Chemogenetic inhibition of central amygdala CRF-expressing neurons decreases alcohol intake but not trauma-related behaviors in a rat model of post-traumatic stress and alcohol use disorder.

Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are often comorbid. Few treatments exist to reduce comorbid PTSD/AUD. Elucidating the mechanisms underlying their comorbidity could reveal new avenues for therapy. Here, we employed a model of comorbid PTSD/AUD, in which rats were subjected to a stressful shock in a familiar context followed by alcohol drinking. We then examined fear overgeneralization and irritability in these rats. Familiar context stress elevated drinking, increased fear overgeneralization, increased alcohol-related aggressive signs, and elevated peripheral stress hormones. We then examined transcripts of stress- and fear-relevant genes in the central amygdala (CeA), a locus that regulates stress-mediated alcohol drinking. Compared with unstressed rats, stressed rats exhibited increases in CeA transcripts for Crh and Fkbp5 and decreases in transcripts for Bdnf and Il18. Levels of Nr3c1 mRNA, which encodes the glucocorticoid receptor, increased in stressed males but decreased in stressed females. Transcripts of Il18 binding protein (Il18bp), Glp-1r, and genes associated with calcitonin gene-related peptide signaling (Calca, Ramp1, Crlr-1, and Iapp) were unaltered. Crh, but not Crhr1, mRNA was increased by stress; thus, we tested whether inhibiting CeA neurons that express corticotropin-releasing factor (CRF) suppress PTSD/AUD-like behaviors. We used Crh-Cre rats that had received a Cre-dependent vector encoding hM4D(Gi), an inhibitory Designer Receptors Exclusively Activated by Designer Drugs. Chemogenetic inhibition of CeA CRF neurons reduced alcohol intake but not fear overgeneralization or irritability-like behaviors. Our findings suggest that CeA CRF modulates PTSD/AUD comorbidity, and inhibiting CRF neural activity is primarily associated with reducing alcohol drinking but not trauma-related behaviors that are associated with PTSD/AUD.

Glucocorticoid receptors regulate central amygdala GABAergic synapses in Marchigian-Sardinian alcohol-preferring rats.

Impairments in the function of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced glucocorticoid receptor (GR) activity in the central amygdala (CeA) are critical mechanisms in the pathogenesis of alcohol use disorder (AUD). The GR antagonist mifepristone attenuates craving in AUD patients, alcohol consumption in AUD models, and decreases CeA γ-aminobutyric acid (GABA) transmission in alcohol-dependent rats. Previous studies suggest elevated GR activity in the CeA of male alcohol-preferring Marchigian-Sardinian (msP) rats, but its contribution to heightened CeA GABA transmission driving their characteristic post-dependent phenotype is largely unknown. We determined Nr3c1 (the gene encoding GR) gene transcription in the CeA in male and female msP and Wistar rats using in situ hybridization and studied acute effects of mifepristone (10 µM) and its interaction with ethanol (44 mM) on pharmacologically isolated spontaneous inhibitory postsynaptic currents (sIPSCs) and electrically evoked inhibitory postsynaptic potentials (eIPSPs) in the CeA using ex vivo slice electrophysiology. Female rats of both genotypes expressed more CeA GRs than males, suggesting a sexually dimorphic GR regulation of CeA activity. Mifepristone reduced sIPSC frequencies (GABA release) and eIPSP amplitudes in msP rats of both sexes, but not in their Wistar counterparts; however, it did not prevent acute ethanol-induced increase in CeA GABA transmission in male rats. In msP rats, GR regulates CeA GABAergic signaling under basal conditions, indicative of intrinsically active GR. Thus, enhanced GR function in the CeA represents a key mechanism contributing to maladaptive behaviors associated with AUD.

IL-18 Signaling in the Rat Central Amygdala Is Disrupted in a Comorbid Model of Post-Traumatic Stress and Alcohol Use Disorder.

