Jaylyn Waddell

A developmental sex difference in hippocampal neurogenesis is mediated by endogenous oestradiol

BackgroundOestradiol is a steroid hormone that exerts extensive influence on brain development and is a powerful modulator of hippocampal structure and function. The hippocampus is a critical brain region regulating complex cognitive and emotional responses and is implicated in the aetiology of several mental health disorders, many of which exhibit some degree of sex difference. Many sex differences in the adult rat brain are determined by oestradiol action during a sensitive period of development. We had previously reported a sex difference in rates of cell genesis in the developing hippocampus of the laboratory rat. Males generate more new cells on average than females. The current study explored the effects of both exogenous and endogenous oestradiol on this sex difference.MethodsNew born male and female rat pups were injected with the mitotic marker 5-bromo-2-deoxyuridine (BrdU) and oestradiol or agents that antagonize oestradiol action. The effects on cell number, proliferation, differentiation and survival were assessed at several time points. Significant differences between groups were determined by two- or thee-Way ANOVA.ResultsNewborn males had higher rates of cell proliferation than females. Oestradiol treatment increased cell proliferation in neonatal females, but not males, and in the CA1 region many of these cells differentiated into neurons. The increased rate of proliferation induced by neonatal oestradiol persisted until at least 3 weeks of age, suggesting an organizational effect. Administering the aromatase inhibitor, formestane, or the oestrogen receptor antagonist, tamoxifen, significantly decreased the number of new cells in males but not females.ConclusionEndogenous oestradiol increased the rate of cell proliferation observed in newborn males compared to females. This sex difference in neonatal neurogenesis may have implications for adult differences in learning strategy, stress responsivity or vulnerability to damage or disease.

Sex-dependent mitochondrial respiratory impairment and oxidative stress in a rat model of neonatal hypoxic-ischemic encephalopathy

Increased male susceptibility to long‐term cognitive deficits is well described in clinical and experimental studies of neonatal hypoxic‐ischemic encephalopathy. While cell death signaling pathways are known to be sexually dimorphic, a sex‐dependent pathophysiological mechanism preceding the majority of secondary cell death has yet to be described. Mitochondrial dysfunction contributes to cell death following cerebral hypoxic‐ischemia (HI). Several lines of evidence suggest that there are sex differences in the mitochondrial metabolism of adult mammals. Therefore, this study tested the hypothesis that brain mitochondrial respiratory impairment and associated oxidative stress is more severe in males than females following HI. Maximal brain mitochondrial respiration during oxidative phosphorylation was two‐fold more impaired in males following HI. The endogenous antioxidant glutathione was 30% higher in the brain of sham females compared to males. Females also exhibited increased glutathione peroxidase (GPx) activity following HI injury. Conversely, males displayed a reduction in mitochondrial GPx4 protein levels and mitochondrial GPx activity. Moreover, a 3–4‐fold increase in oxidative protein carbonylation was observed in the cortex, perirhinal cortex, and hippocampus of injured males, but not females. These data provide the first evidence for sex‐dependent mitochondrial respiratory dysfunction and oxidative damage, which may contribute to the relative male susceptibility to adverse long‐term outcomes following HI.

Changing the rate and hippocampal dependence of trace eyeblink conditioning: Slow learning enhances survival of new neurons

Trace eyeblink conditioning in which a conditioned stimulus and unconditioned stimulus are separated by a gap, is hippocampal dependent and can rescue new neurons in the adult dentate gyrus from death (e.g., Beylin et al., 2001; Gould et al., 1999). Tasks requiring more training trials for reliable expression of the conditioned response are most effective in enhancing survival of neurons (Waddell & Shors, 2008). To dissociate hippocampal dependence from acquisition rate, we facilitated hippocampal-dependent trace eyeblink conditioning in two ways: a shorter trace interval and signaling the intertrial interval with a post-US cue. Trace conditioning with a shorter trace interval (250ms) requires an intact hippocampus, and acquisition is faster relative to rats trained with a 500ms trace interval (e.g., Weiss et al., 1999). Using excitotoxic hippocampal lesions, we confirmed that eyeblink conditioning with the 250 or 500ms trace interval is hippocampal dependent. However, training with the post-US cue was not hippocampal dependent. The majority of lesion rats in this condition reached criterion of conditioned responding. To determine whether hippocampal dependence is sufficient to rescue adult-generated neurons in the dentate gyrus, rats were injected with BrdU and trained in one of the three trace eyeblink arrangements one week later. Of these training procedures, only the 500ms trace interval enhanced survival of new cells; acquisition of this task proceeded slowly relative to the 250ms and post-US cue conditions. These data demonstrate that rate of acquisition and not hippocampal dependence determines the impact of learning on adult neurogenesis.

