Diazepam Inhibits Post-Traumatic Neurogenesis and Blocks Aberrant Dendritic Development.

Traumatic brain injury (TBI) triggers a robust increase in neurogenesis within the dentate gyrus of the hippocampus, but these new neurons undergo aberrant maturation and dendritic outgrowth. Because gamma-aminobutyric acid (GABA)A receptors (GABAARs) modulate dendritic outgrowth during constitutive neurogenesis and GABAAR-modulating sedatives are often administered to human patients after TBI, we investigated whether the benzodiazepine, diazepam (DZP), alters post-injury hippocampal neurogenesis. We used a controlled cortical impact (CCI) model of TBI in adult mice, and administered DZP or vehicle continuously for 1 week after injury via osmotic pump. Although DZP did not affect the neurogenesis rate in control mice, it almost completely prevented the TBI-induced increase in hippocampal neurogenesis as well as the aberrant dendritic growth of neurons born after TBI. DZP did not reduce cortical injury, reactive gliosis, or cell proliferation early after injury, but decreased c-Fos activation in the dentate gyrus at both early and late time-points after TBI, suggesting an association between neuronal activity and post-injury neurogenesis. Because DZP blocks post-injury neurogenesis, further studies are warranted to assess whether benzodiazepines alter cognitive recovery or the development of complications after TBI.

Ketamine Alters Hippocampal Cell Proliferation and Improves Learning in Mice after Traumatic Brain Injury.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Traumatic brain injury induces cellular proliferation in the hippocampus, which generates new neurons and glial cells during recovery. This process is regulated by N-methyl-D-aspartate-type glutamate receptors, which are inhibited by ketamine. The authors hypothesized that ketamine treatment after traumatic brain injury would reduce hippocampal cell proliferation, leading to worse behavioral outcomes in mice. METHODS: Traumatic brain injury was induced in mice using a controlled cortical impact injury, after which mice (N = 118) received either ketamine or vehicle systemically for 1 week. The authors utilized immunohistochemical assays to evaluate neuronal, astroglial, and microglial cell proliferation and survival 3 days, 2 weeks, and 6 weeks postintervention. The Morris water maze reversal task was used to assess cognitive recovery. RESULTS: Ketamine dramatically increased microglial proliferation in the granule cell layer of the hippocampus 3 days after injury (injury + vehicle, 2,800 ± 2,700 cells/mm, n = 4; injury + ketamine, 11,200 ± 6,600 cells/mm, n = 6; P = 0.012). Ketamine treatment also prevented the production of astrocytes 2 weeks after injury (sham + vehicle, 2,400 ± 3,200 cells/mm, n = 13; injury + vehicle, 10,500 ± 11,300 cells/mm, n = 12; P = 0.013 vs. sham + vehicle; sham + ketamine, 3,500 ± 4,900 cells/mm, n = 14; injury + ketamine, 4,800 ± 3,000 cells/mm, n = 13; P = 0.955 vs. sham + ketamine). Independent of injury, ketamine temporarily reduced neurogenesis (vehicle-exposed, 105,100 ± 66,700, cells/mm, n = 25; ketamine-exposed, 74,300 ± 29,200 cells/mm, n = 27; P = 0.031). Ketamine administration improved performance in the Morris water maze reversal test after injury, but had no effect on performance in sham-treated mice. CONCLUSIONS: Ketamine alters hippocampal cell proliferation after traumatic brain injury. Surprisingly, these changes were associated with improvement in a neurogenesis-related behavioral recall task, suggesting a possible benefit from ketamine administration after traumatic brain injury in mice. Future studies are needed to determine generalizability and mechanism.

Paradoxical effects of 137Cs irradiation on pharmacological stimulation of reactive oxygen species in hippocampal slices from apoE2 and apoE4 mice.

