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The objective of this study was to examine the effect of epigenetic treatment using an histone deacetylases (HDAC) inhibitor in addition to aerobic exercise on the epigenetic markers and neurotrophic gene expressions in the motor cortex, to find a more enriched brain pre-conditioning for motor learning in neurorehabilitation. ICR mice were divided into four groups based on two factors: HDAC inhibition and exercise. Intraperitoneal administration of an HDAC inhibitor (1.2 g/kg sodium butyrate, NaB) and treadmill exercise (approximately at 10 m/min for 60 min) were conducted five days a week for four weeks. NaB administration inhibited total HDAC activity and enhanced acetylation level of histones specifically in histone H4, accompanying the increase of transcription levels of immediate-early genes (IEGs) (c-fos and Arc) and neurotrophins (BDNF and NT-4) crucial for neuroplasticity in the motor cortex. However, exercise enhanced HDAC activity and acetylation level of histone H4 and H3 without the modification of transcription levels. In addition, there were no synergic effects between HDAC inhibition and the exercise regime on the gene expressions. This study showed that HDAC inhibition could present more enriched condition for neuroplasticity to the motor cortex. However, exercise-induced neurotrophic gene expressions could depend on exercise regimen based on the intensity, the term etc. Therefore, this study has a novelty suggesting that pharmacological HDAC inhibition could be an alternative potent approach to present a neuronal platform with enriched neuroplasticity for motor learning and motor recovery, however, an appropriate exercise regimen is expected in this approach., (Copyright © 2020 Elsevier B.V. All rights reserved.)

During exercise, multiple sensory information such as visual outflow, proprioception, and vestibular information promote an increase in arousal state, which may convey positive effects on cognitive abilities such as memory. Nevertheless, which of the components of the ascending arousal system (AAS) are engaged during physical activity and which of them are critical for cognitive enhancement, induced by exercise is still unclear. Two experiments were conducted, to answer these questions: in the first one, the neuronal activity of different components of the AAS was evaluated by c-Fos immunoreactivity (Fos-ir) in running rats exposed to a lock or unlock running wheel. We found a specific Fos-ir increase in the tuberomammillary nucleus (TMN) associated with physical exercise. In the second experiment sedentary and exercised rats were challenged to conduct an object recognition memory task, and the activity of the AAS after learning was evaluated by c-Fos immunoreactivity. The exercised group showed a higher performance in the object recognition memory task which gets correlated with an increase on Fos-ir in the TMN, but not with the other components of the AAS, suggesting that the increase on TMN activity induced by exercise may be the foremost contributor of the AAS to memory enhancement observed in exercised animals., (Copyright © 2019. Published by Elsevier B.V.)

There is evidence that neuronal injury can affect uninjured neurons in the same neural circuit. The overall goal of this study was to understand the effects of peripheral nerve injury on uninjured neurons located in the central nervous system (CNS). As a model, we examined whether axotomy (transection of postganglionic axons) of the superior cervical ganglion (SCG) affected the uninjured, preganglionic neurons that innervate the SCG. At 7 days post-injury a reduction in choline acetyltransferase (ChAT) and synaptophysin immunoreactivity in the SCG, both markers for preganglionic axons, was observed, and this reduction persisted at 8 and 12 weeks post-injury. No changes were observed in the number or size of the parent cell bodies in the intermediolateral cell column (IML) of the spinal cord, yet synaptic input to the IML neurons was decreased at both 8 and 12 weeks post-injury. In order to understand the mechanisms underlying these changes, protein levels of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) were examined and reductions were observed at 7 days post-injury in both the SCG and spinal cord. Taken together these results suggest that axotomy of the SCG led to reduced BDNF in the SCG and spinal cord, which in turn influenced ChAT and synaptophysin expression in the SCG and also contributed to the altered synaptic input to the IML neurons. More generally these findings provide evidence that the effects of peripheral injury can cascade into the CNS and affect uninjured neurons., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chronic constriction injury (CCI) of the sciatic nerve elicits changes in neuropeptide expression on the dorsal root ganglia (DRG). The neural mobilization (NM) technique is a noninvasive method that has been proven clinically effective in reducing pain. The aim of this study was to analyze the expression of substance P, transient receptor potential vanilloid 1 (TRPV1) and opioid receptors in the DRG of rats with chronic constriction injury and to compare it to animals that received NM treatment. CCI was performed on adult male rats. Each animal was submitted to 10 sessions of neural mobilization every other day, starting 14 days after the CCI injury. At the end of the sessions, the DRG (L4-L6) were analyzed using Western blot assays for substance P, TRPV1 and opioid receptors (µ-opioid receptor, δ-opioid receptor and κ-opioid receptor). We observed a decreased substance P and TRPV1 expression (48% and 35%, respectively) and an important increase of µ-opioid receptor expression (200%) in the DRG after NM treatment compared to control animals. The data provide evidence that NM promotes substantial changes in neuropeptide expression in the DRG; these results may provide new options for treating neuropathic pain., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Molecular mechanism underlying ischemic stroke remains poorly understood. We previously reported glucose 6-phosphate dehydrogenase (G6PD) activity in pentose phosphate pathway (PPP) is activated via heat shock protein 27 (HSP27) phosphorylation at serine 85 (S85) by ataxia telangiectasia mutated (ATM) kinase during cerebral ischemia. This mechanism seems to be endogenous antioxidative system. To determine whether this system also works during reperfusion, we performed comparative metabolic analysis of reperfusion effect on metabolism in rat cortex using middle cerebral artery occlusion (MCAO). Metabolic profiling using gas-chromatography/mass-spectrometry analysis showed changes in metabolic state that depended on reperfusion time. Enrichment analysis showed PPP was significantly upregulated during ischemia-reperfusion. Significant increases in fructose 6-phosphate and ribulose 5-phosphate after reperfusion also suggested enhancement of PPP. In relation to PPP, ischemia-reperfusion induced an increase of up to 69-fold in HSP27 transcripts after 24-h reperfusion. Immunoblotting showed gradual increase in HSP27 protein and marked increase in HSP27 phosphorylation (S85) that were time-dependent (4.5-fold after 24-h reperfusion). G6PD activity was significantly elevated after 1-h MCAO (20%), reduced after 1-h reperfusion, increased gradually thereafter and significantly elevated after 24-h reperfusion. The NADPH/NAD + ratio displayed similar increasing pattern. Intracerebroventricular injection of ATM kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD activity, significantly increased protein carbonyl, and resulted in increase in infarct size (100%) 24-h after reperfusion following 90-min MCAO. Consequently, G6PD activation via HSP27 phosphorylation by ATM kinase may be part of endogenous antioxidant defense neuroprotection mechanism that is activated during ischemia-reperfusion. These findings have important implications for treatment of stroke., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Enriched environment (EE) has been shown to promote post-stroke neurogenesis and functional recovery. However, the underlying molecular mechanisms remains poorly understood. Male C57BL/6 mice underwent 60-min middle cerebral artery occlusion (MCAO) followed by reperfusion, after which mice were housed in either standard environment (SE) or EE. We found that post-ischemic EE exhibited reduced protein level of nuclear factor κB (NF-κB)/p65 in cytoplasm and increased its expression correspondingly in nucleus at 28 days post-ischemia (dpi). However, post-ischemic EE had no effects on terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive cells in ischemic hemisphere at 28dpi. EE mice treated with NF-kB inhibitor Bay11-7082 had decreased subventricular zone (SVZ) neural precursor cells (NPCs) proliferation, neuronal differentiation and subsequent functional recovery after stroke at 28dpi. Bay11-7082 treatment attenuated the promoting effects of post-ischemic EE on interleukin 17A (IL-17A) messenger RNA (mRNA) and protein expression at 28dpi. Furthermore, our in vitro data