Figures and data
Task associated DG labeled neurons show consistent activation of 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). C-D) Representative epifluorescence image of a section from mice one 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. * indicates p<0.05, *** indicates p=0.0003 by Nested t-test, n=4 subjects/treatment. Schematics were generated using BioRender under license.
Intrinsic differences in frequency adaptation distinguish labeled SGCs.
A-B) Representative images of a biocytin filled GC (A) with a narrow dendritic arbor and a smaller somatic width and a 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 TW-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 −200pA current injections (E) and +400pA current injection (F) in a 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 3Way ANOVA n=9-22 cells/group. H-J) Summary plots of firing frequency at 520pA 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 400pA (I) and spike frequency adaptation (J). # indicates p<0.05, ## p<0.01 for main factor cell type by TW-ANOVA and ** i ndicates p<0.01 for labeled versus unlabeled within cell type by Šídák’s multiple comparisons post hoc test in n=8-19 cells/group.
Tagged DG neurons do not support mutual excitatory drive.
A) Schematic showing dual patch clamp recording from labeled (green) GC-SGC pair. 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 gray scale and inverted and are overexposed to emphasize the intact axonal arbors in the recorded pair. D) Presence of spontaneous 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 a SGC recorded in voltage clamp (top).
Evidence for 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-SGC and Unlabeled-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. 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 EPSCs (F) and 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).
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 EPSCs (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; cooccurrence 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 2xSD = 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 2xSD = 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 2xSD = 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 2xSD = 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; TW-ANOVA with Šídák’s multiple comparisons post hoc tests.