In humans, apoE, which plays a role in repair, is expressed in three isoforms: E2, E3, and E4. E4 is a risk factor for age-related cognitive decline (ACD) and Alzheimer's disease (AD), particularly in women. In contrast, E2 is a protective factor for ACD and AD. E2 and E4 might also differ in their response to cranial 137Cs irradiation, a form of radiation typically used in a clinical setting for the treatment of cancer. This might be mediated by reactive oxygen species (ROS) in an-apoE isoform-dependent fashion. E2 and E4 female mice received sham-irradiation or cranial irradiation at 8 weeks of age and a standard mouse chow or a diet supplemented with the antioxidant alpha-lipoic acid (ALA) starting at 6 weeks of age. Behavioral and cognitive performance of the mice were assessed 12 weeks later. Subsequently, the generation of ROS in hippocampal slices was analyzed. Compared to sham-irradiated E4 mice, irradiated E4 mice showed enhanced spatial memory in the water maze. This was associated with increased hippocampal PMA-induction of ROS. Similar effects were not seen in E2 mice. Irradiation increased endogenous hippocampal ROS levels in E2 mice while decreasing those in E4 mice. NADPH activity and MnSOD levels were higher in sham-irradiated E2 than E4 mice. Irradiation increased NADPH activity and MnSOD levels in hemi brains of E4 mice but not in those of E2 mice. ALA did not affect behavioral and cognitive performance or hippocampal formation of ROS in either genotype. Thus, apoE isoforms modulate the radiation response.

Genotype differences in anxiety and fear learning and memory of WT and ApoE4 mice associated with enhanced generation of hippocampal reactive oxygen species.

Apolipoprotein E (apoE), involved in cholesterol and lipid metabolism, also influences cognitive function and injury repair. In humans, apoE is expressed in three isoforms. E4 is a risk factor for age-related cognitive decline and Alzheimer's disease, particularly in women. E4 might also be a risk factor for developing behavioral and cognitive changes following (56) Fe irradiation, a component of the space environment astronauts are exposed to during missions. These changes might be related to enhanced generation of reactive oxygen species (ROS). In this study, we compared the behavioral and cognitive performance of sham-irradiated and irradiated wild-type (WT) mice and mice expressing the human E3 or E4 isoforms, and assessed the generation of ROS in hippocampal slices from these mice. E4 mice had greater anxiety-like and conditioned fear behaviors than WT mice, and these genotype differences were associated with greater levels of ROS in E4 than WT mice. The greater generation of ROS in the hippocampus of E4 than WT mice might contribute to their higher anxiety levels and enhanced fear conditioning. In E4, but not WT, mice, phorbol-12-myristate-13-acetate-treated hippocampal slices showed more dihydroxy ethidium oxidation in sham-irradiated than irradiated mice and hippocampal heme oxygenase-1 levels were higher in irradiated than sham-irradiated E4 mice. Mice with apolipoprotein E4 (E4), a risk factor for Alzheimer's disease, have greater anxiety-like and conditioned fear behaviors than wild-type (WT) mice. Generation of reactive oxygen species (ROS, in red) 3 months following (56) Fe irradiation, a component of the space environment astronauts are exposed to, is more pronounced in the hippocampus of E4 than WT mice. In E4, but not WT, mice, hippocampal levels of the oxidative stress-relevant marker heme oxygenase-1 are higher in irradiated than sham-irradiated E4 mice.

Functional Integration of Adult-Born Hippocampal Neurons after Traumatic Brain Injury(1,2,3).

Traumatic brain injury (TBI) increases hippocampal neurogenesis, which may contribute to cognitive recovery after injury. However, it is unknown whether TBI-induced adult-born neurons mature normally and functionally integrate into the hippocampal network. We assessed the generation, morphology, and synaptic integration of new hippocampal neurons after a controlled cortical impact (CCI) injury model of TBI. To label TBI-induced newborn neurons, we used 2-month-old POMC-EGFP mice, which transiently and specifically express EGFP in immature hippocampal neurons, and doublecortin-CreER(T2) transgenic mice crossed with Rosa26-CAG-tdTomato reporter mice, to permanently pulse-label a cohort of adult-born hippocampal neurons. TBI increased the generation, outward migration, and dendritic complexity of neurons born during post-traumatic neurogenesis. Cells born after TBI had profound alterations in their dendritic structure, with increased dendritic branching proximal to the soma and widely splayed dendritic branches. These changes were apparent during early dendritic outgrowth and persisted as these cells matured. Whole-cell recordings from neurons generated during post-traumatic neurogenesis demonstrate that they are excitable and functionally integrate into the hippocampal circuit. However, despite their dramatic morphologic abnormalities, we found no differences in the rate of their electrophysiological maturation, or their overall degree of synaptic integration when compared to age-matched adult-born cells from sham mice. Our results suggest that cells born after TBI participate in information processing, and receive an apparently normal balance of excitatory and inhibitory inputs. However, TBI-induced changes in their anatomic localization and dendritic projection patterns could result in maladaptive network properties.

