The Role of Autophagy and Apoptosis in Neuropathic Pain Formation.

Neuropathic pain indicates pain caused by damage to the somatosensory system and is difficult to manage and treat. A new treatment strategy urgently needs to be developed. Both autophagy and apoptosis are critical adaptive mechanisms when neurons encounter stress or damage. Recent studies have shown that, after nerve damage, both autophagic and apoptotic activities in the injured nerve, dorsal root ganglia, and spinal dorsal horn change over time. Many studies have shown that upregulated autophagic activities may help myelin clearance, promote nerve regeneration, and attenuate pain behavior. On the other hand, there is no direct evidence that the inhibition of apoptotic activities in the injured neurons can attenuate pain behavior. Most studies have only shown that agents can simultaneously attenuate pain behavior and inhibit apoptotic activities in the injured dorsal root ganglia. Autophagy and apoptosis can crosstalk with each other through various proteins and proinflammatory cytokine expressions. Proinflammatory cytokines can promote both autophagic/apoptotic activities and neuropathic pain formation, whereas autophagy can inhibit proinflammatory cytokine activities and further attenuate pain behaviors. Thus, agents that can enhance autophagic activities but suppress apoptotic activities on the injured nerve and dorsal root ganglia can treat neuropathic pain. Here, we summarized the evolving changes in apoptotic and autophagic activities in the injured nerve, dorsal root ganglia, spinal cord, and brain after nerve damage. This review may help in further understanding the treatment strategy for neuropathic pain during nerve injury by modulating apoptotic/autophagic activities and proinflammatory cytokines in the nervous system.

Transient receptor potential vanilloid type 4 channels mediate Na-K-Cl-co-transporter-induced brain edema after traumatic brain injury.

Na+ -K+ -2Cl- co-transporter (NKCC1) plays an important role in traumatic brain injury (TBI)-induced brain edema via the MAPK cascade. The transient receptor potential vanilloid type 4 (TRPV4) channel participates in neurogenic inflammation, pain transmission, and edema. In this study, we investigated the relationship between NKCC1 and TRPV4 and the related signaling pathways in TBI-induced brain edema and neuronal damage. TBI was induced by the calibrated weight-drop device. Adult male Wistar rats were randomly assigned into sham and experimental groups for time-course studies of TRPV4 expression after TBI. Hippocampal TRPV4, NKCC1, MAPK, and PI-3K cascades were analyzed by western blot, and brain edema was also evaluated among the different groups. Expression of hippocampal TRPV4 peaked at 8 h after TBI, and phosphorylation of the MAPK cascade and Akt was significantly elevated. Administration of either the TRPV4 antagonist, RN1734, or NKCC1 antagonist, bumetanide, significantly attenuated TBI-induced brain edema through decreasing the phosphorylation of MEK, ERK, and Akt proteins. Bumetanide injection inhibited TRPV4 expression, which suggests NKCC1 activation is critical to TRPV4 activation. Our results showed that hippocampal NKCC1 activation increased TRPV4 expression after TBI and then induced severe brain edema and neuronal damage through activation of the MAPK cascade and Akt-related signaling pathway.

NKCC1 mediates traumatic brain injury-induced hippocampal neurogenesis through CREB phosphorylation and HIF-1α expression.

