Decreased demand for olfactory periglomerular cells impacts on neural precursor cell viability in the rostral migratory stream

The subventricular zone (SVZ) provides a constant supply of new neurons to the olfactory bulb (OB). Different studies have investigated the role of olfactory sensory input to neural precursor cell (NPC) turnover in the SVZ but it was not addressed if a reduced demand specifically for periglomerular neurons impacts on NPC-traits in the rostral migratory stream (RMS). We here report that membrane type-1 matrix metalloproteinase (MT1-MMP) deficient mice have reduced complexity of the nasal turbinates, decreased sensory innervation of the OB, reduced numbers of olfactory glomeruli and reduced OB-size without alterations in SVZ neurogenesis. Large parts of the RMS were fully preserved in MT1-MMP-deficient mice, but we detected an increase in cell death-levels and a decrease in SVZ-derived neuroblasts in the distal RMS, as compared to controls. BrdU-tracking experiments showed that homing of NPCs specifically to the glomerular layer was reduced in MT1-MMP-deficient mice in contrast to controls while numbers of tracked cells remained equal in other OB-layers throughout all experimental groups. Altogether, our data show the demand for olfactory interneurons in the glomerular layer modulates cell turnover in the RMS, but has no impact on subventricular neurogenesis.

The cerebellar nuclei were strongly fate mapped and did not display differences in controls and knockout mice. Scale bar in A is 1 mm (also representative for E). Scale bar in B is 200 µm for (also representative for C, D, F, G and H).

Supplemental figure 4: MT1-MMP deficient mice do not show any signs of neuro-inflammation.
We investigated a range of immune-markers faithfully indicating microglia (Iba1 or CD68) and markers informing on functional states in myeloid cells (CD11c, CD209 or F4/80). In addition we performed immunofluorescence detection of lymphocytes in the CNS with antibodies for CD3 or CD8. Furthermore, we studied the three-dimensional cyto-architecture of microglia, as microglial cells undergo profound morphological changes during activation (see Hanisch UK & Kettenmann H. Nat Neurosci. 2007;11:1387-94.). We found that expression levels for some immune markers in both MT1-MMP-deficient and WT mice was very weak (F4/80) or even absent (CD11c or CD209), supporting the view that MT1-MMP-knockouts have no inflammatory reactions in the brain. In order to corroborate the validity of our immunolabeling paradigms we included samples from an orthotopic Labelling for CD209 (in red) is intense in TAM but absent in control or MT1-MMP Δ/Δ brain sections.
(E) Immunofluorescence-labelling for CD3 or CD8 and confocal microscopy were used to visualize lymphocytes; image stacks (Z-axis is 3µm) are presented as maximum-projection; labeling for CD3 or CD8 was extremely rare in brain samples from control or MT1-MMP Δ/Δ mice and was confined to the perivascular space. (F) Labeling-intensity for the set of immune markers described above was scored (0 = no labeling; 1 = weak; 2 = intermediate; 3 = intense) by confocal microscopy; therefore, identical settings for microscopy recordings were used for each marker and fluorescence raw intensity values were obtained. (G) Immunofluorescently labeled cells were quantified from n = 4 animals per group, 6 sections per animal (horizontal brain sections with fixed stereotactical coordinates in the dorso-ventral axis) and 5 sampling sites per section (in the olfactory bulb, SVZ, cortex, striatum and hippocampus).
Scale bar in A, C and D is 10µm; scale bar in B is 20µm; scale bar in E is 5µm; statistical significance in G is indicated as n.s. = not significant.

Supplemental figure 5: The contribution of distinct NPC subtypes to subventricular proliferation is not altered by MT1-MMP expression. (A) Immunofluorescence labeling for nestin
(green) and GFAP (red) and subsequent confocal microscopy at low magnification provides an overview on the morphology of the SVZ in wild-type (control) and MT1-MMP-deficient (MT1-MMP Δ/Δ ) mice; a horizontal brain section was used to image the SVZ in the left brain hemisphere and the narrow ventricular lumen is indicated by a dotted line; note that the morphology and marker expression appear highly similar in both groups. (B and C) Control and MT1-MMP Δ/Δ mice were intraperitoneally (i.p.) injected with BrdU (1 BrdU pulse per day for 3 consecutive days) followed by an 18 days chase period; then mouse brains were immunolabeled for GFAP (red), nestin (green) and BrdU ( Furthermore we investigated the potential of NPCs from MT1-MMP -/and control mice for differentiation into the neuronal lineage. Therefore, NPC-cultures were maintained without growth factors and supplemented with 0.5% fetal calf serum; after seven days adherent cells were fixed, immunostained for PSA-NCAM or DCX and quantified. Statistical significance in B through E is indicated as n.s. = not significant.