Neuronal activity-related transcription is blunted in immature compared to mature dentate granule cells

Abstract

Immature dentate granule cells (DGCs) generated in the hippocampus during adulthood are believed to play a unique role in dentate gyrus (DG) function. Although immature DGCs have hyperexcitable membrane properties in vitro, the consequences of this hyperexcitability in vivo remain unclear. In particular, the relationship between experiences that activate the DG, such as exploration of a novel environment (NE), and downstream molecular processes that modify DG circuitry in response to cellular activation is unknown in this cell population. We first performed quantification of immediate early gene (IEG) proteins in immature (5-week-old) and mature (13-week-old) DGCs from mice exposed to a NE. Paradoxically, we observed lower IEG protein expression in hyperexcitable immature DGCs. We then isolated nuclei from active and inactive immature DGCs and performed single-nuclei RNA-Sequencing. Compared to mature nuclei collected from the same animal, immature DGC nuclei showed less activity-induced transcriptional change, even though they were classified as active based on expression of ARC protein. These results demonstrate that the coupling of spatial exploration, cellular activation, and transcriptional change differs between immature and mature DGCs, with blunted activity-induced changes in immature cells.

Keywords: Arc; Smart-Seq2; adult neurogenesis; dentate gyrus; hippocampus; immediate early gene; single-nuclei RNA-seq.

FIGURE 1

Reduced immediate early gene (IEG) protein expression in immature DGCs. (a) Experimental timeline. Mice received tamoxifen (TAM) gavage for 7 days to label DGCs and were exposed to a novel environment (NE) to stimulate activation 5 or 13 weeks (wk) later. Untreated littermates collected directly from the home cage (HC) were used as a control. (b) Sample of durable GFP labeling 13 weeks after TAM in the DG. Scale bar: 20 μm. Yellow arrows indicate a cell triple labeled for GFP, ARC, and FOS. Dashed yellow line indicates the boundaries of the granule cell layer (GCL). (c) NE exposure induces significantly more FOS and ARC expression than the home cage control condition (n’s: HC = 6, 5-wk NE = 7, 13-wk NE = 7). (d) Quantification of active 5-week and 13-week DGCs in coronal tissue sections shows greater IEG expression in more mature cells (n’s: 5-wk = 7, 13-wk = 7). (e) Quantification of active 5-week and 13-week DGCs via flow cytometry (FACS) also shows greater IEG expression in more mature cells (n’s: Arc 5-wk = 7, Arc 13-wk = 7, Fos 5-wk = 10, Fos 13-wk = 9). (f) Co-expression of DG marker proteins PROX1 and CTIP2 occurs in the majority of GFP+ nuclei assessed by FACS, but co-expression continues to increase between 5 and 13 weeks (n’s: 5-wk = 10, 13-wk = 9). (g) Sample FACS gating strategy for identifying active immature DGCs. DGCs were identified by co-labeling with marker proteins PROX1 and CTIP2. Cell age was identified by GFP. Cell activation was assessed with ARC or FOS. (h) Experimental timeline for seizure treatment. Mice received TAM gavage for 7 days to label DGCs and were given pentylenetetrazole (PTZ) 5 or 13 weeks later. PBS-injected littermates served as staining controls. Mice were perfused following a 90-min observation period after the injection. (i) Top: Sample of FOS labeling following either PBS (left) or PTZ (right). Scale bar: 50 μm. Bottom: Sample of cell classification in PTZ-treated mice. Turquoise arrows indicate a cell triple labeled for GFP, CTIP2, and FOS. Yellow arrows indicate a cell double labeled for GFP and FOS, negative for CTIP2. Dashed yellow line indicates GCL boundaries. Scale bar: 20 μm. (j) Quantification reveals that CTIP2−GFP+ immature cells activate at significantly lower rates than CTIP2+GFP+ immature cells. CTIP2+GFP+ immature cells still have reduced FOS relative to mature CTIP2+GFP− cells at 5 weeks after TAM even under seizure conditions (n = 4, within-subject comparison). Points and n’s in dot plots represent mice. Error bars are mean ± S.E.M. *p < .05, **p < .01, ***p < .001, ns = not significant.

FIGURE 2

Reduced transcriptional response to activation in immature DG neurons. (a) Experimental timeline. Mice received TAM to label DGCs, were exposed to a NE 5 weeks later, and then were sacrificed for hippocampus dissection and nuclei preparation immediately or after an additional 3-hr delay. (b) Diagram of nuclear cell markers used to isolate specific populations via FACS and total cells per population included in the analysis. (c) Unbiased clustering of nuclei isolated at 1 hr after the start of NE. (d) Arc expression in nuclei isolated at 1 hr identifies active clusters. (e) Mature Active cells preferentially localize to clusters 1 and 4. Immature Active cells are more broadly distributed across clusters despite isolation based on ARC protein expression. (f) Unbiased clustering of nuclei isolated at 4 hr after the start of NE. (g) Arc expression in nuclei isolated at 4 hr identifies active clusters. (h) Mature Active cells preferentially localize to clusters 1 and 3. Immature Active cells are more broadly distributed across clusters despite isolation based on ARC protein expression. DG, dentate granule; DGC, dentate granule cell; NE, novel environment.

FIGURE 3

Immature DG neurons show activity-related changes in transcription of only a subset of genes observed in mature. (a) Number of differentially expressed genes (DEGs) between Active and Inactive nuclei based on maturation status and time point, for example, 1-hr immature indicates the number of DEGs between Immature Active and Immature Inactive cells isolated at 1 hr after the start of NE. Coral bar indicates genes shared between 1-hr immature and 1-hr mature comparisons. Blue bar indicates genes shared between 4-hr immature and 4-hr mature comparisons. (b) Log2 fold change (logFC) between active and inactive nuclei of DEGs common to both immature and mature nuclei from A (ImmMatCommon genes). (c) FC of DEGs uniquely identified in mature, but not immature, active, and inactive nuclei comparisons (Uniq genes). DEGs detected in mature cells at both 1 and 4 hr after the start of NE are labeled in red. No DEGs were uniquely identified in immature cells. (d) GO terms significantly enriched among DEGs uniquely identified in mature cells 4 hr after activation. (e) Violin plots of DEGs presented in both “postsynaptic membrane” and “postsynaptic density” terms from (d). (f) Model for contribution of immature DGCs to activity-dependent changes (created with BioRender.com). Mature cells respond by engaging cell intrinsic mechanisms of transcriptional change, whereas physiologically hyperactive immature cells may contribute more indirectly by influencing other cells. DG, dentate granule; DGC, dentate granule cell; NE, novel environment.