Traumatic brain injury (TBI) is one of the most prevalent causes of worldwide mortality and morbidity. We previously had evidenced that TBI induced Na-K-2Cl co-transporter (NKCC1) upregulation in hippocampus. Here, we aim to investigate the role of NKCC1 in TBI-induced neurogenesis and the detailed mechanisms. The TBI-associated alternations in the expression of NKCC1, HIF-1α, VEGF, MAPK cascade, and CREB phosphorylation were analyzed by Western blot. TBI-induced neurogenesis was determined by immuno-fluorescence labeling. Chromatin immunoprecipitation was used to elucidate whether HIF-1α would activate VEGF gene after TBI. We found that the level of hippocampal NKCC1 and VEGF began to rise 8 h after TBI, and both of them reached maxima at day 7. Along with the upregulation of NKCC1 and VEGF, MAPK cascade was activated and hippocampal neurogenesis was promoted. Administration of CREB antisense oligonucleotide significantly attenuated the expression of HIF-1α, while HIF-1α antisense oligonucleotide exhibited little effect on the expression of CREB. However, HIF-1α antisense oligonucleotide administration did effectively suppress the expression of VEGF. Our results of the chromosome immunoprecipitation also indicated that HIF-1α could directly act on the VEGF promoter and presumably would elevate the VEGF expression after TBI. All these results have illustrated the correlation between NKCC1 upregulation and TBI-associated neurogenesis. The pathway involves the activation of Raf/MEK/ERK cascade, CREB phosphorylation, and HIF-1α upregulation, and finally leads to the stimulation of VEGF expression and the induction of neurogenesis.

Neonatal glucocorticoid treatment increased depression-like behaviour in adult rats.

Synthetic glucocorticoid dexamethasone (DEX) is frequently used as a therapeutic agent to lessen the morbidity of chronic lung disease in premature infants. Previous studies suggested that neonatal DEX treatment altered brain development and cognitive function. It has been recognized that the amygdala is involved in emotional processes and also a critical site of neuronal plasticity for fear conditioning. Little is known about the possible long-term adverse effect of neonatal DEX treatment on amygdala function. The present study was aimed to evaluate the possible effect of neonatal DEX treatment on the synaptic function of amygdala in adult rats. Newborn Wistar rats were subjected to subcutaneous tapering-dose injections of DEX (0.5, 0.3 and 0.1 mg/kg) from post-natal day one to three, PN1-PN3. Animals were then subjected to a forced swimming test (FST) and electrophysiological recording aged eight weeks. The results of the FST showed neonatal DEX treatment increased depression-like behaviour in adulthood. After acute stress evoking, the percentage of time spent free floating is significantly increased in the DEX treated group compared with the control animals. Furthermore, neonatal DEX treatment elevated long-term potentiation (LTP) response and the phosphorylation level of MAPK in the lateral nucleus of amygdala (LA). Intracerebroventricular infusion of the MAPK inhibitor, PD98059, showed significant rescue effects including reduced depression-like behaviour and restoration of LTP to within normal range. In conclusion, our results suggested that MAPK signalling cascade in the LA plays an important role in the adverse effect of neonatal DEX treatment on amygdala function, which may result in adverse consequences in adult age, such as the enhancement of susceptibility for a depressive disorder in later life.

The novel indole compound SK228 induces apoptosis and FAK/Paxillin disruption in tumor cell lines and inhibits growth of tumor graft in the nude mouse.

Drugs in clinical use with indole structure exhibit side effects. Therefore, to search for indole compounds with more efficacy and less side effect for cancer therapy, we developed a novel indole compound SK228 and examined its effects and mechanisms on antitumor growth and invasion inhibition in cell and tumor xenografts in nude mice models. SK228 significantly inhibited growth of different lung and esophageal cancer cell lines at sub-micromolar range, but not normal lung cells. SK228 induced DNA damages mainly by producing reactive oxygen species (ROS) resulting in apoptosis. SK228 treatment increased the release of cytochrome c into the cytosol along with the increased activity of caspase-3 and -9 without affecting caspase-8, whereas these effects were attenuated by ROS inhibitor. The expression levels of BCL-2 family regulators were also affected. Moreover, low-dose SK228 significantly reduced the invasion of cancer cells. The active phosphorylated form of FAK/Paxillin signaling pathway proteins and active form of RhoA were decreased. Moreover, the F-actin cytoskeleton was disrupted after low-dose SK228 treatment. Growth of an A549 tumor cell xenograft was markedly inhibited without significant side effects. SK228-induced apoptosis was confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry of cleaved caspase-3 in tumors from treated mice. Our study provides the first evidence that SK228 exhibits cancer cell-specific cytotoxicity by inducing mitochondria-mediated apoptosis. In addition, SK228 inhibits cancer cell invasion via FAK/Paxillin disruption at noncytotoxic doses. SK228 can be further tested as a pharmaceutical compound for cancer treatment.