ApoE isoform modulates effects of cranial 56Fe irradiation on spatial learning and memory in the water maze.

Apolipoprotein E, which plays an important role in lipid transport and metabolism and neuronal repair, might modulate the CNS risk following (56)Fe irradiation exposure during space missions. In this study, we investigated this risk by behavioral and cognitive testing male E2, E3, and E4 mice 3 months following cranial (56)Fe irradiation. In the open field, mice irradiated with 2 Gy showed higher activity levels than sham-irradiated mice or mice irradiated with 1 Gy. In addition, E2 mice showed higher activity and lower measures of anxiety than E3 and E4 mice in the open field and elevated zero maze. During hidden platform training, sham-irradiated mice showed most robust learning, 1 Gy irradiated mice reduced learning, and 2 Gy irradiated mice no improvement over the four sessions. In the water maze probe trials, sham-irradiated E2, E3, and E4 mice and E2 and E4 mice irradiated with 1 Gy showed spatial memory retention, but E3 mice irradiated with 1 Gy, and E2, E3, and E4 mice irradiated with 2 Gy did not. Thus, cranial (56)Fe irradiation increases activity levels in the open field and impairs spatial learning and memory in the water maze. E3 mice are more susceptible than E2 or E4 mice to impairments in spatial memory retention in the water maze, indicating that apoE isoform modulates the CNS risk following space missions.

Effects of alpha-lipoic acid on associative and spatial memory of sham-irradiated and 56Fe-irradiated C57BL/6J male mice.

Cranial irradiation with (56)Fe, a form of space radiation, causes hippocampus-dependent cognitive changes. (56)Fe irradiation also increases reactive oxygen species (ROS) levels, which may contribute to these changes. Therefore, we investigated the effects of the antioxidant alpha lipoic acid (ALA) on cognition following sham-irradiation and irradiation. Male mice were irradiated (brain only) with (56)Fe (3 Gy) or sham-irradiated at 6-9 months of age. Half of the mice remained fed a regular chow and the other half of the mice were fed a caloric-matched diet containing ALA starting two-weeks prior to irradiation and throughout cognitive testing. Following cognitive testing, levels of 3-nitrotyrosine (3NT), a marker of oxidative protein stress, and levels of microtubule-associated protein (MAP-2), a dendritic protein important for cognition, were assessed using immunohistochemistry and confocal microscopy. ALA prevented radiation-induced impairments in spatial memory retention in the hippocampal and cortical dependent water maze probe trials following reversal learning. However, in sham-irradiated mice, ALA treatment impaired cortical-dependent novel object recognition and amygdala-dependent cued fear conditioning. There was a trend towards lower 3NT levels in irradiated mice receiving a diet containing ALA than irradiated mice receiving a regular diet. In the hippocampal dentate gyrus of mice on regular diet, irradiated mice had higher levels of MAP-2 immunoreactivity than sham-irradiated mice. Thus, ALA might have differential effects on the brain under normal physiological conditions and those involving environmental challenges such as cranial irradiation.

Long-term effects of 56Fe irradiation on spatial memory of mice: role of sex and apolipoprotein E isoform.

