Primary sensory neuron addition in the adult rat trigeminal ganglion: evidence for neural crest glio-neuronal precursor maturation

Abstract

It is debated whether primary sensory neurons of the dorsal root ganglia increase the number in adult animals and, if so, whether the increase is attributable to postnatal neurogenesis or maturation of dormant, postmitotic precursors. Similar studies are lacking in the trigeminal ganglion (TG). Here we demonstrate by stereological methods that the number of neurons in the TG of adult male rats nearly doubles between the third and eighth months of age. The increase is mainly attributable to the addition of small, B-type neurons, with a smaller contribution of large, A-neurons. We looked for possible proliferative or maturation mechanisms that could explain this dramatic postnatal expansion in neuron number, using bromodeoxyuridine (BrdU) labeling, immunocytochemistry for neural precursor cell antigens, retrograde tracing identification of peripherally projecting neurons, and in vitro isolation of precursor cells from adult TG explant cultures. Cell proliferation identified months after an extended BrdU administration was sparse and essentially corresponded to glial cells. No BrdU-labeled cell took up the peripherally injected tracer, and only a negligible number coexpressed BrdU and the pan-neuronal tracer neuron-specific enolase. In contrast, a population of cells not recognizable as mature neurons in the TG and neighboring nerve expressed neuronal precursor antigens, and neural crest glioneuronal precursor cells were successfully isolated from adult TG explants. Our data suggest that a protracted maturation process persists in the TG that can be responsible for the neuronal addition found in the adult rat.

Figure 1.

Number of cells per class and side in the TG at different ages. TG sensory neurons nearly double from 3 to 8 months of age (p < 0.001). This increase is significant for both major classes, A and B, of ganglion neurons (p < 0.01), but it is greater for B-type cells (small neurons), which nearly triple with aging. In 7-month-old animals that sustained a transection of the right ION at 3 months of age, the deafferented TG shows a decrease both in A- and B-type cells, the latter being more severely affected (p < 0.01 for B-type cells; p < 0.05 for all cells). The nondeafferented left TG at 7 months shows fewer B-type cells than controls at 8 months (p < 0.01). Circles and asterisk mark outliers.

Figure 2.

BrdU immunohistochemistry in TG and rat hippocampus. A–F, Photomicrographs of 40-μm-thick BrdU-immunoreacted and thionin-counterstained sections from rat hippocampus (A) and TG (B–F) 3 months after BrdU administration. Immunoreactive nuclei in the TG are all small and are mainly situated around apparently “empty” spaces corresponding to sensory neuron bodies, which stain very weakly with thionin after BrdU processing. Satellite cells display different degrees of immunolabeling. A positive presumed Schwann cell nucleus in a nerve fascicle in the ganglion is shown in F. Scale in E also applies to A, C, D, and F.

Figure 3.

BrdU immunofluorescence in TG combined with Fast Blue and NSE. Photomicrographs of 40-μm-thick sections from rat TG and hippocampus immunostained for BrdU and NSE or after transport of Fast Blue. A, TG after transport of Fast Blue shows intense labeling of large groups of sensory neurons (top left), and dentate gyrus cells show nuclear staining for BrdU in the granular layer (top right); in TG, a group of sensory neurons intensely labeled with Fast Blue (bottom left) are interspersed with many BrdU⁺ nuclei (bottom middle), but none are double labeled (bottom right). B, A confocal image of a rare finding of a BrdU⁺NSE⁺ double-labeled cell. Scale bars: A, bottom series, 100 μm; B, 50 μm.

Figure 4.

Characterization of BrdU⁺ cells in the TG 3 months after axotomy. A–D, E, Bottom row, Three-dimensional constructed images (30 images taken with 60× oil objectives and 0.5 μm Z-step for A–C, and 25 images taken with 40× oil objectives and 1.0 μm Z-step for D and bottom part of E). E, Bottom row, High-magnification image showing the region in the white box in the top row of E. A and B show the double labeling for BrdU (red) with PGP9.5 or GFAP (green), and arrows in B indicate BrdU⁺/GFAP⁺ colabeling cells. C is a representative confocal image showing that the majority of perineuronal cells coexpressed Nestin (red), p75NTR (green), and GFAP (cyan). D and E are representative photomicrographs taken from different deafferented TGs. The arrowhead in D shows a PGP9.5⁺/βIII-tubulin-negative neuron. In D and E, Nestin⁺ neuron-like profiles (thin arrows) are negative for PGP9.5/βIII-tubulin (thick arrows). Scale bars: A–C, 25 μm; D, E, 50 μm.

