Development of early-born γ-Aminobutyric acid hub neurons in mouse hippocampus from embryogenesis to adulthood

Abstract

Early-born γ-aminobutyric acid (GABA) neurons (EBGNs) are major components of the hippocampal circuit because at early postnatal stages they form a subpopulation of "hub cells" transiently supporting CA3 network synchronization (Picardo et al. [2011] Neuron 71:695-709). It is therefore essential to determine when these cells acquire the remarkable morphofunctional attributes supporting their network function and whether they develop into a specific subtype of interneuron into adulthood. Inducible genetic fate mapping conveniently allows for the labeling of EBGNs throughout their life. EBGNs were first analyzed during the perinatal week. We observed that EBGNs acquired mature characteristics at the time when the first synapse-driven synchronous activities appeared in the form of giant depolarizing potentials. The fate of EBGNs was next analyzed in the adult hippocampus by using anatomical characterization. Adult EBGNs included a significant proportion of cells projecting selectively to the septum; in turn, EBGNs were targeted by septal and entorhinal inputs. In addition, most EBGNs were strongly targeted by cholinergic and monoaminergic terminals, suggesting significant subcortical innervation. Finally, we found that some EBGNs located in the septum or the entorhinal cortex also displayed a long-range projection that we traced to the hippocampus. Therefore, this study shows that the maturation of the morphophysiological properties of EBGNs mirrors the evolution of early network dynamics, suggesting that both phenomena may be causally linked. We propose that a subpopulation of EBGNs forms into adulthood a scaffold of GABAergic projection neurons linking the hippocampus to distant structures. J. Comp. Neurol. 524:2440-2461, 2016. © 2016 Wiley Periodicals, Inc.

Keywords: AB_10000240; AB_10550535; AB_2270299; AB_2302603; AB_2531897; AB_477329; AB_887864; inducible genetic fate mapping; long-range projecting GABA neuron; nif-0000-10294; operational hub.

Figure 1

Morphological features of EBGNs in developing hippocampus. Sparse fate‐mapped neurons are visible in the developing hippocampus in a Dlx1/2 ^CreERTM/RCE:LoxP mouse tamoxifen treated at E7.5. A: E18.5; most of the EBGNs have a fusiform soma with a poorly developed dendritic tree (arrows). B: P0; The dendritic (single arrow) and axonal (double arrow) arborizations of EBGNs increase and become more complicated. C: P3; EBGNs show a conspicuous development of the dendritic (single arrow) and axonal (double arrow) arborizations; several axons are seen running through the fimbria (double arrow). D: Filopodia (single arrows) are visible at the soma and dendrites of an EBGN at P3. Scale bars = 200 μm in A–C; 10 μm in D.

Figure 2

Development of the morphometric properties of EBGNs in CA3 during the perinatal period. A: Composite drawings of neurobiotin‐filled interneurons reconstructed with the Neurolucida workstation. The somatodendritic domains of EBGNs are represented in black. The axons are in red; note that some axons are leaving the hippocampus via the fimbria as early as E18.5. H, hilus; sl, stratum lucidum; slm, stratum lacunosum moleculare; sp, stratum pyramidale; so, stratum oriens; sr, stratum radiatum. B: Normalized distribution graphs of the fraction of axonal intersections with concentric circles of increasing radius (Sholl analysis, 20‐μm steps) centered at the soma for the cell populations described in A. Scale bar = 100 μm.

Figure 3

Perisomatic KCC2 expression in hippocampal EBGNs. Immunolabeling for KCC2 is visible along the membrane (single arrows) of EBGNs (asterisks) in CA1 stratum oriens (so) at P3 (A) and in adulthood (B; P45). Note the presence of similar labeling in neighboring neurons (double arrows in B) only at P45. sp, Stratum pyramidale. Scale bars = 20 μm at left; 10 μm at right.

Figure 4

Maturation of correlated neuronal activities in the CA3 region during the perinatal period. A: Two‐photon calcium fluorescence image of the CA3 region of a mouse hippocampal slice at P3. B: Automatically detected contours of cells from the calcium fluorescence image (A1); silent cells (open contours); active cells (solid contours) could display SPAs (red), GDPs (blue), or both (orange). C: Calcium fluorescence traces as a function of time of six active cells from the movie illustrated in A showing calcium plateau characteristics of SPAs (red) or fast calcium transients associated with GDPs (blue). D: The percentage of active cells remains constant throughout the investigated ages. E: Representative histograms showing the percentage of imaged cells detected as being active at each movie frame from E18.5 to P7 (time resolution 100 msec). There is a clear increase in the frequency of GDPs (peaks of synchrony; threshold is 5% of coactive cells) and in their amplitude (i.e. percentage of coactive cells at peaks of synchrony). F: Graph indicates the fraction of SPA cells (red) as well as the frequency of GDPs (blue) for five successive age groups. Error bars indicate SEM. All data were obtained from n = 9 movies/n = 6 mice at E18.5; 6/2 at P0–P1; 13/5 at P3; 4/2 at P5; 26/12 at P7.

Figure 5

Parallel evolution of the properties of EBGN and and the rate of GDPs from embryogenesis to adulthood. Previously reported data were normalized and fitted using ad hoc functions (least square algorithm). Hyperbolic tangent for KCC2 (r² = 0.99) and for Cm (membrane capacitance; r² = 0,84), Gaussian (r² = 0.97) from E18 until P7 then extrapolated for later ages for GDPs, exponential for sEPSPs (r² = 0.99) and for Rm (membrane resistance, r² = 0.98). Axonal length was measured only during the perinatal period. It was fitted using a parabola (r² = 0.86).

