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Impaired inhibitory synapse development is suggested to drive neuronal hyperactivity in autism spectrum disorders (ASD) and epilepsy. We propose a novel mechanism by which astrocytes control the development of parvalbumin (PV)-specific inhibitory synapses in the hippocampus, implicating ephrin-B/EphB signaling. Here, we utilize genetic approaches to assess functional and structural connectivity between PV and pyramidal cells (PC) through whole-cell patch-clamp electrophysiology, optogenetics, immunohistochemical analysis, and behaviors in male and female mice. While inhibitory synapse development is adversely affected by PV-specific expression of EphB2, a strong candidate ASD risk gene, astrocytic ephrin-B1 facilitates PV->PC connectivity through a mechanism involving EphB signaling in PV boutons. In contrast, the loss of astrocytic ephrin-B1 reduces PV->PC connectivity and inhibition, resulting in increased seizure susceptibility and an ASD-like phenotype. Our findings underscore the crucial role of astrocytes in regulating inhibitory circuit development and discover a new role for EphB2 receptors in PV-specific inhibitory synapse development.Significance Statement The findings presented in this study describe a novel mechanism by which astrocytes regulate the establishment of connections between parvalbumin interneurons and pyramidal neurons. We also present new evidence showing the role of presynaptic EphB2 in the formation of inhibitory synapses, specifically between PV-expressing interneurons and pyramidal neurons. Impaired inhibition is suggested to underlie the development of neuronal hyperactivity in several neurodevelopmental disorders (NDDs), and EphB2 receptor itself is also implicated in the pathogenesis of autism. Therefore, this study not only addresses critical gaps in our understanding, but also offers clinical relevance as EphB2 signaling in PV interneurons may be a promising therapeutic target to correct inhibitory circuit dysfunction in NDDs.

Traumatic brain injury leads to cellular and circuit changes in the dentate gyrus, a gateway to hippocampal information processing. Intrinsic granule cell firing properties and strong feedback inhibition in the dentate are proposed as critical to its ability to generate unique representation of similar inputs by a process known as pattern separation. Here we evaluate the impact of brain injury on cellular decorrelation of temporally patterned inputs in slices and behavioral discrimination of spatial locations in vivo one week after concussive lateral fluid percussion injury (FPI) in mice. Despite posttraumatic increases in perforant path evoked excitatory drive to granule cells and enhanced ΔFosB labeling, indicating sustained increase in excitability, the reliability of granule cell spiking was not compromised after FPI. Although granule cells continued to effectively decorrelate output spike trains recorded in response to similar temporally patterned input sets after FPI, their ability to decorrelate highly similar input patterns was reduced. In parallel, encoding of similar spatial locations in a novel object location task that involves the dentate inhibitory circuits was impaired one week after FPI. Injury induced changes in pattern separation were accompanied by loss of somatostatin expressing inhibitory neurons in the hilus. Together, these data suggest that the early posttraumatic changes in the dentate circuit undermine dentate circuit decorrelation of temporal input patterns as well as behavioral discrimination of similar spatial locations, both of which could contribute to deficits in episodic memory.

Axo-axonic cells (AACs) provide specialized inhibition to the axon initial segment (AIS) of excitatory neurons and can regulate network output and synchrony. Although hippocampal dentate AACs are structurally altered in epilepsy, physiological analyses of dentate AACs are lacking. We demonstrate that parvalbumin neurons in the dentate molecular layer express PTHLH, an AAC marker, and exhibit morphology characteristic of AACs. Dentate AACs show high-frequency, non-adapting firing but lack persistent firing in the absence of input and have higher rheobase than basket cells suggesting that AACs can respond reliably to network activity. Early after pilocarpine-induced status epilepticus (SE), dentate AACs receive fewer spontaneous excitatory and inhibitory synaptic inputs and have significantly lower maximum firing frequency. Paired recordings and spatially localized optogenetic stimulation revealed that SE reduced the amplitude of unitary synaptic inputs from AACs to granule cells without altering reliability, short-term plasticity, or AIS GABA reversal potential. These changes compromised AAC-dependent shunting of granule cell firing in a multicompartmental model. These early post-SE changes in AAC physiology would limit their ability to receive and respond to input, undermining a critical brake on the dentate throughput during epileptogenesis.

PURPOSE: Accurately distinguishing renal cell carcinoma (RCC) from normal kidney tissue is critical for identifying positive surgical margins (PSMs) during partial and radical nephrectomy, which remains the primary intervention for localized RCC. Techniques that detect PSM with higher accuracy and faster turnaround time than intraoperative frozen section (IFS) analysis can help decrease reoperation rates, relieve patient anxiety and costs, and potentially improve patient outcomes. MATERIALS AND METHODS: Here, we extended our combined desorption electrospray ionization mass spectrometry imaging (DESI-MSI) and machine learning methodology to identify metabolite and lipid species from tissue surfaces that can distinguish normal tissues from clear cell RCC (ccRCC), papillary RCC (pRCC), and chromophobe RCC (chRCC) tissues. RESULTS: From 24 normal and 40 renal cancer (23 ccRCC, 13 pRCC, and 4 chRCC) tissues, we developed a multinomial lasso classifier that selects 281 total analytes from over 27,000 detected molecular species that distinguishes all histological subtypes of RCC from normal kidney tissues with 84.5% accuracy. On the basis of independent test data reflecting distinct patient populations, the classifier achieves 85.4% and 91.2% accuracy on a Stanford test set (20 normal and 28 RCC) and a Baylor-UT Austin test set (16 normal and 41 RCC), respectively. The majority of the models selected features show consistent trends across data sets affirming its stable performance, where the suppression of arachidonic acid metabolism is identified as a shared molecular feature of ccRCC and pRCC. CONCLUSION: Together, these results indicate that signatures derived from DESI-MSI combined with machine learning may be used to rapidly determine surgical margin status with accuracies that meet or exceed those reported for IFS.

Strong inhibitory synaptic gating of dentate gyrus granule cells (GCs), attributed largely to fast-spiking parvalbumin interneurons (PV-INs), is essential to maintain sparse network activity needed for dentate dependent behaviors. However, the contribution of PV-INs to basal and input-driven sustained synaptic inhibition in GCs and semilunar granule cells (SGCs), a sparse morphologically distinct dentate projection neuron subtype, is currently unknown. In studies conducted in hippocampal slices from mice, we find that although basal IPSCs are more frequent in SGCs and optical activation of PV-INs reliably elicited IPSCs in both GCs and SGCs, optical suppression of PV-INs failed to reduce IPSC frequency in either cell type. Amplitude and kinetics of IPSCs evoked by perforant path (PP) activation were not different between GCs and SGCs. However, the robust increase in sustained polysynaptic IPSCs elicited by paired afferent stimulation was lower in SGCs than in simultaneously recorded GCs. Optical suppression of PV-IN selectively reduced sustained IPSCs in SGCs but not in GCs. These results demonstrate that PV-INs, while contributing minimally to basal synaptic inhibition in both GCs and SGCs in slices, mediate sustained feedback inhibition selectively in SGCs. The temporally selective blunting of activity-driven sustained inhibitory gating of SGCs could support their preferential and persistent recruitment during behavioral tasks.SIGNIFICANCE STATEMENT Our study identifies that feedback inhibitory regulation of dentate semilunar granule cells (SGCs), a sparse and functionally distinct class of projection neurons, differs from that of the classical projection neurons, GCs. Notably, we demonstrate relatively lower activity-dependent increase in sustained feedback inhibitory synaptic inputs to SGCs when compared with GCs which would facilitate their persistent activity and preferential recruitment as part of memory ensembles. Since dentate GC activity levels during memory processing are heavily shaped by basal and feedback inhibition, the fundamental differences in basal and evoked sustained inhibition between SGCs and GCs characterized here provide a framework to reorganize current understanding of the dentate circuit processing.

Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell-line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displays increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay reveals that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic profiling demonstrates that SU086 binds to and decreases HSP90. Metabolomic profiling reveals that SU086 leads to perturbation of glycolysis. Our study identifies SU086 as a treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.

