Circadian Rhythms in Fear Extinction Recall Depend on the Time of Day of Extinction Recall, Not the Time of Day of Extinction Learning.

The recall of conditioned fear extinction exhibits a circadian rhythm in humans and rodents, with optimal extinction recall occurring during the early active phase. However, it remains unclear whether this rhythm depends on the circadian modulation of mechanisms supporting memory consolidation versus memory maintenance and retrieval. Here, adult male rats underwent conditioned fear extinction at one of four times throughout the day and then, starting 24 h after extinction, were repeatedly tested for extinction recall over the next 24 h. Rats undergoing extinction learning during the early active phase tended toward accelerated extinction learning compared with rats in other groups, pointing to rhythms in mechanisms that support extinction memory encoding. The next day, the strength of extinction recall followed a 24-h cycle that depended not on the time of day of extinction learning but, instead, on the time of day of extinction recall. This latter finding indicates a rhythm in mechanisms supporting extinction memory maintenance and/or retrieval. Subsequent testing for fear relapse in the conditioning context suggested reduced fear in rats tested during the early active phase. These results lay the groundwork for mechanistic investigations of circadian rhythms in fear extinction memory.

Association between Altered Cortisol Profiles and Neurobehavioral Impairment after Mild Traumatic Brain Injury in College Students.

Mild traumatic brain injury (mTBI) is the most common form of TBI, with more than 2.5 million TBI cases in the United States annually. Identification of easily obtainable biomarkers that track strongly with mTBI symptoms may improve our understanding of biological factors that contribute to mTBI symptom profiles and long-term outcomes. Notably, some individuals with mTBI exhibit circadian disruptions and elevated stress sensitivity, which in other clinical groups often correlate with disrupted secretion of cortisol, a glucocorticoid hormone that coordinates circadian and stress physiology. Here, we examined whether cortisol profiles could serve as a biomarker to complement the assessment of neurobehavioral sequelae after mTBI. We partnered with our on-campus health clinic to recruit college students seeking medical care after mTBI (n = 46) and compared this population to a well-matched non-injured student control group (n = 44). We collected data at an initial visit (shortly after injury in mTBI subjects) and one week later. At each visit, we evaluated neurobehavioral function using the Automated Neuropsychological Assessment Metric (ANAM). The subjects also provided cortisol samples through at-home saliva collection. We observed strong coherence between ANAM subjective and objective measures, indicating significant multi-dimensional impairment in subjects with mTBI. Further, female mTBI subjects exhibited diminished neurobehavioral function compared with males. Regardless of sex, decreased amplitude of diurnal cortisol and a blunted cortisol awakening response were associated with mTBI symptom severity and neurobehavioral impairment. Taken together, these findings suggest that salivary cortisol profiles may be a sensitive biomarker for studying underlying biological factors that impact mTBI symptoms and outcomes.

Iterative Metaplasticity Across Timescales: How Circadian, Ultradian, and Infradian Rhythms Modulate Memory Mechanisms.

Work in recent years has provided strong evidence for the modulation of memory function and neuroplasticity mechanisms across circadian (daily), ultradian (shorter-than-daily), and infradian (longer-than-daily) timescales. Despite rapid progress, however, the field has yet to adopt a general framework to describe the overarching role of biological rhythms in memory. To this end, Iyer and colleagues introduced the term iterative metaplasticity, which they define as the "gating of receptivity to subsequent signals that repeats on a cyclic timebase." The central concept is that the cyclic regulation of molecules involved in neuroplasticity may produce cycles in neuroplastic capacity-that is, the ability of neural cells to undergo activity-dependent change. Although Iyer and colleagues focus on the circadian timescale, we think their framework may be useful for understanding how biological rhythms influence memory more broadly. In this review, we provide examples and terminology to explain how the idea of iterative metaplasticity can be readily applied across circadian, ultradian, and infradian timescales. We suggest that iterative metaplasticity may not only support the temporal niching of neuroplasticity processes but also serve an essential role in the maintenance of memory function.

Memory and the circadian system: Identifying candidate mechanisms by which local clocks in the brain may regulate synaptic plasticity.

The circadian system is an endogenous biological network responsible for coordinating near-24-h cycles in behavior and physiology with daily timing cues from the external environment. In this review, we explore how the circadian system regulates memory formation, retention, and recall. Circadian rhythms in these memory processes may arise through several endogenous pathways, and recent work highlights the importance of genetic timekeepers found locally within tissues, called local clocks. We evaluate the circadian memory literature for evidence of local clock involvement in memory, identifying potential nodes for direct interactions between local clock components and mechanisms of synaptic plasticity. Our discussion illustrates how local clocks may pervasively modulate neuronal plastic capacity, a phenomenon that we designate here as circadian metaplasticity. We suggest that this function of local clocks supports the temporal optimization of memory processes, illuminating the potential for circadian therapeutic strategies in the prevention and treatment of memory impairment.