Indomethacin protects rats from neuronal damage induced by traumatic brain injury and suppresses hippocampal IL-1ß release through the inhibition of Nogo-A expression.

BACKGROUND: Nogo-A is a member of the reticulon family of membrane-associated proteins and plays an important role in axonal remodeling. The present study aimed to investigate alterations in Nogo-A expression following traumatic brain injury (TBI)-induced inflammation and neuronal damage. METHODS: A weight-drop device was used to deliver a standard traumatic impact to rats. Western blot, RT-PCR and ELISA were used to analyze the expression of Nogo-A and IL-1ß. Nogo-A antisense, and an irrelevant control oligonucleotide was intracerebroventricularly infused. We also performed H & E staining and luxol fast blue staining to evaluate the neuronal damage and demyelination resulting from TBI and various treatments. RESULTS: Based on RT-PCR and western blot analyses, the expression of Nogo-A was found to be significantly upregulated in the hippocampus beginning eight hours after TBI. In addition, TBI caused an apparent elevation in IL-1ß levels and severe neuronal damage and demyelination in the tested animals. All of the TBI-associated molecular and cellular consequences could be effectively reversed by treating the animals with the anti-inflammatory drug indomethacin. More importantly, the TBI-associated stimulation in the levels of both Nogo-A and IL-1ß could be effectively inhibited by a specific Nogo-A antisense oligonucleotide. CONCLUSIONS: Our findings suggest that the suppression of Nogo-A expression appears to be an early response conferred by indomethacin, which then leads to decreases in the levels of IL-1ß and TBI-induced neuron damage.

Effect of MK-801-induced impairment of inhibitory avoidance learning in zebrafish via inactivation of extracellular signal-regulated kinase (ERK) in telencephalon.

N-Methyl-D-aspartate (NMDA) receptors are implicated in a wide range of complex behavioral functions, including cognitive activity. Numerous studies have shown that using the repetitive administration of a noncompetitive NMDA receptor antagonist, MK-801, induces amnesia in rodents. In this study, the effect of a subchronic MK-801 treatment on the cognitive function of zebrafish was evaluated using a novel inhibitory avoidance task. First, we established a new system to investigate the inhibitory avoidance learning of zebrafish where they were trained to refrain from swimming from a shallow compartment to a deep compartment in order to avoid electric shock. Second, we found that blocking NMDA receptors by MK-801 could significantly attenuate the inhibitory avoidance behavior of the zebrafish and alter the telencephalic extracellular signal-regulated kinase (ERK) phosphorylation level 90 min after the inhibitory avoidance training. These results suggest that the formation of long-term emotional memory is possibly mediated by ERK activation in the telencephalon of zebrafish.

A Novel Laser-Based Zebrafish Model for Studying Traumatic Brain Injury and Its Molecular Targets.

Traumatic brain injury (TBI) is a major public health problem. Here, we developed a novel model of non-invasive TBI induced by laser irradiation in the telencephalon of adult zebrafish (Danio rerio) and assessed their behavior and neuromorphology to validate the model and evaluate potential targets for neuroreparative treatment. Overall, TBI induced hypolocomotion and anxiety-like behavior in the novel tank test, strikingly recapitulating responses in mammalian TBI models, hence supporting the face validity of our model. NeuN-positive cell staining was markedly reduced one day, but not seven days, after TBI, suggesting increased neuronal damage immediately after the injury, and its fast recovery. The brain-derived neurotrophic factor (Bdnf) level in the brain dropped immediately after the trauma, but fully recovered seven days later. A marker of microglial activation, Iba1, was elevated in the TBI brain, albeit decreasing from Day 3. The levels of hypoxia-inducible factor 1-alpha (Hif1a) increased 30 min after the injury, and recovered by Day 7, further supporting the construct validity of the model. Collectively, these findings suggest that our model of laser-induced brain injury in zebrafish reproduces mild TBI and can be a useful tool for TBI research and preclinical neuroprotective drug screening.