IntroductionCoronavirus disease 2019 (COVID-19) has caused >3.5 million deaths worldwide and affected >160 million people. At least twice as many have been infected but remained asymptomatic or minimally symptomatic. COVID-19 includes central nervous system manifestations mediated by inflammation and cerebrovascular, anoxic, and/or viral neurotoxicity mechanisms. More than one third of patients with COVID-19 develop neurologic problems during the acute phase of the illness, including loss of sense of smell or taste, seizures, and stroke. Damage or functional changes to the brain may result in chronic sequelae. The risk of incident cognitive and neuropsychiatric complications appears independent from the severity of the original pulmonary illness. It behooves the scientific and medical community to attempt to understand the molecular and/or systemic factors linking COVID-19 to neurologic illness, both short and long term.MethodsThis article describes what is known so far in terms of links among COVID-19, the brain, neurological symptoms, and Alzheimer's disease (AD) and related dementias. We focus on risk factors and possible molecular, inflammatory, and viral mechanisms underlying neurological injury. We also provide a comprehensive description of the Alzheimer's Association Consortium on Chronic Neuropsychiatric Sequelae of SARS-CoV-2 infection (CNS SC2) harmonized methodology to address these questions using a worldwide network of researchers and institutions.ResultsSuccessful harmonization of designs and methods was achieved through a consensus process initially fragmented by specific interest groups (epidemiology, clinical assessments, cognitive evaluation, biomarkers, and neuroimaging). Conclusions from subcommittees were presented to the whole group and discussed extensively. Presently data collection is ongoing at 19 sites in 12 countries representing Asia, Africa, the Americas, and Europe.DiscussionThe Alzheimer's Association Global Consortium harmonized methodology is proposed as a model to study long-term neurocognitive sequelae of SARS-CoV-2 infection.Key pointsThe following review describes what is known so far in terms of molecular and epidemiological links among COVID-19, the brain, neurological symptoms, and AD and related dementias (ADRD)The primary objective of this large-scale collaboration is to clarify the pathogenesis of ADRD and to advance our understanding of the impact of a neurotropic virus on the long-term risk of cognitive decline and other CNS sequelae. No available evidence supports the notion that cognitive impairment after SARS-CoV-2 infection is a form of dementia (ADRD or otherwise). The longitudinal methodologies espoused by the consortium are intended to provide data to answer this question as clearly as possible controlling for possible confounders. Our specific hypothesis is that SARS-CoV-2 triggers ADRD-like pathology following the extended olfactory cortical network (EOCN) in older individuals with specific genetic susceptibility.The proposed harmonization strategies and flexible study designs offer the possibility to include large samples of under-represented racial and ethnic groups, creating a rich set of harmonized cohorts for future studies of the pathophysiology, determinants, long-term consequences, and trends in cognitive aging, ADRD, and vascular disease.We provide a framework for current and future studies to be carried out within the Consortium. and offers a "green paper" to the research community with a very broad, global base of support, on tools suitable for low- and middle-income countries aimed to compare and combine future longitudinal data on the topic.The Consortium proposes a combination of design and statistical methods as a means of approaching causal inference of the COVID-19 neuropsychiatric sequelae. We expect that deep phenotyping of neuropsychiatric sequelae may provide a series of candidate syndromes with phenomenological and biological characterization that can be further explored. By generating high-quality harmonized data across sites we aim to capture both descriptive and, where possible, causal associations.

Ru360, a mitochondrial Ca2+ uptake inhibitor, was tested in a unilateral fluid percussion TBI model in developing rats (P31). Vehicle and Ru360 treated TBI rats underwent sensorimotor behavioral monitoring between 24 and 72 h, thereafter which 185 brain metabolites were analyzed postmortem using LC/MS. Ru360 treatment after TBI improved sensorimotor behavioral recovery, upregulated glycolytic and pentose phosphate pathways, mitigated oxidative stress and prevented NAD+ depletion across both hemispheres. While neural viability improved ipsilaterally, it reduced contralaterally. Ru360 treatment, overall, had a global impact with most benefit near the strongest injury impact areas, while perturbing mitochondrial oxidative energetics in the milder TBI impact areas.

The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2-deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2-/- mice have a reduced number of parvalbumin, somatostatin, and neuropeptide Y cells, mainly in CA1. Whole-cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/- and Nrp2-/- mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2-deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration, and higher severity in Nrp2-/- compared to Nrp2+/+ animals. Finally, Nrp2+/- and Nrp2-/- but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2-/- mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.

Semilunar granule cells (SGCs) have been proposed as a morpho-functionally distinct class of hippocampal dentate projection neurons contributing to feedback inhibition and memory processing in juvenile rats. However, the structural and physiological features that can reliably classify granule cells (GCs) from SGCs through postnatal development remain unresolved. Focusing on postnatal days 11-13, 28-42, and > 120, corresponding with human infancy, adolescence, and adulthood, we examined the somato-dendritic morphology and inhibitory regulation in SGCs and GCs to determine the cell-type specific features. Unsupervised cluster analysis confirmed that morphological features reliably distinguish SGCs from GCs irrespective of animal age. SGCs maintain higher spontaneous inhibitory postsynaptic current (sIPSC) frequency than GCs from infancy through adulthood. Although sIPSC frequency in SGCs was particularly enhanced during adolescence, sIPSC amplitude and cumulative charge transfer declined from infancy to adulthood and were not different between GCs and SGCs. Extrasynaptic GABA current amplitude peaked in adolescence in both cell types and was significantly greater in SGCs than in GCs only during adolescence. Although GC input resistance was higher than in SGCs during infancy and adolescence, input resistance decreased with developmental age in GCs, while it progressively increased in SGCs. Consequently, GCs' input resistance was significantly lower than SGCs in adults. The data delineate the structural features that can reliably distinguish GCs from SGCs through development. The results reveal developmental differences in passive membrane properties and steady-state inhibition between GCs and SGCs which could confound their use in classifying the cell types.

The mechanisms by which the neurophysiological and inflammatory responses to brain injury contribute to memory impairments are not fully understood. Recently, we reported that the innate immune receptor, toll-like receptor 4 (TLR4) enhances AMPA receptor (AMPAR) currents and excitability in the dentate gyrus after fluid percussion brain injury (FPI) while limiting excitability in controls. Here, we examine the cellular mediators underlying TLR4 regulation of dentate excitability and its impact on memory performance. In ex vivo slices, astrocytic and microglial metabolic inhibitors selectively abolished TLR4 antagonist modulation of excitability in controls, but not in rats after FPI, demonstrating that glial signaling contributes to TLR4 regulation of excitability in controls. In glia-depleted neuronal cultures from naïve mice, TLR4 ligands bidirectionally modulated AMPAR charge transfer consistent with neuronal TLR4 regulation of excitability, as observed after brain injury. In vivo TLR4 antagonism reduced early post-injury increases in mediators of MyD88-dependent and independent TLR4 signaling without altering expression in controls. Blocking TNFα, a downstream effector of TLR4, mimicked effects of TLR4 antagonist and occluded TLR4 agonist modulation of excitability in slices from both control and FPI rats. Functionally, transiently blocking TLR4 in vivo improved impairments in working memory observed one week and one month after FPI, while the same treatment impaired memory function in uninjured controls. Together these data identify that distinct cellular signaling mechanisms converge on TNFα to mediate TLR4 modulation of network excitability in the uninjured and injured brain and demonstrate a role for TLR4 in regulation of working memory function.

Reducing the risk of dementia can halt the worldwide increase of affected people. The multifactorial and heterogeneous nature of late-onset dementia, including Alzheimer's disease (AD), indicates a potential impact of multidomain lifestyle interventions on risk reduction. The positive results of the landmark multidomain Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) support such an approach. The World-Wide FINGERS (WW-FINGERS), launched in 2017 and including over 25 countries, is the first global network of multidomain lifestyle intervention trials for dementia risk reduction and prevention. WW-FINGERS aims to adapt, test, and optimize the FINGER model to reduce risk across the spectrum of cognitive decline-from at-risk asymptomatic states to early symptomatic stages-in different geographical, cultural, and economic settings. WW-FINGERS aims to harmonize and adapt multidomain interventions across various countries and settings, to facilitate data sharing and analysis across studies, and to promote international joint initiatives to identify globally implementable and effective preventive strategies.

