Our group previously showed that genetic or pharmacological inhibition of the cystine/glutamate antiporter, system xc-, mitigates excitotoxicity after anoxia by increasing latency to anoxic depolarization, thus attenuating the ischaemic core. Hypoxia, however, which prevails in the ischaemic penumbra, is a condition where neurotransmission is altered, but excitotoxicity is not triggered. The present study employed mild hypoxia to further probe ischaemia-induced changes in neuronal responsiveness from wild-type and xCT KO (xCT-/-) mice. Synaptic transmission was monitored in hippocampal slices from both genotypes before, during and after a hypoxic episode. Although wild-type and xCT-/- slices showed equal suppression of synaptic transmission during hypoxia, mutant slices exhibited a persistent potentiation upon re-oxygenation, an effect we termed 'post-hypoxic long-term potentiation (LTP)'. Blocking synaptic suppression during hypoxia by antagonizing adenosine A1 receptors did not preclude post-hypoxic LTP. Further examination of the induction and expression mechanisms of this plasticity revealed that post-hypoxic LTP was driven by NMDA receptor activation, as well as increased calcium influx, with no change in paired-pulse facilitation. Hence, the observed phenomenon engaged similar mechanisms as classical LTP. This was a remarkable finding as theta-burst stimulation-induced LTP was equivalent between genotypes. Importantly, post-hypoxic LTP was generated in wild-type slices pretreated with system xc- inhibitor, S-4-carboxyphenylglycine, thereby confirming the antiporter's role in this phenomenon. Collectively, these data indicate that system xc- interference enables neuroplasticity in response to mild hypoxia, and, together with its regulation of cellular damage in the ischaemic core, suggest a role for the antiporter in post-ischaemic recovery of the penumbra.

Autism spectrum disorder (ASD) is characterized by social communication impairments with restricted and repetitive behaviors (RRBs). The increase in prevalence of ASD and the heterogeneity of symptom severity may arise from a complex interaction of environmental and genetic factors that alter synaptic plasticity. Maternal stress during pregnancy, which is linked to depression, may be one risk factor for an ASD phenotype in offspring. Selective serotonin reuptake inhibitor (SSRI) treatment can be effective in alleviating maternal depression but prenatal SSRI exposure itself may be a risk factor for autism in offspring. The present study investigated in C57BL/6J pregnant mice whether restraint stress (G4-18) and/or treatment with the SSRI fluoxetine (G8-18) affects autism-related behaviors and hippocampal synaptic plasticity in male and female offspring. The findings indicate that restraint stress reduces preference for sucrose reward in pregnant dams that is reversed by fluoxetine. In adult male offspring, combined prenatal stress and SSRI exposure increased self-grooming and impaired spatial reversal learning. In adult female offspring, the prenatal experiences did not affect self-grooming, but restraint stress alone or SSRI exposure alone impaired spatial reversal learning. Prenatal stress reduced anxiety-related behavior in male and female offspring. Further, LTP induced by theta-burst stimulation of Schaffer-commissural afferents in field CA1 was significantly reduced in female offspring exposed to prenatal stress alone or combination with fluoxetine. Together, these findings suggest that exposure to prenatal stress, SSRI treatment or the combination differentially affects male and female offspring in autism-like behaviors and synaptic plasticity.

IntroductionFragile X Syndrome (FXS) is a monogenic condition that leads to intellectual disability along with behavioral and learning difficulties. Among behavioral and learning difficulties, cognitive flexibility impairments are among the most commonly reported in FXS, which significantly impacts daily living. Despite the extensive use of the Fmr1 knockout (KO) mouse to understand molecular, synaptic and behavioral alterations related to FXS, there has been limited development of translational paradigms to understand cognitive flexibility that can be employed in both animal models and individuals with FXS to facilitate treatment development.MethodsTo begin addressing this limitation, a parallel set of studies were carried out that investigated probabilistic reversal learning along with other behavioral and cognitive tests in individuals with FXS and Fmr1 KO mice. Fifty-five adolescents and adults with FXS (67% male) and 34 age- and sex-matched typically developing controls (62% male) completed an initial probabilistic learning training task and a probabilistic reversal learning task.ResultsIn males with FXS, both initial probabilistic learning and reversal learning deficits were found. However, in females with FXS, we only observed reversal learning deficits. Reversal learning deficits related to more severe psychiatric features in females with FXS, whereas increased sensitivity to negative feedback (lose:shift errors) unexpectedly appear to be adaptive in males with FXS. Male Fmr1 KO mice exhibited both an initial probabilistic learning and reversal learning deficit compared to that of wildtype (WT) mice. Female Fmr1 KO mice were selectively impaired on probabilistic reversal learning. In a prepotent response inhibition test, both male and female Fmr1 KO mice were impaired in learning to choose a non-preferred spatial location to receive a food reward compared to that of WT mice. Neither male nor female Fmr1 KO mice exhibited a change in anxiety compared to that of WT mice.DiscussionTogether, our findings demonstrate strikingly similar sex-dependent learning disturbances across individuals with FXS and Fmr1 KO mice. This suggests the promise of using analogous paradigms of cognitive flexibility across species that may speed treatment development to improve lives of individuals with FXS.