Controlled release of corticosteroid with biodegradable nanoparticles for treating experimental autoimmune uveitis.

Noninfectious uveitis is a potentially blinding ocular condition that often requires treatment with corticosteroids to prevent inflammation-related ocular complications. Severe forms of uveitis such as panuveitis that affects the whole eye often require a combination of topical and either regional or systemic corticosteroid. Regional corticosteroids are currently delivered inside the eye by intravitreal injection (e.g. Ozurdex®, an intravitreal dexamethasone implant). Intravitreal injection is associated with rare but potentially serious side effects, including endophthalmitis, retinal and vitreous hemorrhage, and retinal detachment. Subconjunctival (SCT) injection is a less invasive option that is a common route used for post-surgical drug administration and treatment of infection and severe inflammation. However, it is the water soluble form of dexamethasone, dexamethasone sodium phosphate (DSP), that has been demonstrated to achieve high intraocular penetration with subconjunctival injection. It is difficult to load highly water soluble drugs, such as DSP, and achieve sustained drug release using conventional encapsulation methods. We found that use of carboxyl-terminated poly(lactic-co-glycolic acid) (PLGA) allowed encapsulation of DSP into biodegradable nanoparticles (NP) with relatively high drug content (6% w/w) if divalent zinc ions were used as an ionic "bridge" between the PLGA and DSP. DSP-Zn-NP had an average diameter of 210 nm, narrow particle size distribution (polydispersity index ~0.1), and near neutral surface charge (-9 mV). DSP-Zn-NP administered by SCT injection provided detectable DSP levels in both the anterior chamber and vitreous chamber of the eye for at least 3 weeks. In a rat model of experimental autoimmune uveitis (EAU), inflammation was significantly reduced in both the front and back of the eye in animals that received a single SCT injection of DSP-Zn-NP as compared to animals that received either aqueous DSP solution or phosphate buffered saline (PBS). DSP-Zn-NP efficacy was evidenced by a reduced clinical disease score, decreased expression of various inflammatory cytokines, and preserved retinal structure and function. Furthermore, SCT DSP-Zn-NP significantly reduced microglia cell density in the retina, a hallmark of EAU in rats. DSP-Zn-NP hold promise as a new strategy to treat noninfectious uveitis and potentially other ocular inflammatory disorders.

Circadian misalignment has differential effects on affective behavior following exposure to controllable or uncontrollable stress.

In modern 24 h society, circadian disruption is pervasive, arising from night shift work, air travel across multiple time zones, irregular sleep schedules, and exposure to artificial light at night. Disruption of the circadian system is associated with many adverse health consequences, including mood disorders. Here we investigate whether inducing circadian misalignment using a phase advance protocol interferes with the ability to cope with a stressor, thereby increasing susceptibility to the negative consequences of stress. Male rats were maintained on a standard 12:12 light: dark (LD) cycle or subjected to a chronic phase advance (CPA) protocol involving 4 weekly 6 h phase shifts (earlier light onset) of the LD cycle. Rats were then exposed to escapable stress (ES), inescapable stress (IS), or no stress (home cage control; HC) and performance on juvenile social exploration and active escape learning in the two-way shuttlebox test was assessed 24 h and 48 h following stress, respectively. CPA alone had no effect on pre-stress juvenile social exploration, and it also did not interfere with the protective effect of ES on the stress-induced reduction in juvenile social exploration. In contrast, CPA impaired escape learning in the two-way shuttlebox to the same extent as IS in all subjects, regardless of stress history. Additionally, CPA produced somatic alterations that included increased body mass, increased epididymal adiposity, and decreased adrenal mass. These data indicate that CPA differentially modulated the stress-protective effects of behavioral control depending on the type of affective behavior examined.

Glucocorticoid hormones are both a major circadian signal and major stress signal: How this shared signal contributes to a dynamic relationship between the circadian and stress systems.

Glucocorticoid hormones are a powerful mammalian systemic hormonal signal that exerts regulatory effects on almost every cell and system of the body. Glucocorticoids act in a circadian and stress-directed manner to aid in adaptation to an ever-changing environment. Circadian glucocorticoid secretion provides for a daily waxing and waning influence on target cell function. In addition, the daily circadian peak of glucocorticoid secretion serves as a timing signal that helps entrain intrinsic molecular clock phase in tissue cells distributed throughout the body. Stress-induced glucocorticoid secretion also modulates the state of these same cells in response to both physiological and psychological stressors. We review the strong functional interrelationships between glucocorticoids and the circadian system, and discuss how these interactions optimize the appropriate cellular and systems response to stress throughout the day. We also discuss clinical implications of this dual aspect of glucocorticoid signaling, especially for conditions of circadian and HPA axis dysregulation.

