Extended Interneuronal Network of the Dentate Gyrus

Abstract

Local interneurons control principal cells within individual brain areas, but anecdotal observations indicate that interneuronal axons sometimes extend beyond strict anatomical boundaries. Here, we use the case of the dentate gyrus (DG) to show that boundary-crossing interneurons with cell bodies in CA3 and CA1 constitute a numerically significant and diverse population that relays patterns of activity generated within the CA regions back to granule cells. These results reveal the existence of a sophisticated retrograde GABAergic circuit that fundamentally extends the canonical interneuronal network.

Keywords: GABA; hippocampus; inhibition; network.

Figure 1. Extrinsic interneurons located in CA3 and CA1 form a significant portion of GABAergic inputs to dentate granule cells (GCs)

(A) Representative confocal image from the dentate gyrus (DG) of an animal that received injections of Syn-GTR at P7 and RbV-mCh at P126, and was euthanized 7 days later. GFP⁺/mCh⁺ cells (yellow) represent ‘starter’ DG GCs that were born at P7 (white arrows). Cells that are mCh⁺ only (in red, denoted by yellow arrowheads) represent the first-order presynaptic inputs onto the starter GCs. Cell nuclei are visualized with bisbenzimide (blue). (B–C) Representative confocal images from the DG, CA3 and CA1 showing cells that are double positive for mCh and either PV or SOM. These cells provide monosynaptic inputs from DG, CA3 and CA1 onto neonatally-born DG GCs. (D) Quantification of the percentages of PV⁺ or SOM⁺ interneurons that arise from the DG, CA3 or CA1 and are monosynaptically connected to starter DG GCs (n=6 animals). Bars indicate means. Scale bars: 100μm (A) 25μm (B–C). For juxtacellularly labelled DG_INT and DG_EXT cell reconstructions see also Figs S1 and S2.

Figure 2. Both perisomatic and dendritic inhibition in the dentate gyrus (DG) are modulated by sharp wave-ripples (SWRs)

(A–B) Examples of perisomatic-targeting and dendrite-targeting cells. (A) Fast spiking basket cell (FSBC); (B) Total molecular layer innervating cell. Red: axon; black: soma and dendrite. Schematic drawings indicate the axonal coverage of the interneurons (red). (GC: granule cell layer; ML: molecular layer; Pyr: pyramidal layer). (C–D) Example traces showing SWR-modulated firing of cells in panels A and B. From top to bottom: local field potential, isolated action potentials, field potential recorded in CA1 pyramidal layer. (E) Significantly increased firing rate inside SWRs for PV perisomatic-targeting cells (in black, *P=0.041, t= −3.268, DF=2; n=3 for perisomatic-targeting cells) and at SOM dendrite-targeting cells (in red, *P=0.023, t=−4.547, DF=2, n=3 for SOM dendrite-targeting cells, paired t test; bars indicate means). All cells were significantly modulated individually as well (asterisks next to circles, P<0.05, see Methods). Note that out of five additional dendrite-targeting cells, one NOS cell had similarly increased firing during SWRs (#6 in Fig S1; outside SWR: 0.26Hz; inside SWR: 1.49Hz; see also Fig S3D, P<0.05, see methods), two neurochemically unidentified hilar neurons (n.t.; e.g. #3 in Fig S1E, see also Fig S3C) showed strongly reduced firing (outside SWR: 3.2Hz and 11.3Hz, inside SWR: 0Hz and 0Hz, respectively, P<0.05, see methods), two cannabinoid type I (CB1) receptor expressing cells (#5 in Fig S1B) were not significantly modulated (data not shown). (F–H) Example traces showing dentate spike-modulated firing of the same two cells shown on A and B. Top: local field potential; bottom: isolated action potentials. (H) Significantly increased firing rate of perisomatic- and dendrite-targeting cells inside dentate spikes (*P=0.01, t=−6.943, DF=2, n=3 and *P=0.016, t=−3.231, DF=4, n=5, respectively; paired t test; dendrite-targeting cells include two n.t. hilar neurons; mean of all dendrite-targeting cells indicated by red bars).

Figure 3. Extrinsic GABAergic sources of the DG are modulated by sharp wave-ripples (SWRs)

(A–D) Top: examples of extrinsically located cells expressing PV, SOM, CB1 or NOS (RAD: str. radiatum; LM: str. lacunosum-moleculare; ML: DG molecular layer; GS: granule cell layer; pyr: pyramidal layer). Red: axon; black: soma and dendrite. Bottom: example traces showing SWR-modulated firing of the corresponding cells. (E) Schematic summary of intrinsic and extrinsic sources of GABA in the DG. Color labels indicate neurochemical content and the lengths of arrows indicate the source (short arrow=DG_INT; long: DG_EXT). Cell numbers (#) are referring to cells shown in A–D and Figs S1and S2. Note that cell #8 projected to the hilus (see Fig S2E); n.t.: neurochemical identity not tested. (F) Significantly increased firing rate of extrinsic cells during (inside) versus outside SWRs (P=0.047, t=1.868, DF=9, n=10; paired t test). All cells were significantly modulated individually as well (asterisks next to filled circles, P<0.05, see Methods). (G) DG_EXT cells show stronger SWR-related modulation than DG_INT neurons (median[1st quartile, 3rd quartile]; DG_INT: 100[58.2, 268.6]; DG_EXT: 177.9[98.8, 1451.6]). P=0.04893, n=21, Z =−1.656, Mann-Whitney U test. Bars indicate medians.

Figure 4. Temporal dynamics of SWR-dependent modulation of interneuronal firing

(A–C) Example raster plots (black dots) and spiking rate curves (in color) showing three typical patterns. Note the right-shifted positive peak with respect to SWR center (cyan bar) on B and C. (D) Three groups of interneurons whose discharges prefer distinct temporal windows relative to SWRs. Inset: summary of time of peak firing (vertical dashed lines on main panel) with respect to the middle of the SWRs (bars indicate median values): FSBCs+SOM (brown) 1.5[1^st quartile: −8, 3^rd quartile: 22.5]; NOS (blue): 98.5[56, 132]; CB1&n.t. (green) 296.5[285.5, 307.5] (P=0.0131, KW-ANOVA test, DF=2, n=16; completed with Mann-Whitney U test; *P=0.023, 0.017 and 0.041 (n=9, 2 and 5; Z=−2.00, −2.13 and 1.74) for brown vs. blue, brown vs. green and blue vs. green, respectively). (E) Example raster plot and spiking rate curve of an axo-axonic (chandelier) cell with a reduction in the firing rate that precedes SWRs by >1sec. Note the different time scale compared to C. (F) Experimental design for panels G and H. (G) Representative example of reduced firing rate when the strong current injection delivered through the juxtacellular pipette co-occurred with a SWR. (H) Significantly decreased spiking rate during SWRs in the experiments indicated in panels F and G. n=5, P=0.016, t=4.012, DF=4; paired t test.