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share dysregulated neuroimmune-related pathways. Here, we used our established rat model of comorbid post-traumatic stress disorder (PTSD)/AUD to characterize the interleukin 18 (IL-18) system in the central amygdala (CeA). Male and female rats underwent novel (NOV) and familiar (FAM) shock stress, or no stress (unstressed controls; CTL) followed by voluntary alcohol drinking and PTSD-related behaviors, then all received renewed alcohol access prior to the experiments. In situ hybridization revealed that the number of CeA positive cells for Il18 mRNA increased, while for Il18bp decreased in both male and female FAM stressed rats versus CTL. No changes were observed in Il18r1 expression across groups. Ex vivo electrophysiology showed that IL-18 reduced GABAA-mediated miniature inhibitory postsynaptic currents (mIPSCs) frequencies in CTL, suggesting reduced CeA GABA release, regardless of sex. Notably, this presynaptic effect of IL-18 was lost in both NOV and FAM males, while it persisted in NOV and FAM females. IL-18 decreased mIPSC amplitude in CTL female rats, suggesting postsynaptic effects. Overall, our results suggest that stress in rats with alcohol access impacts CeA IL-18-system expression and, in sex-related fashion, IL-18's modulatory function at GABA synapses.

The glucagon-like peptide-1 (GLP-1) analogue semaglutide reduces alcohol drinking and modulates central GABA neurotransmission.

Growing evidence indicates that the glucagon-like peptide-1 (GLP-1) system is involved in the neurobiology of addictive behaviors, and GLP-1 analogues may be used for the treatment of alcohol use disorder (AUD). Here, we examined the effects of semaglutide, a long-acting GLP-1 analogue, on biobehavioral correlates of alcohol use in rodents. A drinking-in-the-dark procedure was used to test the effects of semaglutide on binge-like drinking in male and female mice. We also tested the effects of semaglutide on binge-like and dependence-induced alcohol drinking in male and female rats, as well as acute effects of semaglutide on spontaneous inhibitory postsynaptic currents (sIPSCs) from central amygdala (CeA) and infralimbic cortex (ILC) neurons. Semaglutide dose-dependently reduced binge-like alcohol drinking in mice; a similar effect was observed on the intake of other caloric/noncaloric solutions. Semaglutide also reduced binge-like and dependence-induced alcohol drinking in rats. Semaglutide increased sIPSC frequency in CeA and ILC neurons from alcohol-naive rats, suggesting enhanced GABA release, but had no overall effect on GABA transmission in alcohol-dependent rats. In conclusion, the GLP-1 analogue semaglutide decreased alcohol intake across different drinking models and species and modulated central GABA neurotransmission, providing support for clinical testing of semaglutide as a potentially novel pharmacotherapy for AUD.

Alcohol Dependence Induces CRF Sensitivity in Female Central Amygdala GABA Synapses.

Alcohol use disorder (AUD) is a chronically relapsing disease characterized by loss of control in seeking and consuming alcohol (ethanol) driven by the recruitment of brain stress systems. However, AUD differs among the sexes: men are more likely to develop AUD, but women progress from casual to binge drinking and heavy alcohol use more quickly. The central amygdala (CeA) is a hub of stress and anxiety, with corticotropin-releasing factor (CRF)-CRF1 receptor and Gamma-Aminobutyric Acid (GABA)-ergic signaling dysregulation occurring in alcohol-dependent male rodents. However, we recently showed that GABAergic synapses in female rats are less sensitive to the acute effects of ethanol. Here, we used patch-clamp electrophysiology to examine the effects of alcohol dependence on the CRF modulation of rat CeA GABAergic transmission of both sexes. We found that GABAergic synapses of naïve female rats were unresponsive to CRF application compared to males, although alcohol dependence induced a similar CRF responsivity in both sexes. In situ hybridization revealed that females had fewer CeA neurons containing mRNA for the CRF1 receptor (Crhr1) than males, but in dependence, the percentage of Crhr1-expressing neurons in females increased, unlike in males. Overall, our data provide evidence for sexually dimorphic CeA CRF system effects on GABAergic synapses in dependence.

Decreased excitability of leptin-sensitive anterior insula pyramidal neurons in a rat model of compulsive food demand.