revealed that in primary astrocyte cultures addition of Bay11-7082 markedly decreased the expression of IL-17A in both the cell lysate and culture supernatant of activated astrocytes. Blockade of IL-17A with neutralizing antibody abrogated the promoting role of EE in NPCs proliferation derived from SVZ, neuronal differentiation and subsequent functional recovery after stroke. Thus, our results reveal a previously uncharacterized property of NF-κB/IL-17A signaling pathway in EE-mediated neurogenesis and functional recovery after ischemic stroke., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Reciprocal interactions closely connect energy metabolism with circadian rhythmicity. Altered clockwork and circadian desynchronization are often linked with impaired energy regulation. Conversely, metabolic disturbances have been associated with altered autonomic and hormonal rhythms. The effects of high-energy (HE) diet on the master clock in the suprachiasmatic nuclei (SCN) remain unclear.This question was addressed in the Sand rat (Psammomys obesus), a non-insulin-dependent diabetes mellitus (NIDDM) animal model. The aim of this work was to determine whether enriched diet in Psammomys affects locomotor activity rhythm, as well as daily oscillations in the master clock of the SCN and in an extra-SCN brain oscillator, the piriform cortex. Sand rats were fed during 3 months with either low or HE diet. Vasoactive intestinal peptide (VIP), vasopressin (AVP) and CLOCK protein cycling were studied by immunohistochemistry and running wheel protocol was used for behavioral analysis. High energy feeding dietary triggered hyperinsulinemia, impaired insulin/glucose ratio and disruption in pancreatic hormonal rhythms. Circadian disturbances in hyper-insulinemic animals include a lengthened rest/activity rhythm in constant darkness, as well as disappearance of daily rhythmicity of VIP, AVP and the circadian transcription factor CLOCK within the suprachiasmatic clock. In addition, daily rhythmicity of VIP and CLOCK was abolished by HE diet in a secondary brain oscillator, the piriform cortex. Our findings highlight a major impact of diabetogenic diet on central and peripheral rhythmicity. The Psammomys model will be instrumental to better understand the functional links between circadian clocks, glucose intolerance and insulin resistance state., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neuronal Per-Arnt-Sim (PAS) domain protein 4 (Npas4) is a key protein that intervenes in GABA synapse scaling and neurotrophicity enhancing. Since GABA and neurotrophicity are implicated in stress response and Npas4-deficient rodents exhibit behavioral alterations, an investigation was designed in rats to verify whether stress-induced spontaneous hippocampus Npas4 mRNA expression would be associated with specific patterns of stress response. The rats were exposed to one of three stressor levels: no stress (CTL, n = 15), exposure to a footshock apparatus (Sham, S, n = 40) and footshock (F, n = 80). After stress exposure the S and F rats were tested in an activity cage, and subsequently in an elevated plus maze (EPM), just prior to the sacrifice. Using cluster analysis, the animals already assigned to a stress level were also distributed into 2 subgroups depending on their Npas4 mRNA levels. The low (L) and high (H) Npas4 expression subgroups were identified in the S and F groups, the CTL group being independent of the Npas4 levels. The Npas4 effect was studied through the interaction between stress (S and F) and Npas4 level (L and H). The biological stress response was similar in H and L rats, except blood corticosterone that was slightly lower in the H rats. The H rats were more active in the actimetry cage and presented higher levels of exploration in the EPM. They also exhibited higher hippocampus activation, as assessed by the c-fos, Egr1 and Arc mRNA levels. Therefore high Npas4 expression favors stress management., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aging brain undergoes several changes leading to a decline in cognitive functions. Memory and learning-related genes such as Creb, Bdnf and its receptor TrkB, are expressed in different brain regions including prefrontal cortex. Those genes' proteins regulate a wide range of functions such as synaptic plasticity and long-term potentiation. In this work, our objectives were: 1) to investigate whether Creb1, Bdnf and TrkB genes display endogenous circadian expression rhythms, in the prefrontal cortex of rats maintained under constant darkness conditions; 2) to study the synchronization of those temporal patterns to the local cellular clock and 3) to evaluate the aging consequences on both cognition-related genes and activating clock transcription factor, BMAL1, rhythms. A bioinformatics analysis revealed clock-responsive (E-box) sites in regulatory regions of Creb1, Bdnf and TrkB genes. Additionally, cAMP response elements (CRE) were found in Bdnf and TrkB promoters. We observed those key cognition-related factors expression oscillates in the rat prefrontal cortex. Creb1 and TrkB mRNAs display a circadian rhythm with their highest levels occurring at the second half of the 24h period. Interestingly, the cosinor analysis revealed a 12-h rhythm of Bdnf transcript levels, with peaks occurring at the second half of the subjective day and night, respectively. As expected, the BMAL1 rhythm's acrophase precedes Creb1 and first Bdnf expression peaks. Noteworthy, Creb1, Bdnf and TrkB expression rhythms are lost in the prefrontal cortex of aged rats, probably, as consequence of the loss of BMAL1 protein circadian rhythm and altered function of the local cellular clock., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Myelin is a specialized subcellular structure that evolved uniquely in vertebrates. A myelinated axon conducts action potentials many times faster than an unmyelinated axon of the same diameter; for the same conduction speed, the unmyelinated axon would need a much larger diameter and volume than its myelinated counterpart. Hence myelin speeds information transfer and saves space, allowing the evolution of a powerful yet portable brain. Myelination in the central nervous system (CNS) is controlled by a gene regulatory program that features a number of master transcriptional regulators including Olig1, Olig2 and Myrf. Olig family genes evolved from a single ancestral gene in non-chordates. Olig2, which executes multiple functions with regard to oligodendrocyte identity and development in vertebrates, might have evolved functional versatility through post-translational modification, especially phosphorylation, as illustrated by its evolutionarily conserved serine/threonine phospho-acceptor sites and its accumulation of serine residues during more recent stages of vertebrate evolution. Olig1, derived from a duplicated copy of Olig2 in early bony fish, is involved in oligodendrocyte development and is critical to remyelination in bony vertebrates, but is lost in birds. The origin of Myrf orthologs might be the result of DNA integration between an invading phage or bacterium and an early protist, producing a fusion protein capable of self-cleavage and DNA binding. Myrf seems to have adopted new functions in early vertebrates - initiation of the CNS myelination program as well as the maintenance of mature oligodendrocyte identity and myelin structure - by developing new ways to interact with DNA motifs specific to myelin genes. This article is part of a Special Issue entitled SI: Myelin Evolution., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Exposure to hand-transmitted vibration in the work-place can result in the loss of sensation and pain in workers. These effects may be exacerbated by pre-existing conditions such as diabetes or the presence of primary Raynaud's phenomena. The goal of these studies was to use an established model of vibration-induced injury in Zucker rats. Lean Zucker rats have a normal metabolic profile, while obese Zucker rats display symptoms of metabolic disorder or Type II diabetes. This study examined the effects of vibration in obese and lean rats. Zucker rats were exposed to 4h of vibration for 10 consecutive days at a frequency of 125 Hz and acceleration of 49 m/s(2) for 10 consecutive days. Sensory function was checked using transcutaneous electrical stimulation on days 1, 5 and 9 of the exposure. Once the study was complete the ventral tail nerves, dorsal root ganglia and spinal cord were dissected, and levels of various transcripts involved in sensorineural dysfunction were measured. Sensorineural dysfunction was assessed using transcutaneous electrical stimulation. Obese Zucker rats displayed very few changes in sensorineural function. However they did display significant changes in transcript levels for factors involved in synapse formation, peripheral nerve remodeling, and inflammation. The changes in transcript levels suggested that obese Zucker rats had some level of sensory nerve injury prior to exposure, and that exposure to vibration activated pathways involved in injury and re-innervation., (Published by Elsevier B.V.)

