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«Strengthening the Nation through Diversity, Innovation & Leadership in STEM San Antonio,Texas · October 3-6, 2013 Get Connected! Connect with the ...»

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1 Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Loss of function of the fmr1 gene results in lack of fragile X mental retardation protein (FMRP), an RNA binding protein. Two homologs of FMR1, FXR1 and FXR2, are expressed in the brain and may have functional redundancy in RNA binding, but little is known about their role in development. Recent studies suggest that neurogenesis, the generation of neurons from progenitor cells, is aberrant in FXS patients. We investigated whether fragile X proteins affect neurogenesis, using Xenopus laevis tadpoles which express homologs of fmr1 and fxr1 genes. We knocked down FMR1 and FXR1 with antisense morpholinos and collected in vivo confocal time-lapse images of GFP-expressing radial glial progenitor cells and their progeny over three days. Animals treated with control morpholinos increase the number of GFP-labeled cells between the first and third days of imaging, but FMR1 or FXR1P knockdown animals significantly decreased the total number of GFP-labeled cells generated over the imaging period. We identified neurons and glia based on their morphology and found that the average number of neurons and radial glia cells on days 2 and 3 is significantly reduced with FMR1 or FXR1 knockdown compared to controls, suggesting that proliferation and survival of neural progenitor cells is compromised by loss of fragile X proteins. Interestingly, knockdown of FMRP increased the proportion of neural progeny compared to progenitors, suggesting that loss of FMRP induces rapid differentiation of neurons from progenitors, adding insight into mechanisms of FXS.



Steve Guzman, V. Reggie Edgerton.

University of California, Los Angeles, Los Angeles, CA.

Considerable levels of upper limb function can be recovered in a non-human primate following a severe cervical spinal cord injury. However, the neural reorganization strategies underlying this recovery remain unclear. We hypothesize that one mechanism that contributes to this recovery can be attributed to an increased excitability of the spinal circuitry that controls the motor pools that innervate the muscles of the upper limb after injury. In our study, 4 rhesus monkeys (Macaca mulatta) underwent C7 hemisection and EMG electrode implantation while video and EMG recordings were taken during a trained hand task, both pre and post-lesion. Following the injury, based on EMG recordings we observed an immediate loss of the ability to activate most muscles of the upper limb as required for reaching, grasping, and retrieving food. However, about 6 to 20 weeks after injury, we found that the level of activation of these muscles in most cases exceeded the normal level of activation when performing these motor tasks. At the same time, there was increased forelimb muscle coactivation as well as motor unit recruitment. These data suggest that, during spinal cord injury recovery, there is a proliferation of aberrant synaptic connections which results in elevated levels of activation and poorly coordinated patterns of activation among agonist and antagonist muscles.

In the more advanced stages of recovery, it appears that the levels of activation and the levels of coordination between motor pools return toward normal levels. Both of these mechanisms probably contributed to the progressive improvement in motor function following the injury.




Victoria Cheung, Olivia Mullins, Phong Nguyen, Andrew Huberman.

University of California, San Diego, La Jolla, CA.

A fundamental goal in neuroscience is to understand the neural circuit organization supporting sensory processing.

Cephalopods, which include squid, octopus, and cuttlefish, are highly visual creatures and possess the most complex invertebrate nervous system. In particular, the cuttlefish exhibits unique visual behaviors including independent eye movements and variable eye positioning giving it a large range of binocular overlap. It is assumed that cuttlefish eyes converge to generate stereopsis for effective prey capture, but this hypothesis has not been adequately explored, and its full range of binocular overlap has not been rigorously tested. Additionally, only the basic neural organization of the visual system has been established. To determine the range of binocular overlap during prey capture, we recorded behaving cuttlefish from multiple angles and tracked their eye movements with semi-automated software to generate positional 3D coordinates. Tentacle dynamics were also analyzed. To determine the necessity of stereopsis for efficient prey capture, cuttlefish were deprived of sight in 1 eye and filmed during hunting episodes. Multiple parameters of prey capture were measured and compared to control animals. What is the neural organization in the cuttlefish supporting these visual behaviors? To answer this question, we are injecting a fluorescent tracer into an eye or an optic lobe of the animal to trace its neuronal projections. Preliminary results indicate that projection neurons in one optic lobe travel to (among other regions) the contralateral optic lobe where they are topographically mapped.

These results will give us a better understanding of the neural circuitry that evolved to support visual behaviors.



Angela Avitua, Angela Echeverri, Mikhail Melnik, Darrin J. Lee, Bruce G. Lyeth, Gene G. Gurkoff.

University of California, Davis, Davis, CA.

Traumatic brain injury (TBI) is associated with an increased prevalence of seizures and accounts for 30% of epilepsy observed in individuals between the ages of 15 and 34. We hypothesized that TBI or TBI with hypoxia would increase the rate of epileptogenesis as compared to sham controls in a kindling model of epilepsy. The amygdala was stimulated daily in 4 groups of animals (sham + normoxia, sham + hypoxia, TBI + normoxia, and TBI + hypoxia) between days 2 and 16 post-injury. Behavioral and electrographic data indicated that sham normoxia rats took 7.2 ± 1.4 days to develop epilepsy, while the TBI normoxia group took 12.3 ± 1.6 days, and the TBI + hypoxia group took 16.6 ± 0.4 days. Therefore, counter to our hypothesis, neither TBI nor TBI + hypoxia increased the rate of epileptogenesis. Brain tissue was sectioned from a subset of animals after 7 days of kindling, stained with GFAP (astrocytes) and IBA-1 (microglia), and is being quantified using stereology. Preliminary data suggests an increase in astrocytic cell death in TBI + hypoxia rats as compared to other groups. Interestingly, microglial activation is more prevalent in animals receiving TBI in the absence of hypoxia. As demonstrated by these data, the pathophysiology of TBI and how TBI relates to epileptogenesis is complex. Better understanding of the relationship between cell death, microglial activation, and epileptogenesis are critical in our long-term goal to determine a therapeutic intervention to reduce post-traumatic epilepsy.



