Toxoplasma parasites predominantly interact with neurons throughout CNS infection.

Cre-reporter mice were infected with II-Cre or III-Cre Toxoplasma parasites as labeled. Brains were harvested, sectioned, and stained for neurons (anti-neuronal cocktail) and astrocytes (anti-GFAP) at specified time points. Stained sections were analyzed by confocal microscopy to identify if GFP co-localized with stains for neurons, astrocytes, or neither (unidentified). (a) Representative stitched-grid image of a brain section from a III-Cre infected mouse at 3 weeks post infection (wpi). White boxed area in left image is enlarged and separated into the different channels, as labeled (right images). White arrowheads denote GFP⁺ cells that co-localized with anti-neuron staining, red arrowheads denote GFP⁺ cells that did not co-localize with either anti-neuron or anti-astrocyte staining. Left image scale bar, 1 mm. Enlarged image scale bar, 50 μm. (b) Quantification of co-localization for II-Cre infected brain sections at different time points post infection. Bars, mean ±SEM. (c) As in (b) but restricting the analysis only to GFP⁺ cells identified as neurons or astrocytes. (d), (e) As in (b), (c) but for III-Cre infected mice. Bars, mean ±SEM. N = 3–4 mice/time point/Toxoplasma strain. The total number of GFP⁺ cells examined at each time point ranged from 232–372/II-Cre and 368–506/III-Cre. No statistical differences were found in the mean percentage of GFP⁺ neurons across time points in either II-Cre or III-Cre infection (one-way ANOVA, p = 0.13 and p = 0.45 respectively). At 6 wpi, one of the III-Cre infected mice had a substantially lower percentage of GFP⁺ neurons compared to the other mice (67 vs. 91,100,100). Exclusion of this mouse from data analysis changes the mean percentage of GFP⁺ neurons at 6 wpi from 90 ±8 (full data set) to 97 ±3 (1 mouse excluded), which results in a suggestion that in III-Cre infected mice the percentage of GFP⁺ neurons is lower at 1.5 wpi compared to 6 and 12 wpi (one-way ANOVA, p < 0.01). No GFP⁺ cells were found in II-Cre or III-Cre infected brain sections from 0.5 wpi (N = 2 mice/Toxoplasma strain, 9 sections/mouse).