Reducing the risk of dementia can halt the worldwide increase of affected people. The multifactorial and heterogeneous nature of late-onset dementia, including Alzheimer's disease (AD), indicates a potential impact of multidomain lifestyle interventions on risk reduction. The positive results of the landmark multidomain Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) support such an approach. The World-Wide FINGERS (WW-FINGERS), launched in 2017 and including over 25 countries, is the first global network of multidomain lifestyle intervention trials for dementia risk reduction and prevention. WW-FINGERS aims to adapt, test, and optimize the FINGER model to reduce risk across the spectrum of cognitive decline-from at-risk asymptomatic states to early symptomatic stages-in different geographical, cultural, and economic settings. WW-FINGERS aims to harmonize and adapt multidomain interventions across various countries and settings, to facilitate data sharing and analysis across studies, and to promote international joint initiatives to identify globally implementable and effective preventive strategies.

Traumatic brain injury often leads to progressive alterations at the molecular to circuit levels resulting in epilepsy and memory impairments. Ex vivo and in vitro models have provided a powerful platform for investigating the multimodal alteration after trauma. Recent ex vivo analyses using voltage sensitive dye imaging, optogenetics, and glutamate uncaging have revealed circuit abnormalities following in vivo brain injury. In vitro injury models have enabled examination of early and progressive changes in activity while development of three-dimensional organoids derived from human induced pluripotent stem cells have opened novel avenues for injury research. Here, we highlight recent advances in ex vivo and in vitro systems, focusing on their potential for advancing mechanistic understandings, possible limitations, and implications for therapeutics.

Traumatic brain injury often leads to progressive alterations at the molecular to circuit levels resulting in epilepsy and memory impairments. Ex vivo and in vitro models have provided a powerful platform for investigating the multimodal alteration after trauma. Recent ex vivo analyses using voltage sensitive dye imaging, optogenetics, and glutamate uncaging have revealed circuit abnormalities following in vivo brain injury. In vitro injury models have enabled examination of early and progressive changes in activity while development of three-dimensional organoids derived from human induced pluripotent stem cells have opened novel avenues for injury research. Here, we highlight recent advances in ex vivo and in vitro systems, focusing on their potential for advancing mechanistic understandings, possible limitations, and implications for therapeutics.

Targeting mitochondrial ion homeostasis using Kaempferol, a mitochondrial Ca2+ uniporter channel activator, improves energy metabolism and behavior soon after a traumatic brain injury (TBI) in developing rats. Because of broad TBI pathophysiology and brain mitochondrial heterogeneity, Kaempferol-mediated early-stage behavioral and brain metabolic benefits may accrue from diverse sources within the brain. We hypothesized that Kaempferol influences TBI outcome by differentially impacting the neural, vascular, and synaptic/axonal compartments. After TBI at early development (P31), functional magnetic resonance imaging and diffusion tensor imaging (DTI) were applied to determine imaging outcomes at adolescence (2 months post-injury). Vehicle and Kaempferol treatments were made at 1, 24, and 48 h post-TBI, and their effects were assessed at adolescence. A significant increase in neural connectivity was observed after Kaempferol treatment as assessed by the spatial extent and strength of the somatosensory cortical and hippocampal resting-state functional connectivity (RSFC) networks. However, no significant RSFC changes were observed in the thalamus. DTI measures of fractional anisotropy (FA) and apparent diffusion coefficient, representing synaptic/axonal and microstructural integrity, showed significant improvements after Kaempferol treatment, with highest changes in the frontal and parietal cortices and hippocampus. Kaempferol treatment also increased corpus callosal FA, indicating measurable improvement in the interhemispheric structural connectivity. TBI prognosis was significantly altered at adolescence by early Kaempferol treatment, with improved neural connectivity, neurovascular coupling, and parenchymal microstructure in select brain regions. However, Kaempferol failed to improve vasomotive function across the whole brain, as measured by cerebrovascular reactivity. The differential effects of Kaempferol treatment on various brain functional compartments support diverse cellular-level mitochondrial functional outcomes in vivo.

ObjectiveTraumatic brain injury is a major risk factor for acquired epilepsies, and understanding the mechanisms underlying the early pathophysiology could yield viable therapeutic targets. Growing evidence indicates a role for inflammatory signaling in modifying neuronal excitability and promoting epileptogenesis. Here we examined the effect of innate immune receptor Toll-like receptor 4 (TLR4) on excitability of the hippocampal dentate gyrus and epileptogenesis after brain injury.MethodsSlice and in vivo electrophysiology and Western blots were conducted in rats subject to fluid percussion brain injury or sham injury.ResultsThe studies identify that TLR4 signaling in neurons augments dentate granule cell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (CP-AMPAR) currents after brain injury. Blocking TLR4 signaling in vivo shortly after brain injury reduced dentate network excitability and seizure susceptibility. When blocking of TLR4 signaling after injury was delayed, however, this treatment failed to reduce postinjury seizure susceptibility. Furthermore, TLR4 signal blocking was less efficacious in limiting seizure susceptibility when AMPAR currents, downstream targets of TLR4 signaling, were transiently enhanced. Paradoxically, blocking TLR4 signaling augmented both network excitability and seizure susceptibility in uninjured controls. Despite the differential effect on seizure susceptibility, TLR4 antagonism suppressed cellular inflammatory responses after injury without impacting sham controls.InterpretationThese findings demonstrate that independently of glia, the immune receptor TLR4 directly regulates post-traumatic neuronal excitability. Moreover, the TLR4-dependent early increase in dentate excitability is causally associated with epileptogenesis. Identification and selective targeting of the mechanisms underlying the aberrant TLR4-mediated increase in CP-AMPAR signaling after injury may prevent epileptogenesis after brain trauma. ANN NEUROL 2020;87:497-515.

Targeting mitochondrial ion homeostasis using Kaempferol, a mitochondrial Ca2+ uniporter channel activator, improves energy metabolism and behavior soon after a traumatic brain injury (TBI) in developing rats. Because of broad TBI pathophysiology and brain mitochondrial heterogeneity, Kaempferol-mediated early-stage behavioral and brain metabolic benefits may accrue from diverse sources within the brain. We hypothesized that Kaempferol influences TBI outcome by differentially impacting the neural, vascular, and synaptic/axonal compartments. After TBI at early development (P31), functional magnetic resonance imaging and diffusion tensor imaging (DTI) were applied to determine imaging outcomes at adolescence (2 months post-injury). Vehicle and Kaempferol treatments were made at 1, 24, and 48 h post-TBI, and their effects were assessed at adolescence. A significant increase in neural connectivity was observed after Kaempferol treatment as assessed by the spatial extent and strength of the somatosensory cortical and hippocampal resting-state functional connectivity (RSFC) networks. However, no significant RSFC changes were observed in the thalamus. DTI measures of fractional anisotropy (FA) and apparent diffusion coefficient, representing synaptic/axonal and microstructural integrity, showed significant improvements after Kaempferol treatment, with highest changes in the frontal and parietal cortices and hippocampus. Kaempferol treatment also increased corpus callosal FA, indicating measurable improvement in the interhemispheric structural connectivity. TBI prognosis was significantly altered at adolescence by early Kaempferol treatment, with improved neural connectivity, neurovascular coupling, and parenchymal microstructure in select brain regions. However, Kaempferol failed to improve vasomotive function across the whole brain, as measured by cerebrovascular reactivity. The differential effects of Kaempferol treatment on various brain functional compartments support diverse cellular-level mitochondrial functional outcomes in vivo.

