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Cea-del Rio C.A.,Center for Neuroscience Research | McBain C.J.,U.S. National Institutes of Health | Pelkey K.A.,U.S. National Institutes of Health
Journal of Physiology | Year: 2012

Information processing and transfer within cortical circuits requires precise spatiotemporal coordination of excitatory principal cell activity by a relatively small population of inhibitory interneurons that exhibit remarkable anatomical, molecular and electrophysiological diversity. One subtype of interneuron, the cholecystokinin-expressing basket cell (CCKBC), is particularly well suited to integrate and impart emotional features of an animal's physiological state to principal cell entrainment through the inhibitory network as CCKBCs are highly susceptible to neuromodulation by local and subcortically generated signals commonly associated with 'mood' such as cannabinoids, serotonin and acetylcholine. Here we briefly review recent studies that have elucidated the cellular mechanisms underlying cholinergic regulation of CCKBCs. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society.

Triplett J.W.,University of California at Santa Cruz | Triplett J.W.,Center for Neuroscience Research | Feldheim D.A.,University of California at Santa Cruz
Seminars in Cell and Developmental Biology | Year: 2012

The axonal connections between the retina and its midbrain target, the superior colliculus (SC), is mapped topographically, such that the spatial relationships of cell bodies in the retina are maintained when terminating in the SC. Topographic map development uses a Cartesian mapping system such that each axis of the retina is mapped independently. Along the nasal-temporal mapping axis, EphAs and ephrin-As, are graded molecular cues required for topographic mapping while the dorsal-ventral axis is mapped in part via EphB and ephrin-Bs. Because both Ephs and ephrins are cell surface molecules they can signal in the forward and reverse directions. Eph/ephrin signaling leads to changes in cytoskeletal dynamics that lead to actin depolymerization and endocytosis guiding axons via attraction and repulsion. © 2011 Elsevier Ltd.

Liu J.S.,Center for Neuroscience Research
Current Neurology and Neuroscience Reports | Year: 2011

Cortical malformations associated with defects in neuronal migration result in severe developmental consequences including intractable epilepsy and intellectual disability. Genetic causes of migration defects have been identified with the advent and widespread use of high-resolution MRI and genetic techniques. Thus, the full phenotypic range of these genetic disorders is becoming apparent. Genes that cause lissencephaly, pachygyria, subcortical band heterotopia, and periventricular nodular heterotopias have been defined. Many of these genes are involved in cytoskeletal regulation including the function of microtubules (LIS1, TUBA1A,TUBB3, and DCX) and of actin (FilaminA). Thus, the molecular pathways regulating neuronal migration including the cytoskeletal pathways appear to be defined by human mutation syndromes. Basic science, including cell biology and animal models of these disorders, has informed our understanding of the pathogenesis of neuronal migration disorders and further progress depends on the continued integration of the clinical and basic sciences. © Springer Science+Business Media, LLC 2011.

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment. Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function. Mitochondrial fission needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary. Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment. A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP). Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the... manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15 nM TMRE in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Kratovac S.,Center for Neuroscience Research | Kratovac S.,University of Maryland University College | Corbin J.G.,Center for Neuroscience Research
Brain Research | Year: 2013

In humans, Fragile X Syndrome (FXS) is characterized by enhanced fear, hyperactivity, social anxiety, and, in a subset of individuals, autism. Many of the emotional and social deficits point to defects in the amygdala. We have previously shown defects in inhibitory neuron drive onto excitatory projection neurons in the basolateral amygdala (BLA) of juvenile Fmr1-/y knockout (KO) mice. Using pharmacological approaches, we have also previously revealed dynamic functional deficits in α1, α2, and α3 subunit-containing GABAA receptors (GABAARs α1, α2, and α3) during early postnatal development. In this study, we sought to determine whether these defects in GABAAR function are accompanied by changes in protein expression of GABAARs α1, α2, and α3 and the post-synaptic GABAAR-clustering protein gephyrin. Interestingly, we found that while the expression of these proteins did not significantly differ between wildtype (WT) and KO mice at each time point, the timing of developmental expression of GABAAR α1, α2, and gephyrin was altered. Collectively, these data reveal novel defects in inhibitory synapse protein expression during critical periods of early postnatal development that could contribute to observed inhibitory neurotransmission deficits in the KO mouse BLA. © 2013 Elsevier B.V.

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