Stress is a potent modulator of hippocampal-dependent memory formation. The aim of the present study was to assess the role of hippocampal signaling pathways in stress-induced memory impairment in male Wistar rats. The animals were exposed to acute elevated platform (EP) stress and memory formation was measured by a step-through type passive avoidance task. The results indicated that post-training or pre-test exposure to EP stress impaired memory consolidation or retrieval respectively. Using western blot analysis, it was found that memory retrieval was associated with the increase in the levels of phosphorylated cAMP-responsive element binding protein (P-CREB), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its downstream targets in the hippocampus. In contrast, the stress exposure decreased the hippocampal levels of these proteins. In addition, stress-induced impairment of memory consolidation or retrieval was associated with the decrease in the P-CREB/CREB ratio and the PGC-1α level in the hippocampus. On the other hand, the hippocampal level of nuclear factor E2-related factor 2 (Nrf2) and gamma-glutamylcysteine synthetase (γ-GCS) which are the master regulators of defense system were decreased by the stress exposure. The increased hippocampal levels of Nrf2 and it׳s downstream was observed during memory retrieval, while stress-induced impairment of memory consolidation or retrieval inhibited this hippocampal signaling pathway. Overall, these findings suggest that down-regulation of CREB/PGC-1α signaling cascade and Nrf2 antioxidant pathways in the hippocampus may be associated with memory impairment induced by stress., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Immune cells have important roles during disease and are known to contribute to secondary, inflammation-induced injury after traumatic brain injury. To delineate the functional role of macrophages during traumatic brain injury, we depleted macrophages using transgenic CD11b-DTR mice and subjected them to controlled cortical impact. We found that macrophage depletion had no effect on lesion size assessed by T2-weighted MRI scans 28 days after injury. Macrophage depletion resulted in a robust increase in proinflammatory gene expression in both the ipsilateral and contralateral hemispheres after controlled cortical impact. Interestingly, this sizeable increase in inflammation did not affect lesion development. We also showed that macrophage depletion resulted in increased proinflammatory gene expression in the brain and kidney in the absence of injury. These data demonstrate that depletion of macrophages in CD11b-DTR mice can significantly modulate the inflammatory response during brain injury without affecting lesion formation. These data also reveal a potentially confounding inflammatory effect in CD11b-DTR mice that must be considered when interpreting the effects of macrophage depletion in disease models., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Early adverse life experiences have been associated with anxiety-like behavior and memory impairment. N-methyl-d-aspartate receptors (NMDARs) play an important role in brain development. Enriched environments are known to positively influence emotional and cognitive functions in the brain. We examined the effects of maternal deprivation (MD) on NMDAR subunits in the hippocampus, locomotor activity, anxiety behaviors, and learning-memory performance of Balb/c mice. We also examined whether these effects could be reversed by raising the offspring in an enriched environment. The mice were separated from their mothers for a single 24h episode on postnatal day (PND) 9. The mice were weaned on day 21 and were housed under either standard (SE) or enriched (EE) environmental conditions. Emotional behaviors and cognitive processes of mice were evaluated using an open field (OF) test, an elevated plus maze (EPM) test, and a Morris water-maze (MWM). NMDAR subunits (GluN1, GluN2A, and GluN2B) mRNA expression levels in the hippocampus were examined by real-time PCR. In OF, MD had no effect on horizontal locomotor activity. MD increased anxiety-like behaviors in the EPM and decreased spatial learning performance in MWM; however, these effects were not reversed by EE. MD (in SE and EE conditions) increased GluN1, GluN2A, and GluN2B mRNA expressions in the hippocampus. In conclusion, MD led to the deterioration of the emotional and cognitive processes during adulthood. Moreover, environmental enrichment did not reverse the deleterious effects of the MD on emotional and cognitive functions and increased the NMDAR levels., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A variety of exercise protocols have been used to promote experimental neuroplasticity. However, the plastic brain responses generated by several aspects of training (types, frequency, regimens, duration) remain undetermined. The aim of this study was to compare the plastic changes in the glutamatergic system and synaptic proteins in motor cortex, striatum and cerebellum promoted by two different treadmill exercise regimens. The present study analyzed by immunohistochemistry and Western blotting the expression of the subunits of AMPA receptors (GluA1 and GluA2/3) and synaptic proteins (synapsin I and synaptophysin) in adult male Wistar rat brains. The animals were divided into animals subjected to two different frequencies of aerobic exercise groups and sedentary animals. The exercise groups were: intermittent treadmill exercise (ITE) - animals that exercised 3 times a week (every other day) during four weeks, and continuous treadmill exercise (CTE) - animals that exercised every day during four weeks. Our results reveal that different protocols of treadmill exercise were able to promote distinct synaptic reorganization processes among the exercised groups. In general, the intermittent exercise regimen induced a higher expression of presynaptic proteins, whereas the continuous exercise regimen increased postsynaptic GluA1 and GluA2/3 receptors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cerebral ischemia is a major health crisis throughout the world, and the currently available thrombolytic therapy is unsatisfactory. Cell death following cerebral ischemia is mediated by a complex pathophysiological interaction of various mechanisms. During an ischemic insult, not only neurons but all of the components of the neurovascular unit, such as glia, endothelia, pericytes and basal membranes, are destroyed. Previous studies have shown that excessive stimulation of poly (ADP-ribose) polymerase (PARP-1) is crucial for cerebral injury after ischemic insult, which is an important cause of cell death in all cell types within the neurovascular unit. To investigate whether PARP-1 plays an important role in protecting the neurovascular unit following cerebral ischemia, we evaluated neurobehavioral deficits, PARP-1 activity, blood brain barrier (BBB) disruption and neurovascular unit deficits using Western blot analysis, TTC staining and electron microscopy in an MCAO rat model. The results revealed that PARP-1 enzymatic activity was dramatically increased after ischemia. Inhibition of PARP-1 significantly reduced the extent of both cerebral infarction and edema, improved neurological scores, and attenuated the damage to the neurovascular unit in cerebral ischemia. Collectively, these findings demonstrate that the down-regulation of PARP-1 activity contributes to reducing post-ischemic brain damage via protection of the neurovascular unit., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Vesicular glutamate transporters (VGLUTs) control the storage and release of glutamate, which plays a critical role in pain processing. The VGLUT2 isoform has been found to be densely distributed in the nociceptive pathways in supraspinal regions, and VGLUT2-deficient mice exhibit an attenuation of neuropathic pain; these results suggest a possible involvement of VGLUT2 in neuropathic pain. To further examine this, we investigated the temporal changes in VGLUT2 expression in different brain regions as well as changes in glutamate release from thalamic synaptosomes in spared nerve injury (SNI) mice. We also investigated the effects of a VGLUT inhibitor, Chicago Sky Blue 6B (CSB6B), on pain behavior, c-Fos expression, and depolarization-evoked glutamate release in SNI mice. Our results showed a significant elevation of VGLUT2 expression up to postoperative day 1 in the thalamus, periaqueductal gray, and amygdala, followed by a return to control levels. Consistent with the changes in VGLUT2 expression, SNI enhanced depolarization-induced glutamate release from thalamic synaptosomes, while CSB6B treatment produced a concentration-dependent inhibition of glutamate release. Moreover, intracerebroventricular administration of CSB6B, at a dose that did not affect motor function, attenuated mechanical allodynia and c-Fos up-regulation in pain-related brain areas during the early stages of neuropathic pain development. These results demonstrate that changes in the expression of supraspinal VGLUT2 may be a new mechanism relevant to the induction of neuropathic pain after nerve injury that acts through an aggravation of glutamate imbalance., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Calorie restriction (CR) is argued to positively affect general health, longevity and normally occurring age-related reduction of cognition. Obesity during adolescence may adversely affect cognition in adulthood but, to date effects of CR have not been investigated. We hypothesized that feeding with as low as 15% low-calorie diet (LCD) during adolescence would increase hippocampal and prefrontal BDNF (Brain-derived neurotrophic factor) levels, proliferative cells and neuron numbers in dentate gyrus (DG), thus positively affecting spatial memory in adulthood. Spatial learning-memory function was improved in adult female Sprague-Dawley rats fed with LCD during adolescence. PCNA (Proliferating cell nuclear antigen-cell proliferation marker) expressing cells and NeuN (Neuronal nuclear antigen-neuron marker) expressing cells in hippocampus DG which are critically involved in memory were increased. Hippocampus and prefrontal cortex BDNF levels were increased while serum glucose levels and level of lipid peroxidation indicator malondialdehyde in serum and hippocampus were reduced. Our unique results suggest that improved cognition in adult rats with LCD feeding during adolescence may result from the increase of neurogenesis and BDNF. These findings reveal the importance of nutrition in adolescence for cognitive function in adulthood. Our results may be useful for further studies aiming to treat age-related cognitive impairments., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Early brain injury has recently been identified as an