Epilepsy, characterized by recurrent seizures and abnormal electrical activity in the brain, is one of the most prevalent brain disorders. Over two million people in the United States have been diagnosed with epilepsy and 3% of the general population will be diagnosed with it at some point in their lives. While most developmental epilepsies occur due to genetic predisposition, a class of "acquired" epilepsies results from a variety of brain insults. A leading etiological factor for epilepsy that is currently on the rise is traumatic brain injury (TBI), which accounts for up to 20% of all symptomatic epilepsies. Remarkably, the presence of an identified early insult that constitutes a risk for development of epilepsy provides a therapeutic window in which the pathological processes associated with brain injury can be manipulated to limit the subsequent development of recurrent seizure activity and epilepsy. Recent studies have revealed diverse pathologies, including enhanced excitability, activated immune signaling, cell death, and enhanced neurogenesis within a week after injury, suggesting a period of heightened adaptive and maladaptive plasticity. An integrated understanding of these processes and their cellular and molecular underpinnings could lead to novel targets to arrest epileptogenesis after trauma. This review attempts to highlight and relate the diverse early changes after trauma and their role in development of epilepsy and suggests potential strategies to limit neurological complications in the injured brain.

Oxidative energy metabolism is depressed after mild/moderate traumatic brain injury (TBI) during early development, accompanied by behavioral debilitation and secondary neuronal death. A TBI metabolome analysis revealed broad effects with a striking impact on energy metabolism. Our studies on mitochondrial modulators and their effects on brain function have shown that kaempferol, a stimulator of the mitochondrial Ca2+ uniporter channel (mCU), enhanced neural and neurovascular activity in the normal brain and improved stimulus-induced brain activation and behavior after TBI during early development. Because kaempferol enhances mitochondrial Ca2+ uptake and cycling, with protective effects after TBI, we tested the hypothesis that kaempferol treatment during the acute/subacute stage after TBI (0-72 h) acted on mitochondria in improving TBI outcome. Developmental age rats (P31) underwent TBI and were treated with vehicle or kaempferol (1 mg/kg intraperitoneally) in three doses at 1, 24, and 48 h after TBI. Brains were harvested at 72 h and subjected to liquid chromatography mass spectrometric measurements. Decrease in pyruvate and tricarboxylic acid (TCA) cycle flux were observed in the untreated and vehicle-treated group, consistent with previously established energy metabolic decline after TBI. Kaempferol improved TCA cycle flux, maintained mitochondrial functional integrity as observed by decreased acyl carnitines, improved neural viability as evidenced by higher N-acetyl aspartate levels. The positive outcomes of kaempferol on metabolic profile corresponded with improved sensorimotor behavior.

Traumatic brain injury (TBI) is a leading cause of morbidity in children. To investigate outcome of early developmental TBI during adolescence, a rat model of fluid percussion injury was developed, where previous work reported deficits in sensorimotor behavior and cortical blood flow at adolescence.1 Based on the nonlocalized outcome, we hypothesized that multiple neurophysiological components of brain function, namely neuronal connectivity, synapse/axonal microstructural integrity, and neurovascular function, are altered and magnetic resonance imaging (MRI) methods could be used to determine regional alterations. Adolescent outcomes of developmental TBI were studied 2 months after injury, using functional MRI (fMRI) and diffusion tensor imaging (DTI). fMRI-based resting-state functional connectivity (RSFC), representing neural connectivity, was significantly altered between sham and TBI. RSFC strength decreased in the cortex, hippocampus, and thalamus, accompanied by decrease in spatial extent of their corresponding RSFC networks and interhemispheric asymmetry. Cerebrovascular reactivity to arterial CO2 changes diminished after TBI across both hemispheres, with a more pronounced decrease in the ipsilateral hippocampus, thalamus, and motor cortex. DTI measures of fractional anisotropy and apparent diffusion coefficient, reporting on axonal and microstructural integrity of the brain, indicated similar interhemispheric asymmetry, with highest change in the ipsilateral hippocampus and regions adjoining the ipsilateral thalamus, hypothalamus, and amygdala. TBI-induced corpus callosal microstructural alterations indicated measurable changes in interhemispheric structural connectivity. Hippocampus, thalamus, and select cortical regions were most consistently affected in multiple imaging markers. The multi-modal MRI results demonstrate cortical and subcortical alterations in neural connectivity, cerebrovascular resistance, and parenchymal microstructure in the adolescent brain, indicating the highly diffuse and persistent nature of the lateral fluid percussion TBI early in development.

Traumatic brain injury (TBI) in general has varied neuropathological consequences depending upon the intensity and biomechanics of the injury. Furthermore, in pediatric TBI, intrinsic developmental changes add further complexity, necessitating a biochemical dimension for improved TBI characterization. In our earlier study investigating the subacute stage TBI metabolome (72 h post-injury) in a developmental rat model, significant ipsilateral brain biochemical changes occurred across 25 metabolite sets as determined by metabolite set enrichment analysis (MSEA). The broad metabolic perturbation was accompanied by behavioral deficits and neuronal loss across the ipsilateral hemisphere containing the injury epicenter. In order to obtain a consolidated biochemical profile of the TBI assessment, a subgrouping of the 190 identified brain metabolites was performed. Metabolites were divided into seven major subgroups: oxidative energy/mitochondrial, glycolysis/pentose phosphate pathway, fatty acid, amino acid, neurotransmitters/neuromodulators, one-carbon/folate and other metabolites. Subgroups were based on the chemical nature and association with critically altered biochemical pathways after TBI as obtained from our earlier untargeted analysis. Each metabolite subgroup extracted from the ipsilateral sham and TBI brains were modeled using multivariate partial least square discriminant analysis (PLS-DA) with the model accuracy used as a measurable index of TBI neurochemical impact. Volcano plots of each subgroup, corrected for multiple comparisons, determined the TBI neurochemical specificity. The results provide a ranked biochemical profile along with specificity of changes after developmental TBI, enabling a consolidated biochemical template for future classification of different TBI intensities and injury types in animal models.

Traumatic brain injury (TBI) in general has varied neuropathological consequences depending upon the intensity and biomechanics of the injury. Furthermore, in pediatric TBI, intrinsic developmental changes add further complexity, necessitating a biochemical dimension for improved TBI characterization. In our earlier study investigating the subacute stage TBI metabolome (72 h post-injury) in a developmental rat model, significant ipsilateral brain biochemical changes occurred across 25 metabolite sets as determined by metabolite set enrichment analysis (MSEA). The broad metabolic perturbation was accompanied by behavioral deficits and neuronal loss across the ipsilateral hemisphere containing the injury epicenter. In order to obtain a consolidated biochemical profile of the TBI assessment, a subgrouping of the 190 identified brain metabolites was performed. Metabolites were divided into seven major subgroups: oxidative energy/mitochondrial, glycolysis/pentose phosphate pathway, fatty acid, amino acid, neurotransmitters/neuromodulators, one-carbon/folate and other metabolites. Subgroups were based on the chemical nature and association with critically altered biochemical pathways after TBI as obtained from our earlier untargeted analysis. Each metabolite subgroup extracted from the ipsilateral sham and TBI brains were modeled using multivariate partial least square discriminant analysis (PLS-DA) with the model accuracy used as a measurable index of TBI neurochemical impact. Volcano plots of each subgroup, corrected for multiple comparisons, determined the TBI neurochemical specificity. The results provide a ranked biochemical profile along with specificity of changes after developmental TBI, enabling a consolidated biochemical template for future classification of different TBI intensities and injury types in animal models.

Pathophysiology of developmental traumatic brain injury (TBI) is unique due to intrinsic differences in the developing brain. Energy metabolic studies of the brain during early development (P13 to P30) have indicated acute oxidative energy metabolic decreases below 24 h after TBI, which generally recovered by 48 h. However, marked neurodegeneration and altered neural functional connectivity have been observed at later stages into adolescence. As secondary neurodegeneration is most prominent during the first week after TBI in the rat model, we hypothesized that the subacute TBI-metabolome may contain predictive markers of neurodegeneration. Sham and TBI metabolomes were examined at 72 h after a mild to moderate intensity TBI in male Sprague-Dawley rats aged P31. Sensorimotor behavior was assessed at 24, 48 and 72 h after injury, followed by 72-hour postmortem brain removal for metabolomics using Liquid Chromatography/Mass Spectrometry (LC-MS) measurement. Broad TBI-induced metabolomic shifts occurred with relatively higher intensity in the injury-lateralized (ipsilateral) hemisphere. Intensity of metabolomic perturbation correlated with the extent of sensorimotor behavioral deficit. N-acetyl-aspartate (NAA) levels at 72 h after TBI, predicted the extent of neurodegeneration assessed histochemically 7-days post TBI. Results from the multivariate untargeted approach clearly distinguished metabolomic shifts induced by TBI. Several pathways including amino acid, fatty acid and energy metabolism continued to be affected at 72 h after TBI, whose collective effects may determine the overall pathological response after TBI in early development including neurodegeneration.