In stroke, the sudden deprivation of oxygen to neurons triggers a profuse release of glutamate that induces anoxic depolarization (AD) and leads to rapid cell death. Importantly, the latency of the glutamate-driven AD event largely dictates subsequent tissue damage. Although the contribution of synaptic glutamate during ischaemia is well-studied, the role of tonic (ambient) glutamate has received far less scrutiny. The majority of tonic, non-synaptic glutamate in the brain is governed by the cystine/glutamate antiporter, system x(c)(-). Employing hippocampal slice electrophysiology, we showed that transgenic mice lacking a functional system x(c)(-) display longer latencies to AD and altered depolarizing waves compared to wild-type mice after total oxygen deprivation. Experiments which pharmacologically inhibited system x(c)(-), as well as those manipulating tonic glutamate levels and those antagonizing glutamate receptors, revealed that the antiporter's putative effect on ambient glutamate precipitates the ischaemic cascade. As such, the current study yields novel insight into the pathogenesis of acute stroke and may direct future therapeutic interventions.

Ischemic stroke remains the third leading cause of death and leading cause of adult disability worldwide. A key event in the pathophysiology of stroke is the anoxic depolarization (AD) of neurons in the ischemic core. Previous studies have established that both the latency to AD and the time spent in AD prior to re-oxygenation are predictors of neuronal death. The present studies used hippocampal slices from male and female mice to investigate the electrophysiological events that affect latency to AD after oxygen deprivation. The results confirm that the epoch between AD and re-oxygenation largely determines the magnitude of synaptic recovery after anoxic challenge. Using a selective antagonist of adenosine A1 receptors, we also confirmed that adenosine released during anoxia (ANOX) suppresses synaptic glutamate release; however, this action has no effect on AD latency or the potential for post-anoxic recovery of synaptic transmission. In contrast, antagonism of AMPA- and NMDA-type glutamate receptors significantly prolongs the latency to AD and alters the speed and synchrony of associated depolarizing waves. Experiments using slices with fields Cornu ammonis 3 (CA3) and Cornu ammonis 1 (CA1) disconnected showed that AD latency is longer in CA1 than in CA3; however, the early AD in CA3 is propagated to CA1 in intact slices. Finally, AD latency in CA1 was found to be longer in slices from female mice than in those from age-matched male mice. The results have implications for stroke prevention and for understanding brain adaptations in hypoxia-tolerant animals.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

Small alterations in the level of extracellular H + can profoundly alter neuronal activity throughout the nervous system. In this study, self-referencing H + -selective microelectrodes were used to examine extracellular H + fluxes from individual astrocytes. Activation of astrocytes cultured from mouse hippocampus and rat cortex with extracellular ATP produced a pronounced increase in extracellular H + flux. The ATP-elicited increase in H + flux appeared to be independent of bicarbonate transport, as ATP increased H + flux regardless of whether the primary extracellular pH buffer was 26 mM bicarbonate or 1 mM HEPES, and persisted when atmospheric levels of CO 2 were replaced by oxygen. Adenosine failed to elicit any change in extracellular H + fluxes, and ATP-mediated increases in H + flux were inhibited by the P2 inhibitors suramin and PPADS suggesting direct activation of ATP receptors. Extracellular ATP also induced an intracellular rise in calcium in cultured astrocytes, and ATP-induced rises in both calcium and H + efflux were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin. Replacement of extracellular sodium with choline did not significantly reduce the size of the ATP-induced increases in H + flux, and the increases in H + flux were not significantly affected by addition of EIPA, suggesting little involvement of Na + /H + exchangers in ATP-elicited increases in H + flux. Given the high sensitivity of voltage-sensitive calcium channels on neurons to small changes in levels of free H + , we hypothesize that the ATP-mediated extrusion of H + from astrocytes may play a key role in regulating signaling at synapses within the nervous system.