indicator of poor prognosis after subarachnoid hemorrhage (SAH). Calmodulin-dependent protein kinase IIα (CaMKIIα) has been shown to phosphorylate neuronal NOS (nNOS) at Ser(847), resulting in a reduction in nNOS activity. In this study, we revealed chronological changes in the phosphorylation of nNOS at Ser(847) in the hippocampus and cortex immediately after SAH. In a rat single-hemorrhage model of SAH, the hippocampus and adjacent cortex were collected up to 24h after SAH. Samples from rats that were not injected with blood were used as controls. NOS was partially purified from the crude samples using ADP-agarose affinity chromatography. Western blot analysis revealed that nNOS phosphorylated (p-nNOS) at Ser(847) was significantly increased in the hippocampus, but not in the cortex, at 1h after SAH compared with that resulting from the control treatment. Immunoreactivity of p-nNOS at Ser(847) was observed in interneurons of the hippocampus at 1h after SAH. Injection of saline instead of blood also significantly induced p-nNOS at Ser(847) levels in the hippocampus at 1h after injection. The colocalization of CaMKIIα and nNOS was transiently increased in the hippocampus at 0.5h after SAH. Our data suggest that immediately after SAH, an increase in intracranial pressure might induce transient cerebral ischemia, potentially promoting the phosphorylation of nNOS at Ser(847) by CaMKIIα in the hippocampus. The activation of p-nNOS at Ser(847) in the hippocampus may alleviate ischemic insults immediately after SAH to exert a neuroprotective effect against early brain injury., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The hippocampal interneurons are very diverse by chemical profiles and rather inconsistent by sensitivity to CI. Some hippocampal GABAergic interneurons survive certain time after ischemia while ischemia-sensitive interneurons and pyramidal neurons are damaged. GABAergic signaling, nicotinic receptors expressing α7-subunit (α7nAChRs(+)) and connexin-36 (Cx36(+), electrotonic gapjunctions protein) contradictory modulate post-ischemic environment. We hypothesized that hippocampal ischemia-resistant GABAergic interneurons coexpressing glutamate decarboxylase-67 isoform (GAD67(+)), α7nAChRs(+), Cx36(+) are able to enhance neuronal viability. To check this hypothesis the histochemical and electrophysiological investigations have been performed using rat hippocampal organotypic culture in the condition of 30-min oxygen-glucose deprivation (OGD). Post-OGD reoxygenation (4h) revealed in CA1 pyramidal layer numerous damaged cells, decreased population spike amplitude and increased pair-pulse depression. In these conditions GAD67(+) interneurons displayed the OGD-resistance and significant increase of GABA synthesis/metabolism (GAD67-immunofluorescence, mitochondrial activity). The α7nAChRs(+) and Cx36(+) co-localizations were revealed in resistant GAD67(+) interneurons. Under OGD: GABAA-receptors (GABAARs) blockade increased cell damage and exacerbated the pair-pulse depression in CA1 pyramidal layer; α7nAChRs and Cx36-channels separate blockades sufficiently decreased cell damage while interneuronal GAD67-immunofluorescence and mitochondrial activity were similar to the control. Thus, hippocampal GABAergic interneurons co-expressing α7nAChRs and Cx36 remained resistant certain time after OGD and were able to modulate CA1 neuron survival through GABAARs, α7nAChRs and Cx36-channels activity. The enhancements of the neuronal viability together with GABA synthesis/metabolism normalization suggest cooperative neuroprotective mechanism that could be used for increase in efficiency of therapeutic strategies against post-ischemic pathology., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Estrogen deficiency worsens ischemic stroke outcomes. In ovariectomized (OVX(+)) rats fed a high-salt diet (HSD), an increase in the body Na(+)/water ratio, which characterizes water-free Na(+) accumulation, was associated with detrimental vascular effects independent of the blood pressure (BP). We hypothesized that an increase in brain water-free Na(+) accumulation is associated with ischemic brain damage in OVX(+)/HSD rats. To test our hypothesis we divided female Wistar rats into 4 groups, OVX(+) and OVX(-) rats fed HSD or a normal diet (ND), and subjected them to transient cerebral ischemia. The brain Na(+)/water ratio was increased even in OVX(+)/ND rats and augmented in OVX(+)/HSD rats. The increase in the brain Na(+)/water ratio was positively correlated with expansion of the cortical infarct volume without affecting the BP. Interestingly, OVX(+) was associated with the decreased expression of ATP1α3, a subtype of the Na(+) efflux pump. HSD increased the expression of brain Na(+) influx-related molecules and the mineralocorticoid receptor (MR). The pretreatment of OVX(+)/HSD rats with the MR antagonist eplerenone reduced brain water-free Na(+) accumulation, up-regulated ATP1α3, down-regulated MR, and reduced the cortical infarct volume. Our findings show that the increase in the brain Na(+)/water ratio elicited by estrogen deficiency or HSD is associated with ischemic brain damage BP-independently, suggesting the importance of regulating the accumulation of brain water-free Na(+). The up-regulation of ATP1α3 and the down-regulation of MR may provide a promising therapeutic strategy to attenuate ischemic brain damage in postmenopausal women., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Recent studies have shown that Onecut (Oc) transcription factors may be involved in the early development of midbrain dopaminergic neurons (mdDA). The expression profile of Oc factors matches that of Lmx1a, an important intrinsic transcription factor in the development of mDA neuron. Moreover, the Wnt1-Lmx1a pathway controls the mdDA differentiation. However, their expression dynamics and molecular mechanisms remain to be determined. To address these issues, we hypothesize that cross-talk between Oc-1 and Lmx1a regulates the mdDA specification and differentiation through the canonical Wnt-β-catenin pathway. We found that Oc-1 and Lmx1a displayed a very similar expression profile from embryonic to adult ventral midbrain (VM) tissues. Oc-1 regulated the proliferation and differentiation of ventral midbrain neural stem cells (vmNSCs). Downregulation of Oc-1 decreased both transcript and protein level of Lmx1a. Oc-1 interacted with lmx1a in vmNSCs in vitro and in VM tissues in vivo. Knockdown of Lmx1a reduced the expression of Oc-1 and Wnt1 in vmNSCs. Inhibiting Wnt1 signaling in vmNSCs provoked similar responses. Our data suggested that Oc-1 interacts with Lmx1a to promote vmNSCs differentiation into dopamine neuron through Wnt1-Lmx1a pathway., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Apoptosis signal-regulating kinase-1 (ASK1) is the mitogen-activated protein kinase kinase kinase (MAPKKK) and participates in the various central nervous system (CNS) signaling pathways. In cerebral ischemia, vascular permeability in the brain is an important issue because regulation failure of it results in edema formation and blood-brain barrier (BBB) disruption. To determine the role of ASK1 on vascular permeability and edema formation following cerebral ischemia, we first investigated ASK1-related gene expression using microarray analyses of ischemic brain tissue. We then measured protein levels of ASK1 and vascular endothelial growth factor (VEGF) in brain endothelial cells after hypoxia injury. We also examined protein expression of ASK1 and VEGF, edema formation, and morphological alteration through cresyl violet staining in ischemic brain tissue using ASK1-small interference RNA (ASK1-siRNA). Finally, immunohistochemistry was performed to examine VEGF and aquaporin-1 (AQP-1) expression in ischemic brain injury. Based on our findings, we propose that ASK1 is a regulating factor of vascular permeability and edema formation in cerebral ischemia., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

There are currently few approaches to transiently manipulate the expression of specific proteins in microglia of the brain. An antibody directed against an extracellular epitope of scavenger receptor class B, type I (SR-BI) was found to be selectively taken up by these cells in the brain. Other antibodies tested were not internalised by microglia. A vector was produced by linking the SR-BI antibody to polyethyleneimine and binding a DNA plasmid encoding green fluorescent protein. Infusions of this vector into the hippocampus produced a widespread transfection of cells, more than 80% of which were immunoreactive for microglial/macrophage markers. Transfection was not detected in cells expressing markers for astrocytes or neurons. Reporter gene expression was most prominent near the infusion site but was seen in tissue up to 4mm away. DNA bound to polyethyleneimine alone or to a vector containing a different antibody did not produce transfection in the brain. Single injections of the vector containing the SR-BI antibody into the brain also resulted in transfection of microglia, albeit with lower efficiency. Vector modifications to promote lysis of endosomes or entry of DNA into the nucleus did not increase efficiency. The findings clearly demonstrate the capacity of the SR-BI antibody to selectively target brain microglia. This approach offers considerable potential to deliver DNA and other molecules capable of modifying the function of these cells in vivo., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Notch signaling is a highly conserved pathway that regulates cell fate decisions during embryonic development. We have recently identified that in ischemic stroke, activity of γ-secretase and the resulting Notch activation may endanger neurons by modulating NF-κB and HIF-1α pathways. Notch signaling can also modulate MAPK-related pathways. However, the role of γ-secretase-mediated Notch signaling in activating MAPK following ischemic stroke has not been investigated. We used control and NICD1-overexpressing HEK and SH-SY5Y cell lines, and inhibitors of γ-secretase and JNK, to explore novel roles of Notch in modulating cell death following ischemic stress in vitro. Our findings indicate that expression of NICD1, JNK/cJun, p38-MAPK and the pro-apoptotic marker, cleaved caspase-3, increased during ischemic conditions. γ-Secretase inhibitors reduced ischemia-induced increase in NICD1 and JNK/p-cJun. Furthermore, NICD overexpression augmented JNK/cJun levels and cell death under these conditions. These results suggest that Notch signaling contributes to the pathogenesis of ischemic stroke, in part by promoting JNK/cJun signaling. These results provide further support for the potential use of γ-secretase inhibitors as therapy for ischemic stroke., (Copyright © 2014 Elsevier B.V. All rights reserved.)