Dynamic glucocorticoid-dependent regulation of Sgk1 expression in oligodendrocytes of adult male rat brain by acute stress and time of day.

Recent studies support plasticity in adult brain white matter structure and myelination in response to various experiential factors. One possible contributor to this plasticity may be activity-dependent modulation of serum- and glucocorticoid-inducible kinase 1 (Sgk1) expression in oligodendrocytes. We examined whether Sgk1 expression in adult rat brain white matter is increased by acute stress-induced elevations in endogenous corticosterone and whether it fluctuates with diurnal variations in corticosterone. We observed rapid increases (within 30 min) in Sgk1 mRNA in the corpus callosum in response to acute stress, as well as large increases at the beginning of the rat's active period (the time of peak corticosterone secretion). These increases were absent in adrenalectomized rats. Corticosterone treatment of adrenalectomized rats also rapidly increased corpus callosum Sgk1 mRNA. The majority of Sgk1 mRNA in corpus callosum was co-localized with myelin basic protein mRNA, suggesting that mature oligodendrocytes respond dynamically to acute stress and circadian rhythms. The regulation of Sgk1 expression by acute stress and time of day was selective for white matter, with limited alteration of Sgk1 expression by these factors in hippocampus and somatosensory cortex. These results indicate a unique sensitivity of oligodendrocyte Sgk1 expression to activity-dependent fluctuations in corticosterone hormone secretion, and raises the prospect that hypothalamic-pituitary-adrenal axis dysregulation or glucocorticoid pharmacotherapy may compromise the normal activity-dependent interactions between oligodendrocytes and neurons.

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

Monomethyl fumarate promotes Nrf2-dependent neuroprotection in retinal ischemia-reperfusion.

BACKGROUND: Retinal ischemia results in neuronal degeneration and contributes to the pathogenesis of multiple blinding diseases. Recently, the fumaric acid ester dimethyl fumarate (DMF) has been FDA-approved for the treatment of multiple sclerosis, based on its neuroprotective and anti-inflammatory effects. Its potential role as a neuroprotective agent for retinal diseases has received little attention. In addition, DMF's mode of action remains elusive, although studies have suggested nuclear factor erythroid 2-related factor 2 (Nrf2) activation as an important mechanism. Here we investigated the neuroprotective role of monomethyl fumarate (MMF), the biologically active metabolite of DMF, in retinal ischemia-reperfusion (I/R) injury, and examined the role of Nrf2 in mediating MMF action. METHODS: Wild-type C57BL/6J and Nrf2 knockout (KO) mice were subjected to 90 min of retinal ischemia followed by reperfusion. Mice received daily intraperitoneal injection of MMF. Inflammatory gene expression was measured using quantitative reverse transcription PCR (qRT-PCR) at 48 h after I/R injury. Seven days after I/R, qRT-PCR for Nrf2 target gene expression, immunostaining for Müller cell gliosis and cell loss in the ganglion cell layer (GCL), and electroretinography for retinal function were performed. RESULTS: The results of this study confirmed that MMF reduces retinal neurodegeneration in an Nrf2-dependent manner. MMF treatment significantly increased the expression of Nrf2-regulated antioxidative genes, suppressed inflammatory gene expression, reduced Müller cell gliosis, decreased neuronal cell loss in the GCL, and improved retinal function measured by electroretinogram (ERG) after retinal I/R injury in wild-type mice. Importantly, these MMF-mediated beneficial effects were not observed in Nrf2 KO mice. CONCLUSIONS: These results indicate that fumaric acid esters (FAEs) exert a neuronal protective function in the retinal I/R model and further validate Nrf2 modulation as a major mode of action of FAEs. This suggests that DMF and FAEs could be a potential therapeutic agent for activation of the Nrf2 pathway in retinal and possibly systemic diseases.

Neuroprotective role of Nrf2 for retinal ganglion cells in ischemia-reperfusion.