Naked mole-rats are extremely tolerant to low concentrations of oxygen (hypoxia) and high concentrations of carbon dioxide (hypercapnia), which is consistent with the environment that they inhabit. Naked mole-rats combine subterranean living with living in very densely populated colonies where oxygen becomes depleted and carbon dioxide accumulates. In the laboratory, naked mole-rats fully recover from 5 h exposure to 5% O-2 and 5 h exposure to 80% CO2, whereas both conditions are rapidly lethal to similarly sized laboratory mice. During anoxia (0% O-2) naked mole-rats enter a suspended animation-like state and switch from aerobic metabolism of glucose to anaerobic metabolism of fructose. Additional fascinating characteristics include that naked mole-rats show intrinsic brain tolerance to anoxia; a complete lack of hypoxia-induced and CO2-induced pulmonary edema; and reduced aversion to high concentrations of CO2 and acidic fumes. Here we outline a constellation of physiological and molecular adaptations that correlate with the naked mole-rat's hypoxic/hypercapnic tolerance and which offer potential targets for ameliorating pathological conditions in humans, such as the damage caused during cerebral ischemia.

The African naked mole-rat's (Heterocephalus glaber) social and subterranean lifestyle generates a hypoxic niche. Under experimental conditions, naked mole-rats tolerate hours of extreme hypoxia and survive 18 minutes of total oxygen deprivation (anoxia) without apparent injury. During anoxia, the naked mole-rat switches to anaerobic metabolism fueled by fructose, which is actively accumulated and metabolized to lactate in the brain. Global expression of the GLUT5 fructose transporter and high levels of ketohexokinase were identified as molecular signatures of fructose metabolism. Fructose-driven glycolytic respiration in naked mole-rat tissues avoids feedback inhibition of glycolysis via phosphofructokinase, supporting viability. The metabolic rewiring of glycolysis can circumvent the normally lethal effects of oxygen deprivation, a mechanism that could be harnessed to minimize hypoxic damage in human disease.

Clinical failures singularly targeting amyloid-β pathology indicate a critical need for alternative Alzheimer’s disease (AD) therapeutic strategies. The mixed pathology reported in a large population of AD patients demands a multifunctional drug approach. Since activation of cAMP response element binding protein (CREB) plays a crucial role in synaptic strengthening and memory formation, we retooled a clinical drug with known neuroprotective and anti-inflammatory activity to activate CREB, and validated this novel multifunctional drug, NMZ, in 4 different mouse models of AD.

Activity-dependent plasticity in NMDA receptor-containing synapses can be regulated by phosphorylation of serines and tyrosines in the C-terminal domain of the receptor subunits by various kinases. We have previously identified S1291/S1312 as important sites for PKC phosphorylation; while Y1292/Y1312 are the sites indirectly phosphorylated by PKC via Src kinase. In the oocyte expression system, mutation of those Serine sites to Alanine (that cannot be phosphorylated) in the GluN2A subunit, resulted in a decreased PKC stimulated current enhancement through the receptors compared to wild-type NMDA receptors. To investigate the behavioral and physiological significance of those PKC-mediated phosphorylation sites in vivo, the Grin2a∆PKC mouse expressing GluN2A with four mutated amino acids: S1291A, S1312A, Y1292F and Y1387F was generated using homologous recombination. The Grin2a∆PKC mice exhibit reduced anxiety in the open field test, light dark emergence test, and elevated plus maze. The mutant mice show reduced alternation in a Y maze spontaneous alternation task and a in a non-reinforced T maze alternation task. Interestingly, when the mutant mice were exposed to novel environments, there was no increase in context-induced Fos levels in hippocampal CA1 and CA3 compared to home-cage Fos levels, while the Fos increased in the WT mice in CA1, CA3 and DG. When the SC-CA1 synapses in slices from mutant mice were stimulated using a theta-burst protocol, there was no impairment in LTP. Overall, these results suggest that at least one of those PKC-mediated phosphorylation sites regulates NMDAR-mediated signaling that modulates anxiety.

