zippering, involving interactions between neurite shafts. Our study indicates that biophysical principles from differential adhesion govern neurite placement and synaptic specificity in vivo in developing neuropil bundles.

Voltage Imaging in Drosophila Using a Hybrid Chemical-Genetic Rhodamine Voltage Reporter

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.

Diversified expression of gamma-protocadherins regulates synaptic specificity in the mouse neocortex

Synaptic specificity is the basis of forming neural microcircuits. However, how a neuron chooses which neurons out of many potentials to form synapses remains largely unknown. Here we identified that the diversified expression of clustered protocadherin γs (cPCDHγs) plays an essential role in regulating such specificity. Our 5-prime end single-cell sequencing data revealed the diversified expression pattern of cPCDHγs in neocortical neurons. Whole-cell recording of neuron pairs in developing mouse brain slices showed that knocking out PCDHγs significantly increased the local connection rate of nearby pyramidal neurons. By contrast, neurons overexpressing the same group of clustered PCDHγ isoforms through in utero electroporation dramatically decreased their synaptic connectivity. Finally and more importantly, decreasing the similarity level of PCDHγ isoforms over-expressed in neuron pairs through sequential in utero electroporation led to a progressive elevation of synaptic connectivity. Our observations provide strong evidence to support that the existence of diversely expressed cPCDHγs allows a neuron to choose which neurons not to form a synapse, rather than choosing which neurons to make synapses.

Voltage imaging in Drosophila using a hybrid chemical-genetic rhodamine voltage reporter

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.

A neurite-zippering mechanism, mediated by layer-specific expression of IgCAMs, regulates synaptic laminar specificity in the C. elegans nerve ring neuropil

A fundamental design principle of nervous systems is the grouping of neuronal contacts into layers within nerve bundles. The layered arrangement of neurites requires nanoscale precision in their placement within bundles, and this precision, which can not be exclusively explained by simple tip-directed outgrowth dynamics, underpins synaptic specificity and circuit architecture. Here we implement novel imaging methods and deep learning approaches to document the specific placement of single neurites during the assembly of the C. elegans nerve ring. We uncover a zippering mechanism that controls precise placement of neurites onto specific layer subdomains. Nanoscale precision in neurite placement is orchestrated via temporally-regulated expression of specific Ig adhesion molecules, such as SYG-1. Ig adhesion molecules act as instructive signals, defining sublaminar regions and guiding neurite zippering onto target neurons. Our study reveals novel developmental mechanisms that coordinate neurite placement and synaptic specificity within layered brain structures.

Intrinsic control of neuronal diversity and synaptic specificity in a proprioceptive circuit

Relay of muscle-derived sensory information to the CNS is essential for the execution of motor behavior, but how proprioceptive sensory neurons (pSNs) establish functionally appropriate connections is poorly understood. A prevailing model of sensory-motor circuit assembly is that peripheral, target-derived, cues instruct pSN identities and patterns of intraspinal connectivity. To date no known intrinsic determinants of muscle-specific pSN fates have been described in vertebrates. We show that expression of Hox transcription factors defines pSN subtypes, and these profiles are established independently of limb muscle. The Hoxc8 gene is expressed by pSNs and motor neurons (MNs) targeting distal forelimb muscles, and sensory-specific depletion of Hoxc8 in mice disrupts sensory-motor synaptic matching, without affecting pSN survival or muscle targeting. These results indicate that the diversity and central specificity of pSNs and MNs are regulated by a common set of determinants, thus linking early rostrocaudal patterning to the assembly of limb control circuits.

R7 photoreceptor axon targeting requires matching levels of lost and found expression in R7 and its synaptic partners

The formation of neural circuits requires growing processes to select the correct synaptic partners from numerous possible choices through interactions between cell surface proteins. The limited number of such proteins suggests that quantitative comparisons of their relative levels may contribute to synaptic specificity. Here we show that the level of the Drosophila CUB-LDL protein Lost and found (Loaf) in the UV-sensitive R7 photoreceptor relative to its synaptic partners is critical for R7 axon targeting. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or restoring it to the postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more transmembrane proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections.