Alpha herpesvirus exocytosis from neuron cell bodies uses constitutive secretory mechanisms, and egress and spread from axons is independent of neuronal firing activity.

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system, causing severe debilitating or deadly disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous activity, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. Using live-cell fluorescence microscopy, we show that Pseudorabies Virus (PRV) particles use the constitutive Rab6 post-Golgi secretory pathway to exit from the cell body of primary neurons, independent of local calcium signaling. Some PRV particles colocalize with Rab6 in the proximal axon, but we did not detect colocalization/co-transport in the distal axon. Thus, the specific secretory mechanisms used for viral egress from axons remains unclear. To address the role of neuronal activity more generally, we used a compartmentalized neuron culture system to measure the egress and spread of PRV from axons, and pharmacological and optogenetics approaches to modulate neuronal activity. Using tetrodotoxin to silence neuronal activity, we observed no inhibition, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread from axons. We conclude that PRV egress from neurons uses constitutive secretory mechanisms: generally, activity-independent mechanisms in axons, and specifically, the constitutive Rab6 post-Golgi secretory pathway in cell bodies.

Quantitative Characterization of Output from the Directionally Selective Visual Interneuron H1 in the Grey Flesh Fly Sarcophaga bullata.

H1, a very well-studied insect visual interneuron, has a panoramic receptive field and is directionally selective in responding to optic flow. The synaptic basis for the directional selectivity of the H1 neuron has been studied using both theoretical and cellular approaches. Extracellular single-unit recordings are readily obtained by beginning students using commercially available adults of the grey flesh fly Sarcophaga bullata. We describe an apparatus which allows students to present a series of moving visual stimuli to the eye of the restrained, minimally dissected adult Sarcophaga, while recording both the single unit responses of the H1 neuron and the position and velocity of the moving stimulus. Students obtain quantitative and reproducible responses of H1, probing the response properties of the neuron by modulating stimulus parameters such as: direction and speed of movement, visual contrast, spatial wavelength, or the extent of the visual field occupied. Students learn to perform quantitative analysis of their data and to generate graphical representations of their results characterizing the tuning and receptive field of this neuron. This exercise demonstrates the utility of single unit recording of an identified interneuron in an awake restrained insect and promotes interpretation of these results in terms of the visual stimuli normally encountered by freely flying flies in their natural environment.

Reproducible Quantitative Stimulation Allows New Analysis of Crayfish Muscle Receptor Organ Responses.

The crustacean muscle receptor organ (MRO) has provided a particularly accessible preparation for the study of sensory coding, which has been widely used in introductory laboratory courses incorporating extracellular recording from sensory nerves in living preparations. We describe three innovations to the standard laboratory exercise using the MRO: (1) a new form of suction electrode to facilitate extracellular recording; (2) a new, Arduino-driven actuator to allow reproducible and quantifiable mechanical stimulation of the MRO; and (3) a new approach to the crayfish abdomen preparation that allows linear extension of the MRO muscles. These novel approaches allow the collection of data sets comprised of sensory cell spike trains under software control as important mechanical stimulus parameters are varied systematically through software. This additional level of user control facilitates a more robust quantitative approach to the analysis of MRO sensory neuron spike trains, which is facilitated by training in data analysis using python.

Kinesin-3 mediates axonal sorting and directional transport of alphaherpesvirus particles in neurons.

During infection of the nervous system, alphaherpesviruses-including pseudorabies virus (PRV)-use retrograde axonal transport to travel toward the neuronal cell body and anterograde transport to traffic back to the cell periphery upon reactivation from latency. The PRV protein Us9 plays an essential but unknown role in anterograde viral spread. To determine Us9 function, we identified viral and host proteins that interact with Us9 and explored the role of KIF1A, a microtubule-dependent kinesin-3 motor involved in axonal sorting and transport. Viral particles are cotransported with KIF1A in axons of primary rat superior cervical ganglion neurons, and overexpression or disruption of KIF1A function, respectively, increases and reduces anterograde capsid transport. Us9 and KIF1A interact early during infection with the aid of additional viral protein(s) but exhibit diminished binding at later stages, when capsids typically stall in axons. Thus, alphaherpesviruses repurpose the axonal transport and sorting pathway to spread within their hosts.