A circuit supporting concentration-invariant odor perception in Drosophila

<p>Abstract</p> <p>Background</p> <p>Most odors are perceived to have the same quality over a large concentration range, but the neural mechanisms that permit concentration-invariant olfactory perception are unknown. In larvae of the vinegar fly <it>Drosophila melanogaster</it>, odors are sensed by an array of 25 odorant receptors expressed in 21 olfactory sensory neurons (OSNs). We investigated how subsets of larval OSNs with overlapping but distinct response properties cooperate to mediate perception of a given odorant across a range of concentrations.</p> <p>Results</p> <p>Using calcium imaging, we found that ethyl butyrate, an ester perceived by humans as fruity, activated three OSNs with response thresholds that varied across three orders of magnitude. Whereas wild-type larvae were strongly attracted by this odor across a 500-fold range of concentration, individuals with only a single functional OSN showed attraction across a narrower concentration range corresponding to the sensitivity of each ethyl butyrate-tuned OSN. To clarify how the information carried by different OSNs is integrated by the olfactory system, we characterized the response properties of local inhibitory interneurons and projection neurons in the antennal lobe. Local interneurons only responded to high ethyl butyrate concentrations upon summed activation of at least two OSNs. Projection neurons showed a reduced response to odors when summed input from two OSNs impinged on the circuit compared to when there was only a single functional OSN.</p> <p>Conclusions</p> <p>Our results show that increasing odor concentrations induce progressive activation of concentration-tuned olfactory sensory neurons and concomitant recruitment of inhibitory local interneurons. We propose that the interplay of combinatorial OSN input and local interneuron activation allows animals to remain sensitive to odors across a large range of stimulus intensities.</p

Rabies Internalizes into Primary Peripheral Neurons via Clathrin Coated Pits and Requires Fusion at the Cell Body

The single glycoprotein (G) of rabies virus (RABV) dictates all viral entry steps from receptor engagement to membrane fusion. To study the uptake of RABV into primary neuronal cells in culture, we generated a recombinant vesicular stomatitis virus in which the G protein was replaced with that of the neurotropic RABV CVS-11 strain (rVSV CVS G). Using microfluidic compartmentalized culture, we examined the uptake of single virions into the termini of primary neurons of the dorsal root ganglion and ventral spinal cord. By pharmacologically disrupting endocytosis at the distal neurites, we demonstrate that rVSV CVS G uptake and infection are dependent on dynamin. Imaging of single virion uptake with fluorescent endocytic markers further identifies endocytosis via clathrin-coated pits as the predominant internalization mechanism. Transmission electron micrographs also reveal the presence of viral particles in vesicular structures consistent with incompletely coated clathrin pits. This work extends our previous findings of clathrin-mediated uptake of RABV into epithelial cells to two neuronal subtypes involved in rabies infection in vivo. Chemical perturbation of endosomal acidification in the neurite or somal compartment further shows that establishment of infection requires pH-dependent fusion of virions at the cell body. These findings correlate infectivity to existing single particle evidence of long-range endosomal transport of RABV and clathrin dependent uptake at the plasma membrane

Alpha-Herpesvirus Infection Induces the Formation of Nuclear Actin Filaments

Herpesviruses are large double-stranded DNA viruses that replicate in the nuclei of infected cells. Spatial control of viral replication and assembly in the host nucleus is achieved by the establishment of nuclear compartments that serve to concentrate viral and host factors. How these compartments are established and maintained remains poorly understood. Pseudorabies virus (PRV) is an alpha-herpesvirus often used to study herpesvirus invasion and spread in the nervous system. Here, we report that PRV and herpes simplex virus type 1 infection of neurons results in formation of actin filaments in the nucleus. Filamentous actin is not found in the nucleus of uninfected cells. Nuclear actin filaments appear physically associated with the viral capsids, as shown by serial block-face scanning electron micropscopy and confocal microscopy. Using a green fluorescent protein-tagged viral capsid protein (VP26), we show that nuclear actin filaments form prior to capsid assembly and are required for the efficient formation of viral capsid assembly sites. We find that actin polymerization dynamics (e.g., treadmilling) are not necessary for the formation of these sites. Green fluorescent protein-VP26 foci co-localize with the actin motor myosin V, suggesting that viral capsids travel along nuclear actin filaments using myosin-based directed transport. Viral transcription, but not viral DNA replication, is required for actin filament formation. The finding that infection, by either PRV or herpes simplex virus type 1, results in formation of nuclear actin filaments in neurons, and that PRV infection of an epithelial cell line results in a similar phenotype is evidence that F-actin plays a conserved role in herpesvirus assembly. Our results suggest a mechanism by which assembly domains are organized within infected cells and provide insight into how the viral infectious cycle and host actin cytoskeleton are integrated to promote the infection process