Sofia Campos1, Alex Bender2, Pierre-Pascal Lenck-Santini2.

University of California, San Diego, La Jolla, CA, 2Geisel School of Medicine at Dartmouth College, Hanover, NH.

1 Dravet syndrome (DS) is a childhood disorder in which symptoms develop as the patient ages. These characteristics include seizures that are anti-epileptic drug resistant, severely cognitive deficit, and abnormal in EEG. It has been observed that many DS patients as well as patients with other neurological disorders have a loss-of-function genetic mutation on the SCN1a gene that affects the coding for the voltage-gated sodium channel type I (Nav1.1). To better understand the age-dependent neurological consequences of Nav1.1 loss of function, we characterized its expression in rats at various ages ranging from birth to early adulthood. Fluorescent and DAB histological staining techniques were used. We found that brain Nav1.1 expression increases with age in a spatial gradient where hindbrain structures express the channel first and neo cortical areas last. These results are validated through two different methods of histology protocols. We are working to expand these results using more quantitative methods including real-time PCR. Such developmental patterns could be directly linked to the progression of seizure types and neurological impairments in Dravet syndrome, a hypothesis that we will test in animal models of the disease. Knowing the timing

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FRI-588 MEF2 IN NEURONS Huwate Yeerna, Shing Chan.

Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, Sanford-Burnham Medical Research Institute, La Jolla, CA.

MEF2, along with various transcription factors, is known to govern important aspects of neural development and synaptic plasticity. Many target genes of such transcription factors have also been identified. Nevertheless, the genome-wide mechanisms that are directly regulated by MEF2 remain unclear. In this study, we attempted to confirm the target genes identified by ChIP-sequencing using RT-qPCR. Also, we have analyzed the genetic program and mechanisms of MEF2, a crucial regulator of activity-depended synapse development. Overall, our analyses suggest that the ubiquitously expressed transcription factor MEF2 regulates a broad and intricate transcriptional program in neurons that control synapse development.




Zuleirys Santana Rodriguez1, Alexandra Colon-Rodriguez2, William Atchison2.

University of Puerto Rico, Rio Piedras, San Juan, PR, 2Michigan State University, East Lansing, MI.

1 Methylmercury (MeHg) is an important environmental neurotoxicant that causes significant damage to the developing and mature nervous system. Studies have demonstrated that MeHg causes alterations in synaptic transmission, [Ca2+]i homeostasis, and GABAA receptor (GABAAR) mediated synaptic inhibition. However the mechanisms underlying MeHg toxicity are not yet understood. In this study, we are focused on determining the effects of chronic MeHg exposure on mRNA expression and protein levels of the α1, β1, β3, γ2, and δ subunits of the GABAAR; and the NR1 and NR2A subunits of the NMDA receptor (NMDAR) in mouse brainstem. The brainstem is an area rich in motor neurons, a recently identified target of MeHg toxicity. Balb/c mice were exposed to 0 ppm (control) or 5 ppm MeHg ad lib in drinking water for 12 months, starting when they were 90 days old. RNA and protein were isolated from 10 mg of brainstem tissue, and quantitative real-time PCR was performed in reverse transcript of the isolated RNA. Relative expression was calculated using the 2ΔΔCt method, which we normalized to the endogenous control GAPDH and the untreated controls. Chronic MeHg exposure caused alterations in the expression of the GABAAR and NMDAR subunits studied. The α1 and β1 of the GABAAR were downregulated as well as the NR1 of the NMDAR.

However the β3, γ2, and δ of GABAAR; and NR2A of NMDAR were close to control levels. Downregulation of GABAAR and NMDAR subunits could be an indication of the mechanism of MeHg-induced alterations on Ca2+ homeostasis on motor neurons.



Danner Peter, Joshua Bonkowsky.

University of Utah School of Medicine, Salt Lake City, UT.

Pelizaeus-Merzbacher disease (PMD) is an X-linked disease that is caused by duplication of the proteolipid protein-1 (PLP1) gene. PMD mostly affects young children and infants at an incidence of 1 in 200,000 to 500,000 newborn males. PMD is a leukodystrophy, or disease of the myelin (white matter), with failure of normal myelin development.

Our goal is to develop a small, inexpensive vertebrate model using Danio rerio (zebrafish) in order to screen for drugs or novel compounds that stop and/or reverse disease progression. The project requires creation of 3 plasmid and transgenic lines. First, we generated a line of zebrafish in which green fluorescent protein (GFP) is expressed and targeted in the myelin (mbp:egfp-caax). These transgenic fish will allow live visualization of myelin integrity.

Second, we will make a line of transgenic zebrafish in which the PLP1 gene can be conditionally overexpressed (mbp:loxP[mCherry]-PLP1-egfp). Third, we will make a line to control the conditional overexpression by induction of a tamoxifen-dependent (ERT) Cre enzyme event (mbp:Cre-ERT). Expression of the different plasmids will use the mbp (myelin basic protein) enhancer. We will generate 3 stable transgenic lines of zebrafish for each of the plasmids, and then will intercross them to make a triple transgenic fish. Tamoxifen will then be added to induce Cre function and

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characterize the time-course and pathology of myelin degeneration to see if the triple transgenic fish phenocopies PMD. We expect that our model will provide a novel approach to discover drugs to treat PMD.




Guinevere Ashley, Emma Hiolski, Donald Smith.

University of California, Santa Cruz, Santa Cruz, CA.

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