Sex-based differences in the long-term fate of hippocampal neurons born after a traumatic brain injury

Abstract

Introduction: Moderate-to-severe traumatic brain injury (TBI) results in an early loss of immature hippocampal granule cells and the activation of typically quiescent neural stem cells (NSCs) in the dentate gyrus. Activation of NSCs leads to a robust increase in proliferation and generation of neural progenitor cells (NPCs), supporting restoration of the immature neuron population of over a period of 1-2 weeks. However, it is unclear if neurons born early after injury develop normally, survive long-term and functionally integrate into the hippocampal network. Although adult hippocampal neurogenesis is regulated in a sex-dependent manner, the majority of pre-clinical TBI studies lack the inclusion of both sexes. The goal of this study was to examine sex differences in hippocampal neurogenesis in response to a moderate controlled cortical impact brain injury.

Methods: In-vivo labeling of NPCs and tracking of their morphological development into a granule cell was achieved using an inducible Cre recombinase driven by the Ascl1 promoter in a CAG-floxStopTom reporter mouse. Ascl1 is a basic helix-loop-helix transcription factor transiently expressed in NPCs and activated NSCs in the dentate gyrus of the adult mammalian brain. To specifically label NPCs born acutely after TBI, tamoxifen was delivered to mice on days 2 and 3 postinjury. Mice survived to 6 weeks after TBI to allow for full neuronal maturation of tdTomato-labeled NPCs.

Results: At 6 weeks postinjury, numbers of tdTomato-positive granule cells were significantly reduced in the ipsilateral hippocampus of brain-injured mice compared to controls, with a more pronounced decrease in males. Further, posttrauma-born neurons in males, but not females, exhibited impaired dendritic development. Neurons born after injury extended axons which formed synaptic terminals within the CA3 region. Numbers of mossy fiber boutons were significantly decreased in injured males compared to naïve males or to injured females. Potential forms of plasticity were observed in brain-injured females, including increased neurogenesis in the contralateral hippocampus and increased mossy fiber bouton volume. Together these data suggest a neurogenic advantage in females after injury.

Discussion: This study is the first to report sex differences in posttraumatic hippocampal neurogenesis and to demonstrate modification of synaptic terminals formed by neurons born after TBI.

Keywords: dendrite morphology; dentate gyrus; hippocampus; mossy fiber boutons; neurogenesis; sex differences; synapses; traumatic brain injury.

Figure 1

Neuronal maturation of Ascl1-derived neural progenitor cells (NPCs) in naïve mice. tdTomato (tdTom) conditionally induced in Ascl1-expressing NPCs allows visualization of granule cell (GC) development in the dentate gyrus during adult neurogenesis. (A) Diagram illustrating the transient expression of Ascl1 in early Type-2A cells and a subset of Type-1 cells in the adult dentate gyrus. ML, molecular layer; GCL, granule cell layer; SGZ, subgranular zone. (B) Ten days after tamoxifen (TAM) induction the majority of tdTom+ (red) cells co-labeled with doublecortin (DCX, green). (C, D) By 6 weeks after TAM induction tdTom+ cells had matured and no longer expressed DCX (green, in C) but co-labeled with the mature neuronal marker NeuN (green, in D). DAPI (blue). Scale bar = 50 μm. (E, F) Mice homozygous for Ascl1 (+/+) and transgenic reporter mice heterozygous for Ascl1 (+/–) have equivalent numbers of proliferating (EdU+) cells in the ipsilateral dentate gyrus during days 3–5 (E) and during days 6–8 (F) after CCI injury.

Figure 2

Cortical tissue loss is equivalent in age-matched male and female mice following CCI. (A, B) Nissl-stained coronal sections of male (A) and female (B) CCI-injured mice. (C) Quantification of cortical contusion volume at 6 weeks after injury.

Figure 3

Regional neurogenic responses to TBI are sex-dependent. (A, D) Adult-born granule neurons in naïve male and female mice visualized by tdTomato expression (red) at 6 weeks after tamoxifen administration. (B, C, E, F) CCI injury results in a deficit in neurogenesis in the ipsilateral dentate gyrus (C, F) as compared to the contralateral dentate gyrus (B, E) with fewer posttrauma-born neurons at 6 weeks after injury in male and female mice. DAPI = blue. Scale bar = 50 μm. (G) Counts of tdTom+ neurons demonstrate that injured females have more surviving posttrauma-born neurons than injured males in both the ipsilateral and contralateral hippocampus. ^**p < 0.01, ^***p < 0.005, ^****p < 0.0001.

Figure 4

Neurons born after injury in male mice demonstrate impairments in dendritic arbor morphology not observed in posttrauma-born neurons in female mice. Representative images demonstrating tdTom+ (red) dendrites digitally traced (green) and reconstructed for Sholl analysis of (A) male naïve, (B) female naïve, (C) male CCI-injured, and (D) female CCI-injured mice. Scale bar = 100 μm. (E) CCI-injured males show a greater decrease in dendritic complexity than CCI-injured females. ^*M: CCI-injured males different than naïve males, ^*F: CCI-injured females different than naïve females. (F) Posttrauma-born granule neurons in male mice develop with shorter primary dendrites than adult-born neurons in male naïve mice. (G) The total dendritic branch length of tdTom+ labeled GCs in CCI-injured males is significantly decreased compared to CCI-injured females and to naïve male controls. ^*p < 0.05, ^***p < 0.005.

Figure 5

Mossy fiber boutons are altered after CCI in a sex-dependent manner. Representative images demonstrating tdTom+ (red) pre-synaptic terminals in the CA3 region of (A) naïve male, (B) naïve female, (A') CCI-injured male, and (B') CCI-injured female mice. DAPI = blue. Scale bar = 10 μm. (C) The average number of mossy fiber boutons (MFBs) is significantly less in CCI-injured males than in CCI-injured females or in male naïve controls. (D) The average surface volume of MFBs is significantly increased only in CCI-injured female mice. (E) Numbers of MFBs per GC did not differ significantly across groups. ^***p < 0.005, ^****p < 0.0001.