After spinal cord injury (SCI), the rapidly upregulated chondroitin sulfate proteoglycans (CSPGs), the prominent chemical constituents and main repulsive factors of the glial scar, play an important role in the extremely limited ability to regenerate in adult mammals. Although many methods to overcome the inhibition have been tested, no successful method with clinical feasibility has been devised to date. It was recently discovered that receptor protein tyrosine phosphatase sigma (RPTPσ) is a functional receptor for CSPGs-mediated inhibition. In view of the potential clinical application of RNA interference (RNAi), here we investigated whether silencing RPTPσ via lentivirus-mediated RNA interference can promote axon regeneration and functional recovery after SCI. Neurites of primary rat cerebral cortical neurons with depleted RPTPσ exhibited a significant enhancement in elongation and crossing ability when they encountered CSPGs in vitro. A contusion model of spinal cord injury in Wistar rats (the New York University (NYU) impactor) was used for in vivo experiments. Local injection of lentivirus encoding RPTPσ shRNA at the lesion site promoted axon regeneration and synapse formation, but did not affect the scar formation. Meanwhile, in vivo functional recovery (motor and sensory) was also enhanced after RPTPσ depletion. Therefore, strategies directed at silencing RPTPσ by RNAi may prove to be a beneficial, efficient and valuable approach for the treatment of SCI., (Copyright © 2014 Elsevier B.V. All rights reserved.)

An active lifestyle involving regular exercise reduces the deleterious effects of the aging process. At the cerebral level, both synaptic plasticity and neurogenesis are modulated by exercise, although the molecular mechanisms underlying these effects are not clearly understood. In the mature nervous system, the canonical Wnt (Wnt/β-catenin) signaling pathway is implicated in neuroprotection and synaptic plasticity. Here, we examined whether the Wnt pathway could be modulated in adult male rat hippocampus by long-term moderate exercise (treadmill running) or enrichment (handling/environmental stimulation). Sedentary animals showed higher protein levels of the Wnt antagonist, Dkk-1, the lowest levels being found in the exercised group. Although there was no evidence of any changes in activation of the LRP6 receptor, the total levels of LRP6 were higher in exercised and enriched animals. Analysis of some of the components implicated in the phosphorylation of β-catenin, which leads ultimately to its proteasomal degradation, revealed higher levels and activation of Axin1 and GSK-3α/β respectively in sedentary animals. However neither different phosphorylated forms nor total β-catenin protein levels differed between the experimental groups. Higher protein levels of Axin2 and the antiapoptotic protein, Bcl-2, were found with exercise and handling, whereas the proapototic, Bax, was unaffected. Thus, our results suggest activation of the Wnt pathway not only with moderate exercise, but also with the handling of the animals., (© 2013 Elsevier B.V. All rights reserved.)

The nucleus incertus (NI), a brainstem nucleus found in the pontine periventricular grey, is the primary source of the neuropeptide relaxin-3 in the mammalian brain. The NI neurons have also been previously reported to express several receptors and neurotransmitters, including corticotropin releasing hormone receptor 1 (CRF₁) and gamma-aminobutyric acid (GABA). The NI projects widely to putative neural correlates of stress, anxiety, depression, feeding behaviour, arousal and cognition leading to speculation that it might be involved in several neuropsychiatric conditions. On the premise that relaxin-3 expressing neurons in the NI predominantly co-express CRF₁ receptors, a novel method for selective ablation of the rat brain NI neurons using corticotropin releasing factor (CRF)-saporin conjugate is described. In addition to a behavioural deficit in the fear conditioning paradigm, reverse transcriptase polymerase chain reaction (RT-PCR), western blotting (WB) and immunofluorescence labelling (IF) techniques were used to confirm the NI lesion. We observed a selective and significant loss of CRF₁ expressing cells, together with a consistent decrease in relaxin-3 and GAD65 expression. The significant ablation of relaxin-3 positive neurons of the NI achieved by this lesioning approach is a promising model to explore the neuropsychopharmacological implications of NI/relaxin-3 in behavioural neuroscience., (© 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Sensory deprivation, such as whisker deprivation, is one of the most common paradigms used to examine experience-dependent plasticity. Many of these studies conducted during development have demonstrated anatomical and synaptic neocortical plasticity with varying lengths of deprivation (for review, see Holtmaat and Svoboda, 2009). However, to date, there have been few studies exploring brief periods of experience-dependent neocortical plasticity in adulthood, similar to that observed from learning and memory paradigms (Siucinska and Kossut, 1996, 2004; Galvez et al., 2006; Chau et al., 2013). Examining both synapsin I and Golgi-Cox stained neurons in primary somatosensory cortex of unilaterally whisker-deprived adult mice, the current study demonstrates that 5 days of whisker deprivation results in more synapses in spared barrels and reduced synapses in deprived barrels. To our knowledge, this is the first study to characterize anatomical changes in layer IV of primary somatosensory cortex after a brief period of sensory deprivation in adulthood. Furthermore, findings from the present study suggest that analyses from prolonged periods of either sensory deprivation or stimulation during adulthood are missing forms of plasticity that could provide better insight into various cognitive processes, such as learning and memory., (© 2013 Published by Elsevier B.V.)

Impaired spinal GABAergic inhibitory function is known to be pivotal in neuropathic pain (NPP). At present, data concerning time-dependent alterations in cell type and cell death in the spinal dorsal horn are highly controversial, likely related to the experimental NPP model used. In this study, we examined the expression of autophagy using a L5 spinal nerve ligation (SNL)-induced neuropathic pain rat model. Following ligation of the spinal nerve, neuropathic pain behavior, such as mechanical allodynia, was induced rapidly and maintained for 14 days. After testing for mechanical allodynia, we assessed the changes in expression of LC3 and Beclin 1 in the spinal cord following SNL. Immunohistochemical analysis showed that the levels of LC3 and Beclin 1 protein in the ipsilateral L5 spinal dorsal horn were significantly elevated on day 14 following SNL. Double immunohistochemical analysis further confirmed increases in LC3 and Beclin 1 in mostly neurons and a few astrocytes following SNL. LC3 and Beclin 1 expressions were upregulated in GABAergic interneurons of spinal dorsal horn after SNL, while the loss of GABAergic interneurons did not change significantly. Our results suggest that autophagic disruption in GABAergic interneurons and astrocytes following peripheral nerve injury might be involved in the induction and maintenance of neuropathic pain., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In the hippocampus, ovarian hormones and sex can alter the trafficking of delta opioid receptors (DORs) and the proportion of DORs that colocalize with the stress hormone, corticotropin releasing factor. Here, we assessed the effects of acute immobilization stress (AIS) and sex on the phosphorylation of DORs in the rat hippocampus. We first localized an antibody to phosphorylated DOR (pDOR) at the SER363 carboxy-terminal residue, and demonstrated its response to an opioid agonist. By light microscopy, pDOR-immunoreactivity (ir) was located predominantly in CA2/CA3a pyramidal cell apical dendrites and in interneurons in CA1-3 stratum oriens and the dentate hilus. By electron microscopy, pDOR-ir primarily was located in somata and dendrites, associated with endomembranes, or in dendritic spines. pDOR-ir was less frequently found in mossy fibers terminals. Quantitative light microscopy revealed a significant increase in pDOR-ir in the CA2/CA3a region of male rats 1h following an injection of the opioid agonist morphine (20mg/kg, I.P). To look at the effects of stress on pDOR, we compared pDOR-ir in males and cycling females after AIS. The level of pDOR-ir in stratum radiatum of CA2/CA3a was increased in control estrus (elevated estrogen and progesterone) females compared to proestrus and diestrus females and males. However, immediately following 30min of AIS, no significant differences in pDOR levels were seen across estrous cycle phase or sex. These findings suggest that hippocampal levels of phosphorylated DORs vary with estrous cycle phase and that acute stress may dampen the differential effects of hormones on DOR activation in females., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cerebral vasospasm (CVS) is the most treatable component of subarachnoid hemorrhage (SAH), which can be reduced by endothelin receptor antagonists. Endothelin-evoked vasospasm is considered to be mediated by Ca(2+) influx in the smooth muscle through voltage-dependent Ca(2+) channel (VDCC) and nonselective cation channels (NSCC). Because VDCC antagonists such as nimodipine have been shown to be relatively less effective than the endothelin receptor antagonists, it is assumed that NSCC maybe a more important component in mediating Ca(2+) influx during CVS. In this study, we used the basilar arteries from a "two-hemorrhage" rat model of SAH to investigate expressions of transient receptor potential channel 1 (TRPC1), transient receptor potential channel 3 (TRPC3) and stromal interaction molecule 1 (STIM1), which are considered as the promising candidates constituting NSCC. To investigate the possible role of NSCC in phenotypic switching, we performed immunohistochemical staining to examine expressions of SMα-actin and PCNA, markers of smooth muscle phenotypic switching. We found that the basilar arteries exhibited vasospasm after SAH and that vasospasm became more severe on days 5 and 7 after SAH. Elevated mRNA and protein expressions of TRPC1 and STIM1 were detected after SAH and peaked on days 5 and 7, which was in a parallel time course to the development of cerebral vasospasm. The mRNA and protein expressions of TRPC3 were not changed in the SAH group when compared with those in the control. Results of immunohistochemical staining with anti-PCNA and anti-SMα-actin antibodies also showed enhanced expression of PCNA and disappearance of SMα-actin from day 1 to day 7. Taken together, the above results supported a novel mechanism that the components of store-operated calcium channels, TRPC1 and STIM1 mediated the Ca(2+) influx and phenotypic switching in smooth muscle cells, which promoted the development of vasospasm after SAH. TRPC3, which is a component of receptor-operated calcium channels, was not involved in the above-mentioned mechanism., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Recent findings implicate the calcium-permeable transient receptor potential (TRP) melastatin subtype 2 (TRPM2) and canonical subtype 3 (TRPC3) channels in the pathogenesis of bipolar disorder (BD). As both channels are involved in calcium and oxidative stress signaling, thought to be disrupted in BD, we sought to determine the effects of elevated oxidative stress on their expression and function. Primary rat cortical neurons and astrocytes were treated with oxidative stressors for 1 (acute) and 4 days (chronic). Expression of TRPC3 and TRPM2 were determined by immunoblotting and real-time PCR. Channel functionality was assessed using a TRPC3 activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and live cell, ratiometric fluorometry with the calcium sensitive dye, Fura-2. Neurons treated with rotenone (15-30nM) for 4 days but not 24h showed significant dose-dependent decreases in TRPC3 mRNA (31%, p<0.001) and protein levels (60%, p<0.001). Similar dose-dependent attenuation of TRPC3-mediated calcium fluxes was demonstrated upon chronic rotenone exposure relative to vehicle controls. In contrast, TRPM2 mRNA but not protein levels increased (47%, p=0.017) after acute and chronic rotenone treatment. Chronic exposure of neurons to paraquat (1-2µM), an alternate oxidative stressor, similarly decreased TRPC3 expression (mRNA: 41%; protein: 61%). Unlike neurons, rotenone treatment incurred no changes in astrocyte TRPC3 levels. These findings demonstrate that TRPC3 and TRPM2 channel expression and/or function is sensitive to the redox status of rat primary neurons and that these changes are time dependent. This provides a critical mechanistic link between altered oxidative stress markers, dysfunction of these TRP channels and calcium dyshomeostasis in BD., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In the present study, we aimed to investigate the protective effect of puerarin (PR) on the substantia nigra (SN) of 6-hydroxydopamine (6-OHDA)-lesioned rats. Our results show that glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities were increased, while the malonaldehyde (MDA) content was reduced in the SN following PR treatment. Furthermore, examination of specific immunostaining revealed that histopathological lesions in SN tissue were alleviated. Similarly, the mRNA level of heme oxygenase-1 (HO-1) was notably decreased, and the expression of NAD(P)H: quinone oxidoreductase-1 (NQO-1) mRNA was increased. As revealed by Western blot analysis, PR therapy contributed to a marked expression of DJ-1 and superoxide dismutase-2 (SOD2) at the protein levels in the SN of 6-OHDA-lesioned rats. Taken together, these findings suggest that puerarin effectively exerts its neuroprotective action against 6-OHDA-induced Parkinsonian rats through the enhancement of antioxidant capability and attenuation of oxidative stress injury, thereby inhibiting neurodegeneration in SN tissue., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The inwardly rectifying potassium channel subunit Kir4.1 is expressed in brain astrocytes and involved in spatial K(+) buffering, regulating neural activity. To explore the pathophysiological alterations of Kir4.1 channels in epileptic disorders, we analyzed interictal expressional levels of Kir4.1 in the Noda epileptic rat (NER), a hereditary animal model for generalized tonic-clonic (GTC) seizures. Western blot analysis showed that Kir4.1 expression in NERs was significantly reduced in the occipito-temporal cortical region and thalamus. However, the expression of Kir5.1, another Kir subunit mediating spatial K(+) buffering, remained unaltered in any brain regions examined. Immunohistochemical analysis revealed that Kir4.1 was primarily expressed in glial fibrillary acidic protein (GFAP)-positive astrocytes (somata) and foot processes clustered around neurons proved with anti-neuronal nuclear antigen (NeuN) antibody. In NERs, Kir4.1 expression in astrocytic processes was region-selectively diminished in the amygdaloid nuclei (i.e., medial amygdaloid nucleus and basomedial amygdaloid nucleus) while Kir4.1 expression in astrocytic somata was unchanged. Furthermore, the amygdala regions with reduced Kir4.1 expression showed a marked elevation of Fos protein expression following GTC seizures. The present results suggest that reduced activity of astrocytic Kir4.1 channels in the amygdala is involved in limbic hyperexcitability in NERs., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The sensorimotor striatum is critical for the acquisition and consolidation of skilled learning-related motor sequences. Excitatory corticostriatal synapses undergo neuroplastic changes that impact signal transmission efficacy. Modification of N-methyl d-aspartate (NMDA) and α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit composition and phosphorylation is critical for bidirectional experience-driven plasticity observed at these synapses. Metaplastic regulation of the ratio of NR2A to NR2B subunits of the NMDA receptor controls the threshold for the induction of subsequent plasticity. However, little is known about how repeated practice effects the differential regulation of glutamate receptors during the acquisition of a unilateral motor skill. Using immunoblot analysis, we assessed changes in NMDA and AMPA receptors during the associative stage of skill acquisition in synaptoneurosome preparations from the rat sensorimotor striatum. We found that the NR2A/B subunit ratio in the striatum contralateral to the trained limb decreased during skill acquisition optimizing the threshold for inducing subsequent synaptic plasticity during learning of the lateralized motor skill. In contrast, there was a significant increase in the NR2A/B subunit ratio in the ipsilateral striatum making the induction of subsequent plasticity more difficult. In addition, there was a selective decrease in AMPAR phosphorylation levels at serine site 831 but not 845 on the GluR1 subunit ipsilaterally with a trend toward a decrease contralaterally. These findings suggest that the successful acquisition of a lateralized motor skill necessitates the integration of motor programs in both striata, each of which reflects unique changes in the NR2A/B ratio that modulate the different task demands on the associated limb., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Gem is an atypical protein of the Ras superfamily that plays a role in regulating voltage-gated Ca(2+) channels and cytoskeletal reorganization. To elucidate the certain expression and biological function in central nervous system (CNS), we performed an acute spinal cord contusion injury model in adult rats. Western blot analysis showed a marked up-regulation of Gem after spinal cord injury (SCI). Immunohistochemistry revealed wide distribution of Gem in spinal cord, including neurons and glial cells. Double immunofluorescent staining for proliferating cell nuclear antigen (PCNA) and phenotype-specific markers indicated increases of Gem expression in proliferating microglia and astrocytes. Our data suggest that Gem may be implicated in the proliferation of microglia and astrocytes after SCI., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Protein translation controlled through activation of mammalian target of rapamycin (mTOR) participates in many physiological and pathological processes. However, whether such activation is required for chronic pain is still unknown. Here, we examined activation of the mTOR signaling pathway during complete Freund's adjuvant (CFA)-induced chronic inflammatory pain and L5 spinal nerve ligation (SNL)-induced neuropathic pain in rats. Western blot analysis showed significantly increased levels of phosphorylated mTOR (p-mTOR) and phosphorylated p70S6 kinase 1 (p-S6K1, a downstream effector of mTOR) in the ipsilateral L4/5 spinal cord 2h, 1 day, 3 days, and 7 days after intraplantar CFA injection and in the ipsilateral L4/5 dorsal root ganglions (DRGs) 1 and 3 days after CFA injection. Immunohistochemistry also demonstrated increases in number of p-mTOR-labeled neurons in the ipsilateral L4/5 DRGs and in density of p-mTOR-labeled immunoreactivity in the ipsilateral L4/5 superficial dorsal horn 1 day after CFA injection. Moreover, intrathecal administration of rapamycin, a selective inhibitor of mTOR, significantly blocked CFA-induced mechanical allodynia and thermal hyperalgesia 1 day post-CFA injection. Interestingly, expression of neither p-mTOR nor p-S6K1 was markedly altered on days 3, 7, or 14 