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.

The Potential Role of Krüppel-Like Zinc-Finger Protein Glis3 in Genetic Diseases and Cancers.

Gli-similar 3 (Glis3) belongs to a Glis subfamily of Krüppel-like zinc-finger transcription factors characterized to regulate a set of downstream targets essential for cellular functions, including pancreatic development, ß-cell maturation and maintenance, and insulin production. Examination of the DNA-binding domain of Glis3 reveals that this domain contains a repeated cysteine 2/histidine 2 (Cys2/His2) zinc-finger motif in the central region where the recognized DNA sequence binds. The loss of the production of pancreatic hormones, such as insulin 1 and 2, is linked to the down-regulation of ß cells-related genes and promotes the apoptotic death of ß cells found in mutant Glis3. Although accumulating studies converge on the Glis3 functioning in ß cells, recently, there have been developments in the field of Glis3 using knockdown/mutant mice to better understand the role of Glis3 in diseases. The Glis3 mutant mice have been characterized for their propensity to develop congenital hypothyroidism, polycystic kidney disease, and some types of cancer. In this review, we attempt to comprehensively summarize the knowledge of Glis3, including its structure and general function in cells. We also collected and organized the academic achievements related to the possible mechanisms of Glis3-related diseases.

Stage-Dependent Axon Transport of Proteasomes Contributes to Axon Development.

Axon extension at the growing tip requires elevated local protein supply, with a capability sustainable over long axons in varying environments. The exact mechanisms, however, remain elusive. Here we report that axon-promoting factors elicited a retrograde transport-dependent removal of proteasomes from nascent axon terminals, thereby increasing protein stability at axon tips. Such an effect occurred through phosphorylation of a dynein-interacting proteasome adaptor protein Ecm29. During the transition from immature neurites to nascent axons in cultured hippocampal neurons, live-cell imaging revealed a significant increase of the retrograde axonal transport of fluorescently labeled 20S proteasomes. This retrograde proteasome transport depended on neuron stage and increased with axon lengths. Blockade of retrograde transport caused accumulation of proteasomes, reduction of axon growth, and attenuation of growth-associated Par6 at the axon tip of newly polarized neurons. Our results delineate a regulatory mechanism that controls proteasome abundance via preferential transport required for axon development in newborn neurons.

BubR1 Acts as a Promoter in Cellular Motility of Human Oral Squamous Cancer Cells through Regulating MMP-2 and MMP-9.

BubR1 is a critical component of spindle assembly checkpoint, ensuring proper chromatin segregation during mitosis. Recent studies showed that BubR1 was overexpressed in many cancer cells, including oral squamous cell carcinomas (OSCC). However, the effect of BubR1 on metastasis of OSCC remains unclear. This study aimed to unravel the role of BubR1 in the progression of OSCC and confirm the expression of BubR1 in a panel of malignant OSCC cell lines with different invasive abilities. The results of quantitative real-time PCR showed that the mRNA level of BubR1 was markedly increased in four OSCC cell lines, Ca9-22, HSC3, SCC9 and Cal-27 cells, compared to two normal cells, normal human oral keratinocytes (HOK) and human gingival fibroblasts (HGF). Moreover, the expression of BubR1 in these four OSCC cell lines was positively correlated with their motility. Immunofluorescence revealed that BubR1 was mostly localized in the cytosol of human gingival carcinoma Ca9-22 cells. BubR1 knockdown significantly decreased cellular invasion but slightly affect cellular proliferation on both Ca9-22 and Cal-27 cells. Consistently, the activities of metastasis-associated metalloproteinases MMP-2 and MMP-9 were attenuated in BubR1 knockdown Ca9-22 cells, suggesting the role of BubR1 in promotion of OSCC migration. Our present study defines an alternative pathway in promoting metastasis of OSCC cells, and the expression of BubR1 could be a prognostic index in OSCC patients.

Activation of mGluR2/3 underlies the effects of N-acetylcystein on amygdala-associated autism-like phenotypes in a valproate-induced rat model of autism.

Autism-like phenotypes in male valproate (VPA)-exposed offspring have been linked to high glutamatergic neurotransmission in the thalamic-amygdala pathway. Glial cystine/glutamate exchange (system Xc(-)), which exchanges extracellular cystine for intracellular glutamate, plays a significant role in the maintenance of extracellular glutamate. N-acetylcysteine (NAC) is a cystine prodrug that restores extracellular glutamate by stimulating system Xc(-). In this study, we examined the effects of NAC on autism-like phenotypes and neurotransmission in the thalamic-amygdala synapses, as well as the involvement of metabotropic glutamate receptors 2/3 (mGluR2/3). Valproate-treated rats received a single intraperitoneal injection of 500 mg/kg NaVPA on E12.5. On postnatal day 21 (P21), NAC or saline was administered once daily for 10 days. From day 8 to 10, NAC was given 1/2 h prior to behavioral testing. Chronic administration of NAC restored the duration and frequency of social interaction and ameliorated anxiety-like behaviors in VPA-exposed offspring. In amygdala slices, NAC treatment normalized the increased frequency of mEPSCs and decreased the paired pulse facilitation (PPF) induced by VPA exposure. The effects of NAC on social interaction and anxiety-like behavior in the VPA-exposed offspring were blocked after intra-amygdala infusion of mGluR2/3 antagonist LY341495. The expressions of mGluR2/3 protein and mGluR2 mRNA were significantly lower in the VPA-exposed offspring. In contrast, the mGluR3 mRNA level did not differ between the saline- and VPA-exposed offspring. These results provide the first evidence that the disruption of social interaction and enhanced presynaptic excitatory transmission in VPA-exposed offspring could be rescued by NAC, which depends on the activation of mGluR2/3.

