Axonal retrograde transport kinetics. Dissociated SCG neurons were cultured on glass-bottom MatTek dishes for 2 weeks. Cultures were infected with PRV GS443 or PRV 765. The movies in the supplemental material were captured 16 h postinfection on a Perkin-Elmer R30 spinning disk confocal microscope. (A) Color-coded arrowheads track the movement of individual capsid puncta in 5-s intervals. (B) Distribution of capsid puncta run lengths is shown in box plots (n = 120). (C) Distribution of average run velocities of capsid puncta (n = 85). The open squares in box plots designate the minimum value, first quartile, median, and third quartile of the data set; filled squares are mean values.

Retrograde infection time course in vitro. (A) Trichamber culture system for study of directional infection of neurons. Dissociated SCG neurons are plated in the S-compartment of the trichamber. Axonal growth is guided into the N-compartment by a series of grooves etched in the dish surface. Inoculum is applied to the N-compartment, and contents of the S-compartment are harvested, and the virus titers were determined. (B and C) Time course of retrograde neuronal infection by PRV Becker (B) or PRV Bartha (C). Filled squares indicate mean values at each time point.

PRV Bartha does not spread efficiently to second-order neurons in vitro. DiI was added to the N-compartment immediately following infection with PRV Becker (A), PRV Bartha (B), or PRV 43/25 aB4 (C). Samples were processed for immunofluorescence against the major capsid protein VP5 at 24 hpi using a green secondary antibody. Arrows mark dually fluorescent cells. Arrowheads mark cells exhibiting VP5 fluorescence only. Bars = 40 μm. (D) The percentage of cells exhibiting both DiI and VP5 fluorescence is represented by filled bars; empty bars are cells exhibiting VP5 fluorescence only (n = 200). The chi-square test was used on raw data to determine significance. *, P < 0.05.

PRV 43/25 aB4 restores wild-type PRV retrograde titers. (A) Diagram of genomes used to map the PRV Bartha retrograde defect. (B) Viral inoculum was added to the N-compartment of neuronal cultures. The contents of the S-compartment were harvested, and virus titers were determined at 24 hpi. Retrograde titers achieved by each strain are shown by filled symbols (n = 4). Empty squares denote the mean values for each data set.

PRV 326 is defective for neuron-to-cell spread, but it undergoes efficient retrograde infection of neurons. (A) The epithelial PK15 detector cells were plated in the N-compartment of neuronal cultures prior to infection. Viral inoculum was applied to the S-compartment, the contents of the S- and N-compartments were harvested at 24 hpi (n = 7), and virus titers were determined. (B) Viral inoculum was adsorbed to axons in the N-compartment. The contents of the S-compartment were harvested at 24 hpi (n = 7), and virus titers were determined. Filled squares represent the mean value of each data set.

PRV 326 invades sympathetic and parasympathetic circuits at a faster rate than PRV Bartha does. The organization of polysynaptic circuits innervating the stomach is illustrated in the midsagittal schematic diagram of the rat brain. Virus injected into the ventral wall of the stomach invades the central nervous system through sympathetic and parasympathetic circuits. Virus injected into the stomach wall is retrogradely transported to the dorsal motor vagal complex (DVC) in the caudal brain stem through parasympathetic circuits. Infection of sympathetic neurons in the intermediolateral cell column (IML) of the thoracic spinal cord is temporally delayed compared to that resulting from invasion of parasympathetic circuits due to the need for first-order replication in sympathetic ganglia. First-order parasympathetic and sympathetic neurons are represented in black in the diagram. Neurons presynaptic to parasympathetic circuits are denoted in red, and those linked to the IML are represented in blue. Note that the paraventricular nucleus (PVN) of hypothalamus is synaptically linked to both. (A) The synaptic organization of circuits in the DVC is illustrated in coronal section. Neurons of the dorsal motor vagal nucleus (DMV) receive synaptic contact from neurons in the immediately adjacent nucleus of the solitary tract (nts) and the area postrema (AP). (B to E) The magnitude of retrograde transneuronal passage of PRV Bartha (B and D) and PRV 326 (C and E) through the DVC (B and C) and IML (D and E) 72 h following inoculation of the stomach is illustrated in the photomicrographs. PRV 326 exhibits more extensive retrograde transneuronal infection of the DVC than PRV Bartha does, and larger numbers of IML neurons are infected in the IML of PRV 326-infected animals. Abbreviations: A5, midbrain catecholamine cell group; BNST, bed nucleus of stria terminalis; CeA, central nucleus of the amygdala; IC, insular cortex; LHA, lateral hypothalamic area; PFC, prefrontal cortex; R, raphe; RVLM, rostroventrolateral medulla; VMM, ventromedial medulla.