after L5 SNL in L5 spinal cord or DRG. These findings indicate that in DRG and spinal cord, mTOR and S6K1 are activated during chronic inflammatory pain, but not during neuropathic pain. Our results strongly suggest that mTOR and its downstream pathway contribute to the development of chronic inflammatory pain., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The forebrain cholinergic neurons project to cortex, including the hippocampus and the cingulate cortex (Cg). However, the relative influence of these neurons on behavior-linked neural processing in the two cortical areas remains unclear. We have now examined the effect of destruction of the cholinergic neurons with microinjection of the immunotoxin 192 IgG-saporin into the medial septum on the induction of c-Fos protein, an index of neuronal synaptic excitation, in the two forebrain areas to varied episodic experiences. Separate groups of rats were (a) re-exposed to the laboratory where they had previously undergone a surgery for intraseptal microinjection or (b) exposed to a novel environment. Re-exposure evoked a differential increase in the number of c-Fos positive neurons in dorsal CA1 compared to novelty, while a robust increase was observed in the Cg selectively in the novel environment. Both the differential and the selective increases were strongly attenuated by the cholinergic destruction with intraseptal-immunotoxin. These findings suggest that the cholinergic modulation of the neural processing in the two forebrain areas varies partly in an environment-dependent fashion affecting CA1 neural activation on repeat exposure to an environment where they had a relatively complex aversive experience while favoring Cg neural activation more during novelty., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Stroke is a devastating brain injury that is a leading cause of adult disability with limited treatment options. We examined the effects of prohormone thyroxine (T4) and the underlying mechanisms in the post-ischaemic rat brain after transient focal cerebral ischemia-induced brain injury. Ischaemic injury was induced for 2h by middle cerebral artery occlusion (MCAo) followed by 24-h reperfusion. T4 (1.1μg/100g BW) was administered by intraperitoneally injection twice, at 1 after the onset of ischemia and 6h after reperfusion. Cerebral infarct area and infarct volume were measured 24h after MCAo. Furthermore, the mechanism of neuroprotective effect of T4 was investigated with a focus on inflammatory cells, neurotrophins, and transcriptional factors. T4 significantly reduced cerebral infarction, which were accompanied by decreased expression of proapotptic Bax and increased antiapoaptotic Bcl-2 protein. T4 suppressed the activation of astrocytes and microglia, increased the expression of neurotrophic factors (BDNF, GDNF), and altered inflammatory-related prooxidative enzymes (iNOS and COX-2) in ischaemic brain. Moreover, T4 downregulated the phosphorylation of p38 and prevented injury-induced increase of PKCδ. These results revealed that T4 has a promising therapeutic effect in ischaemic stroke treatment protecting the brain from I/R injury, probably by its anti-apoptotic, and anti-inflammatory mechanism., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The serotonin transporter (5-HTT) regulates the extracellular concentration of serotonin, influencing neurotransmission. Evidence suggests that 5-HTT is altered during depression, but the precise changes in 5-HTT expression in the pathogenesis and treatment of depression are not clear. We investigated the protein expression of hippocampal 5-HTT in CD-1 mice exposed to unpredictable chronic mild stress for 10 continuous weeks. Following 6 weeks of the stress procedure, the mice were separated into anhedonic and non-anhedonic groups, which were then treated with fluoxetine (FLX, 10mg/kg/day, i.p.) for 4 weeks. Behavioral state and therapeutic efficacy of the drug treatment were assessed using sucrose preference, physical state of the coat and body weight. Our results show that changes in hippocampal 5-HTT protein expression correlated with stress-induced behavioral states. Decreases in 5-HTT expression were associated with the stress-induced anhedonic state, whereas increases were associated with the stress-induced non-anhedonic state. Following FLX treatment, the changes in 5-HTT expression were reversed in a subpopulation of anhedonic mice, i.e., the treatment-responsive anhedonic mice. The treatment did not alter the changes in the treatment-resistant anhedonic mice or in the non-anhedonic mice. The data indicate that down-regulation of hippocampal 5-HTT protein expression is a signature change associated with anhedonia, a key endophenotype of clinical depression. Differential changes in 5-HTT expression may contribute to variations in the susceptibility to anhedonia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In mammals Orexin-A and -B are neuropeptides involved in the hypothalamic regulation of diverse physiological functions including food intake and the sleep-wake cycle. This generalisation was investigated in meat-(broiler) and layer-type juvenile domestic chickens by immunocytochemical localisation of orexin A/B in the hypothalamus, and by measurements of hypothalamic hypocretin mRNA which encodes for orexin A/B after chronic food restriction, and during the sleep-wake cycle. Orexin immunoreactive fibres were observed throughout the hypothalamus with cell bodies in and around the paraventricular nucleus. No differences were observed in the pattern of immunoreactivity using anti- human orexin-A, or -B antisera. The amount of hypothalamic hypocretin mRNA in food -restricted broilers was higher than in broilers fed ad libitum, but the same as in layer- type hens fed ad libitum. Hypothalamic hypocretin mRNA was increased (P<0.01) in 12-week-old broilers fed 25% of their ad libitum intake between 6-12 weeks of age. No difference in hypothalamic hypocretin mRNA was seen in 12-week-old layer- type hens when they were awake (1-2h after lights on) or sleeping (1-2h after lights off). It is concluded that in the chicken, we could not find evidence that hypothalamic orexin plays a role in the sleep-wake cycle and it may be involved in aspects of energy balance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study was done to investigate whether chronic intermittent hypoxia (CIH) induced changes in leptin and leptin receptor protein levels, and known downstream mediators of leptin receptor signaling in the carotid body. Rats were subjected to CIH (120s normoxia, 80s hypoxia) or normoxia for 8h/day to either short term (7 days) or long term CIH exposure (95 days). After both 7 and 95 days of CIH, carotid body leptin protein expression was decreased, while protein levels of the long form leptin receptor (OB-Rb) were elevated. On the other hand, protein expression levels of the short form leptin receptor (OB-R100) were unchanged. Furthermore, phosphorylated signal transducer and activator of transcription 3 (pSTAT3) protein levels were found to be significantly decreased at only the 7 day period. On the other hand, suppressor of cytokine signaling 3 (SOCS3) protein levels were elevated at only the 7 day period, while phosphorylated extracellular-signal-regulated kinase 1/2 (pERK1/2) was elevated only at the 95 day period. In both the normoxia and the CIH groups, carotid body leptin was decreased at the 95 day period compared to 7 days. However, OB-Rb or Ob-R100 protein levels were not changed in the normoxic or CIH group at either time point. Furthermore, pSTAT3 protein levels were found to be significantly higher, while SOCS3 levels were significantly lower in the 95 day CIH group compared to the 7 day CIH group. Taken together, these data indicate that CIH induces changes in leptin and leptin downstream signaling proteins within the carotid bodies which may contribute to alterations in carotid chemoreceptor sensitivity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Expectation of salient rewards and novelty seeking are processes implicated in substance use disorders but the neurobiological substrates underlying these associations are not well understood. To better understand the regional circuitry of novelty and reward preference, rats were conditioned to pair unique cues with bacon, an initially novel food, or chow, a familiar food. In the same animals, after training, cue-induced brain activity was measured, and the relationships between activity and preference for three rewards, the conditioned foods and ethanol (EtOH), were separately determined. Activity in response to the food paired cues was measured using brain glucose metabolism (BGluM). Rats favoring bacon-paired (BAP) cues had increased BGluM in mesocorticolimbic brain regions after exposure to these cues, while rats favoring chow-paired (CHP) cues showed relative deactivation in these regions. Rats exhibiting BAP cue-induced activation in prefrontal cortex (PFC) also consumed more EtOH while rats with cortical activation in response to CHP cues showed lower EtOH consumption. Additionally, long-term stable expression levels of PFC Grin2a, a subunit of the NMDA receptor, correlated with individual differences in EtOH preference insomuch that rats with high EtOH preference had enduringly low PFC Grin2a mRNA expression. No other glutamatergic, dopaminergic or endocannabinoid genes studied showed this relationship. Overall, these results suggest that natural variation in mesocorticolimbic sensitivity to reward-paired cues underlies behavioral preferences for and vulnerability to alcohol abuse, and support the notion of common neuronal circuits involved in food- and drug-seeking behavior. The findings also provide evidence that PFC NMDA-mediated glutamate signaling may modulate these associations., (Published by Elsevier B.V.)

