Cellular and circuit features distinguish mouse dentate gyrus semilunar granule cells and granule cells activated during contextual memory formation
- Biomedical Sciences Graduate Program,University of California Riverside, United States
- Department of Molecular, Cell and Systems Biology, University of California Riverside, United States
- Neuroscience Graduate Program, University of California Riverside, United States
- Department of Psychology, University of California Riverside, United States
Figures
Figure 1 with 1 supplement
Task-associated dentate gyrus (DG) labeled neurons show consistent activation of semilunar granule cells (SGCs) and paradigm-specific reactivation.
(A–B) Schematic of experimental timeline for animals trained in the Barnes maze (BM) task followed by exposure to enriched environment (EE), the BM-EE cohort (BM) group (A) and mice housed in EE followed by reintroduction of EE, the EE-EE cohort (EE) group (B), created with BioRender.com. (C–D) Representative epifluorescence image of a section from mice 1 week after induction of tdT labeling (Ci, Di) following BM testing (C) or EE testing (D) and c-Fos immunostaining (Cii, Dii) following subsequent EE exposure. (E–F) Quantification of number of tdT-labeled cells per slice (E) and summary of proportion of tdT-labeled cells in the upper blade of the DG per slice (F). (G) Summary of proportion of tdT cells co-labeled with c-Fos (green). (H) Representative TRAP-tdT section showing distinct SGC morphology (white arrowhead). (I) Plot of % of tdT cells that had morphology consistent with SGCs.Data are presented as mean ± SEM. * indicates p<0.05, *** indicates p=0.0003 by nested t-test, n=4 subjects/treatment.
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Figure 1—source data 1
Data underlying Figure 1E, F, G and I.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Search strategies adopted in the Barnes maze task.
(A–B) Plot of primary latency (A) and number of errors (B) to find the escape hole over training days. (C) Visualization of search strategies used in the Barnes maze paradigm. (D) Summary cognitive score based on search strategy. Data are presented as mean ± SEM based on data from 4 mice/group.
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Figure 1—figure supplement 1—source data 1
Data underlying Figure 1—figure supplement 1A-D.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
Intrinsic differences in frequency adaptation distinguish labeled semilunar granule cells (SGCs).
(A–B) Representative images of a biocytin-filled granule cell (GC) (A) with a narrow dendritic arbor and a smaller somatic width and an SGC (B) with wide dendritic span, greater somatic width than height, and axonal projections throughout the molecular and granule cell layer (arrowheads). Maximum intensity projections of confocal image stacks are presented as gray scale, inverted images. (C–D) Summary plots of resting membrane potential (RMP in C) and input resistance (Rin in D) between labeled and unlabeled GCs and SGCs. # indicates p<0.05 for main factor cell type by two-way ANOVA and * indicates p<0.05 for labeled versus unlabeled within cell type by Šídák’s multiple comparisons post hoc test in n=11–19 cells/group. (E–F) Representative cell membrane voltage traces in response to +120 and –200 pA current injections (E) and +400 pA current injection (F) in GC (top) and SGC (bottom). (G) Summary plot of firing frequency in response to increasing current injections in labeled and unlabeled SGCs and GCs. #### indicates p<0.0001 for main factor cell type by three-way ANOVA, n=9–22 cells/group. (H–J) Summary plots of firing frequency at 520 pA compared to max frequency (H), spike amplitude attenuation calculated as ratio between the amplitude of the 15th spike and 1st spike at a current injection of 400 pA (I) and spike frequency adaptation (J). # indicates p<0.05, ##p<0.01 for main factor cell type by two-way ANOVA and ** indicates p<0.01 for labeled versus unlabeled within cell type by Šídák’s multiple comparisons post hoc test in n=8–19 cells/group.
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Figure 2—source data 1
Data underlying Figure 2C, D, G, H, I and J.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Active properties of labeled and unlabeled granule cells (GCs) and semilunar granule cells (SGCs).
(A) Pie chart showing the proportion of labeled and unlabeled GCs and SGCs included for analysis of active membrane properties. Note the greater proportion of SGCs represented among labeled neurons. (B–G) Summary histograms of threshold of action potential (B), amplitude (C), half-width (D), fast (E) and medium afterhyperpolarizations (F) and latency (G). Data are presented as mean ± SEM.
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Figure 2—figure supplement 1—source data 1
Data underlying Figure 2—figure supplement 1A-G.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig2-figsupp1-data1-v1.xlsx
Figure 3
Tagged dentate gyrus (DG) neurons do not support mutual excitatory drive.
