Functional consequences following infection of the olfactory system by intranasal infusion of the olfactory bulb line variant (OBLV) of mouse hepatitis strain JHM

Abstract

The present study assessed the functional consequences of viral infection with a neurotropic coronavirus, designated MHV OBLV, that specifically targets central olfactory structures. Using standard operant techniques and a 'go, no-go' successive discrimination paradigm, six BALB/c mice were trained to discriminate between the presentation of an air or odor stimulus (three mice for each of the odorants propanol and propyl acetate). Two additional BALB/c mice were trained to discriminate between the presentation of air and the presentation of either vanillin or propionic acid. Following criterion performance, each mouse received an additional 2000 trials of overtraining. At completion of overtraining one mouse from the propanol and propyl acetate groups were allocated as untreated. The remaining six mice were inoculated with 300 microl of the OBLV stock per nostril for a total of 1.5 x 10(6) p.f.u. in 600 microl. Following a 1 month rest, untreated and inoculated animals were again tested on their respective air versus odor discrimination task. Untreated animals immediately performed at criterion levels. In contrast, inoculated animals varied in their capacity to discriminate between air and odorant. Five of the six inoculated mice showed massive disruption of the olfactory bulb, including death of mitral cells; the other was more modestly affected. In addition, the density of innervation of the olfactory mucosa by substance P-containing trigeminal fibers is also affected by inoculation. Those mice that remained anosmic to the training odorants had the most severe reduction in mitral cell number and substance P fiber density among the inoculated animals.

Figure 5

The distribution of substance P-labeled axons is also different between normal versus osmic versus anosmic mice. The map was constructed by assembling a mosaic of digital images taken of the stained sections and then highlighting the fiber labeling. Note the marked reduction in tangential extent and in density of the labeled fibers in the anosmic mouse.

Figure 1

After intranasal inoculation with MHV OBLV, infectious virus is cleared from both brain (A) and lung (B) within 2 weeks after infection. Prolonged food restriction (circles) did not substantially delay clearance relative to mice that were fed ad libitum throughout (triangles). Values of 0 p.f.u./ml are represented as 0.1 p.f.u./ml for the sake of clarity.

Figure 2

Comparison of the structure of the bulb in normal and OBLV-inoculated animals trained on the air versus odor discrimination task. (A, B) Olfactory bulb of normal mouse MV131 who retained the task following the 1 month rest period. Note the precise lamination of the bulb and numerous mitral cells, a few of which are indicated by the arrows. (C, D) Bulbs from virus-infected mouse MV132 who initially performed poorly after lesion, but soon reached the pre-lesion performance. The external plexiform layer (epl) is scant and only a very few mitral cells remain; only two, which are indicated by arrows, are evident in this high magnification field (D). (E, F) Bulbs from MV130 that was incapable of suprathreshold detection after recovery from inoculation. The bulbs are not markedly more affected than in the osmic animal and some mitral cells have survived in this case; one in this field is indicated by the arrow. Scale bar in (E), 500 μm, also applies to (A) and (C); in (F), 50 μm, also applies to (B) and (D).

Figure 3

The number of mitral cells that survived infection correlates weakly with the behavioral capacity of the inoculated mice. Raw uncorrected counts of mitral cell profiles summed across the five sampled sections are plotted for each of the three behaviorally defined groups: normal; OBLV-inoculated but able to perform the air—odor discrimination at the pre-infection criterion level (OBLV-Osmic) either immediately on re-initiating testing 1 month after infection or shortly thereafter; OBLV-inoculated and unable to recover their performance of the air—odor discrimination to the pre-infection criterion level (OBLV-Anosmic). The numbers in parentheses record the performance during the first post-lesion testing session. MV129 and 132 recovered to criterion level at the second testing session. MV133 recovered to criterion level at the third. Odorants were: MV130 and 131, propyl acetate; MV128, 129, 132 and 133, 2-propanol; MV135 and 136, vanillin and propionic acid.

Figure 4

Substance P staining demonstrates differences in the extent of trigeminal fibers across the three behaviorally defined groups. In all three cases, the labeling is concentrated at the basal lamina. (A, B) The density of labeling is roughly equivalent in the normal and OBLV-inoculated, osmic mouse. The arrow in (A) indicates a fiber that ascends through the epithelium toward its apical surface. (C) Substance P-labeled fibers are much reduced in the OBLV-inoculated, anosmic mouse. Scale bar in (C) 50 μm, also applies to (A) and (B).