Rabies Glycoprotein-Mediated Uptake Into Epithelial Cells and Compartmentalized Primary Neuronal Culture

Rabies virus (RABV) subverts host neuronal circuitry to gain access to the brain where it causes generally incurable, lethal encephalitis. The single glycoprotein (G) dictates two defining steps for infection and neuroinvasion: receptor-mediated endocytosis and transport of virus. We generate two recombinant VSV (rVSV) clones that genetically incorporate G (rVSV RABV G) from the fixed RABV strains, SAD B19 and CVS, to study internalization into epithelial cells and compartmentalized primary cultures of peripheral neurons. Through the use of chemical inhibitors and markers for specific endocytic routes, we demonstrate that the predominant RABV entry route in both epithelial and neuronal cells is dynamin- and clathrin- dependent. Viral endocytosis is mediated by actin-dependent, partially coated clathrin pits as evidenced by live high resolution confocal microscopy of envelopment in epithelial cells and transmission electron micrographs in neuronal and non-neuronal cells. Thus, we corroborate the hypothesis that particle size is the sole viral determinant of actin-dependence of coated pits. Through a combination of high resolution microscopy and infectivity-based approaches, we link molecular mechanisms of viral uptake at the single particle level to productive infection. Targeted pharmacological disruption of endosomal acidification at the neurites or cell bodies of peripheral neurons demonstrates that fusion and viral genome release at the cell body, the site of replication, is a prerequisite for infection. This work extends the current understanding of RABV entry by providing a detailed characterization of endocytosis from the plasma membrane to the site of fusion and correlating it with establishment of infection into neuronal populations relevant for pathogenesis in vivo.Biology, Molecular and Cellula

Dynasore and EIPA inhibit rVSV CVS G infection in compartmentalized neuronal culture.

<p>Fluorescence microscopy of MF cultures of <b>A.</b> DRG neurons or <b>B.</b> V SC neurons infected with rVSV CVS G in the N compartment. Neurons were additionally treated with the dynamin inhibitor, dynasore (150 μM), or the macropinocytosis inhibitor, EIPA (25 μM), as indicated at either 0 or 2 hpi and monitored for viral eGFP (green) expression at 26 hpi. Cells are further stained against phosphorylated neurofilament H (NF; red) and with DAPI (blue). <b>C.</b> Neurites in the N compartment at 26hpi and continuous treatment with the indicated inhibitor, stained against neurofilament H. <b>D.</b> Quantitation of percentage eGFP positive neurons in three iterations of the experiment in DRG culture. Error bars indicate the standard deviation for each condition.</p

Somal endosomal acidification is required for rVSV CVS G infection.

<p>Fluorescence microscopy of compartmentalized cultures of <b>A.</b> DRG neurons or <b>B.</b> V SC neurons infected with rVSV CVS G in the N compartment in the presence or absence (U) of 1 μM bafilomycin A1 (BAF A1). BAF A1, a block of endosomal acidification, was administered to either the N or S compartment at various timepoints relative to the start of infection. In the N compartment, BAF A1 was added at 0 or 2 hpi. In the S compartment, BAF A1 was added at 0, 2, 9 or 12 hpi as indicated. Infection was assessed by expression of viral eGFP at 26hpi. DRG neurons in A. were pre-stained with CellTracker (CT, red) and NucBlue (NucB, blue) prior to the start of the experiments. In B., infected V SC neurites were fixed and permeabilized at 26hpi. V SC neurites were detected by immunofluorescence against phosphorylated neurofilament (NF, red) and nuclei were stained with DAPI (blue). <b>C.</b> Quantitation of percentage eGFP positive cells following a representative experiment in DRG (left) and V SC (right) culture. All conditions, except the untreated control, were tested in duplicate. Averages are provided above each individual bar. D. Fluorescence microscopy of non-compartmentalized DRG cultures infected with rVSV CVS G and treated with BAF A1 at 0 or 2 hpi. U indicates untreated controls. Presence of the infection marker, eGFP (green), was assessed at 8 hpi. Merged images combine the eGFP signal and phase microscopy of the DRG neurons in culture.</p

Model of the predominant RABV G-dependent entry mechanism into neurons of the peripheral nervous system.

<p>Rabies G association with neuronal receptors at the synapse or neurite membrane results in clathrin-mediated endocytosis and transport via microtubules to the somatodendritic compartment. <b>A.</b> Rhabdoviruses are internalized within endosomes only partially coated with clathrin, which require actin to complete envelopment and scission from the plasma membrane. <b>B.</b> Following internalization, early endosomes containing virus engage the motors and travel retrogradely through the axon. Viruses are transported intact within endosomes from the neuronal synapse. Endosomes acidify upon arrival at the soma releasing RNPs and leading to productive infection.</p