(A) Schematic showing dual patch clamp recording from labeled (green) granule cell (GC)-semilunar granule cell (SGC) pair. Created in BioRender.com. (B) Summary breakdown of cell-type-specific connections tested in dual recordings from labeled neurons. (C) Representative maximum intensity projection of a confocal image stack of a pair of biocytin-filled SGC (left) and GC (right). Images are grayscale and inverted and are overexposed to emphasize the intact axonal arbors in the recorded pair. (D) Presence of spontaneous excitatory postsynaptic currents (EPSCs) in the SGC-GC pair in E–G to verify the presence of excitatory inputs and a healthy circuit. (E) Light-evoked inward currents validate expression of ChR2 in labeled cell pair. (F) Representative traces from a labeled SGC and labeled GC show that depolarization-induced firing in SGC (top) failed to evoke EPSCs in a GC (bottom) recorded in voltage clamp. Individual traces are in gray with average trace overlaid in black. (G) Depolarization-induced firing in GC (bottom) fails to evoke EPSCs in an SGC recorded in voltage clamp (top).
Figure 4 with 1 supplement
Evidence for dentate gyrus (DG) engram neurons supporting sparse feedback inhibition onto non-engram neurons.
(A–C) Representative confocal image of eYFP-labeled neurons in a TRAP-ChR2-eYFP mouse (A) shows biocytin staining (B) in a pair of recorded labeled-semilunar granule cell (SGC) and unlabeled-granule cell (GC). Note co-labeling for eYFP and biocytin in the SGC, while the GC does not colocalize eYFP (C). (D) Summary of cell-type-specific connections tested in dual recordings from labeled and unlabeled neurons. Inset depicts a schematic showing dual patch clamp recording from a labeled (green) SGC and an unlabeled (blue) GC pair. Created with BioRender.com. (E) Light-evoked currents validate the expression of ChR2 in the labeled-SGC and lack of response in the unlabeled-GC. (F–G) Representative traces from a labeled-SGC and an unlabeled-GC show that depolarization-induced firing in the labeled-SGC (top) failed to evoke excitatory postsynaptic currents (EPSCs) (F) and inhibitory postsynaptic currents (IPSCs) (G) in the unlabeled-GC. (H) Schematic of recording configuration illustrated wide-field optical illumination with labeled neurons (green), unlabeled neurons (blue), and local circuit interneuron (yellow). (I) Example traces from a recording in which wide-field optical stimulation evoked inhibitory responses in the unlabeled-GC and firing in the labeled-SGC. Note that the SGC firing by depolarization in the absence of light failed to elicit IPSCs in the same GC. (J–K) Schematic with labeled-GC (green), unlabeled-SGC (blue), and local circuit interneuron (yellow) (J) and traces from a recorded pair where depolarization of a labeled-GC elicited inhibitory responses in an unlabeled-SGC (K). Panels H and J were created with BioRender.com.
Figure 4—figure supplement 1
Robust feedback inhibition in response to focal activation of a random cohort of granule cells.
(A) Example of optically evoked inhibitory postsynaptic currents (IPSCs) in slices from mice injected with AAV5-CaMKIIa-hChR2(H134A)-EYFP in response to activation of three progressively smaller regions of interest (ROIs), the largest spanning the granule cell layer. Inset: Schematic of ROI selection in the dentate gyrus (DG). (B) Summary plot of excitatory postsynaptic current (eIPSC) amplitude in response to optical activations of the three ROIs. Data are presented as mean ± SEM. p-Values indicated in the one-way ANOVA, with a significance threshold set at p<0.05. Recordings were obtained from 5–8 cells from 3 mice.
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Figure 4—figure supplement 1—source data 1
IPSC ampltide data used to generate plots in Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig4-figsupp1-data1-v1.xlsx
Figure 5 with 1 supplement
Labeled granule cells (GCs) and semilunar granule cells (SGCs) receive more frequent spontaneous excitatory inputs than unlabeled cells.