Brain neurons of the deep-diving hooded seal (Cystophora cristata) are known to be inherently hypoxia tolerant. Here, we have used in vitro field potential recordings in hippocampal slices to compare effects of severe hypoxia on synaptic transmission in hooded seals vs. non-diving mammals. Synaptic responses of mice (Mus musculus) to hypoxia were in accordance with previously published data. Hippocampal slices of reindeer (Rangifer tarandus), an alternative large-mammal non-diving model, behaved in a similar way as mouse slices, in that synaptic activity disappeared rapidly without recovery after >20min in hypoxia. The synaptic activity of hooded seal slices decreased in hypoxia, but unlike mice and reindeer, it remained at >30% of the normoxic amplitude throughout 3h of severe hypoxia. Also, upon reoxygenation, the signal recovered to ∼50% of the pre-challenge (normoxic) amplitude. The AMPA-type glutamate receptor antagonist CNQX eliminated this signal, showing that it was not an artifact. Paired pulse facilitation (PPF), typically associated with increased presynaptic calcium (Ca2+) levels, was significantly reduced in the seal slices. We propose that the build-up of Ca2+ concentration is limited in seal presynaptic terminals, possibly due to a high Ca2+ buffering capacity, which could explain both the attenuated PPF and the remarkable neural hypoxia tolerance of this species. Although we found no significant hypoxia-induced upregulation of mRNA for the Ca2+ binding proteins calbindin d28k or parvalbumin in hooded seal hippocampal slices, a recent study reports very high transcript levels of the Ca2+ binding protein S100B in this species, which is in support of the hypothesis.

This review covers the spatial and temporal rules governing induction of hippocampal long-term potentiation (LTP) by theta-burst stimulation. Induction of LTP in field CA1 by high frequency stimulation bursts that resemble the burst discharges (complex-spikes) of hippocampal pyramidal neurons involves a multiple-step mechanism. A single burst is insufficient for LTP induction because it evokes both excitatory and inhibitory currents that partially cancel and limit postsynaptic depolarization. Bursts repeated at the frequency (~5 Hz) of the endogenous theta rhythm induce maximal LTP, primarily because this frequency disables feed-forward inhibition and allows sufficient postsynaptic depolarization to activate voltage-sensitive NMDA receptors. The disinhibitory process, referred to as "priming", involves presynaptic GABA autoreceptors that inhibit GABA release. Activation of NMDA receptors allows a calcium flux into dendritic spines that serves as the proximal trigger for LTP. We include new data showing that theta-burst stimulation is more efficient than other forms of stimulation for LTP induction. In addition, we demonstrate that associative interactions between synapses activated during theta-bursts are limited to major dendritic domains since such interactions occur within apical or basal dendritic trees but not between them. We review evidence that recordings of electrophysiological responses during theta burst stimulation can help to determine if experimental manipulations that affect LTP do so by affecting events antecedent to the induction process, such as NMDA receptor activation, or downstream signaling cascades that result from postsynaptic calcium fluxes. Finally, we argue that theta-burst LTP represents a minimal model for stable, non-decremental LTP that is more sensitive to a variety of experimental manipulations than is LTP induced by other stimulation paradigms. This article is part of a Special Issue entitled SI: Brain and Memory.

Extreme longevity requires the continuous and large-scale adaptation of organ systems to delay senescence. Naked mole rats are the longest-living rodents, whose nervous system likely undergoes life-long adaptive reorganization. Nevertheless, neither the cellular organization of their cerebral cortex nor indices of structural neuronal plasticity along extreme time-scales have been established. We find that adult neurogenesis and neuronal migration are not unusual in naked mole rat brains. Instead, we show the prolonged expression of structural plasticity markers, many recognized as being developmentally controlled, and multi-year-long postnatal neuromorphogenesis and spatial synapse refinement in hippocampal and olfactory structures of the naked mole rat brain. Neurophysiological studies on identified hippocampal neurons demonstrated that morphological differentiation is disconnected from the control of excitability in all neuronal contingents regardless of their ability to self-renew. Overall, we conclude that naked mole rats show an extremely protracted period of brain maturation that may permit plasticity and resilience to neurodegenerative processes over their decades-long life span. This conclusion is consistent with the hypothesis that naked mole rats are neotenous, with retention of juvenile characteristics to permit survival in a hypoxic environment, with extreme longevity a consequence of greatly retarded development.