Hippocampal dentate gyrus is a focus of enhanced neurogenesis and excitability after traumatic brain injury. Increased neurogenesis has been proposed to aid repair of the injured network. Our data show that an early increase in neurogenesis after fluid percussion concussive brain injury is transient and is followed by a persistent decrease compared with age-matched controls. Post-injury changes in neurogenesis paralleled changes in neural precursor cell proliferation and resulted in a long-term decline in neurogenic capacity. Targeted pharmacology to restore post-injury neurogenesis to control levels reversed the long-term decline in neurogenic capacity. Limiting post-injury neurogenesis reduced early increases in dentate excitability and seizure susceptibility. Our results challenge the assumption that increased neurogenesis after brain injury is beneficial and show that early post-traumatic increases in neurogenesis adversely affect long-term outcomes by exhausting neurogenic potential and enhancing epileptogenesis. Treatments aimed at limiting excessive neurogenesis can potentially restore neuroproliferative capacity and limit epilepsy after brain injury.

Hippocampal dentate gyrus is a focus of enhanced neurogenesis and excitability after traumatic brain injury. Increased neurogenesis has been proposed to aid repair of the injured network. Our data show that an early increase in neurogenesis after fluid percussion concussive brain injury is transient and is followed by a persistent decrease compared with age-matched controls. Post-injury changes in neurogenesis paralleled changes in neural precursor cell proliferation and resulted in a long-term decline in neurogenic capacity. Targeted pharmacology to restore post-injury neurogenesis to control levels reversed the long-term decline in neurogenic capacity. Limiting post-injury neurogenesis reduced early increases in dentate excitability and seizure susceptibility. Our results challenge the assumption that increased neurogenesis after brain injury is beneficial and show that early post-traumatic increases in neurogenesis adversely affect long-term outcomes by exhausting neurogenic potential and enhancing epileptogenesis. Treatments aimed at limiting excessive neurogenesis can potentially restore neuroproliferative capacity and limit epilepsy after brain injury.

We investigated the hypothesis that high Ca2+ influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca2+ level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca2+ level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1β. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca2+ influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca2+ to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1β, and intrinsic apoptotic pathways. We conclude that excess IL-1β production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.

Strong perisomatic inhibition by fast-spiking basket cells (FS-BCs) regulates dentate throughput. Homotypic FS-BC interconnections that support gamma oscillations, and heterotypic inputs from diverse groups of interneurons that receive extensive neurochemical regulation, together, shape FS-BC activity patterns. However, whether seizures precipitate functional changes in inhibitory networks and contribute to abnormal network activity in epilepsy is not known. In the first recordings from dentate interneuronal pairs in a model of temporal lobe epilepsy, we demonstrate that status epilepticus (SE) selectively compromises GABA release at synapses from dentate accommodating interneurons (AC-INs) to FS-BCs, while efficacy of homotypic FS-BC synapses is unaltered. The functional decrease in heterotypic cannabinoid receptor type 1 (CB1R)-sensitive inhibition of FS-BCs resulted from enhanced baseline GABAB-mediated suppression of synaptic release after SE. The frequency of CB1R-sensitive inhibitory synaptic events in FS-BCs was depressed early after SE induction and remained reduced in epileptic rats. In biologically based simulations of heterogeneous inhibitory networks and excitatory-inhibitory cell networks, experimentally identified decrease in reliability of AC-IN to FS-BCs synaptic release reduced theta power and theta-gamma coupling and enhanced gamma coherence. Thus, the experimentally identified functional reduction in heterotypic inhibition of FS-BCs can contribute to compromised network oscillations in epilepsy and could precipitate memory and cognitive co-morbidities.

Altered inhibition is a salient feature of hippocampal network reorganization in epilepsy. Hippocampal pyramidal cells and dentate granule cells show specific reduction in cannabinoid receptor type 1 (CB1R)-sensitive GABAergic inputs in experimental epilepsy. In the dentate gyrus, CB1Rs regulate synaptic release from accommodating interneurons (AC-INs) with adapting firing characteristics and axonal projections in the molecular layer, but not from fast-spiking basket cells (FS-BCs). However, it is not known whether the intrinsic physiology and synaptic inhibition of AC-INs responsible for CB1R-sensitive inhibition is altered in epilepsy. Using the pilocarpine-induced status epilepticus (SE) model of epilepsy, we find that the basic physiological characteristics of AC-INs in epileptic rats are not different from age-matched controls. In paired interneuronal recordings, the amplitude of unitary inhibitory synaptic currents (uIPSCs) between AC-INs doubled after SE. Non-stationary noise analysis revealed that the post-SE strengthening of synapses between AC-INs resulted from an increase in postsynaptic receptors. Baseline synaptic release and CB1R antagonist enhancement of release at synapses between AC-INs were not different between control and post-SE rats. Additionally, uIPSC amplitude in FS-BCs to AC-INs pairs was unchanged after SE indicating input-specific microcircuit alterations in inhibitory inputs to AC-INs. At the network level, AC-INs showed no reduction in spontaneous and miniature inhibitory synaptic current (sIPSC or mIPSC) frequency or amplitude after SE. However, AC-IN mIPSC amplitude was persistently enhanced in post-SE and epileptic rats. CB1R agonist reduced the amplitude and suppressed a greater proportion of sIPSCs in AC-INs from post-SE and epileptic rats demonstrating a novel, cell-type specific increase in CB1R-sensitive inhibition of AC-INs after SE. This unique post-SE strengthening of inhibition between AC-INs could lead to activity-dependent suppression of AC-IN firing and compromise dentate CB1R-sensitive inhibition in epilepsy.

Candidate gene and genome-wide association studies (GWAS) have identified 15 independent genomic regions associated with bladder cancer risk. In search for additional susceptibility variants, we followed up on four promising single-nucleotide polymorphisms (SNPs) that had not achieved genome-wide significance in 6911 cases and 11 814 controls (rs6104690, rs4510656, rs5003154 and rs4907479, P < 1 × 10(-6)), using additional data from existing GWAS datasets and targeted genotyping for studies that did not have GWAS data. In a combined analysis, which included data on up to 15 058 cases and 286 270 controls, two SNPs achieved genome-wide statistical significance: rs6104690 in a gene desert at 20p12.2 (P = 2.19 × 10(-11)) and rs4907479 within the MCF2L gene at 13q34 (P = 3.3 × 10(-10)). Imputation and fine-mapping analyses were performed in these two regions for a subset of 5551 bladder cancer cases and 10 242 controls. Analyses at the 13q34 region suggest a single signal marked by rs4907479. In contrast, we detected two signals in the 20p12.2 region-the first signal is marked by rs6104690, and the second signal is marked by two moderately correlated SNPs (r(2) = 0.53), rs6108803 and the previously reported rs62185668. The second 20p12.2 signal is more strongly associated with the risk of muscle-invasive (T2-T4 stage) compared with non-muscle-invasive (Ta, T1 stage) bladder cancer (case-case P ≤ 0.02 for both rs62185668 and rs6108803). Functional analyses are needed to explore the biological mechanisms underlying these novel genetic associations with risk for bladder cancer.