(A–B) Representative images of a biocytin-filled unlabeled GC (left panel) and SGC (right panel) (A) and image of a slice in which an unlabeled GC was recorded alongside a labeled GC and SGC. (B) Inset in B shows biocytin fill, tdT labeling, and merge of the somata to illustrate co-labeling. (C) Representative current traces illustrate spontaneous excitatory postsynaptic currents (sEPSCs) in an unlabeled (top) and labeled (bottom) GC. Panels to the right: Representative average sEPSCs trace. (D) Cumulative probability plot of sEPSC inter-event interval (left panel) and amplitude (right panel) in labeled (black) and unlabeled (blue) GC. (E) Representative current traces illustrate sEPSCs in an unlabeled (top) and labeled (bottom) SGC. Panels to the right: Representative average sEPSCs trace. (F) Cumulative probability plot of sEPSC inter-event interval (left panel) and amplitude (right panel) in labeled (black) and unlabeled (blue) SGC. p-Value by Kolmogorov-Smirnov test is indicated in the figure, n=5–6 cells/group. Effect size estimate using Cohen’s d is indicated in the plots.
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Figure 5—source data 1
sIPSC interevent interval and amplitude data used to generate Figure 5D and F.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Spontaneous excitatory postsynaptic currents (EPSCs) in dentate granule cells (GCs) include action potential-driven events.
(A) Representative current traces from a GC illustrate spontaneous EPSCs (in aCSF, above) and miniature EPSCs (in tetrodotoxin [TTX], below). (B) Summary of EPSC inter-event interval (IEI) in aCSF and after perfusion of TTX. Data presented as mean ± SEM. *** indicates p=0.0005 by paired t-test.
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Figure 5—figure supplement 1—source data 1
IPSC interevent interval data used to generate Figure 5—figure supplement 1B.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 2 supplements
Correlated spontaneous excitatory inputs to labeled pairs.
(A) Representative confocal image of eYFP-labeled and biocytin-stained neurons in a TRAP-ChR2-eYFP mouse. (B) Schematic for labeled-labeled (L–L) dual recordings with representative example of spontaneous excitatory postsynaptic currents (sEPSCs) in an L-L pair below. (C) Schematic for labeled-unlabeled (L–U) dual recordings with representative example of sEPSCs in an L-U pair below. (D) Schematic for session-wise cross-correlation profiles (CCPs) defined by correlations exceeding a 2 standard deviation (SD) threshold above the total mean correlation: EPSC peri-occurrence was tested as event time CCP exceeding threshold within full detection window; co-occurrence was defined as event time CCP exceeding threshold within center bin of detection window. (E) CCP from recordings from L-L pairs analyzed with ±100 ms detection window (bright blue, n=7). Overlaid jittered data (black) was developed by appending the event timing of one cell with a randomized lead/lag of ±0.5 s for 100 iterations (top panel). Inset: Plot of maximum correlations (Rmax) in relation to the dashed line representing 2×SD = 0.15. CCP in recordings from L-U pairs analyzed with ±100 ms detection window (dark blue, n=8). Corresponding jittered data, developed as detailed above, is overlaid in black (bottom panel). Inset: Plot of Rmax in relation to the dashed line representing 2×SD = 0.15. (F) CCP from sessions with recordings from L-L pairs analyzed with ±50 ms detection window from L-L pairs (bright purple, n=7) with jittered data developed as detailed above is overlaid in black (top panel). Inset: Plot of Rmax in relation to the dashed line representing 2×SD = 0.10. CCP from recordings in L-U pairs analyzed with ±50 ms detection window (dark purple, n=8) with corresponding jittered data overlaid in black (bottom panel). Inset: Rmax in relation to the dashed line representing 2×SD = 0.10. (G) Comparison of center bin correlation between L-L versus L-U pairs in aligned (align-recorded) versus jittered (Jitter-simulated) data, analyzed using ±100 ms detection window (left, colors as in E) and using ±50 ms detection window (right, colors as in F). (H) Center bin classifier performance (solid line) compared to chance performance (dashed line, colors as in E and F, respectively) plotted as area under the receiver operating characteristic (ROC) curve (AUROC) between L-L (true positive rate) and L-U (false positive rate) for analysis using ±100 ms detection window (left panel) and for analysis using ±50 ms detection window (right panel). Data presented as mean ± SEM (dual recording sessions), * indicate p<0.05, ** indicates p<0.01; *** indicates p<0.001, **** indicates p<0.0001; two-way ANOVA with Šídák’s multiple comparisons post hoc tests. Panels B and C were created with BioRender.com.
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Figure 6—source data 1
Data used to generate Figure 6E, F and H.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Labeled-labeled (L-L) and labeled-unlabeled (L-U) sessions do not differ in event rates.