Many vertebrates are challenged by either chronic or acute episodes of low oxygen availability in their natural environments. Brain function is especially vulnerable to the effects of hypoxia and can be irreversibly impaired by even brief periods of low oxygen supply. This review describes recent research on physiological mechanisms that have evolved in certain vertebrate species to cope with brain hypoxia. Four model systems are considered: freshwater turtles that can survive for months trapped in frozen-over lakes, arctic ground squirrels that respire at extremely low rates during winter hibernation, seals and whales that undertake breath-hold dives lasting minutes to hours, and naked mole-rats that live in crowded burrows completely underground for their entire lives. These species exhibit remarkable specializations of brain physiology that adapt them for acute or chronic episodes of hypoxia. These specializations may be reactive in nature, involving modifications to the catastrophic sequelae of oxygen deprivation that occur in non-tolerant species, or preparatory in nature, preventing the activation of those sequelae altogether. Better understanding of the mechanisms used by these hypoxia-tolerant vertebrates will increase appreciation of how nervous systems are adapted for life in specific ecological niches as well as inform advances in therapy for neurological conditions such as stroke and epilepsy.

s 1. Keil, S.A. & Larson, J. Impaired olfactory discrimination learning in fragile X knockout mice. Abstracts, Society for Neuroscience (2014) Program no. 699.10. (APPENDIX 1) Manuscripts 1. Keil, S.A. & Larson, J. Impaired olfactory discrimination learning in fragile X knockout mice. Manuscript in preparation (APPENDIX 2) Degrees obtained with support from this award 1. Patricia Dykas, B.S., with Distinction in Biological Sciences, University of Illinois at Chicago (2014). Research Opportunities supported by this award 1. Samantha Keil, B.S. was accepted into the UIC Graduate Program in Neuroscience, in part because of research experience obtained working on this project (2013-2014). 2. Tiffany Cheng, UIC undergraduate student, is assisting with the behavioral studies of Fmr1KO mice.

It has been suggested that age-related impairments in learning and memory may be due to age-related deficits in long-term potentiation of glutamatergic synaptic transmission. For example, olfactory discrimination learning is significantly affected by aging in mice and this may be due, in part, to diminished synaptic plasticity in piriform cortex. In the present study, we tested for alterations in electrophysiological properties and synaptic transmission in this simple cortical network. Whole-cell recordings were made from principal neurons in slices of anterior piriform cortex from young (3-6 months old) and old (24-28 months) C57Bl/6 mice. Miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were collected from cells in presence of tetrodotoxin (TTX) and held at -80 mV in voltage-clamp. Amplitudes of mEPSCs were significantly reduced in aged mice, suggesting that synaptic AMPA receptor expression is decreased during aging. In a second set of experiments, spontaneous excitatory postsynaptic currents (s/mEPSCs) were recorded in slices from different cohorts of young and old mice, in the absence of TTX. These currents resembled mEPSCs and were similarly reduced in amplitude in old mice. The results represent the first electrophysiological evidence for age-related declines in glutamatergic synaptic function in the mammalian olfactory system.

Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3(rd) generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer's transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events.

Results and Conclusion The hypothesis that activation of CREB through NO/ cGMP signaling might modify the amyloid-b (Ab) neuropathology, linked to AD pathogenesis, was demonstrated in both APP/PS1 and 3xTg transgenic mouse models of AD using small molecules, termed nomethiazoles, also designed to provide neuroprotection and attenuate pro-inflammatory cytokine release. Functional restoration of long-term potentiation was shown in hippocampal slices from AD transgenic mice in accord with observation of restoration of cognitive function in vivo, and was dependent upon soluble guanylyl cyclase (sGC) activation. Levels of pCREB and BDNF were significantly elevated, whereas TNFa, Ab, oligomeric Ab142, and also tau protein were significantly lowered after drug treatment. In the absence of neuronal loss in animal models of AD, neuroprotection was demonstrated in rat primary neurons after oxygen-glucose deprivation or application of oligomeric Ab. The lead nomethiazole was also studied in a novel transgenic mouse model, E4FAD, which incorporates familial AD mutations, but also has targeted replacement of mouse apolipoproteinE with human ApoE4, the major genetic risk factor for sporadic and age-related AD.