ContextThe College of American Pathologists periodically surveys laboratories to determine changes in cytopathology practices. We report the results of a 2011 gynecologic cytology survey.ObjectiveTo provide a cross-sectional survey of gynecologic cytology practices in 2010.DesignIn 2011, a survey was sent to 1604 laboratories participating in the College of American Pathologists gynecologic cytology interlaboratory comparison education program and proficiency testing programs requesting data from 2010 on the following topics: terminology/reporting, cytotechnologist workload, quality assurance, reagents, and ancillary testing.ResultsSix hundred and twenty-five laboratories (39%) replied to the survey. The nonstandard use of "low-grade squamous intraepithelial lesion cannot exclude high-grade squamous intraepithelial lesion" is used by most laboratories to report the presence of low-grade squamous intraepithelial lesion with possibility of high-grade squamous intraepithelial lesion. Most laboratories also report the presence or absence of cells from the transformation zone. Most respondents do not limit cytotechnologist screening workload during the work shift. Only about one-third of laboratories (188 of 582; 32%) use image-assisted screening devices. Rapid prescreening as a quality assurance measure is used by only 3.5% (21 of 594) of the laboratories. When used for screening, most laboratories use the imager for retrospective review of slides to detect human locator and interpretive errors. Most laboratories receive both liquid-based cytology samples (mainly ThinPrep, Hologic, Marlborough, Massachusetts) and conventional Papanicolaou tests. Expiration dates of liquid-based cytology test vials are not usually recorded.ConclusionsThe field of gynecologic cytology is evolving rapidly. These survey results offer a snapshot of national gynecologic cytology practices in 2010.

Mild to moderate traumatic brain injury (mTBI) leads to secondary neuronal loss via excitotoxic mechanisms, including mitochondrial Ca(2+) overload. However, in the surviving cellular population, mitochondrial Ca(2+) influx, and oxidative metabolism are diminished leading to suboptimal neuronal circuit activity and poor prognosis. Hence we tested the impact of boosting neuronal electrical activity and oxidative metabolism by facilitating mitochondrial Ca(2+) uptake in a rat model of mTBI. In developing rats (P25-P26) sustaining an mTBI, we demonstrate post-traumatic changes in cerebral blood flow (CBF) in the sensorimotor cortex in response to whisker stimulation compared to sham using functional Laser Doppler Imaging (fLDI) at adulthood (P67-P73). Compared to sham, whisker stimulation-evoked positive CBF responses decreased while negative CBF responses increased in the mTBI animals. The spatiotemporal CBF changes representing underlying neuronal activity suggested profound changes to neurovascular activity after mTBI. Behavioral assessment of the same cohort of animals prior to fLDI showed that mTBI resulted in persistent contralateral sensorimotor behavioral deficit along with ipsilateral neuronal loss compared to sham. Treating mTBI rats with Kaempferol, a dietary flavonol compound that enhanced mitochondrial Ca(2+) uptake, eliminated the inter-hemispheric asymmetry in the whisker stimulation-induced positive CBF responses and the ipsilateral negative CBF responses otherwise observed in the untreated and vehicle-treated mTBI animals in adulthood. Kaempferol also improved somatosensory behavioral measures compared to untreated and vehicle treated mTBI animals without augmenting post-injury neuronal loss. The results indicate that reduced mitochondrial Ca(2+) uptake in the surviving populations affect post-traumatic neural activation leading to persistent behavioral deficits. Improvement in sensorimotor behavior and spatiotemporal neurovascular activity following kaempferol treatment suggests that facilitation of mitochondrial Ca(2+) uptake in the early window after injury may sustain optimal neural activity and metabolism and contribute to improved function of the surviving cellular populations after mTBI.

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 µm or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

ContextThe College of American Pathologists periodically surveys laboratories to determine changes in cytopathology practices. We report the results of a 2011 gynecologic cytology survey.ObjectiveTo provide a cross-sectional survey of gynecologic cytology practices in 2010.DesignIn 2011, a survey was sent to 1604 laboratories participating in the College of American Pathologists gynecologic cytology interlaboratory comparison education program and proficiency testing programs requesting data from 2010 on the following topics: terminology/reporting, cytotechnologist workload, quality assurance, reagents, and ancillary testing.ResultsSix hundred and twenty-five laboratories (39%) replied to the survey. The nonstandard use of "low-grade squamous intraepithelial lesion cannot exclude high-grade squamous intraepithelial lesion" is used by most laboratories to report the presence of low-grade squamous intraepithelial lesion with possibility of high-grade squamous intraepithelial lesion. Most laboratories also report the presence or absence of cells from the transformation zone. Most respondents do not limit cytotechnologist screening workload during the work shift. Only about one-third of laboratories (188 of 582; 32%) use image-assisted screening devices. Rapid prescreening as a quality assurance measure is used by only 3.5% (21 of 594) of the laboratories. When used for screening, most laboratories use the imager for retrospective review of slides to detect human locator and interpretive errors. Most laboratories receive both liquid-based cytology samples (mainly ThinPrep, Hologic, Marlborough, Massachusetts) and conventional Papanicolaou tests. Expiration dates of liquid-based cytology test vials are not usually recorded.ConclusionsThe field of gynecologic cytology is evolving rapidly. These survey results offer a snapshot of national gynecologic cytology practices in 2010.

Mild to moderate traumatic brain injury (mTBI) leads to secondary neuronal loss via excitotoxic mechanisms, including mitochondrial Ca(2+) overload. However, in the surviving cellular population, mitochondrial Ca(2+) influx, and oxidative metabolism are diminished leading to suboptimal neuronal circuit activity and poor prognosis. Hence we tested the impact of boosting neuronal electrical activity and oxidative metabolism by facilitating mitochondrial Ca(2+) uptake in a rat model of mTBI. In developing rats (P25-P26) sustaining an mTBI, we demonstrate post-traumatic changes in cerebral blood flow (CBF) in the sensorimotor cortex in response to whisker stimulation compared to sham using functional Laser Doppler Imaging (fLDI) at adulthood (P67-P73). Compared to sham, whisker stimulation-evoked positive CBF responses decreased while negative CBF responses increased in the mTBI animals. The spatiotemporal CBF changes representing underlying neuronal activity suggested profound changes to neurovascular activity after mTBI. Behavioral assessment of the same cohort of animals prior to fLDI showed that mTBI resulted in persistent contralateral sensorimotor behavioral deficit along with ipsilateral neuronal loss compared to sham. Treating mTBI rats with Kaempferol, a dietary flavonol compound that enhanced mitochondrial Ca(2+) uptake, eliminated the inter-hemispheric asymmetry in the whisker stimulation-induced positive CBF responses and the ipsilateral negative CBF responses otherwise observed in the untreated and vehicle-treated mTBI animals in adulthood. Kaempferol also improved somatosensory behavioral measures compared to untreated and vehicle treated mTBI animals without augmenting post-injury neuronal loss. The results indicate that reduced mitochondrial Ca(2+) uptake in the surviving populations affect post-traumatic neural activation leading to persistent behavioral deficits. Improvement in sensorimotor behavior and spatiotemporal neurovascular activity following kaempferol treatment suggests that facilitation of mitochondrial Ca(2+) uptake in the early window after injury may sustain optimal neural activity and metabolism and contribute to improved function of the surviving cellular populations after mTBI.

Activity of the dentate gyrus, which gates information flow to the hippocampus, is under tight inhibitory regulation by interneurons with distinctive axonal projections, intrinsic and synaptic characteristics and neurochemical identities. Total molecular layer cells (TML-Cs), a class of morphologically distinct GABAergic neurons with axonal projections across the molecular layer, are among the most frequent interneuronal type in the dentate subgranular region. However, little is known about their synaptic and neurochemical properties. We demonstrate that synapses from morphologically identified TML-Cs to dentate interneurons are characterized by low release probability, facilitating short-term dynamics and asynchronous release. TML-Cs consistently show somatic and axonal labeling for the cannabinoid receptor type 1 (CB1 R) yet fail to express cholecystokinin (CCK) indicating their distinctive neurochemical identity. In paired recordings, the release probability at synapses between TML-Cs was increased by the CB1 R antagonist AM251, demonstrating baseline endocannabinoid regulation of TML-C synapses. Apart from defining the synaptic and neurochemical features of TML-Cs, our findings reveal the morphological identity of a class of dentate CB1 R-positive neurons that do not express CCK. Our findings indicate that TML-Cs can mediate cannabinoid sensitive feed-forward and feedback inhibition of dentate perforant path inputs.

