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. 2017 Sep 7;7(1):10903.
doi: 10.1038/s41598-017-11268-z.

Running Reorganizes the Circuitry of One-Week-Old Adult-Born Hippocampal Neurons

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Free PMC article

Running Reorganizes the Circuitry of One-Week-Old Adult-Born Hippocampal Neurons

Nirnath Sah et al. Sci Rep. .
Free PMC article

Abstract

Adult hippocampal neurogenesis is an important form of structural and functional plasticity in the mature mammalian brain. The existing consensus is that GABA regulates the initial integration of adult-born neurons, similar to neuronal development during embryogenesis. Surprisingly, virus-based anatomical tracing revealed that very young, one-week-old, new granule cells in male C57Bl/6 mice receive input not only from GABAergic interneurons, but also from multiple glutamatergic cell types, including mature dentate granule cells, area CA1-3 pyramidal cells and mossy cells. Consistently, patch-clamp recordings from retrovirally labeled new granule cells at 7-8 days post retroviral injection (dpi) show that these cells respond to NMDA application with tonic currents, and that both electrical and optogenetic stimulation can evoke NMDA-mediated synaptic responses. Furthermore, new dentate granule cell number, morphology and excitatory synaptic inputs at 7 dpi are modified by voluntary wheel running. Overall, glutamatergic and GABAergic innervation of newly born neurons in the adult hippocampus develops concurrently, and excitatory input is reorganized by exercise.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Circuitry tracing of one-week-old dentate granule cells in control and running mice. (A) Dorsal and ventral dentate gyrus injection of retrovirus (RV-SYN-GTRgp) and rabies virus (EnvA-ΔG-MCh). (B) Timeline of the experiment. (C,D) Photomicrographs of coronal sections derived from (C) control and (D) runner mice showing retrovirally infected cells expressing nuclear green fluorescent protein (GFP+) in the dentate gyrus. (E,F) Photomicrographs showing BrdU+ cells in coronal sections derived from (E) control and (F) runner mice. (G) Representative photomicrograph showing “starter cells” [retrovirus expressing nuclear GFP+- rabies virus expressing cytoplasmic MCh+ double labelled cells (green + red = yellow)] and retrovirally infected cells only (nuclear GFP+ only). Inset shows overview of the labeling. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), blue. (H) Photomicrograph of a coronal section derived from a mouse injected with retrovirus missing Rgp (RV-SYN-HT) followed by rabies virus (EnvA-ΔG-MCh) in the dentate gyrus at the same inter-injection interval. Rabies virus infection did not spread beyond the “starter cells” (GFP+ - MCh+). Inset shows overview of the labeling. (IK) Running increased (I) BrdU+ cell number, (J) GFP+ cell number and (K) “Starter cells” (GFP+ - MCh+) number in the dentate gyrus as compared to controls. (L) Running did not modify the total traced cell (MCh+ only) number. (M) Ratio of connectivity between traced and starter cells was not significantly different between controls and runners. *P < 0.05. Figure 1A: the drawings of the brain sections were modified from ref. , with permission from Elsevier to the Nature Publishing Group to publish these images under an Open Access license.
Figure 2
Figure 2
Monosynaptic inputs to one-week-old adult-born granule cells. (A) Running did not modify the numbers of intra-hippocampal cells [mature granule cells (mGC), interneurons (INT), pyramidal cells (PYR) and mossy cells (MC)]. (B) Ratio of connectivity of intra-hippocampal (mGCs, INT, PYR and MC), subcortical [medial septum (MS) and diagonal band of Broca (DBB)] and cortical [somatosensory, auditory and visual cortex (CTX)] cells to one-week old adult-born GCs. The ratio of connectivity from PYR to starter cells was significantly modified by running. (C) Percentage of contribution per cell type to the initial wiring of immature granule cells in controls and runners. (D) Representative photomicrograph showing GFP+ cells (green), “starter cells” (nuclear GFP+-cytoplasmic MCh+; yellow nucleus) and hippocampal traced cells (MCh+ only, red), including traced CA1 pyramidal cells (PYR) with their characteristic pyramidal shaped soma and apical and basal dendritic arborizations, and a traced interneuron localized in the molecular layer (ML) of the dentate gyrus (DG). Inset shows a traced mature granule cell (mGC) characterized by its elliptical cell body and its apical dendritic tree projecting throughout the entire ML of the dentate gyrus. (E) Traced mossy cells (MC) localized in the hilus of the dentate gyrus, with dendritic branches covered by large thorny excrescences. (F) Traced interneuron localized in the hilus of the dentate gyrus. Nuclei were stained with DAPI (blue). SL-M, stratum lacunosum-moleculare; SR, stratum radiatum; SP, stratum pyramidale; SO, stratum oriens, SUB, subiculum. *P < 0.05.