Retinal ischemia plays a critical role in multiple vision-threatening diseases and leads to death of retinal neurons, particularly ganglion cells. Oxidative stress plays an important role in this ganglion cell loss. Nrf2 (NF-E2-related factor 2) is a major regulator of the antioxidant response, and its role in the retina is increasingly appreciated. We investigated the potential retinal neuroprotective function of Nrf2 after ischemia-reperfusion (I/R) injury. In an experimental model of retinal I/R, Nrf2 knockout mice exhibited much greater loss of neuronal cells in the ganglion cell layer than wild-type mice. Primary retinal ganglion cells isolated from Nrf2 knockout mice exhibited decreased cell viability compared to wild-type retinal ganglion cells, demonstrating the cell-intrinsic protective role of Nrf2. The retinal neuronal cell line 661W exhibited reduced cell viability following siRNA-mediated knockdown of Nrf2 under conditions of oxidative stress, and this was associated with exacerbation of increase in reactive oxygen species. The synthetic triterpenoid CDDO-Im (2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide), a potent Nrf2 activator, inhibited reactive oxygen species increase in cultured 661W under oxidative stress conditions and increased neuronal cell survival after I/R injury in wild-type, but not Nrf2 knockout mice. Our findings indicate that Nrf2 exhibits a retinal neuroprotective function in I/R and suggest that pharmacologic activation of Nrf2 could be a therapeutic strategy. Oxidative stress is thought to be an important mediator of retinal ganglion cell death in ischemia-reperfusion injury. We found that the transcription factor NF-E2-related factor 2 (Nrf2), a major regulator of oxidative stress, is an important endogenous neuroprotective molecule in retinal ganglion cells in ischemia-reperfusion, exerting a cell-autonomous protective effect.  The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) reduces neurodegeneration following ischemia-reperfusion in an Nrf2-dependent fashion. This suggests that Nrf2-activating drugs including triterpenoids could be a therapeutic strategy for retinal neuroprotection.

PAC1 receptor antagonism in the bed nucleus of the stria terminalis (BNST) attenuates the endocrine and behavioral consequences of chronic stress.

Chronic or repeated stressor exposure can induce a number of maladaptive behavioral and physiological consequences and among limbic structures, the bed nucleus of the stria terminalis (BNST) has been implicated in the integration and interpretation of stress responses. Previous work has demonstrated that chronic variate stress (CVS) exposure in rodents increases BNST pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) and PAC1 receptor (Adcyap1r1) transcript expression, and that acute BNST PACAP injections can stimulate anxiety-like behavior. Here we show that chronic stress increases PACAP expression selectively in the oval nucleus of the dorsolateral BNST in patterns distinct from those for corticotropin releasing hormone (CRH). Among receptor subtypes, BNST PACAP signaling through PAC1 receptors not only heightened anxiety responses as measured by different behavioral parameters but also induced anorexic-like behavior to mimic the consequences of stress. Conversely, chronic inhibition of BNST PACAP signaling by continuous infusion with the PAC1 receptor antagonist PACAP(6-38) during the week of CVS attenuated these stress-induced behavioral responses and changes in weight gain. BNST PACAP signaling stimulated the hypothalamic-pituitary-adrenal (HPA) axis and heightened corticosterone release; further, BNST PACAP(6-38) administration blocked corticosterone release in a sensitized stress model. In aggregate with recent associations of PACAP/PAC1 receptor dysregulation with altered stress responses including post-traumatic stress disorder, these data suggest that BNST PACAP/PAC1 receptor signaling mechanisms may coordinate the behavioral and endocrine consequences of stress.

Induction of endothelial RAGE expression in pterygium.

PURPOSE: Chronic inflammation is a critical process in pterygium development and progression, including promotion of angiogenesis. Vascular endothelial cells (ECs) actively participate in and regulate inflammation. Pterygium research has uncovered multiple inflammatory cytokines that are upregulated, but there has been minimal focus on EC activation. The Receptor for Advanced Glycation Endproducts (RAGE), a major proinflammatory molecule expressed in the vascular endothelium and other cell types, is a major instigator of endothelial cell activation. In this study, we explored the hypothesis that RAGE is upregulated in ECs in pterygium. To this end, we examined RAGE expression and immunolocalization in human pterygium and normal conjunctival tissue, with a particular interest in assessing endothelial RAGE. METHODS: Pterygium specimens were obtained from 25 patients during surgery at the King Khaled Eye Specialist Hospital (KKESH). In the same patients, conjunctiva were obtained from the autograft during surgery. Tissue specimens were formalin-fixed and paraffin-embedded. Tissue sections were analyzed with immunohistochemistry with anti-RAGE antibody. Expression and localization of RAGE were evaluated in pterygium and corresponding conjunctiva. RESULTS: RAGE expression was detected in the vascular endothelium in all pterygium tissue specimens and most conjunctival specimens. Other cell types exhibited expression, notably epithelial cells, fibroblasts, and possibly macrophages. Strikingly, endothelial RAGE expression was increased in 19 of 25 pterygium tissue specimens, compared to the corresponding control conjunctiva. CONCLUSIONS: Our data reveal that RAGE expression is upregulated in vascular endothelial cells in pterygium. RAGE upregulation is an important mechanism by which endothelial cells amplify the overall inflammatory response, and suppression of RAGE has been shown to prevent the progression of some systemic disease processes in experimental models. This suggests that pharmacologic targeting of RAGE, which is already being attempted in clinical trials for some diseases, could be useful in treating pterygium.