Activity of the dentate gyrus, which gates information flow to the hippocampus, is under tight inhibitory regulation by interneurons with distinctive axonal projections, intrinsic and synaptic characteristics and neurochemical identities. Total molecular layer cells (TML-Cs), a class of morphologically distinct GABAergic neurons with axonal projections across the molecular layer, are among the most frequent interneuronal type in the dentate subgranular region. However, little is known about their synaptic and neurochemical properties. We demonstrate that synapses from morphologically identified TML-Cs to dentate interneurons are characterized by low release probability, facilitating short-term dynamics and asynchronous release. TML-Cs consistently show somatic and axonal labeling for the cannabinoid receptor type 1 (CB1 R) yet fail to express cholecystokinin (CCK) indicating their distinctive neurochemical identity. In paired recordings, the release probability at synapses between TML-Cs was increased by the CB1 R antagonist AM251, demonstrating baseline endocannabinoid regulation of TML-C synapses. Apart from defining the synaptic and neurochemical features of TML-Cs, our findings reveal the morphological identity of a class of dentate CB1 R-positive neurons that do not express CCK. Our findings indicate that TML-Cs can mediate cannabinoid sensitive feed-forward and feedback inhibition of dentate perforant path inputs.

BackgroundInjury to the brain can occur from a variety of physical insults and the degree of disability can greatly vary from person to person. It is likely that injury outcome is related to the biomechanical parameters of the traumatic event such as magnitude, direction and speed of the forces acting on the head.New methodTo model variations in the biomechanical injury parameters, a voice coil driven fluid percussion injury (FPI) system was designed and built to generate fluid percussion waveforms with adjustable rise times, peak pressures, and durations. Using this system, pathophysiological outcomes in the rat were investigated and compared to animals injured with the same biomechanical parameters using the pendulum based FPI system.Results in comparison with existing methodsImmediate post-injury behavior shows similar rates of seizures and mortality in adolescent rats and similar righting times, toe pinch responses and mortality rates in adult rats. Interestingly, post injury mortality in adult rats was sensitive to changes in injury rate. Fluoro-Jade labeling of degenerating neurons in the hilus and CA2-3 hippocampus were consistent between injuries produced with the voice coil and pendulum operated systems. Granule cell population spike amplitude to afferent activation, a measure of dentate network excitability, also showed consistent enhancement 1 week after injury using either system.ConclusionsOverall our results suggest that this new FPI device produces injury outcomes consistent with the commonly used pendulum FPI system and has the added capability to investigate pathophysiology associated with varying rates and durations of injury.

Acoustic startle response (ASR) is a defensive reflex that is largely ignored unless greatly exaggerated. ASR is suppressed after moderate and severe traumatic brain injury (TBI), but the effect of mild TBI (mTBI) on ASR has not been investigated. Because the neural circuitry for ASR resides in the pons in all mammals, ASR may be a good measure of brainstem function after mTBI. The present study assessed ASR in Sprague-Dawley rats after mTBI using lateral fluid percussion and compared these effects to those on spatial working memory. mTBI caused a profound, long-lasting suppression of ASR. Both probability of emitting a startle and startle amplitude were diminished. ASR suppression was observed as soon as 1 day after injury and remained suppressed for the duration of the study (21 days after injury). No indication of recovery was observed. mTBI also impaired spatial working memory. In contrast to the suppression of ASR, working memory impairment was transient; memory was impaired 1 and 7 days after injury, but recovered by 21 days. The long-lasting suppression of ASR suggests long-term dysfunction of brainstem neural circuits at a time when forebrain neural circuits responsible for spatial working memory have recovered. These results have important implications for return-to-activity decisions because recovery of cognitive impairments plays an important role in these decisions.

BackgroundInjury to the brain can occur from a variety of physical insults and the degree of disability can greatly vary from person to person. It is likely that injury outcome is related to the biomechanical parameters of the traumatic event such as magnitude, direction and speed of the forces acting on the head.New methodTo model variations in the biomechanical injury parameters, a voice coil driven fluid percussion injury (FPI) system was designed and built to generate fluid percussion waveforms with adjustable rise times, peak pressures, and durations. Using this system, pathophysiological outcomes in the rat were investigated and compared to animals injured with the same biomechanical parameters using the pendulum based FPI system.Results in comparison with existing methodsImmediate post-injury behavior shows similar rates of seizures and mortality in adolescent rats and similar righting times, toe pinch responses and mortality rates in adult rats. Interestingly, post injury mortality in adult rats was sensitive to changes in injury rate. Fluoro-Jade labeling of degenerating neurons in the hilus and CA2-3 hippocampus were consistent between injuries produced with the voice coil and pendulum operated systems. Granule cell population spike amplitude to afferent activation, a measure of dentate network excitability, also showed consistent enhancement 1 week after injury using either system.ConclusionsOverall our results suggest that this new FPI device produces injury outcomes consistent with the commonly used pendulum FPI system and has the added capability to investigate pathophysiology associated with varying rates and durations of injury.

Concussive brain injury results in neuronal degeneration, microglial activation and enhanced excitability in the hippocampal dentate gyrus, increasing the risk for epilepsy and memory dysfunction. Endogenous molecules released during injury can activate innate immune responses including toll-like receptor 4 (TLR4). Recent studies indicate that immune mediators can modulate neuronal excitability. Since non-specific agents that reduce TLR4 signaling can limit post-traumatic neuropathology, we examined whether TLR4 signaling contributes to early changes in dentate excitability after brain injury. Concussive brain injury caused a transient increase in hippocampal TLR4 expression within 4h, which peaked at 24h. Post-injury increase in TLR4 expression in the dentate gyrus was primarily neuronal and persisted for one week. Acute, in vitro treatment with TLR4 ligands caused bidirectional modulation of dentate excitability in control and brain-injured rats, with a reversal in the direction of modulation after brain injury. TLR4 antagonists decreased, and agonist increased, afferent-evoked dentate excitability one week after brain injury. NMDA receptor antagonist did not occlude the ability of LPS-RS, a TLR4 antagonist, to decrease post-traumatic dentate excitability. LPS-RS failed to modulate granule cell NMDA EPSCs but decreased perforant path-evoked non-NMDA EPSC peak amplitude and charge transfer in both granule cells and mossy cells. Our findings indicate an active role for TLR4 signaling in early post-traumatic dentate hyperexcitability. The novel TLR4 modulation of non-NMDA glutamatergic currents, identified herein, could represent a general mechanism by which immune activation influences neuronal excitability in neurological disorders that recruit sterile inflammatory responses.

Traumatic brain injury (TBI) can occur from physical trauma from a wide spectrum of insults ranging from explosions to falls. The biomechanics of the trauma can vary in key features, including the rate and magnitude of the insult. Although the effect of peak injury pressure on neurological outcome has been examined in the fluid percussion injury (FPI) model, it is unknown whether differences in rate of rise of the injury waveform modify cellular and physiological changes after TBI. Using a programmable FPI device, we examined juvenile rats subjected to a constant peak pressure at two rates of injury: a standard FPI rate of rise and a faster rate of rise to the same peak pressure. Immediate postinjury assessment identified fewer seizures and relatively brief loss of consciousness after fast-rise injuries than after standard-rise injuries at similar peak pressures. Compared with rats injured at standard rise, fewer silver-stained injured neuronal profiles and degenerating hilar neurons were observed 4-6 hr after fast-rise FPI. However, 1 week postinjury, both fast- and standard-rise FPI resulted in hilar cell loss and enhanced perforant path-evoked granule cell field excitability compared with sham controls. Notably, the extent of neuronal loss and increase in dentate excitability were not different between rats injured at fast and standard rates of rise to peak pressure. Our data indicate that reduced cellular damage and improved immediate neurological outcome after fast rising primary concussive injuries mask the severity of the subsequent cellular and neurophysiological pathology and may be unreliable as a predictor of prognosis.