Interactions between Autophagy, Proinflammatory Cytokines, and Apoptosis in Neuropathic Pain: Granulocyte Colony Stimulating Factor as a Multipotent Therapy in Rats with Chronic Constriction Injury.

Our previous studies have shown that early systemic granulocyte colony-stimulating factor (G-CSF) treatment can attenuate neuropathic pain in rats with chronic constriction injury (CCI) by modulating expression of different proinflammatory cytokines, microRNAs, and proteins. Besides the modulation of inflammatory mediators' expression, previous studies have also reported that G-CSF can modulate autophagic and apoptotic activity. Furthermore, both autophagy and apoptosis play important roles in chronic pain modulation. In this study, we evaluated the temporal interactions of autophagy, and apoptosis in the dorsal root ganglion (DRG) and injured sciatic nerve after G-CSF treatment in CCI rats. We studied the behaviors of CCI rats with or without G-CSF treatment and the various levels of autophagic, proinflammatory, and apoptotic proteins in injured sciatic nerves and DRG neurons at different time points using Western blot analysis and immunohistochemical methods. The results showed that G-CSF treatment upregulated autophagic protein expression in the early phase and suppressed apoptotic protein expression in the late phase after nerve injury. Thus, medication such as G-CSF can modulate autophagy, apoptosis, and different proinflammatory proteins in the injured sciatic nerve and DRG neurons, which have the potential to treat neuropathic pain. However, autophagy-mediated regulation of neuropathic pain is a time-dependent process. An increase in autophagic activity in the early phase before proinflammatory cytokines reach the threshold level to induce neuropathic pain can effectively alleviate further neuropathic pain development.

Chronic Administration of 7,8-DHF Lessens the Depression-like Behavior of Juvenile Mild Traumatic Brain Injury Treated Rats at Their Adult Age.

Traumatic brain injury (TBI) is a leading cause of mortality and morbidity among the global youth and commonly results in long-lasting sequelae, including paralysis, epilepsy, and a host of mental disorders such as major depressive disorder. Previous studies were mainly focused on severe TBI as it occurs in adults. This study explored the long-term adverse effect of mild TBI in juvenile animals (mTBI-J). Male Sprague Dawley rats received mTBI-J or sham treatment at six weeks old, then underwent behavioral, biochemical, and histological experiments three weeks later (at nine weeks old). TTC staining, H&E staining, and brain edema measurement were applied to evaluate the mTBI-J induced cerebral damage. The forced swimming test (FST) and sucrose preference test (SPT) were applied for measuring depression-like behavior. The locomotor activity test (LAT) was performed to examine mTBI-J treatment effects on motor function. After the behavioral experiments, the dorsal hippocampus (dHip) and ventral hippocampus (vHip) were dissected out for western blotting to examine the expression of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB). Finally, a TrkB agonist 7,8-DHF was injected intraperitoneally to evaluate its therapeutic effect on the mTBI-J induced behavioral abnormalities at the early adult age. Results showed that a mild brain edema occurred, but no significant neural damage was found in the mTBI-J treated animals. In addition, a significant increase of depression-like behaviors was observed in the mTBI-J treated animals; the FST revealed an increase in immobility, and a decrease in sucrose consumption was found in the mTBI-J treated animals. There were no differences observed in the total distance traveled of the LAT and the fall latency of the rotarod test. The hippocampal BDNF expression, but not the TrkB, were significantly reduced in mTBI-J, and the mTBI-J treatment-induced depression-like behavior was lessened after four weeks of 7,8-DHF administration. Collectively, these results indicate that even a mild juvenile TBI treatment that did not produce motor deficits or significant histological damage could have a long-term adverse effect that could be sustained to adulthood, which raises the depression-like behavior in the adult age. In addition, chronic administration of 7,8-DHF lessens the mTBI-J treatment-induced depression-like behaviors in adult rats. We suggest the potential usage of 7,8-DHF as a therapeutic agent for preventing the long-term adverse effect of mTBI-J.