Figure 5.

DCX immunocytochemistry in TG, trigeminal nerves, and hippocampus. Photomicrographs of 40-μm-thick sections from rat TG and hippocampus immunostained for DCX. A–D, Immunoreactive cells in the maxillary (A, B) and mandibular (C, D) nerves; they are arranged in cords parallel to or interspersed with nerve fibers (B–D) or near the perineurium of nerve fascicles (A). D shows a detail from C. E, Cells in the basal area of the granular layer of the dentate gyrus show cytoplasmic immunostaining for DCX that extends to the dendrites. Scale bars: A, 500 μm; C, 200 μm; D (also applies to B), E, 50 μm.

Figure 6.

Double immunofluorescence for DCX–NSE and DCX–Fast Blue in the TG and trigeminal nerve branches. Confocal microscopy of 40-μm-thick sections from rat TG. A–C, Examples of DCX⁺/NSE-negative cells interspersed with NSE⁺ nerve fibers. D, A small DCX⁺ cell is surrounded by Fast Blue⁺ cells. No double-labeled cells were found in any case. Scale bar (in top left): A, C, 34 μm; B, 64 μm; D, 48 μm.

Figure 7.

Cells emigrated from adult TG explants form clusters and spheres that could be transfected with retroviral vector. Phase-contrast morphology of emigrating cells and spheres from adult cultured TG. Top, Cells emigrate from TG explants at 1–3 d (A, arrows) in vitro. Sprouting-like cells are seen at the edges of the tissue blocks (B, arrows). After 1 week in culture, loose-packed suspended cellular aggregates (clusters) can be found (C, arrows), and there are floating single cells and small clusters in the medium. After 2 weeks in culture, a few spherical structures can be found in flasks; the size of these multicellular aggregates varies from several cells (D, arrowhead) to solid spheres (the tight-packed floating cellular aggregates marked by an arrow in D). With extension of the culture period, larger size spheres are formed as shown by a bold arrow in E. Clusters (arrowheads) and free-floating cells (slim arrows) are still predominant after 4 weeks in culture. Scale bar, 100 μm. Bottom, Green fluorescent primary spheres (arrows)/clusters (arrowheads) are found 1 week after retroviral EGFP vector transfection (G–I). The dissociated cells from EGFP-transfected sphere/clusters also form secondary GFP⁺ spheres/clusters as shown by arrows and arrowheads, respectively (J–L). Scale bar, 100 μm.

Figure 8.

Characterization by immunofluorescence of the secondary spheres derived from adult TG explants. Immunohistochemical characterization of neurospheres derived from adult rat TG explant cultures. Top, A, Representative photomicrographs show that spheres express Nestin and p75NTR; B, another sphere was stained for GFAP, and nuclei were counterstained by DAPI. Bottom, Immunocytochemistry staining for lineage markers on 2 week differentiation culture of hand-picked secondary spheres in DM. A, The same field image showing S100 (A1, red), NF200 (A2, green), and SMA (A5, black by DAB development) labeling; A4 is the merged image of A1–A3, and A6 is the overlay micrograph of A4 and A6. B, A representative staining of GFAP (B1, red), Nestin (B2, green), and DCX (B4, purple); B5 is the merged image of B1–B4, and B6 is the enlarged image of the white box inset in B5. C, Double staining of the differentiation culture with βIII-tubulin (C1, red) and GFAP (C2, green); C3 is the merged image of C1 and C2. Scale bar, 50 μm.

Figure 9.

Molecular profile of adult TG-derived neurospheres. Gene expression profiles of TG-derived progenitor cells characterized by RT-PCR. Total RNA was isolated from adult TG-derived secondary spheres. Neurospheres express a set of markers of neural crest stem cell and also express distinct genes involved in proliferation, self-renewal, and self-clearing/autophagy. A–C, The representative bands of the PCR products. The right lane indicates positive control using E12 cDNA, and the left lane indicates the result of the secondary spheres derived from adult TG. RT-PCR for G3PDH was used as a loading control. D, Tabularized results of RT-PCR for the tested genes in total RNA isolated from E12 rat tissue compared with adult TG-derived spheres: −, no signal; ±, minor signal; +, weak signal; ++, moderate signal; +++, strong signal. Hes, Hairy and enhancer of split 1.