Compulsive eating is an overlapping construct with binge eating that shares many characteristics with substance use disorders. Compulsive eating may impact millions of Americans; presenting in some cases of binge eating disorders, overweight/obesity, and among individuals who have not yet been diagnosed with a recognized eating disorder. To study the behavioral and neurobiological underpinnings of compulsive eating, we employ a published rodent model using cyclic intermittent access to a palatable diet to develop a self-imposed binge-withdrawal cycle. Here, we further validated this model of compulsive eating in female Wistar rats, through the lens of behavioral economic analyses and observed heightened demand intensity, inelasticity and essential value as well as increased food-seeking during extinction. Using electrophysiological recordings in the anterior insular cortex, a region previously implicated in modulating compulsive-like eating in intermittent access models, we observed functional adaptations of pyramidal neurons. Within the same neurons, application of leptin led to further functional adaptations, suggesting a previously understudied, extrahypothalamic role of leptin in modulating feeding-related cortical circuits. Collectively, the findings suggest that leptin may modulate food-related motivation or decision-making via a plastic cortical circuit that is influenced by intermittent access to a preferred diet. These findings warrant further study of whether behavioral economics analysis of compulsive eating can impact disordered eating outcomes in humans and of the translational relevance of a leptin-sensitive anterior insular circuit implicated in these behaviors.

Sex Differences in Acute Alcohol Sensitivity of Naïve and Alcohol Dependent Central Amygdala GABA Synapses.

AIMS: Alcohol use disorder (AUD) is linked to hyperactivity of brain stress systems, leading to withdrawal states which drive relapse. AUD differs among the sexes, as men are more likely to have AUD than women, but women progress from casual use to binge and heavy alcohol use more quickly and are more likely to relapse into repetitive episodes of heavy drinking. In alcohol dependence animal models of AUD, the central amygdala (CeA) functions as a hub of stress and anxiety processing and gamma-Aminobutyric acid (GABA)ergic signaling within the CeA is involved in dependence-induced increases in alcohol consumption. We have shown dysregulation of CeA GABAergic synaptic signaling in alcohol dependence animal models, but previous studies have exclusively used males. METHODS: Here, we used whole-cell patch clamp electrophysiology to examine basal CeA GABAergic spontaneous inhibitory postsynaptic currents (sIPSC) and the effects of acute alcohol in both naïve and alcohol dependent rats of both sexes. RESULTS: We found that sIPSC kinetics differ between females and males, as well as between naïve and alcohol-dependent animals, with naïve females having the fastest current kinetics. Additionally, we find differences in baseline current kinetics across estrous cycle stages. In contrast to the increase in sIPSC frequency routinely found in males, acute alcohol (11-88 mM) had no effect on sIPSCs in naïve females, however the highest concentration of alcohol increased sIPSC frequency in dependent females. CONCLUSION: These results provide important insight into sex differences in CeA neuronal function and dysregulation with alcohol dependence and highlight the need for sex-specific considerations in the development of effective AUD treatment.

Importance of sex and trauma context on circulating cytokines and amygdalar GABAergic signaling in a comorbid model of posttraumatic stress and alcohol use disorders.

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share mechanisms that could be therapeutic targets. To facilitate mechanistic studies, we adapted an inhibitory avoidance-based "2-hit" rat model of posttraumatic stress disorder (PTSD) and identified predictors and biomarkers of comorbid alcohol (ethanol)/PTSD-like symptoms in these animals. Stressed Wistar rats received a single footshock on two occasions. The first footshock occurred when rats crossed into the dark chamber of a shuttle box. Forty-eight hours later, rats received the second footshock in a familiar (FAM) or novel (NOV) context. Rats then received 4 weeks of two-bottle choice (2BC) ethanol access. During subsequent abstinence, PTSD-like behavior responses, GABAergic synaptic transmission in the central amygdala (CeA), and circulating cytokine levels were measured. FAM and NOV stress more effectively increased 2BC drinking in males and females, respectively. Stressed male rats, especially drinking-vulnerable individuals (≥0.8 g/kg average 2-h ethanol intake with >50% ethanol preference), showed higher fear overgeneralization in novel contexts, increased GABAergic transmission in the CeA, and a profile of increased G-CSF, GM-CSF, IL-13, IL-6, IL-17a, leptin, and IL-4 that discriminated between stress context (NOV > FAM > Control). However, drinking-resilient males showed the highest G-CSF, IL-13, and leptin levels. Stressed females showed increased acoustic startle and decreased sleep maintenance, indicative of hyperarousal, with increased CeA GABAergic transmission in NOV females. This paradigm promotes key features of PTSD, including hyperarousal, fear generalization, avoidance, and sleep disturbance, with comorbid ethanol intake, in a sex-specific fashion that approximates clinical comorbidities better than existing models, and identifies increased CeA GABAergic signaling and a distinct pro-hematopoietic, proinflammatory, and pro-atopic cytokine profile that may aid in treatment.