(A) Spontaneous excitatory postsynaptic current (sEPSC) event counts (L-L n=14, L-U n=16), (B) recording durations (L-L n=7, L-U n=8), and (C) event frequency (L-L n=14, L-U n=16) for data used in correlation analysis. Data presented as mean ± SEM. (D–E) Distribution of average sEPSC inter-event interval (D) and amplitude (C) in labeled and unlabeled GCs and semilunar granule cells (SGCs) included among the L-L and L-U pairs. Data are presented as mean ± SEM.
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Figure 6—figure supplement 1—source data 1
Raw data for plots in Figure 6—figure supplement 1B and C.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Example spontaneous excitatory postsynaptic current (sEPSC) cross-correlation profiles (CCPs).
(A) Representative CCP for labeled to labeled (L-L) dual recording session with maximum correlation in the center bin (co-occurrence). (B) Representative CCP for labeled to unlabeled (L-U) dual recording session with no maximum correlation within detection window (no coincidence, same session as in Figure 5C). (C) Representative CCP of sEPSCs for L-L dual recording session with maximum correlation within detection window (peri-occurrence, same session as in Figure 5B). (D–E) Examples of EPSC cross-correlation histograms generated using the CCP method (above) and using the MATLAB cross-correlation function xcorr (below).
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Figure 6—figure supplement 2—source data 1
Raw data used to generate plots in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/101428/elife-101428-fig6-figsupp2-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J | The Jackson Laboratory | MSR_JAX: 000664 | Male and female |
| Strain, strain background (Mus musculus) | Fos-creer (Fostm1.1(Cre/Ert2)Luo/J) | The Jackson Laboratory | JAX Stock: 030323 | Male and female |
| Strain, strain background (Mus musculus) | B6;129S6-Gt(ROSA) 26Sor t m14(CAG-tdTomato) Hze/J | The Jackson Laboratory | JAX Stock: 007908 | Male and female |
| Strain, strain background (Mus musculus) | B6;129S-Gt(ROSA)26Sortm32(CAGB6;129S-Gt(ROSA)COP4*H134R/EYFP)Hze/J | The Jackson Laboratory | JAX Stock: 12659 | Male and female |
| Strain, strain background (AAV) | AAV5-CaMKIIa- hChR2(H134A)-EYFP | Addgene | Plasmid # 26969 | |
| Antibody | c-Fos (9f6) rabbit antibody (Monoclonal) | Cell Signaling Technology | 2250S /RRID:AB_2247211 | 1:750 |
| Antibody | Goat anti-rabbit Alexa Fluor 488 secondary antibody (Polyclonal) | Abcam | 150077/ RRID:AB_2630356 | 1:500 |
| Antibody | Chicken Anti-Green Fluorescent Protein Antibody (Polyclonal) | Aves Labs | AB_2307313 | 1:500 |
| Antibody | Goat anti-Chicken Alexa Fluor 488 (Polyclonal) | Abcam | 150169/ RRID:AB_2636803 | 1:500 |
| Chemical compound, drug | 4-Hydroxytamoxifen | Sigma | H7904-25MG | |
| Chemical compound, drug | Tetrodotoxin | Tocris | Tetrodotoxin | Male and female |
| Chemical compound, drug | Gabazine-SR95531 | Tocris | SR95531 | Male and female |
| Software, algorithm | Easy Electrophysiology | Easy Electrophysiology Ltd | v2.6.3 | https://www.easyelectrophysiology.com/ |
| Software, algorithm | BUNS analysis software | Illouz et al., 2016 | http://okunlab.wix.com/okunlab | |
| Software, algorithm | pClamp10-Data Acquisition | Molecular Devices | https://www.moleculardevices.com | |
| Software, algorithm | Anymaze | Stoelting Co. | https://www.any-maze.com/ | |
| Software, algorithm | MATLAB | MathWorks | R2024a | |
| Software, algorithm | Prism 10 | GraphPad | 10.4.1 | |
| Other | Custom code for correlation analysis | VijiSanthakumarLab, 2025 | https://github.com/VijiSanthakumarLab/eLife_Correlation_Cells_2025 | |
| Other | Alexa-594 streptavidin conjugate | Thermo Fisher | S11227 | 1:1000 |
| Other | Vectashield | Vector Labs | NC9524612 | |
| Other | Goat serum | Sigma | SIAL-G6767-100ML | |
| Other | Barnes maze table | Maze Engineers | https://conductscience.com/maze/ |
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Cellular and circuit features distinguish mouse dentate gyrus semilunar granule cells and granule cells activated during contextual memory formation
eLife 13:RP101428.
https://doi.org/10.7554/eLife.101428.3