Quantitative analysis reveals a statistically significant increase of PRV 326 transport through preautonomic circuits compared to controls. The number of infected neurons observed in selected areas of the CNS 72 h following injection of PRV Bartha or PRV 326 into the stomach is illustrated. In each area, the number of PRV 326-infected neurons shows a statistically significant increase relative to PRV Bartha. The dramatic increase in the number of infected neurons in the PVN is likely related to the fact that this nucleus is synaptically linked to both the dorsal motor vagal complex (DVC) and IML, whereas the rostroventrolateral medulla (RVLM), midbrain catecholamine cell group (A5), central nucleus of the amygdala (CeA), and bed nucleus of stria terminalis (BNST) are selectively linked to the IML. Values that are statistically significantly different for PRV Bartha and PRV 326 are indicated with an asterisk (Student's t test, P ≤ 0.05).

Distribution of infected neurons at three comparable levels of the neuraxis 72 h following injection of PRV Bartha or PRV 326 into the ventral wall of the stomach. The gray bars on the sagittal section at the top of the figure illustrate the positions of the coronal sections shown immediately below. The coronal sections sample the caudal brain stem at the level of the dorsal motor vagal complex (DMV) (nucleus of the solitary tract [nts] and area postrema [AP]), through the cardiovascular regulatory cell group linked to the sympathetic outflow (rostroventrolateral medulla [RVLM]), and through a level of the forebrain through the paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA). Infected neurons were mapped by systematic examination of the section with a 40× objective. The position of each cell was recorded using an image analysis system. Each red dot represents an infected neuron. The maps demonstrate the dramatic increase in transport of PRV 326 relative to PRV Bartha in preautonomic circuits synaptically linked to the sympathetic and parasympathetic outflow.

The increase in the magnitude of infection of preautonomic circuits resulting from infection with PRV 326 compared to PRV Bartha is striking and unambiguous. The extent of infection of four cell groups following identical inoculation of PRV Bartha (A, C, E, and G) or PRV 326 (B, D, F, and H) is illustrated. Comparisons of infection in the rostral portion of the nucleus of the solitary tract (A and B), rostroventrolateral medulla (C and D), midbrain catecholamine cell group (E and F), and paraventricular nucleus (G and H) are illustrated. Each comparison reveals larger numbers of infected neurons, a finding that was confirmed by statistical analysis. The yellow areas in the schematic insets illustrate the locations of the areas that were photographed. The third ventricle of the hypothalamus (3V) is indicated. The magnification of all images in this figure is the same. Bar = 100 μm.

Virus-induced recruitment of immune cells into the brain is increased in PRV 326-infected animals. The extent of ED1+ immune cell recruitment into the dorsal motor vagal complex 72 h after infection with PRV Bartha (A) or PRV 326 (B) is illustrated. Note the larger number of immunopositive cells within the DMV and immediately adjacent nucleus of the solitary tract (nts) of PRV 326-infected animals. The area postrema (AP) is indicated. The yellow area in the schematic inset illustrates the location of the area illustrated in the photomicrographs. The magnification of both images is the same. Bar = 100 μm.