The changes of excitability in affected neural networks can be used as a marker to study the temporal course of traumatic brain injury (TBI). The cerebellum is an ideal platform to study brain injury mechanisms at the network level using the electrophysiological methods. Within its crystalline morphology, the cerebellar cortex contains highly organized topographical subunits that are defined by two main inputs, the climbing (CFs) and mossy fibers (MFs). Here we demonstrate the use of cerebellar evoked potentials (EPs) mediated through these afferent systems for monitoring the injury progression in a rat model of fluid percussion injury (FPI). A mechanical tap on the dorsal hand was used as a stimulus, and EPs were recorded from the paramedian lobule (PML) of the posterior cerebellum via multi-electrode arrays (MEAs). Post-injury evoked response amplitudes (EPAs) were analyzed on a daily basis for 1 week and compared with pre-injury values. We found a trend of consistently decreasing EPAs in all nine animals, losing as much as 72 ± 4% of baseline amplitudes measured before the injury. Notably, our results highlighted two particular time windows; the first 24 h of injury in the acute period and day-3 to day-7 in the delayed period where the largest drops (~50% and 24%) were observed in the EPAs. In addition, cross-correlations of spontaneous signals between electrode pairs declined (from 0.47 ± 0.1 to 0.35 ± 0.04, p < 0.001) along with the EPAs throughout the week of injury. In support of the electrophysiological findings, immunohistochemical analysis at day-7 post-injury showed detectable Purkinje cell loss at low FPI pressures and more with the largest pressures used. Our results suggest that sensory evoked potentials (SEPs) recorded from the cerebellar surface can be a useful technique to monitor the course of cerebellar injury and identify the phases of injury progression even at mild levels.

Tumor necrosis factor (TNF) plays an important role in containing mycobacterial infections. With the rapidly increasing role of TNF inhibitors in dermatology, tuberculosis (TB) is becoming an important and worrisome concern to dermatologists. This paper aims to provide a comprehensive review on the incidence of TB in patients treated with anti-TNF, the variety of TB screening methods, and management of these cases. Various national recommendations have been highlighted. The monoclonal antibodies, infliximab and adalimumab, appear to be more associated with the risk of TB reactivation than the soluble receptor etanercept. Tuberculosis associated with TNF inhibitors, in contrast to classical TB, is more likely to be disseminated, atypical, extra pulmonary, and life threatening. Vigilance for typical and atypical presentations of active TB is mandatory until the end of therapy. Although tuberculin standard test (TST) has been the gold standard for screening of latent TB infection (LTBI) for close to a century, it has several inadequacies and may be unreliable in patients with widespread psoriasis. Interferon gamma release assays (IGRAs) with better diagnostic specificity and sensitivity are a promising adjunct to diagnose LTBI at present. Although appropriate screening and treatment of LTBI will lower the risk of reactivation to a great extent, no chemoprophylactic regimen is fully protective.

The changes of excitability in affected neural networks can be used as a marker to study the temporal course of traumatic brain injury (TBI). The cerebellum is an ideal platform to study brain injury mechanisms at the network level using the electrophysiological methods. Within its crystalline morphology, the cerebellar cortex contains highly organized topographical subunits that are defined by two main inputs, the climbing (CFs) and mossy fibers (MFs). Here we demonstrate the use of cerebellar evoked potentials (EPs) mediated through these afferent systems for monitoring the injury progression in a rat model of fluid percussion injury (FPI). A mechanical tap on the dorsal hand was used as a stimulus, and EPs were recorded from the paramedian lobule (PML) of the posterior cerebellum via multi-electrode arrays (MEAs). Post-injury evoked response amplitudes (EPAs) were analyzed on a daily basis for 1 week and compared with pre-injury values. We found a trend of consistently decreasing EPAs in all nine animals, losing as much as 72 ± 4% of baseline amplitudes measured before the injury. Notably, our results highlighted two particular time windows; the first 24 h of injury in the acute period and day-3 to day-7 in the delayed period where the largest drops (~50% and 24%) were observed in the EPAs. In addition, cross-correlations of spontaneous signals between electrode pairs declined (from 0.47 ± 0.1 to 0.35 ± 0.04, p < 0.001) along with the EPAs throughout the week of injury. In support of the electrophysiological findings, immunohistochemical analysis at day-7 post-injury showed detectable Purkinje cell loss at low FPI pressures and more with the largest pressures used. Our results suggest that sensory evoked potentials (SEPs) recorded from the cerebellar surface can be a useful technique to monitor the course of cerebellar injury and identify the phases of injury progression even at mild levels.

Gamma frequency oscillations have been proposed to contribute to memory formation and retrieval. Fast-spiking basket cells (FS-BCs) are known to underlie development of gamma oscillations. Fast, high amplitude GABA synapses and gap junctions have been suggested to contribute to gamma oscillations in FS-BC networks. Recently, we identified that, apart from GABAergic synapses, FS-BCs in the hippocampal dentate gyrus have GABAergic currents mediated by extrasynaptic receptors. Our experimental studies demonstrated two specific changes in FS-BC GABA currents following experimental seizures [Yu et al., J. Neurophysiol. 109, 1746 (2013)]: increase in the magnitude of extrasynaptic (tonic) GABA currents and a depolarizing shift in GABA reversal potential (E(GABA)). Here, we use homogeneous networks of a biophysically based model of FS-BCs to examine how the presence of extrasynaptic GABA conductance (g(GABA-extra)) and experimentally identified, seizure-induced changes in g(GABA-extra) and E(GABA) influence network activity. Networks of FS-BCs interconnected by fast GABAergic synapses developed synchronous firing in the dentate gamma frequency range (40-100 Hz). Systematic investigation revealed that the biologically realistic range of 30 to 40 connections between FS-BCs resulted in greater coherence in the gamma frequency range when networks were activated by Poisson-distributed dendritic synaptic inputs rather than by homogeneous somatic current injections, which were balanced for FS-BC firing frequency in unconnected networks. Distance-dependent conduction delay enhanced coherence in networks with 30-40 FS-BC interconnections while inclusion of gap junctional conductance had a modest effect on coherence. In networks activated by somatic current injections resulting in heterogeneous FS-BC firing, increasing g(GABA-extra) reduced the frequency and coherence of FS-BC firing when E(GABA) was shunting (-74 mV), but failed to alter average FS-BC frequency when E(GABA) was depolarizing (-54 mV). When FS-BCs were activated by biologically based dendritic synaptic inputs, enhancing g(GABA-extra) reduced the frequency and coherence of FS-BC firing when E(GABA) was shunting and increased average FS-BC firing when E(GABA) was depolarizing. Shifting E(GABA) from shunting to depolarizing potentials consistently increased network frequency to and above high gamma frequencies (>80 Hz). Since gamma oscillations may contribute to learning and memory processing [Fell et al., Nat. Neurosci. 4, 1259 (2001); Jutras et al., J. Neurosci. 29, 12521 (2009); Wang, Physiol. Rev. 90, 1195 (2010)], our demonstration that network oscillations are modulated by extrasynaptic inhibition in FS-BCs suggests that neuroactive compounds that act on extrasynaptic GABA receptors could impact memory formation by modulating hippocampal gamma oscillations. The simulation results indicate that the depolarized FS-BC GABA reversal, observed after experimental seizures, together with enhanced spillover extrasynaptic GABA currents are likely to promote generation of focal high frequency activity associated with epileptic networks.

TERT-locus SNPs and leukocyte telomere measures are reportedly associated with risks of multiple cancers. Using the Illumina custom genotyping array iCOGs, we analyzed ∼480 SNPs at the TERT locus in breast (n = 103,991), ovarian (n = 39,774) and BRCA1 mutation carrier (n = 11,705) cancer cases and controls. Leukocyte telomere measurements were also available for 53,724 participants. Most associations cluster into three independent peaks. The minor allele at the peak 1 SNP rs2736108 associates with longer telomeres (P = 5.8 × 10(-7)), lower risks for estrogen receptor (ER)-negative (P = 1.0 × 10(-8)) and BRCA1 mutation carrier (P = 1.1 × 10(-5)) breast cancers and altered promoter assay signal. The minor allele at the peak 2 SNP rs7705526 associates with longer telomeres (P = 2.3 × 10(-14)), higher risk of low-malignant-potential ovarian cancer (P = 1.3 × 10(-15)) and greater promoter activity. The minor alleles at the peak 3 SNPs rs10069690 and rs2242652 increase ER-negative (P = 1.2 × 10(-12)) and BRCA1 mutation carrier (P = 1.6 × 10(-14)) breast and invasive ovarian (P = 1.3 × 10(-11)) cancer risks but not via altered telomere length. The cancer risk alleles of rs2242652 and rs10069690, respectively, increase silencing and generate a truncated TERT splice variant.

GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants.

GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants.