Fig 1.
PRV particles use constitutive secretory pathway to exit from the cell body of primary SCG neurons.
(A) PRV particles (mCherry-VP26 capsid tag) colocalize with EmGFP-Rab6a in the cell body of SCG neurons. Colocalization quantified from 40 cells over 5 independent experiments. Pearson’s correlation between red and green channels was significantly greater (p<0.01, student’s t-test) at intracellular membranes compared to control conditions. Scale bar represents 4 μm. (B) PRV particles and EmGFP-Rab6a co-transport in the cell body. Kymograph represents particle movement along the indicated track (magenta). Data are representative of >56 cells over 7 independent experiments. Scale bar represents 4 μm. (C) Virus particle exocytosis from neuron cell bodies occurs as early as 5 hpi. Image is a maximum difference projection over a 5.3 min time course. Exocytosis of gM-pHluorin particles that do not contain capsids (green circles) and particles containing both gM-pHluorin and capsids (yellow circles) are indicated. Scale bar represents 4 μm. (D) Ensemble average of gM-pHluorin fluorescence (green line) and mRFP capsid (red line) over 26 exocytosis events. Shaded area represents standard deviation. (E-F) SCG neurons were transduced to express mCherry-Rab proteins, infected with PRV expressing gM-pHluorin, and imaged beginning at 5 hr after PRV infection. Images show representative exocytosis events at the moment of exocytosis (time = 0). Each image is 5 μm square. Line plots are ensemble averages of gM-pHluorin (top, green line) and mCherry-Rab protein (bottom, red line) relative fluorescence. Shaded area represents standard deviation. (E) Rab6a is associated with virus particle exocytosis from neuron cell bodies. Data represent 72 exocytosis events. (F) Rab8a is associated with virus particle exocytosis from neuron cell bodies. Data represent 35 exocytosis events.
Fig 2.
PRV particles exocytosis is not associated with Rab11a, Rab3a, nor local Ca2+ signaling.
(A-B) SCG neurons were transduced to express mCherry-Rab proteins, infected with PRV expressing gM-pHluorin, and imaged beginning at 5 hr after PRV infection. Images show representative exocytosis events at the moment of exocytosis (time = 0). Each image is 5 μm square. Line plots are ensemble averages of gM-pHluorin (top, green line) and mCherry-Rab protein (bottom, red line) relative fluorescence. Shaded area represents standard deviation. (A) Rab11a is not associated with virus particle exocytosis in neurons. Data represent 41 exocytosis events. (B) Rab3a is not associated with virus particle exocytosis in neurons. Data represent 64 exocytosis events. (C-E) Neurons were infected with PRV expressing gM-pHluorin, loaded with Ca2+ sensitive fluorescent dye Rhod-2-AM, and imaged beginning at 5 hpi. (C) Image is a maximum intensity projection of Rhod-2-AM fluorescence over a 6 min time course. Scale bar represents 4 μm. (D) Rhod-2-AM fluorescence intensity at the region of interest indicated in panel C (black circle) over 6 min time course. (E) Virus particle exocytosis in neurons is not associated with local Ca2+ transients. Ensemble average of gM-pHluorin (top, green line) and Rhod-2-AM (bottom, red line) relative fluorescence over 27 exocytosis events. Shaded area represents standard deviation.
Fig 3.
Some PRV particles colocalize with Rab6a in the proximal axon, but Rab6a is not detected with PRV particles in distal axons.
(A-B) SCG neurons were infected with PRV expressing mCherry-VP26 and EmGFP-Rab6a, and imaged beginning at 5 hr post-infection. Data are representative of 50 cells over 7 independent experiments. Scale bars represent 4 μm. (A) EmGFP-Rab6a vesicles undergo sorting and transport into the proximal axon. Kymograph (inset) represents particle movement along the indicated track (magenta). (B) PRV particles (mCherry-VP26) colocalize with EmGFP-Rab6a in the proximal axon (arrowheads). (C) EmGFP-Rab6a vesicles transport to the distal axon (top set of kymographs), but EmGFP-Rab6a is not detectable on PRV particles (bottom kymographs). Kymographs represent particle movement along the indicated tracks (magenta). Data are representative of ~500 capsid tracks, from 36 independent fields of view, over 4 independent experiments. Scale bar represents 4 μm.