Sex differences in cell genesis, hippocampal volume and behavioral outcomes in a rat model of neonatal HI.

Hypoxia-ischemia (HI) of the brain in near-term and term infants is a leading cause of infant mortality and lifelong disability but current therapeutic approaches remain limited. Males consistently display greater vulnerability to the deleterious consequences of HI in both humans and animal models. Neurogenesis increases after neonatal HI and offers a potential therapeutic target for recovery. The steroid hormone estradiol has been extensively explored as a neuroprotectant in adult models of stroke but with mixed results. Less consideration has been afforded to this naturally occurring agent in the developing brain, which has unique challenges from the adult. Using a model of term HI in the rat we have explored the impact of this insult on cell genesis in the hippocampus of males and females and the ability of estradiol treatment immediately after insult to restore function. Both short-term (3 days) and long-term (7 days) post-injury were assessed and revealed that only females had markedly increased cell genesis on the short-term but both sexes were increased long-term. A battery of behavioral tests revealed motor impairment in males and compromised episodic memory while both sexes were modestly impaired in spatial memory. Juvenile social play was also depressed in both sexes after HI. Estradiol therapy improved behavioral performance in both sexes but did not reverse a deficit in hippocampal volume ipsilateral to the insult. Thus the effects of estradiol do not appear to be via cell death or proliferation but rather involve other components of neural functioning.

Sex-dependent mitophagy and neuronal death following rat neonatal hypoxia-ischemia.

Males are more susceptible than females to long-term cognitive deficits following neonatal hypoxic-ischemic encephalopathy (HIE). Mitochondrial dysfunction is implicated in the pathophysiology of cerebral hypoxia-ischemia (HI), but the influence of sex on mitochondrial quality control (MQC) after HI is unknown. Therefore, we tested the hypothesis that mitophagy is sexually dimorphic and neuroprotective 20-24h following the Rice-Vannucci model of rat neonatal HI at postnatal day 7 (PN7). Mitochondrial and lysosomal morphology and degree of co-localization were determined by immunofluorescence in the cerebral cortex. No difference in mitochondrial abundance was detected in the cortex after HI. However, net mitochondrial fission increased in both hemispheres of female brain, but was most extensive in the ipsilateral hemisphere of male brain following HI. Basal autophagy, assessed by immunoblot for the autophagosome marker LC3BI/II, was greater in males suggesting less intrinsic reserve capacity for autophagy following HI. Autophagosome formation, lysosome size, and TOM20/LAMP2 co-localization were increased in the contralateral hemisphere following HI in female, but not male brain. An accumulation of ubiquitinated mitochondrial protein was observed in male, but not female brain following HI. Moreover, neuronal cell death with NeuN/TUNEL co-staining occurred in both hemispheres of male brain, but only in the ipsilateral hemisphere of female brain after HI. In summary, mitophagy induction and neuronal cell death are sex dependent following HI. The deficit in elimination of damaged/dysfunctional mitochondria in the male brain following HI may contribute to male vulnerability to neuronal death and long-term neurobehavioral deficits following HIE.

In vivo longitudinal proton magnetic resonance spectroscopy on neonatal hypoxic-ischemic rat brain injury: Neuroprotective effects of acetyl-L-carnitine.

This study evaluated the longitudinal metabolic alterations after neonatal hypoxia‐ischemia (HI) in rats and tested the neuroprotective effect of acetyl‐L‐carnitine (ALCAR) using in vivo proton short‐TE Point‐RESolved Spectroscopy method.