Granulocyte Colony Stimulating Factor (GCSF) Can Attenuate Neuropathic Pain by Suppressing Monocyte Chemoattractant Protein-1 (MCP-1) Expression, through Upregulating the Early MicroRNA-122 Expression in the Dorsal Root Ganglia.

Our previous animal studies and several human clinical trials have shown that granulocyte-colony stimulating factor (GCSF) can attenuate neuropathic pain through various mechanisms. GCSF itself is also a multipotent cytokine that can modulate microribonucleic acid (microRNA) expression profiles in vitro. In this study, we used the NanoString nCounter analysis system to screen the expression of different rodent microRNAs at early stage after nerve injury and studied the expression of related cytokines/chemokines in the dorsal root ganglia (DRGs) of rats that underwent chronic constriction injury (CCI) to explore the underlying mechanisms of the analgesic effects of GCSF. We found that microRNA-122 expression was downregulated by CCI; in contrast, GCSF treatment significantly upregulated microRNA-122 expression in the DRGs of CCI rats on the 1st day after nerve injury. We further studied the expression of different cytokines/chemokines (IL-1ß, IL-6, and monocyte chemoattractant protein-1 (MCP-1)) that were modulated by microRNA-122. MCP-1 has been reported to participate in neuropathic pain development, and its expression on the DRGs of vehicle-treated CCI rats was significantly higher than that on the DRGs of sham-operated rats; in contrast, GCSF-treated rats exhibited significantly lower MCP-1 expression in the DRG than vehicle-treated rats on the 7th day after nerve injury. An early GCSF treatment can suppress MCP-1 expressions, through upregulating microRNA-122 expressions in the DRGs of CCI rats at an earlier stage, thus indirectly attenuating neuropathic pain development.

Neonatal Dexamethasone Treatment Suppresses Hippocampal Estrogen Receptor α Expression in Adolescent Female Rats.

Previous studies showed that neonatal dexamethasone treatment (NDT) transiently impaired hippocampal function in male rats. Hippocampal estrogen receptors (ERs) participate in avoidance learning. As previous studies focused on males only, this study was aimed to investigate the NDT effects on the hippocampal function of female rats. Newborn Wistar female rats were subjected to a tapering dose of dexamethasone (0.5 mg, 0.3 mg, and 0.1 mg/kg, subcutaneously) from postnatal days 1 to 3 and were subjected to experiments at the age of 6 weeks (adolescence). Brain slice extracellular recording and the inhibitory avoidance (IA) test were used to evaluate the NDT effects on hippocampal function. The results showed that NDT completely blocked the hippocampal long-term potentiation (LTP) formation and IA learning of adolescents. The expression of hippocampal estrogen receptor alpha (ERα) was attenuated in NDT subjects. Reduced histone acetylation of the ERα gene was found, possibly explaining the reduced hippocampal ERα expression in NDT female rats. Suprafusion of estradiol (E2) partially restored the hippocampal LTP formation in adolescent NDT female rats. Coadministration of the histone deacetylase inhibitor trichostatin-A restored the hippocampal ERα expression, hippocampal LTP formation, and IA learning in adolescent NDT female rats. Collectively, these results suggested that NDT has an epigenetic modulation effect on the expression of hippocampal ERα, which is responsible for its adverse effect on hippocampal function.

NKCC1-mediated traumatic brain injury-induced brain edema and neuron death via Raf/MEK/MAPK cascade.

