Functional Integration of Adult-Born Hippocampal Neurons after Traumatic Brain Injury

Granule cells born after CCI have accelerated dendritic growth. A, Confocal image of BrdU⁺ cells expressing GFP in POMC-EGFP mice, used to precisely date the birth of cells for morphologic analysis. In this example, BrdU was administered to POMC-EGFP mice 5 d after CCI or sham procedure and perfusion was fixed at 14 d, so that BrdU⁺ cells were 9 d postmitosis. Traced cells are outlined in white. B, Representative images (top) of dendritic tracings of 7-, 9-, and 12-d-old GFP⁺ cells in sham and CCI-treated mice, and the Sholl analyses (bottom) for group data at each time point. In each cohort, CCI increased the number of dendritic branch points of immature cells born during post-traumatic neurogenesis (p = 0.0027¹³). Branches also occurred closer to the cell body in CCI-treated mice in each cohort, which was independent of cell age (p = 0.76¹⁵). C, Representative images of GFP⁺ cells and their dendritic outgrowth. White arrows point to the first dendritic branch points. D–F, GFP⁺ cells from CCI treated mice branched closer to their cell body (***p = 0.0012¹⁶; D), had more cumulative branches (***p = 0.0005¹⁷; E), and had greater total dendritic length (***p = 0.001¹⁸; F).

Immature granule cells born after TBI are excitable and integrated into the hippocampal circuitry. A, Current-clamp recordings from GFP-positive immature granule cells born after sham or CCI surgery demonstrate that cells born after TBI fire action potentials during depolarizing current injection. Respective voltage traces during current injections of +5 pA (red), +10 pA (green), +30 pA (blue), and +40 pA (black) for both sham and CCI-treated animals are shown. Summary data are given in the text. B, C, Single-cell, voltage-clamp recordings demonstrate that immature granule cells have similar voltage-dependent action currents. B, Averaged action currents from example cells during steps of +20, +40, and +60 mV from a holding potential of −70 mV. C, Summary data of the amplitude of the action current response in response to the various steps between groups (sham = 7 cells; CCI = 7 cells; p = 0.65⁵⁴ with a +40 mV step; p = 0.75⁵⁵ with a +60 mV step). D, Synaptic currents elicited in immature adult-born neurons in response to stimulation at the IML/MML border, while voltage clamping the cells to −70, −40, 0, and +40 mV. E, Summary data of response amplitudes of evoked currents (recorded while clamping cells at +40 mV), demonstrating almost complete block of the evoked synaptic response by the GABA_AR antagonist SR95531 (10 μm; n = 5 cells per group). F, G, High-power imaging of dendrites from newborn neurons in sham and CCI-treated mice show no evidence of dendritic spines on immature GFP⁺ cells in either condition.

Tamoxifen administration pulse labels adult born neurons in DcxCre/tdTomato mice. A, Tamoxifen (TAM) was administered to adult DcxCre/tdTom mice, and they were fixed at two different time intervals thereafter. Early after TAM administration (left), tdTom⁺ cells have immature dendritic morphologies and stain for the immature marker Dcx, indicating recent mitosis. After 3 weeks (right), tdTom⁺ cells no longer costain for Dcx, suggesting that labeled cells mature, and that no new immature cells have been labeled. The minor overlap of red and green in the projected image at 26 d after TAM administration reflects the overlap of separate cells in the stack and not coexpression. B, DcxCre/tdTom mice received BrdU to mark cells born either before (left) or after (right) adult TAM administration, and were fixed 3 weeks later. Cells born before TAM administration are efficiently labeled by tdTom expression, but cells born after TAM did not express tdTom, indicating that neurons born just prior to tamoxifen administration are selectively pulse labeled. Scale bar, 50 μm.

Neurons born shortly after CCI survive and maintain their aberrant localization in maturity. A, Representative images of ∼4-week-old tdTom⁺ cells from the ipsilateral hippocampus of DcxCre/tdTom sham and CCI-treated mice, in which cells born after CCI were permanently pulse labeled with tdTomato. B, One month after injury, CCI-treated mice had more tdTom⁺ cells in the ipsilateral granule cell layer of the dentate gyrus compared with sham mice (**p = 0.0087²⁹, n = 6 mice/group). C, Representative images of tdTom⁺ cell dispersion in the granule cell layer of the ipsilateral dentate gyrus. D, CCI-treated mice had greater cell migration in the ipsilateral dentate gyrus compared with sham mice (*p = 0.042³⁰, n = 6 mice/group).

CCI-induced changes in the dendritic morphology of newborn neurons persist as these cells mature. A, Representative images of 4-week-old tdTom⁺ cells from sham and CCI-treated mice. Arrows point to the first dendritic branch point of selected cells. B, Representative tracings of tdTom⁺ cells from sham and CCI-treated mice. C, Sholl analyses of dendritic arborization of tdTom⁺ cells reveal a CCI-induced persistent increase in the number of branch points observed near the soma (*p = 0.0001, 0.003, 0.004, 0.003, and 0.049 for each 10 μm increment between 10 and 50 μm from the soma³⁴), but a reduction in more distal regions (**p = 0.038, 0.002, 0.001, 0.009, 0.003, 0.003, 0.004, and 0.038 for each 10 μm increment between 150 and 220 μm from the soma³⁴; n = 26 cells from 7 sham mice; 28 cells from 8 CCI-treated mice). D–F, tdTom⁺ cells from CCI-treated mice branched closer to their cell body (***p = 0.0001³⁵; D), and had wider angles (***p = 0.0007³⁶; E) and area (*p = 0.040³⁷; F) between the dendrites that were farthest apart. G, tdTom⁺ cells from sham and CCI-treated mice had similar cumulative dendritic lengths (p = 0.50⁵⁶).

CCI increased the heterogeneity of dendritic branch morphology in cells born after injury. A, Representative morphology of tdTom⁺ cells categorized according to their dendritic phenotype. B, The majority of tdTom⁺ cells in sham mice were characterized by a single apical dendrite that branched farther away from the cell body (>10 μm from soma, typical cell), whereas the majority of tdTom⁺ cells in CCI-treated mice branched more proximal to their somata (<10 μm), had more than one apical dendrite, or had an apical dendrite that projected laterally from their soma (<30° from GCL). There were fewer typical tdTom⁺ cells in CCI-treated mice compared with sham mice (p = 0.021³⁸, n = 5 mice/group).

Granule cells born after TBI become functionally integrated into the hippocampal circuit. A, At 1 month after injury, single-cell recordings demonstrate normal firing patterns and the excitability of cells born early after TBI. Voltage traces show the response of adult-born granule cells to current injections of −10, +10, and +50 pA. B, C, Whole cell, voltage-clamp recordings of mEPSCs demonstrate that cells born after TBI acquire excitatory synaptic connections (B), and that mEPSC amplitude and frequency (C) are similar to those recorded from adult-born cells of similar age in sham animals (n = 8 and 9 cells for sham and CCI, respectively; p > 0.4 each). Each graph depicts summary data (bars) and individual cell data (points). D, At 1 month after injury, cells born early following TBI possess dendritic spines within the dentate molecular layer, and dendritic spine density is similar in cells born after sham or CCI procedures (sham, n = 16 cells; CCI, n = 22 cells; p = 0.60⁵³). E, Images of dendritic spines from cells born after CCI or sham procedure.