Figure 2: Septal-hippocampal inputs and quantification of traced cells.

(a) Horizontal section through the DG and area CA3 at 30 d.p.i. Newborn 'starter' cells express nuclear hGFP (green) and MCh (red). Trans-synaptic rabies-virus-mediated labelling (red) was observed in area CA3 pyramidal cells suggesting a direct 'back projection' to DG. Newborn neurons receive input from mature GCs (right inset, scale bar, 50 μm). Astrocytes surrounded the dendritic arborization of newborn 'starter' cells (left inset, scale bar 25 μm). (b) Traced cells labelled with MCh in the medial septum (MS) and horizontal nucleus of the diagonal band (HDB) at 21 d.p.i. (coronal section). (c) MCh⁺- HC at 30 d.p.i. (horizontal section) and a characteristic mossy cell (inset, scale bar, 25 μm). Overview panels a,b,c, scale bar, 200 μm. Nuclei are labelled with DAPI (blue). (d–j) Quantification and characterization of afferent input to newborn GCs. (d) hGFP⁺ new GCs and, starter cells (dual-labelled hGFP⁺-MCh⁺ new GCs), remained stable over time. (e) The total number of traced cells did not differ across time points. (f) The ratio of traced to 'starter' cells was significantly increased at 90 d.p.i compared with 21 d.p.i. and 30 d.p.i., suggesting a strengthening of inputs to new GCs. (g) Upon evaluation across time points and brain regions a transient input from mature to newborn GCs was observed, whereas hilar afferents increased significantly compared to 21 d.p.i. (h) The ratio of traced HC to 'starter' cells was increased significantly at 90 d.p.i. compared with all other time points. Similarly, distal cortical input differed from 21 d.p.i. and 30 d.p.i. at 90 d.p.i. (i) Percentage input from mature GC was high at 21 d.p.i. but was absent at 60 and 90 d.p.i. Both hilar (60 d.p.i. and 90 d.p.i. versus 21 d.p.i. and 30 d.p.i.) and distal cortical (30, 60, 90 d.p.i. versus 21 d.p.i.) inputs increased over time. The septal and area CA3 cells remained stable over time. (j) Quantification of intra-hippocampal MCh⁺ cells double labelled with calretinin (CR), a marker for mossy cells, or GABA, a marker for inhibitory interneurons, at 60 d.p.i. and 90 d.p.i. *P<0.05, One-way ANOVA followed by Fisher's post-hoc t-tests, 21 d.p.i. (n=4), 30 d.p.i. (n=6), 60 d.p.i. (n=4), 90 d.p.i. (n=5). Data are means±s.e.m. HC, hilar cells; GC, granule cells; PYR, area CA3 pyramidal cells; SEP, septal cells; CTX, cortical cells.