Although many studies regarding ischemic brain damage in the gerbil have been reported, studies on neuronal damage according to various durations of ischemia-reperfusion (I-R) have been limited. In this study, we examined neuronal damage/death and glial changes in the somatosensory cortex 4 days after 5, 10 and 15 min of transient cerebral ischemia using the gerbil. To examine neuronal damage, we used Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining as well as cresyl violet (CV) staining and neuronal nuclei (NeuN, neuronal marker) immunohistochemistry. In the somatosensory cortex, some CV and NeuN positive (+) neurons were slightly decreased only in layers III and VI in the 5 min ischemia-group, and the number of CV+ and NeuN+ neurons were decreased with longer ischemic time. The F-J B histofluorescence staining showed a clear neuronal damage in layers III and VI, and the number of F-J B+ neurons was increased with time of ischemia-reperfusion: in the 15 min ischemia-group, the number of F-J B+ neurons was much higher in layer III than in layer VI. In addition, we immunohistochemically examined gliosis of astrocytes and microglia using anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1) antibody, respectively. In the 5 min ischemia-group, GFAP+ astrocytes and Iba-1+ microglia were distinctively increased in number, and their immunoreactivity was stronger than that in the sham-group. In the 10 and 15 min ischemia-groups, numbers of GFAP+ and Iba-1+ glial cells were much more increased with time of ischemia-reperfusion; in the 15 min ischemia-group, their distribution patterns of GFAP+ and Iba-1+ glial cells were similar to those in the 10 min ischemia-group. Our fining indicates that neuronal death/damage and gliosis of astrocytes and microglia were apparently increased with longer time of ischemia-reperfusion., (Copyright © 2013 Elsevier B.V. All rights reserved.)

MicroRNAs are abundantly expressed in the brain and play an important role in disorders of the brain, including cognitive impairment and Alzheimer's disease (AD). A growing body of evidence suggests that the janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays a key role in the pathogenesis of AD. However, it is unclear whether miRNAs are involved in this process. Therefore, we characterized the expression and role of miR-106a and JAK/STAT signaling in an ovariectomized (OVX) mouse model of cognitive impairment. Cognitive impairment, as indicated by escape latency and time spent in the platform quadrant in the Morris water maze test, was significantly reduced at 12 weeks post-OVX, compared to age-matched controls. Quantitative real-time PCR and Western blotting demonstrated that miR-106a was upregulated, and STAT3 and phospho-STAT3 were downregulated in the hippocampus at 12 weeks post-OVX, compared with age matched controls and the 6 and 8 weeks post-OVX groups. Transfection of human neuroblastoma SH-SY5Y cells with a miR-106a mimic reduced the expression of STAT3 mRNA, compared to control cells transfected with a scrambled mimic. STAT3 and phospho-STAT3 protein expression was upregulated or downregulated by a miR-106a inhibitor or miR-106a mimic, respectively, indicating that miR-106a negatively regulates STAT3. Luciferase reporter gene assays confirmed that miR-106a directly targets the 3' untranslated region (UTR) of STAT3. This study suggests that miR-106a negatively regulates STAT3 activation, and also that miR-106a may provide a marker of onset or potential therapeutic target for cognitive disturbances., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hyaluronan is a large glycosaminoglycan, which is abundant in the extracellular matrix of the developing rodent brain. In the adult brain however, levels of hyaluronan are significantly reduced. In this study, we used neurocan-GFP as a histochemical probe to analyze the distribution of hyaluronan in the adult mouse subventricular zone (SVZ), as well as in the rostral migratory stream (RMS). Interestingly, we observed that hyaluronan is generally downregulated in the adult brain, but notably remains at high levels in the SVZ and RMS; areas in which neural stem/progenitor cells (NSPCs) persist, proliferate and migrate throughout life. In addition, we found that the receptor for hyaluronan-mediated motility (Rhamm) was expressed in migrating neuroblasts in these areas, indicating that Rhamm could be involved in regulating hyaluronan-mediated cell migration. Hyaluronan levels are balanced by synthesis through hyaluronan synthases (Has) and degradation by hyaluronidases (Hyal). We found that Has1 and Has2, as well as Hyal1 and Hyal2 were expressed in GFAP positive cells in the adult rodent SVZ and RMS, indicating that astrocytes could be regulating hyaluronan-mediated functions in these areas. We also demonstrate that hyaluronan levels are substantially increased at six weeks following a photothrombotic stroke lesion to the adult mouse cortex. Furthermore, GFAP positive cells in the peri-infarct area express Rhamm. Thus, hyaluronan may be involved in regulating cell migration in the normal SVZ and RMS and could also be responsible for priming the peri-infarct area following an ischemic lesion for cell migration., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To investigate whether fibronectin, a high-molecular weight glycoprotein of the extracellular matrix (ECM), plays a role in the activation of microglia/macrophages after brain injury, we examined the changes in fibronectin and arginase-1, a marker for alternatively activated macrophages, in a rat cryoinjury model using Western blot analysis, real-time reverse transcription PCR and immunohistochemistry. The protein and mRNA level of fibronectin and arginase-1 significantly increased in the injury site of the ipsilateral cerebral cortex at days 4 and 7 after cryoinjury but was decreased at day 14. The immunohistochemical analysis revealed fibronectin expression in ED1-positive microglia/macrophages and reactive astrocytes, in the lesion core and periphery, respectively. Fibronectin immunoreactivity in the lesion was similar to arginase-1 except that fibronectin was detected in the ECM after cryoinjury. The present results suggest that fibronectin was extravasated into injured brain lesions via an impaired blood-brain barrier and stimulated glial cells including microglia and infiltrating macrophages in the lesion core and periphery to become alternatively activated microglia/macrophages, which modulated CNS inflammation after brain injury., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Chronic restraint stress can induce depressive and anxiety-like behavior and neurophysiological disturbances. The social living shows the health-promoting and stress-protective effects on both human and animal. However, whether group housing exerts effects on development of depression and anxiety induced by chronic restraint treatments and its detailed neuroendocrine mechanism remain unclear. Following repeated restraint, the anxiety and depression-like behaviors of single and group housing mice were examined using the elevated plus-maze, open field test and forced swimming test. The levels of central oxytocin (OT) expression in the paraventricular nucleus (PVN), glucocorticoid receptors (GR) in the hippocampus and serum OT and corticosterone (CORT) were also measured using immunohistochemistry and ELISA methods. Our results show that chronic restraint significantly decreased time in open arm of elevated plus maze and increased immobility time in forced swimming test in single-housed mice. However, chronic restraint exerted no effects on these aspects in group-housed mice. Accompanying the changes of behaviors, chronic restraint up-regulated levels of serum CORT and reduced the hippocampus GR in single-housed animals, but did not change these measures in group-housed mice. Furthermore, repeated restraint had no effect on OT levels in these two housing conditions although group-housing significantly increased the PVN OT levels. Taken together, these results provide substantial evidence that group housing can reduce levels of anxiety and depression induced by chronic restraint stress in mice. The elevation of central GR and OT, and decrease of circulating CORT may possibly be involved in these buffering effects., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Experiencing early life stress increases the risk of developing a psychiatric disorder later in life, possibly by altering neural networks, such as the locus-coeruleus norepinephrine (LC-NE) system. Whether early life stress affects the LC-NE system directly, or whether the effects are via changes in glutamate and GABA modulation of the LC-NE system, is unclear. Early life stress has been shown to alter glutamate and GABA transmission, and in particular, to alter GABA(A) receptor expression. The LC-NE system has been implicated in attention-deficit/hyperactivity disorder (ADHD), amongst other disorders, and is over-responsive to glutamate stimulation in a validated rat model of ADHD, the spontaneously hypertensive rat (SHR). It is plausible that the LC-NE system, or glutamate and GABA modulation thereof, in an individual already genetically predisposed to develop ADHD, or in SHR, may respond in a unique way to early life stress. To investigate this we applied a mild developmental stressor, maternal separation, onto SHR, and onto their control strain, Wistar-Kyoto rats (WKY), from post-natal day (P)2-14. On P50-52, in early adulthood, we assayed glutamate and potassium stimulated release of radio-actively labelled NE ((3)[H]NE) from hippocampal slices using an in vitro superfusion technique, in the presence or absence of a GABA(A) receptor antagonist, bicuculline. Our results show that maternal separation altered GABA(A) receptor-mediated modulation of NE release in the hippocampus of the two strains in opposite directions, increasing it in SHR and decreasing it in WKY. Our findings indicate that effects of early life stress are highly dependent on genetic predisposition, since opposite changes in GABA(A) receptor-mediated modulation of NE release were observed in the rat model of ADHD, SHR, and their control strain, WKY., (Copyright © 2012 Elsevier B.V. All rights reserved.)