Microglia Control Escalation of Drinking in Alcohol-Dependent Mice: Genomic and Synaptic Drivers.

BACKGROUND: Microglia, the primary immune cells of the brain, are implicated in alcohol use disorder. However, it is not known if microglial activation contributes to the transition from alcohol use to alcohol use disorder or is a consequence of alcohol intake. METHODS: We investigated the role of microglia in a mouse model of alcohol dependence using a colony stimulating factor 1 receptor inhibitor (PLX5622) to deplete microglia and a chronic intermittent ethanol vapor two-bottle choice drinking procedure. Additionally, we examined anxiety-like behavior during withdrawal. We then analyzed synaptic neuroadaptations in the central nucleus of the amygdala (CeA) and gene expression changes in the medial prefrontal cortex and CeA from the same animals used for behavioral studies. RESULTS: PLX5622 prevented escalations in voluntary alcohol intake and decreased anxiety-like behavior associated with alcohol dependence. PLX5622 also reversed expression changes in inflammatory-related genes and glutamatergic and GABAergic (gamma-aminobutyric acidergic) genes in the medial prefrontal cortex and CeA. At the cellular level in these animals, microglia depletion reduced inhibitory GABAA and excitatory glutamate receptor-mediated synaptic transmission in the CeA, supporting the hypothesis that microglia regulate dependence-induced changes in neuronal function. CONCLUSIONS: Our multifaceted approach is the first to link microglia to the molecular, cellular, and behavioral changes associated with the development of alcohol dependence, suggesting that microglia may also be critical for the development and progression of alcohol use disorder.

Altered Cognitive Flexibility and Synaptic Plasticity in the Rat Prefrontal Cortex after Exposure to Low (≤15 cGy) Doses of 28Si Radiation.

This study has established the impact that 1-15 cGy 600 MeV/n 28Si radiation had on cognitive flexibility performance, glutamatergic synaptic transmission and plasticity in the prelimbic area (PrL) of the medial prefrontal cortex (mPFC) of ∼10-month-old (at the time of irradiation) male Wistar rats. Exposure to 1 cGy 600 MeV/n 28Si ions resulted in significantly impaired performance in the simple (SD) and compound discrimination (CD) stages of the attentional set shifting (ATSET) task. However, there was a pronounced non-linear dose response for cognitive impairment. Should similar effects occur in astronauts, the impairment of SD performance would result in a decreased ability to identify and learn the "rules" required to respond to new tasks/situations, while the impaired CD performance would result in a decreased ability to identify and maintain focus on relevant aspects of the task being conducted. The irradiated rats were also screened for performance in a task for unconstrained cognitive flexibility (UCFlex), often referred to as creative problem solving. Exposure to 1, 5 and 10 cGy resulted in a significant reduction in UCFlex performance, in an apparent all-or-none responsive manner. Importantly, performance in the ATSET test was not indicative of UCFlex performance. From a risk assessment perspective, these findings suggest that a value based on a single behavioral end point may not fully represent the cognitive deficits induced by space radiation, even within the cognitive flexibility domain. After completion of the cognitive flexibility testing, in vitro electrophysiological assessments of glutamatergic synaptic transmission and plasticity were performed in slices of the PrL cortex of 10 cGy irradiated rats. Extracellular recordings of field excitatory postsynaptic potentials revealed that radiation significantly decreased long-term depression in layer L5. Patch-clamp whole cell recordings in pyramidal neurons of the L2-3 revealed reduced frequency of spontaneous excitatory postsynaptic currents indicating alterations in presynaptic glutamate release and impaired neuronal spiking (e.g., decreased action potential amplitudes) in irradiated neurons. However, there was no obvious correlation between magnitudes of these electrophysiological decrements and the cognitive performance status of the irradiated rats. These data suggest that while radiation-induced changes in synaptic plasticity in the PrL cortex may be associated with cognitive impairment, they are most likely not the sole determinant of the incidence and severity of such impairments.

Role of MyD88 in IL-1ß and Ethanol Modulation of GABAergic Transmission in the Central Amygdala.

Myeloid differentiation primary response protein (MyD88) is a critical neuroimmune adaptor protein in TLR (Toll-like receptor) and IL-1R (Interleukin-1 receptor) signaling complexes. These two pro-inflammatory families play an important role in the neurobiology of alcohol use disorder, specifically MyD88 regulates ethanol drinking, ethanol-induced sedation, and ethanol-induced deficits in motor coordination. In this study, we examined the role of MyD88 in mediating the effects of IL-1ß and ethanol on GABAergic transmission in the central amygdala (CeA) of male mice using whole-cell patch-clamp recordings in combination with pharmacological (AS-1, a mimetic that prevents MyD88 recruitment by IL-1R) and genetic (Myd88 knockout mice) approaches. We demonstrate through both approaches that IL-1ß and ethanol's modulatory effects at CeA GABA synapses are not dependent on MyD88. Myd88 knockout potentiated IL-1ß's actions in reducing postsynaptic GABAA receptor function. Pharmacological inhibition of MyD88 modulates IL-1ß's action at CeA GABA synapses similar to Myd88 knockout mice. Additionally, ethanol-induced CeA GABA release was greater in Myd88 knockout mice compared to wildtype controls. Thus, MyD88 is not essential to IL-1ß or ethanol regulation of CeA GABA synapses but plays a role in modulating the magnitude of their effects, which may be a potential mechanism by which it regulates ethanol-related behaviors.

Increased IL-6 expression in astrocytes is associated with emotionality, alterations in central amygdala GABAergic transmission, and excitability during alcohol withdrawal.

Accumulating evidence from preclinical and clinical studies has implicated a role for the cytokine IL-6 in a variety of CNS diseases including anxiety-like and depressive-like behaviors, as well as alcohol use disorder. Here we use homozygous and heterozygous transgenic mice expressing elevated levels of IL-6 in the CNS due to increased astrocyte expression and non-transgenic littermates to examine a role for astrocyte-produced IL-6 in emotionality (response to novelty, anxiety-like, and depressive-like behaviors). Our results from homozygous IL-6 mice in a variety of behavioral tests (light/dark transfer, open field, digging, tail suspension, and forced swim tests) support a role for IL-6 in stress-coping behaviors. Ex vivo electrophysiological studies of neuronal excitability and inhibitory GABAergic synaptic transmission in the central nucleus of the amygdala (CeA) of the homozygous transgenic mice revealed increased inhibitory GABAergic signaling and increased excitability of CeA neurons, suggesting a role for astrocyte produced IL-6 in the amygdala in exploratory drive and depressive-like behavior. Furthermore, studies in the hippocampus of activation/expression of proteins associated with IL-6 signal transduction and inhibitory GABAergic mechanisms support a role for astrocyte produced IL-6 in depressive-like behaviors. Our studies indicate a complex and dose-dependent relationship between IL-6 and behavior and implicate IL-6 induced neuroadaptive changes in neuronal excitability and the inhibitory GABAergic system as important contributors to altered behavior associated with IL-6 expression in the CNS.

IL-1ß expression is increased and regulates GABA transmission following chronic ethanol in mouse central amygdala.

The interleukin-1 system (IL-1) is a prominent pro-inflammatory pathway responsible for the initiation and regulation of immune responses. Human genetic and preclinical studies suggest a critical role for IL-1ß signaling in ethanol drinking and dependence, but little is known about the effects of chronic ethanol on the IL-1 system in addiction-related brain regions such as the central amygdala (CeA). In this study, we generated naïve, non-dependent (Non-Dep) and dependent (Dep) male mice using a paradigm of chronic-intermittent ethanol vapor exposure interspersed with two-bottle choice to examine 1) the expression of IL-1ß, 2) the role of the IL-1 system on GABAergic transmission, and 3) the potential interaction with the acute effects of ethanol in the CeA. Immunohistochemistry with confocal microscopy was used to assess expression of IL-1ß in microglia and neurons in the CeA, and whole-cell patch clamp recordings were obtained from CeA neurons to measure the effects of IL-1ß (50 ng/ml) or the endogenous IL-1 receptor antagonist (IL-1ra; 100 ng/ml) on action potential-dependent spontaneous inhibitory postsynaptic currents (sIPSCs). Overall, we found that IL-1ß expression is significantly increased in microglia and neurons of Dep compared to Non-Dep and naïve mice, IL-1ß and IL-1ra bi-directionally modulate GABA transmission through both pre- and postsynaptic mechanisms in all three groups, and IL-1ß and IL-1ra do not alter the facilitation of GABA release induced by acute ethanol. These data suggest that while ethanol dependence induces a neuroimmune response in the CeA, as indicated by increased IL-1ß expression, this does not significantly alter the neuromodulatory role of IL-1ß on synaptic transmission.