PURPOSE: To assess whether the effects of cranial (56)Fe irradiation on the spatial memory of mice in the water maze are sex and apolipoprotein E (apoE) isoform dependent and whether radiation-induced changes in spatial memory are associated with changes in the dendritic marker microtubule-associated protein 2 (MAP-2) and the presynaptic marker synaptophysin. METHODS AND MATERIALS: Two-month-old male and female mice expressing human apoE3 or apoE4 received either a 3-Gy dose of cranial (56)Fe irradiation (600 MeV/amu) or sham irradiation. Mice were tested in a water maze task 13 months later to assess effects of irradiation on spatial memory retention. After behavioral testing, the brain tissues of these mice were analyzed for synaptophysin and MAP-2 immunoreactivity. RESULTS: After irradiation, spatial memory retention of apoE3 female, but not male, mice was impaired. A general genotype deficit in spatial memory was observed in sham-irradiated apoE4 mice. Strikingly, irradiation prevented this genotype deficit in apoE4 male mice. A similar but nonsignificant trend was observed in apoE4 female mice. Although there was no change in MAP-2 immunoreactivity after irradiation, synaptophysin immunoreactivity was increased in irradiated female mice, independent of genotype. CONCLUSIONS: The effects of (56)Fe irradiation on the spatial memory retention of mice are critically influenced by sex, and the direction of these effects is influenced by apoE isoform. Although in female mice synaptophysin immunoreactivity provides a sensitive marker for effects of irradiation, it cannot explain the apoE genotype-dependent effects of irradiation on the spatial memory retention of the mice.

Early changes in the apparent diffusion coefficient (ADC) in a mouse model of Sandhoff's disease occur prior to disease symptoms and behavioral deficits.

Sandhoff's disease is a lysosomal storage disease in which the ganglioside GM2 accumulates in lysosomes. It has been reported that MRI cannot detect abnormalities in spin echo images in clinically presymptomatic Sandhoff's disease patients. Because one of the results of GM2 accumulation is cell swelling and lysosomal distension, our goal was to determine if changes in the diffusion of water is perturbed. We utilized the MRI imaging modality diffusion-weighted imaging to measure the apparent diffusion coefficient in a mouse models of Sandhoff's disease, the hexb-/- mouse, and determined if diffusion-weighted imaging could be utilized to detect early changes prior to behavioral or overt disease symptom onset. Here we report for the first time a comprehensive behavioral characterization of the hexb-/- mouse in conjunction with the apparent diffusion coefficient measurement. Our data indicate that the apparent diffusion coefficient decreases in the hexb-/- mouse in many but not all brain regions prior to disease symptoms (<3.5 to 4 months of age) and behavioral deficits (3 months of age). The magnitude of the decrease ranged from 4-18%.

NMDA receptor activation induces translocation and activation of Rac in mouse hippocampal area CA1.

Neuronal development requires several discrete morphological steps that are believed to involve the small GTPase Rac. For example, neural activity, through NMDA receptors and/or AMPA receptors, activates Rac leading to elaboration of dendritic arbors. In the current study, we have conducted studies which indicate that Rac might be an important molecule involved in morphological plasticity in the adult mouse. We demonstrate that Rac is expressed at synapses in the adult mouse hippocampus. We also demonstrate that treatment of hippocampal slices with NMDA induces membrane translocation and activation of Rac in area CA1. Interestingly, we also find that there is an increase in Rac that is associated with NMDA receptor complexes following NMDA receptor activation. Taken together, our data are consistent with the idea that Rac could be participating in NMDA receptor-dependent changes in morphology that occur during synaptic plasticity and memory formation in the adult mouse hippocampus.

Synaptic localization of a functional NADPH oxidase in the mouse hippocampus.

Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91phox and p22phox, and four cytosolic proteins, p40phox, p47phox, p67phox, and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the membrane components, which results in production of superoxide. Here, we determined the presence, localization, and functionality of a NADPH oxidase in mouse hippocampus by examining the NADPH oxidase proteins as well as the production of superoxide. All of the NADPH oxidase proteins were present in hippocampal homogenates and enriched in synaptoneurosome preparations. Immunocytochemical analysis of cultured hippocampal neurons indicated that all NADPH oxidase proteins were localized in neuronal cell bodies as well as dendrites. Furthermore, double labeling analysis using antibodies to p67phox and the presynaptic marker synaptophysin suggest a close association of the NADPH oxidase subunits with synaptic sites. Finally, stimulation of hippocampal slices with phorbol esters triggered translocation of the cytoplasmic NADPH oxidase proteins to the membrane and an increase in superoxide production that was blocked by inhibitors of NADPH oxidase. Taken together, our data suggest that NADPH oxidase is present in mouse hippocampus and might be the source of superoxide production required for LTP and memory function.