Figure 6

Distribution of EBGNs in the adult mouse hippocampus. Sparse EGBNs are visible in CA1, CA3 and the hilus (H) of the dentate gyrus (arrows in A,B).They have horizontally or vertically oriented dendrites (double arrowheads in A). Their axons are found in all sublayers (e.g., double arrows in B). Numerous EGFP‐labeled EGBNs' axons are observed in the fimbria (arrows in C). Electron microscopic view of the fimbria (D) showing a myelinated EBGN axon (arrows in D) following EGFP immunoperoxydase treatment. This axon is running along with unlabeled myelinated axons (asterisks). DG, dentate gyrus; H, hilus; sl, stratum lucidum; slm, stratum lacunosum moleculare; sp, stratum pyramidale; so, stratum oriens; sr, stratum radiatum; Sub, subiculum. Scale bars = 100 μm in A,B; 25 μm in C, 10 μm in D.

Figure 7

A subpopulation of EBGNs projects to the septum. A,B: Two retrogradely labeled EBGNs are in the stratum oriens of CA3 (arrows in A) and CA1 (arrows in B) in the hippocampus after injection of fluorogold (FG) in the septum. Both are immunopositive for somatostatin (SOM); one is also labeled for mGluR1α; one is negative for parvalbumin (PV). C: Histogram showing the fraction of hippocamposeptal EBGNs (HS‐EBGNs) immunoreactive for the various neurochemicals tested in comparison with the entire EBGN population. Note that 100% of HS‐EBGNs are positive for SOM. D: Histogram of the distribution of EBGNs and HS‐EBGNs along the rostrocaudal axis of the hippocampus. E: Histogram showing that the percentage of HS‐EBGNs among the EBGNs population decreases in hippocampal sublayers from the rostral to the caudal hippocampus; note that HS‐EBGNs represent half of the EBGNs in the rostral CA1 so. F: Histogram of the distribution of EBGNs and HS‐EBGNs in the various hippocampal areas and their sublayers. Alv, alveus; CB, calbindin; DG, dentate gyrus; gl, granular layer; H, hilus; ml, molecular layer; sl, stratum lucidum; slm, stratum lacunosum moleculare; sp, stratum pyramidale; so, stratum oriens; sr, stratum radiatum. *P < 0.05 as given by nonparametric bootstrap method. Scale bars = 50 μm.

Figure 8

Septal and entorhinal afferents to EBGNs. A–E: Injection of Phaseolus vulgaris (PhaV) within the medial septum results in anterograde labeling of numerous fibers within the hippocampus. Numerous septal fibers are found in the hippocampus at the intermediate level in the stratum oriens of CA1 (arrows in A) and the hilus (H, arrows in A). B and D are confocal maximal‐intensity z stacks showing EBGNs in the stratum oriens of CA1 (B) and CA3 (D) targeted by anterogradely labeled septal fibers (arrows) on their soma and dendrites (outlined areas in C and E; C and E are high‐magnification single optical section from B and D z stacks, respectively). F–J: PhaV injection in the entorhinal cortex results in the anterograde labeling of numerous fibers within the stratum lacunosum moleculare (slm) of CA1 and CA3 (arrows in F); labeled fibers are also found in the stratum oriens (so) of CA1 (G). Confocal maximal‐intensity z stacks showing EBGNs in the stratum oriens of CA3 (arrows in G) and in the stratum radiatum of CA1 (arrow in I) targeted by entorhinal afferent fibers on dendrites and soma (outlined areas, H and J are high‐magnification single optical section from G and I z stacks, respectively). alv, Alveus; DG, dentate gyrus. Scale bars = 1 mm in A,F; 50 μm in B,D,G,I; 10 μm in H,J.

Figure 9

Subcortical afferents to EBGNs. A: EBGNs are covered by cholinergic fibers labeled for the vesicular acetylcholine transporter (VAchT). B–D: EBGNs are contacted by monoaminergic fibers (vesicular monoamine transporter VMAT2) on their soma (outlined areas, C) and dendrites (outlined areas, D). Scale bars = 10 μm in A–C; 5 μm in D.

Figure 10

EBGNs form a subpopulation of GABA long‐range projecting neurons. A–C: Numerous EBGNs are observed in the medial septum (MS) and in the horizontal diagonal band (HDB). One of them (single arrows in A,C) is retrogradely labeled after hippocampal fluorogold injection (FG). It is not positive for parvalbumin (PV; single arrows in C), although other retrogradely labeled neurons are positive (double arrow in C). Note the presence of EBGN medium spiny neurons in the caudate putamen (CPu, in A,B). D–F: EBGNs are present in the entorhinal cortex (Ent Cx); two of them (single arrows in D–F) are retrogradely labeled; one is positive for PV (arrows in E). Note the presence of a neighboring PV retrogradely neuron (double arrows in F). ac, Anterior commissure; cc, corpus callosum; DG, dentate gyrus. Scale bars = 500 μm in A,D, 100 μm in B,C; 10 μm in E,F.

Figure 11

Hippocampal afferents to septal EBGNs. A: PhaV injection in CA1 of dorsal hippocampus results in the presence of labeled fibers within the medial septum (MS) and the ventral diagonal band nucleus (VDB) beside septal EBGNs (arrow in A). B: One hippocampal fiber contacts a septum EBGN (arrow in B and inset). Scale bars = 500 μm in A; 50 μm in B; 5 μm in inset.