Low-dose proton radiation effects in a transgenic mouse model of Alzheimer's disease - Implications for space travel.

Space radiation represents a significant health risk for astronauts. Ground-based animal studies indicate that space radiation affects neuronal functions such as excitability, synaptic transmission, and plasticity, and it may accelerate the onset of Alzheimer's disease (AD). Although protons represent the main constituent in the space radiation spectrum, their effects on AD-related pathology have not been tested. We irradiated 3 month-old APP/PSEN1 transgenic (TG) and wild type (WT) mice with protons (150 MeV; 0.1-1.0 Gy; whole body) and evaluated functional and biochemical hallmarks of AD. We performed behavioral tests in the water maze (WM) before irradiation and in the WM and Barnes maze at 3 and 6 months post-irradiation to evaluate spatial learning and memory. We also performed electrophysiological recordings in vitro in hippocampal slices prepared 6 and 9 months post-irradiation to evaluate excitatory synaptic transmission and plasticity. Next, we evaluated amyloid ß (Aß) deposition in the contralateral hippocampus and adjacent cortex using immunohistochemistry. In cortical homogenates, we analyzed the levels of the presynaptic marker synaptophysin by Western blotting and measured pro-inflammatory cytokine levels (TNFα, IL-1ß, IL-6, CXCL10 and CCL2) by bead-based multiplex assay. TG mice performed significantly worse than WT mice in the WM. Irradiation of TG mice did not affect their behavioral performance, but reduced the amplitudes of population spikes and inhibited paired-pulse facilitation in CA1 neurons. These electrophysiological alterations in the TG mice were qualitatively different from those observed in WT mice, in which irradiation increased excitability and synaptic efficacy. Irradiation increased Aß deposition in the cortex of TG mice without affecting cytokine levels and increased synaptophysin expression in WT mice (but not in the TG mice). Although irradiation with protons increased Aß deposition, the complex functional and biochemical results indicate that irradiation effects are not synergistic to AD pathology.

A Single Low Dose of Proton Radiation Induces Long-Term Behavioral and Electrophysiological Changes in Mice.

Astronauts traveling outside Earth's magnetosphere risk exposure to charged particle radiation that may cause neurophysiological changes and behavioral deficits. Although proton particles comprise a large portion of the space radiation environment, little has been published on the effects of low-dose proton radiation on central nervous system function. In the current study, we irradiated young male mice with 0.5 Gy 150 MeV protons and assessed the effects on behavior and hippocampal neurophysiology. Spatial learning ability, a sensitive behavioral marker of hippocampal damage, was assessed using the water maze and Barnes maze before irradiation and repeatedly 3 and 6 months after irradiation. Evoked field excitatory postsynaptic potentials (fEPSPs) and population spikes, long-term potentiation (LTP) and spontaneous oscillations (SOs) triggered by incubation with Mg(2+)-free media (reflecting interictal epileptiform activity) were assessed 9 months after irradiation in vitro in hippocampal slice preparations. Irradiated mice exhibited impaired reversal learning in the water maze compared to control mice 6 months after irradiation. Proton radiation did not affect LTP, but significantly increased fEPSP slopes and reduced the incidence of SOs 9 months after irradiation. These findings suggest that a single exposure to low-dose proton radiation can increase synaptic excitability and suppress the propensity for epileptiform activity. Such findings of functional alterations in the irradiated mouse hippocampus have implications for extended manned space missions planned in the near future.