Figure 3
Figure 3
Subcortical and cortical connectivity to immature adult-born granule cells. (A) Coronal section derived from a control mouse showing traced cells expressing MCh (red) in the medial septum (MS) and horizontal nucleus of the diagonal band of Broca (DBB). MCh+ cells are immunoreactive for choline-acetyl-transferase (ChAT, green). Nuclei were stained with DAPI, blue. (B) Photomicrograph of a coronal section stained for the neuronal marker NeuN (blue), showing traced cells expressing MCh (red) in the visual (VC) and auditory (AC) cortex. Inset shows traced auditory cortex cells.
Figure 4
Figure 4
Running increases arborization of immature adult-born granule cells. (A,B) Retrovirally labeled GFP+ granule cells at 7 dpi from (A) control and (B) runner mice. Insets show the overview of the dentate gyrus. Morphometric analysis revealed that running significantly increases (C) cell body area, (D) total dendritic length, (E) dendritic maximum reach, and (F) mean number of branch points of new granule cells. Sections were co-labeled with neuronal marker NeuN (red). Nuclei were stained with DAPI, blue. *P < 0.05.
Figure 5
Figure 5
Glutamatergic connectivity is functional and NMDA receptor-dependent in adult born granule cells. (A) NMDA (500 μM) evokes an outward current (holding potential, Vh = +50 mV) that could be blocked by NMDA receptor antagonist AP5 (100 μM) in immature GCs (7dpi). (B) Running did not modify the shift in holding current induced by the application of NMDA (500 µM) in GFP+ cells at 7 dpi. (C) Schematic representation of the location of GFP+ recorded cells (dashed box) in acute hippocampal slices. Stimulation was performed in the inner molecular layer (ML) and whole-cell recordings were obtained from GFP+ at Vh = +50 mV. (D) Representative traces of evoked postsynaptic responses under control conditions (normal ACSF; black trace). Application of picrotoxin (PTX), an antagonist of GABAA receptors, reduced the amplitude of the postsynaptic current (purple trace). The remaining current was completely blocked by AP5 (gray trace), an antagonist of the NMDA receptor. (E) Running did not modify the percentage of GABAAR-mediated and NMDAR-mediated currents. (F). Input-output curve of evoked NMDAR-mediated responses of GFP+ cells from control and runner mice. Significant differences between groups are observed at 0.6 and 0.8 mA stimulus intensity. *P < 0.02.
Figure 6
Figure 6
Optogenetic stimulation of dentate gyrus cells induces NMDAR-mediated responses in immature adult-born GCs. (A) Schematic representation of the viral injection. AAV5-hSyn-hChR2-EYFP viral vector was injected into the molecular layer of the dentate gyrus to express ChR2 in hippocampal neurons. Two to 3 weeks later, CAG-RFP retrovirus was injected into the same dentate gyrus to label dividing cells. (B) Photomicrograph of a horizontal section showing robust YFP expression in hippocampal neurons (green) and retroviral expression of RFP in immature adult-born granule cells (red) in the dentate gyrus. Nuclei stained with DAPI, blue. (C) Schematic representation of the experimental design. Granule cell layer (GCL) of the dentate gyrus was stimulated with brief blue light pulses (465 nm LED light, 10 ms, 0.1 Hz). Granule cell expressing YFP (green) and immature adult-born GCs expressing RFP (red) were electrophysiologically recorded. (D) High power photomicrograph of immature adult-born GCs (RFP+) in panel B (box) surrounded by YFP expressing fibers; dashed line between GCL and H, hilus. (E) Patch-clamp recordings under current-clamp configuration from a glutamatergic mature granule cell expressing ChR2-YFP. Brief light pulses trigger action potential. (F) Patch-clamp recordings under voltage-clamp configuration (Vh = +50 mV) of immature adult-born GCs (7 dpi). Optogenetic stimulation of the granule cell layer in presence of GABA receptor blockers (Picrotoxin [PTX] and CGP55845 [CGP]) evokes an outward current that is blocked by AP5, a selective antagonist of NMDA receptors. Figure 6A: the drawings of the brain sections were modified from ref. , with permission from Elsevier to the Nature Publishing Group to publish these images under an Open Access license.

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