OBJECTIVE: Brain edema is one of the characteristic features of patients with severe traumatic brain injury. The aim of this study was to examine the effects of Na+-K+-2Cl- co-transporter on traumatic brain injury-induced brain edema and neuron damage and to elucidate the relationship between Na+-K+-2Cl- co-transporter and mitogen-activated protein kinase (MAPK) cascade. DESIGN: Laboratory investigation. SETTING: University research laboratory. SUBJECTS: Male Wistar rats weighing 350-400 g. INTERVENTIONS: Anesthetized animals were subjected to a weight-drop device (450-g weight, 1.8-m height) to induce traumatic brain injury. MEASUREMENTS AND MAIN RESULTS: The expression of Na+-K+-2Cl- co-transporter and phosphorylation of MAPK cascade were determined by Western blot test. We also analyzed the degree of brain edema and neuronal damage in this study. We found that the messenger RNA and protein of Na+-K+-2Cl- co-transporter were up-regulated mainly in hippocampus neurons from 2 to 24 hrs after traumatic brain injury. After traumatic brain injury, animals displayed severe brain edema and neuron damage. The phosphorylation of extracellular signal-regulated kinase, MAPK kinase, and Raf also was significantly elevated after traumatic brain injury. Bumetanide (15.2 mg/kg), a specific Na+-K+-2Cl- co-transporter inhibitor, significantly attenuated the neuronal damage and brain edema after traumatic brain injury by decreasing the phosphorylation of Raf/MEK/ERK cascade proteins. CONCLUSIONS: The present study suggests that Na+-K+-2Cl- co-transporter plays an important role in TBI-induced brain edema and neuronal damage via activation of MAPK cascade.

Bumetanide blocks the acquisition of conditioned fear in adult rats.

BACKGROUND AND PURPOSE: Bumetanide has anxiolytic effects in rat models of conditioned fear. As a loop diuretic, bumetanide blocks cation-chloride co-transport and this property may allow bumetanide to act as an anxiolytic by modulating GABAergic synaptic transmission in the CNS. Its potential for the treatment of anxiety disorders deserves further investigation. In this study, we evaluated the possible involvement of the basolateral nucleus of the amygdala in the anxiolytic effect of bumetanide. EXPERIMENTAL APPROACH: Brain slices were prepared from Wistar rats. extracellular recording, stereotaxic surgery, fear-potentiated startle response, locomotor activity monitoring and Western blotting were applied in this study. KEY RESULTS: Systemic administration of bumetanide (15.2 mg·kg-1 , i.v.), 30 min prior to fear conditioning, significantly inhibited the acquisition of the fear-potentiated startle response. Phosphorylation of ERK in the basolateral nucleus of amygdala was reduced after bumetanide administration. In addition, suprafusion of bumetanide (5 or 10 µM) attenuated long-term potentiation in the amygdala in a dose-dependent manner. Intra-amygdala infusion of bumetanide, 15 min prior to fear conditioning, also blocked the acquisition of the fear-potentiated startle response. Finally, the possible off-target effect of bumetanide on conditioned fear was excluded by side-by-side control experiments. CONCLUSIONS AND IMPLICATIONS: These results suggest the basolateral nucleus of amygdala plays a critical role in the anxiolytic effects of bumetanide.

Extract of Ginkgo biloba EGb761 facilitates extinction of conditioned fear measured by fear-potentiated startle.

A standard extract of Ginkgo biloba (EGb761) has been used in the treatment of various common geriatric complaints including vertigo, short-term memory loss, hearing loss, lack of attention, or vigilance. We demonstrated that acute systemic administration of EGb761 facilitated the acquisition of conditioned fear. Many studies suggest the neural mechanism underlies extinction is similar to the acquisition. This raises a possibility that EGb761 may modulate and accelerate the fear extinction process. We tested this possibility by using fear-potentiated startle (FPS) on laboratory rats. Acute systemic injection of EGb761 (10, 20, or 50 mg/kg) 30 min before extinction training facilitated extinction in a dose-dependent manner. Intra-amygdaloid infusion of EGb761 (28 ng/side, bilaterally) 10 min before extinction training also facilitated extinction. Control experiments showed that facilitation effect of EGb761 was not the result of impaired expression of conditioned fear or accelerated forgetting. Rats previously injected with EGb761 showed significant FPS after retraining. Extinction of conditioned fear appeared to result from acute drug effects rather than from toxic action. Systemic administration of EGb761 immediately after extinction training did not facilitate extinction, suggested the EGb761 facilitation effect is contributed to the acquisition phase of extinction learning. Western blot results showed that extinction induced amygdaloid extracellular signal-regulated kinase (ERK1/2) phosphorylation was significantly elevated by EGb761 treatment. Intra-amygdala injection of ERK1/2 inhibitor PD98059 completely blocked the EGb761 effect. Therefore, acute EGb761 administration modulated extinction of conditioned fear by activating ERK1/2.