Proton radiation alters intrinsic and synaptic properties of CA1 pyramidal neurons of the mouse hippocampus.

High-energy protons constitute at least 85% of the fluence of energetic ions in interplanetary space. Although protons are only sparsely ionizing compared to higher atomic mass ions, they nevertheless significantly contribute to the delivered dose received by astronauts that can potentially affect central nervous system function at high fluence, especially during prolonged deep space missions such as to Mars. Here we report on the long-term effects of 1 Gy proton irradiation on electrophysiological properties of CA1 pyramidal neurons in the mouse hippocampus. The hippocampus is a key structure for the formation of long-term episodic memory, for spatial orientation and for information processing in a number of other cognitive tasks. CA1 pyramidal neurons form the last and critical relay point in the trisynaptic circuit of the hippocampal principal neurons through which information is processed before being transferred to other brain areas. Proper functioning of CA1 pyramidal neurons is crucial for hippocampus-dependent tasks. Using the patch-clamp technique to evaluate chronic effects of 1 Gy proton irradiation on CA1 pyramidal neurons, we found that the intrinsic membrane properties of CA1 pyramidal neurons were chronically altered at 3 months postirradiation, resulting in a hyperpolarization of the resting membrane potential (VRMP) and a decrease in input resistance (Rin). These small but significant alterations in intrinsic properties decreased the excitability of CA1 pyramidal neurons, and had a dramatic impact on network function in a computational model of the CA1 microcircuit. We also found that proton-radiation exposure upregulated the persistent Na(+) current (INaP) and increased the rate of miniature excitatory postsynaptic currents (mEPSCs). Both the INaP and the heightened rate of mEPSCs contribute to neuronal depolarization and excitation, and at least in part, could compensate for the reduced excitability resulting from the radiation effects on the VRMP and the Rin. These results show long-term alterations in the intrinsic properties of CA1 pyramidal cells after realistic, low-dose proton irradiation.

Targeted overexpression of mitochondrial catalase prevents radiation-induced cognitive dysfunction.

AIMS: Radiation-induced disruption of mitochondrial function can elevate oxidative stress and contribute to the metabolic perturbations believed to compromise the functionality of the central nervous system. To clarify the role of mitochondrial oxidative stress in mediating the adverse effects of radiation in the brain, we analyzed transgenic (mitochondrial catalase [MCAT]) mice that overexpress human catalase localized to the mitochondria. RESULTS: Compared with wild-type (WT) controls, overexpression of the MCAT transgene significantly decreased cognitive dysfunction after proton irradiation. Significant improvements in behavioral performance found on novel object recognition and object recognition in place tasks were associated with a preservation of neuronal morphology. While the architecture of hippocampal CA1 neurons was significantly compromised in irradiated WT mice, the same neurons in MCAT mice did not exhibit extensive and significant radiation-induced reductions in dendritic complexity. Irradiated neurons from MCAT mice maintained dendritic branching and length compared with WT mice. Protected neuronal morphology in irradiated MCAT mice was also associated with a stabilization of radiation-induced variations in long-term potentiation. Stabilized synaptic activity in MCAT mice coincided with an altered composition of the synaptic AMPA receptor subunits GluR1/2. INNOVATION: Our findings provide the first evidence that neurocognitive sequelae associated with radiation exposure can be reduced by overexpression of MCAT, operating through a mechanism involving the preservation of neuronal morphology. CONCLUSION: Our article documents the neuroprotective properties of reducing mitochondrial reactive oxygen species through the targeted overexpression of catalase and how this ameliorates the adverse effects of proton irradiation in the brain.

(28)Silicon radiation-induced enhancement of synaptic plasticity in the hippocampus of naïve and cognitively tested mice.