Fig 4.
Tetrodotoxin and KCl affect activity of infected neurons.
Primary SCG neurons in modified Campenot trichambers were infected with a recombinant PRV expressing the fluorescent Ca2+ sensor GCaMP3, and treated with 4μM tetrodotoxin (TTX), pulses of 55mM KCl, or untreated, as indicated. (A) Relative Ca2+-dependent GCaMP3 fluorescence of 5 representative cells over time. Relative fluorescence units (RFU) are scaled identically between experimental conditions. (B) Steady-state Ca2+ accumulation indicated by GCaMP3 fluorescence. Scale bar represents 40 μm.
Fig 5.
Neuronal activity does not correlate with PRV spread from neurons to non-neuronal cells.
(A) Schematic of modified Campenot tri-chamber. SCG neurons were seeded in the left soma compartment and extended axons under the chamber walls to the right neurite compartment. A detector cell monolayer (PK15) was then added to the neurite compartment. PRV infection was initiated in the soma compartment, and neurons were treated with 4μM TTX, or pulses of 55mM KCl, or untreated, as indicated. (B) Tiled image of neurite chambers (area indicated by red box in panel A). Scale bar represents 1mm. (C) Relative mRFP-VP26 capsid fluorescence of entire neurite chambers over time. (D) Virus titer measured in neurite chambers by serial dilution plaque assay.
Fig 6.
Neuronal activity does not correlate with PRV spread from neuron to neuron.
(A) Schematic of modified Campenot tri-chamber. SCG neurons were seeded in the left soma compartment and extended axons under the chamber walls to the right neurite compartment. Recipient SCG neurons were then added to the neurite compartment. PRV infection was initiated in the soma compartment, and neurons were treated with 4μM TTX, or pulses of 55mM KCl, or untreated, as indicated. (B) Recipient neurons in the right neurite chamber do not penetrate the left soma compartment. Lipophilic fluorescent dye, DiD (blue), was added to the soma chamber, and chambered cultures were imaged by live-cell fluorescence microscopy. Out of many chambers, only a single DiD-labeled cell body was detected (zoom). Scale bars represent 0.5 mm (left) and 30 μm (zoom). (C) A defective leaky chamber demonstrates extensive DiD-labeling of cells, demonstrating that these imaging parameters can readily detect DiD-positive cells in the neurite chamber. (D) Tiled image of neurite chambers (area indicated by red box in Fig 2A). Scale bar represents 1mm. (E) Mean relative mRFP-VP26 capsid fluorescence of entire neurite chambers over time. Mean of 8 independent experiments. Shaded area represents standard deviation. No significant difference (p>0.05) at all time points.
Fig 7.
Neuronal activity does not correlate with PRV spread from neurons: optogenetics approach.
(A-B) SCG neurons were seeded onto a multi-electrode array, transduced with an AAV vector expressing ChR2-mCherry. (A) Scale bar is 500μm. (B) Scale bar is 100μm. (C) Extracellular electrical recordings of SCG neurons exhibiting spontaneous spiking activity (no stimulus), or evoked activity synchronized to 12 Hz blue light pulses. (D) Superimposed spikes (n = 100) demonstrating increases in spike peak-to-peak amplitude with optogenetic stimulation. (E) Schematic of modified Campenot tri-chamber. SCG neurons were seeded in the left soma compartment and extended axons under the chamber walls to the right neurite compartment. A detector cell monolayer (PK15) was then added to the neurite compartment. SCGs were transduced with a ChR2 AAV and exposed to pulsed blue light, as indicated. PRV infection was initiated in the soma compartment at a multiplicity of infection of 10 or 100, as indicated. (F) Virus titer measured in neurite chambers by serial dilution plaque assay. No significant difference (p>0.05) at 24 hpi.