Glutamate NMDA receptors within the amygdala participate in the modulatory effect of glucocorticoids on extinction of conditioned fear in rats.

Recent results show that brain glucocorticoids are involved in the dysregulation of fear memory extinction in post-traumatic stress disorder patients. The present study was aimed to elucidate the possible mechanism of glucocorticoids on the conditioned fear extinction. To achieve these goals, male SD rats, fear-potentiated startle paradigm, and Western blot were used. We found that (1) systemic administration of the synthetic glucocorticoid agonist dexamethasone (DEX) facilitated extinction of conditioned fear in a dose-dependent manner (0.05, 0.1, 0.5, or 1.0 mg/kg, i.p.); (2) systemic administration of the glutamate NMDA receptor antagonist (+/-)-HA966 (6.0 mg/kg, i.p.) and intra-amygdala infusion of the NMDA receptor antagonists MK801 (0.5 ng/side, bilaterally) or D,L-2-amino-5-phosphonovaleric acid (AP5, 2.0 ng/side, bilaterally) blocked the DEX facilitation effect; (3) the corticosteroid synthesis inhibitor metyrapone (25 mg/kg. s.c.) blocked extinction and this was prevented by co-administration of NMDA receptor agonist D-cycloserine (DCS, 5.0 mg/kg, i.p.); (4) co-administration of DEX and DCS in subthreshold doses provided a synergistic facilitation effect on extinction (0.2 and 5 mg/kg, respectively). Control experiments indicated that co-administration of DEX and DCS did not alter the expression of conditioned fear and the effect was not due to lasting damage to the amygdala. These results suggest that glutamate NMDA receptors within the amygdala participate in the modulatory effect of glucocorticoids on extinction.

Bumetanide administration attenuated traumatic brain injury through IL-1 overexpression.

OBJECTIVE: To examine the effects of administration of bumetanide, a specific NKCC1 inhibitor, on traumatic brain injury (TBI)-induced interleukin-1 (IL-1) expression. METHODS: TBI model was induced by the calibrated weight drop device (450 g in weight, 2.0 m in height) in adult rats based on procedures previously reported. One hundred and sixty Wistar rats were divided into sham-control group and experimental group for time course works of TBI. The expression of IL-1beta brain edema and neuronal damage were determined in these animals after TBI. RESULTS: We found that both mRNA and protein of IL-1beta were up-regulated in the hippocampus 3-24 hours after TBI. Animals displayed severe brain edema and neuron damage after TBI. Bumetanide (15 mg/kg), a specific Na(+) -K(+) -2Cl(-) cotransporter inhibitor, significantly attenuated the TBI-induced neuronal damage by IL-1beta overexpression. The present study suggests that administration of bumetanide could significantly decreased TBI-induced inflammatory response and neuronal damage.

Unilateral stimulation of the lateral division of the dorsal telencephalon induces synaptic plasticity in the bilateral medial division of zebrafish.