The space radiation environment consists of multiple species of high-energy charge particles (HZE), including (56)Fe and (28)Si nuclei, that may impact neuronal cells, but their damaging effects on the central nervous system (CNS) have been poorly defined. Hippocampus-dependent memory functions have been shown to be highly sensitive to (56)Fe HZE particles, which poses a significant risk to the cognitive performance of astronauts during space missions. While low doses of (56)Fe radiation do not induce cell death of mature neurons, they affect synaptic plasticity in the CA1 region, the principal neuronal output of the hippocampal formation involved in memory formation. The effects of (28)Si on the CNS have not been defined. Compared to behaviorally naïve mice, cognitive testing might affect synaptic plasticity and the effects of (28)Si radiation on synaptic plasticity might be modulated by prior cognitive testing. Therefore, in the current study, we quantified the effects of whole-body (28)Si radiation (600 MeV/n, 0.25 and 1 Gy) on hippocampus-dependent contextual freezing and synaptic plasticity in the CA1 region of animals not exposed (behaviorally naïve mice) and animals exposed to the contextual freezing test (cognitively tested mice). In behaviorally naïve mice exposed to 0.25 and 1 Gy of (28)Si radiation, the magnitude of long-term potentiation (LTP) was enhanced. However, in mice irradiated with 0.25 Gy contextual fear conditioning was enhanced and was associated with a further enhancement of the LTP magnitude. Such increase in synaptic plasticity was not seen in cognitively tested mice irradiated with 1 Gy. Thus, low dose (28)Si radiation has effects on synaptic plasticity in the CA1 region of the hippocampus and these effects are modulated by cognitive testing in a contextual fear-conditioning test.

(28)silicon radiation impairs neuronal output in CA1 neurons of mouse ventral hippocampus without altering dendritic excitability.

An unavoidable complication of space travel is exposure to radiation consisting of high-energy charged particles (HZE), such as Fe and Si nuclei. HZE radiation can affect neuronal functions at the level of the synapse or neuronal soma without inducing significant neuronal death. Different radiation species impart distinct patterns of radiation damage depending on their track structure, dose rate and fluence. Moreover, structural differences exist along the dorsoventral axis of the hippocampus that may underlie different radiosensitivities within the same neuronal field (e.g., the CA1 pyramidal cell population of the hippocampus). In this study we tested the functional effects of low doses of (28)Si radiation on excitability and synaptic plasticity in hippocampal slices prepared strictly from the ventral hippocampus. We used extracellular electrophysiological techniques to record field excitatory postsynaptic potentials (EPSPs) and population spikes in hippocampal CA1 neurons from C57BL/6J male mice 3 months after exposure to (28)Si radiation (600 MeV/n; 0.25 and 1 Gy, whole body). In irradiated mice we found prominent decrements in population spike amplitudes and reduced maximal neuronal output without changes in dendritic field EPSP. Reduced field EPSP vs. population spike ratios indicate radiation-induced impairment of the EPSP-spike (E-S) coupling. This effect was not associated with significant changes in the magnitude of short- and long-term synaptic plasticity [long-term potentiation (LTP)]. These data confirm that irradiation with (28)Si particles at relatively low doses alters the properties of the hippocampal network, which can limit its connectivity with other brain centers.

Traumatic brain injury in young rats leads to progressive behavioral deficits coincident with altered tissue properties in adulthood.

Traumatic brain injury (TBI) affects many infants and children, and results in enduring motor and cognitive impairments with accompanying changes in white matter tracts, yet few experimental studies in rodent juvenile models of TBI (jTBI) have examined the timeline and nature of these deficits, histologically and functionally. We used a single controlled cortical impact (CCI) injury to the parietal cortex of rats at post-natal day (P) 17 to evaluate behavioral alterations, injury volume, and morphological and molecular changes in gray and white matter, with accompanying measures of electrophysiological function. At 60 days post-injury (dpi), we found that jTBI animals displayed behavioral deficits in foot-fault and rotarod tests, along with a left turn bias throughout their early developmental stages and into adulthood. In addition, anxiety-like behaviors on the zero maze emerged in jTBI animals at 60 dpi. The final lesion constituted only ∼3% of brain volume, and morphological tissue changes were evaluated using MRI, as well as immunohistochemistry for neuronal nuclei (NeuN), myelin basic protein (MBP), neurofilament-200 (NF200), and oligodendrocytes (CNPase). White matter morphological changes were associated with a global increase in MBP immunostaining and reduced compound action potential amplitudes at 60 dpi. These results suggest that brain injury early in life can induce long-term white matter dysfunction, occurring in parallel with the delayed development and persistence of behavioral deficits, thus modeling clinical and longitudinal TBI observations.