Behavioral and synaptic circuit features in a zebrafish model of fragile X syndrome.

Fragile X syndrome (FXS) is the most frequent inherited form of human mental retardation. It is characterized by cognitive impairment and physical and behavioral problems and is caused by the silencing of fmr1 transcription and the absence of the fmr1 protein (FMRP). Recently, animal models of FXS have greatly facilitated the investigation of the molecular and cellular mechanisms of this loss-of-function disorder. The present study was aimed to further characterize the role of FMRP in behavior and synaptic function by using fmr1 knockout zebrafish. In adult zebrafish, we found that fmr1 knockout produces the anxiolytic-like responses of increased exploratory behavior in light/dark and open-field tests and avoidance learning impairment. Furthermore, electrophysiological recordings from telencephalic slice preparations of knockout fish displayed markedly reduced long-term potentiation and enhanced long-term depression compared to wild-type fish; however, basal glutamatergic transmission and presynaptic function at the lateral (Dl) and medial (Dm) division of the dorsal telencephalon synapse remained normal. Taken together, our study not only evaluates the mechanism of FRMP but also suggests that zebrafish have valuable potential as a complementary vertebrate model in studying the molecular pathogenesis of human fragile X syndrome.

Stimulation of the lateral division of the dorsal telencephalon induces synaptic plasticity in the medial division of adult zebrafish.

In ray-finned fishes, the lateral (Dl) and medial (Dm) division of the dorsal telencephalon are important in learning and memory formation. Tract-tracing studies revealed that neural connections are formed between these regions via afferent Dl fibers projecting to the Dm. However, research analyzing Dl-Dm synaptic transmission is scant. We have used electrophysiological techniques to assess neurotransmission and synaptic plasticity in projections from the Dl to the Dm in zebrafish. We demonstrate that electrical stimulation of the Dl division evoked a negative field potential in the Dm division that could be inhibited by application of the AMPA/kainate receptor antagonist, CNQX (5µM). Pairs of stimuli, when delivered at brief inter-pulse intervals (IPI), elicited paired pulse facilitation (PPF). Long-term potentiation (LTP), induced through the application of three trains of high frequency stimulation (HFS; 100Hz for 1s), lasted for more than 1h and could be inhibited with DL-AP5 (40µM), an N-methyl-d-aspartate (NMDA) receptor antagonist. Our results suggest that the intratelencephalic connection between Dl and Dm may play an important role in the synaptic plasticity of the zebrafish brain. It also provides a new electrophysiological model for studying the neural mechanisms underlying learning and memory in zebrafish.

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.

Hippocampal neurogenesis after traumatic brain injury is mediated by vascular endothelial growth factor receptor-2 and the Raf/MEK/ERK cascade.

Adult neurogenesis occurs in the subgranular zone of the hippocampal dentate gyrus, and can be modulated by physiological and pathological events. We examined the effect of vascular endothelial growth factor (VEGF), and the correlation between VEGF and the Raf/MEK/ERK cascade in neurogenesis after traumatic brain injury (TBI). The expression of VEGF and the phosphorylation level of Raf/MEK/ERK were analyzed by Western blot, and TBI-induced neurogenesis was determined by immunofluorescence labeling and confocal microscopic detection. Hippocampal VEGF began to increase after 12 h, and reached a peak at day 7. Along with the upregulation of VEGF, neurogenesis in the hippocampus also increased. Administration of the VEGF antisense oligodeoxynucleotide, or the VEGF receptor-2 antagonist SU1498 (10 µg, ICV), attenuated the phosphorylation of the MAPK cascade proteins and caused a decrease in neurogenesis in the hippocampus. Similarly, administration of the ERK inhibitor PD98059 (500 ng, ICV) also exhibited a suppressive effect on neurogenesis. Our results indicate that VEGF plays an important role in neurogenesis after TBI, and that the process involves VEGF receptor-2 and the Raf/MEK/ERK cascade.

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.

Extract of Ginkgo biloba EGb 761 facilitates fear conditioning measured by fear-potentiated startle.

Extract of Ginkgo biloba EGb 761 has been used in the treatment of various common geriatric complaints including vertigo, short-term memory loss, hearing loss, lack of attention, vigilance and cerebral vascular disorder. Recent results suggest that it can serve as a cognitive enhancer and anti-stress buffer. It raises a possibility that EGb 761 may be involved in the fear conditioning. In this study, we used fear-potentiated startle (FPS) to evaluate the possible effects of EGb 761 on the acquisition stage of fear conditioning. Our results showed that administration of EGb 761 30 min prior to the conditioning facilitated acquisition of conditioned fear in a dose dependent manner. No significant differences had been observed in either basal startle response or shock activity. These results indicated that the facilitation effect of EGb 761 was not the result of impaired basal startle response or enhanced pain perception. Subsequent control experiment results indicated that the facilitation effect of EGb 761 on the acquisition was not due to anxiogenic effect or non-specific effect. Our data present the first evidence that EGb 761 can enhance fear memory formation rather than serve as an anti-stress buffer.