This study was aimed to evaluate the synaptic plasticity in projections from the dorsal lateral region (Dl) to the bilateral dorsal medial region (Dm) of the zebrafish telencephalon. The results showed that unilateral electrical stimulation of the Dl evokes a negative field potential (FP) in both the contralateral and ipsilateral side of the Dm. We tested synaptic plasticity, including high-frequency stimulation-induced LTP (HFS-LTP) and low-frequency stimulation-induced LTD (LFS-LTD). We demonstrated that HFS-induced bilateral LTP is NMDAR-dependent by the application of an NMDAR antagonist, DL-AP5 (30 µM, suprafused for 10 min), which blocked the HFS-induced LTP in both the contralateral and ipsilateral Dm. In addition, LTP was restored after DL-AP5 was washed out by continuous aCSF suprafusion. These results suggested that the potentiation is NMDAR-dependent. Either LFS (1 Hz for 20 min) or applying the mGluR agonist, DHPG (40 µM, suprafused for 10 min) successfully induced bilateral LTD for at least 1 h. Furthermore, both the contralateral fEPSP and LTP vanished after ablation of the anterior commissure. In conclusion, the results of the present study suggested that the projection between the Dl and contralateral Dm in the telencephalon of zebrafish is via the anterior commissure and possesses synaptic plasticity.

Fragile X Mental Retardation-1 Knockout Zebrafish Shows Precocious Development in Social Behavior.

Fragile X syndrome (FXS) is a generally hereditary form of human mental retardation that is caused by triplet repeat expansion (CGG) mutation in fragile X mental retardation 1 (fmr1) gene promoter and that results in the absence of the fragile X mental retardation protein (FMRP) expression. The common symptoms of FXS patients include learning disabilities, anxiety, autistic behaviors, as well as other behavioral abnormalities. Our previous results demonstrated the behavioral abnormalities in fmr1 knockout (KO) zebrafish such as fear memory impairment and autism-like behavior. Here, we studied the functional role of fmr1 gene on the development of social behavior by behavioral experiments, including shoaling behavior, shoaling preference, light/dark test, and novel tank task. Our results demonstrated that precocious development of shoaling behavior is found in fmr1 KO zebrafish without affecting the shoaling preference on conspecific zebrafish. The shoaling behavior appeared after 14 days postfertilization (dpf), and the level of shoaling elevated in fmr1 KO zebrafish. Furthermore, the fmr1 KO zebrafish at 28 dpf expressed higher anxiety level in novel tank task. These results suggest that the change of shoaling behavior in fmr1 KO zebrafish may result from hyperactivity and an increase of anxiety.

Systemic and intra-amygdala administration of glucocorticoid agonist and antagonist modulate extinction of conditioned fear.

We examined the effect of glucocorticoid agonists on the extinction of conditioned fear in rats by using fear-potentiated startle. Systemic injection of glucocorticoid receptor agonists dexamethasone (DEX) (0.1, 0.5, and 1.0 mg/kg) and intra-amygdala infusion of RU28362 (0.5, 1.0, and 3.0 ng/side) prior to extinction training facilitated extinction of conditioned fear in a dose-dependent manner. Extinction of conditioned fear and circulating corticosterone levels were attenuated by administration of corticosteroid synthesis inhibitor metyrapone (25 mg/kg s.c.) 90 min before extinction training. The facilitation effect of DEX was dependent on repeated presentation of the conditioned stimulus rather than exposure to the experimental context, indicating this effect did not result from impaired expression of conditioned fear or accelerated forgetting. Intra-amygdaloid administration of the glucocorticoid receptor antagonist mifepristone (0.1, 0.2, and 0.5 ng/side, bilaterally) blocked extinction of conditioned fear and the facilitation effect of DEX in a dose-dependent manner. Mifepristone (2 ng/side) did not affect extinction but blocked the facilitating effect of DEX. Systemic administration of DEX after extinction training also facilitated extinction, suggesting that DEX may influence the memory consodilation phase of extinction. The Dose of dexamethsone or metyrapone used here did not influence fear-potentiated startle when administered before testing. Thus, it is unlikely that these drugs influenced extinction by increasing or disrupting CS processing. All results suggested that amygdaloid glucocorticoid receptors were involved in the extinction of conditioned fear.