Dysregulation of neonatal hippocampal cell genesis in the androgen insensitive Tfm rat

The first two weeks of life are a critical period for hippocampal development. At this time gonadal steroid exposure organizes sex differences in hippocampal sensitivity to activational effects of steroids, hippocampal cell morphology and hippocampus dependent behaviors. Our laboratory has characterized a robust sex difference in neonatal neurogenesis in the hippocampus that is mediated by estradiol. Here, we extend our knowledge of this sex difference by comparing the male and female hippocampus to the androgen insensitive testicular feminized mutant (Tfm) rat. In the neonatal Tfm rat hippocampus, fewer newly generated cells survive compared to males or females. This deficit in cell genesis is partially recovered with the potent androgen DHT, but is more completely recovered following estradiol administration. Tfm rats do not differ from males or females in the level of endogenous estradiol in the neonatal hippocampus, suggesting other mechanisms mediate a differential sensitivity to estradiol in male, female and Tfm hippocampus. We also demonstrate disrupted performance on a hippocampal-dependent contextual fear discrimination task. Tfm rats generalize fear across contexts, and do not exhibit significant loss of fear during extinction exposure. These results extend prior reports of exaggerated response to stress in Tfm rats, and following gonadectomy in normal male rats.

Longitudinal in vivo developmental changes of metabolites in the hippocampus of Fmr1 knockout mice

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is studied in the Fmr1 knockout (KO) mouse, which models both the anatomical and behavioral changes observed in FXS patients. In vitro studies have shown many alterations in synaptic plasticity and increased density of immature dendritic spines in the hippocampus, a region involved in learning and memory. In this study, magnetic resonance imaging (MRI) and 1H magnetic resonance spectroscopy (MRS) were used to determine in vivo longitudinal changes in volume and metabolites in the hippocampus during the critical period of early myelination and synaptogenesis at post‐natal days (PND) 18, 21, and 30 in Fmr1 KO mice compared with wild‐type (WT) controls. MRI demonstrated an increase in volume of the hippocampus in the Fmr1 KO mouse compared with controls. MRS revealed significant developmental changes in the ratios of hippocampal metabolites N‐acetylaspartate (NAA), myo‐inositol (Ins), and taurine to total creatine (tCr) in Fmr1 KO mice compared with WT controls. Ins was decreased at PND 30, and taurine was increased at all ages studied in Fmr1 KO mice compared with controls. An imbalance of brain metabolites in the hippocampus of Fmr1 KO mice during the critical developmental period of synaptogenesis and early myelination could have long‐lasting effects that adversely affect brain development and contribute to ongoing alterations in brain function.

The Depolarizing Action of GABA in Cultured Hippocampal Neurons Is Not Due to the Absence of Ketone Bodies

Two recent reports propose that the depolarizing action of GABA in the immature brain is an artifact of in vitro preparations in which glucose is the only energy source. The authors argue that this does not mimic the physiological environment because the suckling rats use ketone bodies and pyruvate as major sources of metabolic energy. Here, we show that availability of physiologically relevant levels of ketone bodies has no impact on the excitatory action of GABA in immature cultured hippocampal neurons. Addition of β-hydroxybutyrate (BHB), the primary ketone body in the neonate rat, affected neither intracellular calcium elevation nor membrane depolarizations induced by the GABA-A receptor agonist muscimol, when assessed with calcium imaging or perforated patch-clamp recording, respectively. These results confirm that the addition of ketone bodies to the extracellular environment to mimic conditions in the neonatal brain does not reverse the chloride gradient and therefore render GABA hyperpolarizing. Our data are consistent with the existence of a genuine “developmental switch” mechanism in which GABA goes from having a predominantly excitatory role in immature cells to a predominantly inhibitory one in adults.

Neuroprotective Effects of Acetyl-L-Carnitine on Neonatal Hypoxia Ischemia-Induced Brain Injury in Rats

Perinatal hypoxia ischemia (HI) is a significant cause of brain injury in surviving infants. Although hypothermia improves outcomes in some infants, additional therapies are needed since about 40% of infants still have a poor outcome. Acetyl-L-carnitine (ALCAR), an acetylated derivative of L-carnitine, protected against early changes in brain metabolites and mitochondrial function after HI on postnatal day (PND) 7 in a rat pup model of near-term HI injury. However, its efficacy in long-term structural and functional outcomes remains unexplored. We determined the efficacy of ALCAR therapy administered to rat pups after HI at PND 7, using both longitudinal in vivo magnetic resonance imaging and behavioral tests, in male and female rats. HI led to sex-specific behavioral impairment, with males exhibiting more global functional deficits than females. Interestingly, HI reduced the volume of the contralateral hemisphere in males only, suggesting that the brain injury is more diffuse in males than in females. Treatment with ALCAR improved both morphological and functional outcomes in both male and female rats. These results suggest that ALCAR may be a potential therapy for clinical use since the treatment attenuated the moderate injury produced under the experimental conditions used and improved the functional outcome in preclinical studies.