Borna disease virus replication in organotypic hippocampal slice cultures from rats results in selective damage of dentate granule cells

Abstract

In the hippocampus of Borna disease virus (BDV)-infected newborn rats, dentate granule cells undergo progressive cell death. BDV is noncytolytic, and the pathogenesis of this neurodevelopmental damage in the absence of immunopathology remains unclear. A suitable model system to study early events of the pathology is lacking. We show here that organotypic hippocampal slice cultures from newborn rat pups are a suitable ex vivo model to examine BDV neuropathogenesis. After challenging hippocampal slice cultures with BDV, we observed a progressive loss of calbindin-positive granule cells 21 to 28 days postinfection. This loss was accompanied by reduced numbers of mossy fiber boutons when compared to mock-infected cultures. Similarly, the density of dentate granule cell axons, the mossy fiber axons, appeared to be substantially reduced. In contrast, hilar mossy cells and pyramidal neurons survived, although BDV was detectable in these cells. Despite infection of dentate granule cells 2 weeks postinfection, the axonal projections of these cells and the synaptic connectivity patterns were comparable to those in mock-infected cultures, suggesting that BDV-induced damage of granule cells is a post-maturation event that starts after mossy fiber synapses are formed. In summary, we find that BDV infection of rat organotypic hippocampal slice cultures results in selective neuronal damage similar to that observed with infected newborn rats and is therefore a suitable model to study BDV-induced pathology in the hippocampus.

FIG. 1.

Disappearance of calbindin-positive granule cells after long-term infection of rat hippocampal slice cultures with BDV. (A) Calbindin-immunofluorescence stained hippocampal cultures at 28 days in vitro. In both untreated and mock-infected cultures, a typical C-shaped layer of calbindin-immunoreactive granule cells developed, and numerous mossy fibers projected to the CA3 region of the hippocampus (arrows). Cultures infected with ca. 100 focus-forming units of BDV stock harvested from either persistently infected cells or from infected rat brain showed severely reduced immunoreactivity. Note that there are only a few calbindin-positive cells. Mossy fiber axons have almost disappeared. Scale bar, 100 μm. (B) Levels of BDV-N and calbindin were compared by Western blot analysis using complete extracts of BDV-infected and uninfected slice cultures shown in panel A. Levels of tubulin served as a loading control. gcl, granule cell layer.

FIG. 2.

Pyramidal cells of the CA3 region of the hippocampus tolerate BDV infection without any obvious damage. Slice cultures were infected with cell culture-derived virus as described in the legend to Fig. 1A. At 28 days postinfection, 50-μm sections were prepared and immunostained for the nucleoprotein of BDV (green) and calbindin (red). (A) A survey of a hippocampus depicting the CA3 region with the pyramidal cell layer (pcl) and the rudimentary granule cell layer (rgcl). (B) High-power magnification of the CA3 pyramidal layer shown in panel A (inset B). Note the typical nuclear dot-like signals in BDV-infected cells (arrows). (C) Magnification of the pyramidal layer, showing several dendrites covered with spines of obviously infected pyramidal neurons from the micrograph in panel A (inset C). Arrowheads point to postsynaptic spines of BDV-positive dendrites. (D) Magnification from the micrograph in panel A (inset D) illustrating calbindin-positive granule cells (arrows) in the residual granule cell layer of the dentate gyrus that are also BDV infected. Scale bars: 40 μm (A); 20 μm (B to D).

FIG. 3.

BDV-induced decline of calbindin-positive granule cells occurs after maturation of the dentate granule cell layer. Slice cultures were infected with BDV as described in the legend to Fig. 1A and cultured for either 14 or 21 days. Sections were analyzed by immunofluorescence for the presence of calbindin (A and B) and by Hoechst 33251 nuclear staining (C and D). (E and F) Panels A to D, triple stained for calbindin (red), nuclei (blue), and BDV-N (green). Note that calbindin-positive granule cells are already infected at day 14 postinfection. gcl, granule cell layer; rgcl, rudimentary gcl; pcl, pyramidal cell layer. Scale bars: 100 μm (A to D); 20 μm (E and F).

FIG. 4.

BDV infection results in a severe decrease of mossy fiber boutons. (A) Cartoon showing the hippocampus, including the pyramidal cells (open triangles) and the granule cells (open circles). The axons of the granular cells establish giant mossy fiber boutons with dendrites of the CA3 pyramidal cells. These large boutons contain many synaptic vesicles and can be identified by synaptophysin immunofluorescence (large red dots). Presynaptic terminals from neurons other than granule cells in this area are significantly smaller and are indicated by small dots. (B) Slice cultures were BDV infected as described in the legend to Fig. 1A, cultured for either 14 or 21 days, and analyzed by immunofluorescence for the presence of synaptophysin, a marker protein for synaptic vesicles. The area of the pyramidal cell layer (pcl) region CA3 is shown where mossy fiber boutons (mf) are visible as large red dots (some indicated by arrowheads). Note that compared to mock-infected cultures the number of mossy fiber boutons is significantly lower at 21 days p.i. than at 14 days p.i. Scale bars, 25 μm.

FIG. 5.

Biocytin-traced mossy fiber axons at 3 weeks postinfection. Tracer application into the hilus (h) revealed intensively labeled hilar neurons and their axonal projections above the Nissl stain-treated granule cell layer in two representative examples of BDV-infected cultures 21 days p.i. (A and C) and a control culture (E). Nissl stain-treated pyramidal cell layers of the hippocampus are clearly visible; few biocytin-stained pyramidal neurons are intermingled. In the mock-infected culture, several granule cells with dendrites in the granule cell layer (gcl) and a fine bundle of traced axons in the CA3 region are visible (E). The boxed areas are shown at higher magnification in panels B, D, and F. In mock-infected cultures, numerous large biocytin-filled synaptic mossy fiber boutons are visible (arrows). Biocytin-traced axons run above the Nissl-stained pyramidal cell layer (pcl). In contrast to mock-infected cells, few biocytin-stained mossy fiber axons bearing giant boutons (arrows) were identified in infected cultures (D). In other infected cultures, no mossy fiber axons could be traced (B). Note that the arrowheads in panel B mark labeled axons that are different from the mossy fibers in the CA3 region. Scale bars: 150 μm (A, C, and E); 50 μm, B, D, and F.

FIG. 6.

Model of BDV-induced neurodevelopmental damage of hippocampal cells by altered neuronal connectivity of granule cells. (Left) During the first week after birth, only a few pyramidal (open triangles) and some other cells get infected with BDV (indicated by green dots). At this point in hippocampal development, the maturation of granule cells (open circles) is not complete, and the outgrowing mossy fiber axons still harbor growth cones at their termini (indicated by black dots). Only a few mossy fiber axons establish synaptic contacts with target neurons (large red dots). (Middle) After 2 weeks, despite strong infection of pyramidal cells, as well as of most granule cells, differentiation of the latter cells is almost complete and most mossy fiber boutons are formed. At this developmental stage, a functional connectivity has been established. (Right) During the following week, mossy fiber axons retract and granule cells undergo cell death (dashed open circles).