Augustine George J.

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George J.

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Now showing 1 - 9 of 9
  • Preprint
    Probing the function of neuronal populations : combining micromirror-based optogenetic photostimulation with voltage-sensitive dye imaging
    ( 2012-12-04) Tsuda, Sachiko ; Kee, Michelle Z. L. ; Cunha, Catarina ; Kim, Jinsook ; Yan, Ping ; Loew, Leslie M. ; Augustine, George J.
    Recent advances in our understanding of brain function have come from using light to either control or image neuronal activity. Here we describe an approach that combines both techniques: a micromirror array is used to photostimulate populations of presynaptic neurons expressing channelrhodopsin-2, while a red-shifted voltage-sensitive dye allows optical detection of resulting postsynaptic activity. Such technology allowed us to control the activity of cerebellar interneurons while simultaneously recording inhibitory responses in multiple Purkinje neurons, their postsynaptic targets. This approach should substantially accelerate our understanding of information processing by populations of neurons within brain circuits.
  • Article
    Defining a critical period for inhibitory circuits within the somatosensory cortex
    (Nature Publishing Group, 2017-08-04) Lo, Shun Qiang ; Sng, Judy C. G. ; Augustine, George J.
    Although experience-dependent changes in brain inhibitory circuits are thought to play a key role during the “critical period” of brain development, the nature and timing of these changes are poorly understood. We examined the role of sensory experience in sculpting an inhibitory circuit in the primary somatosensory cortex (S1) of mice by using optogenetics to map the connections between parvalbumin (PV) expressing interneurons and layer 2/3 pyramidal cells. Unilateral whisker deprivation decreased the strength and spatial range of inhibitory input provided to pyramidal neurons by PV interneurons in layers 2/3, 4 and 5. By varying the time when sensory input was removed, we determined that the critical period closes around postnatal day 14. This yields the first precise time course of critical period plasticity for an inhibitory circuit.
  • Article
    Structural domains involved in the regulation of transmitter release by synapsins
    (Society for Neuroscience, 2005-03-09) Hilfiker, Sabine ; Benfenati, Fabio ; Doussau, Frederic ; Nairn, Angus C. ; Czernik, Andrew J. ; Augustine, George J. ; Greengard, Paul
    Synapsins are a family of neuron-specific phosphoproteins that regulate neurotransmitter release by associating with synaptic vesicles. Synapsins consist of a series of conserved and variable structural domains of unknown function. We performed a systematic structure-function analysis of the various domains of synapsin by assessing the actions of synapsin fragments on neurotransmitter release, presynaptic ultrastructure, and the biochemical interactions of synapsin. Injecting a peptide derived from domain A into the squid giant presynaptic terminal inhibited neurotransmitter release in a phosphorylation-dependent manner. This peptide had no effect on vesicle pool size, synaptic depression, or transmitter release kinetics. In contrast, a peptide fragment from domain C reduced the number of synaptic vesicles in the periphery of the active zone and increased the rate and extent of synaptic depression. This peptide also slowed the kinetics of neurotransmitter release without affecting the number of docked vesicles. The domain C peptide, as well as another peptide from domain E that is known to have identical effects on vesicle pool size and release kinetics, both specifically interfered with the binding of synapsins to actin but not with the binding of synapsins to synaptic vesicles. This suggests that both peptides interfere with release by preventing interactions of synapsins with actin. Thus, interactions of domains C and E with the actin cytoskeleton may allow synapsins to perform two roles in regulating release, whereas domain A has an actin-independent function that regulates transmitter release in a phosphorylation-sensitive manner.
  • Preprint
    Photolysis of a caged peptide reveals rapid action of N-ethylmaleimide sensitive factor before neurotransmitter release
    ( 2007-08-01) Kuner, T. ; Li, Y. ; Gee, K. R. ; Bonewald, L. F. ; Augustine, George J.
    The time at which the N-ethylmaleimide-sensitive factor (NSF) acts during synaptic vesicle trafficking was identified by time-controlled perturbation of NSF function with a photo-activatable inhibitory peptide. Photolysis of this caged peptide in the squid giant presynaptic terminal caused an abrupt (0.2 s) slowing of the kinetics of the postsynaptic current (PSC) and a more gradual (2-3 s) reduction in PSC amplitude. Based on the rapid rate of these inhibitory effects relative to the speed of synaptic vesicle recycling, we conclude that NSF functions in reactions that immediately precede neurotransmitter release. Our results indicate the locus of SNARE protein recycling in presynaptic terminals and reveal a new target for rapid regulation of transmitter release.
  • Article
    Presynaptic nanodomains : a tale of two synapses
    (Frontiers Media, 2015-01-26) Wang, Lu-Yang ; Augustine, George J.
    Here we summarize the evidence from two “giant” presynaptic terminals—the squid giant synapse and the mammalian calyx of Held—supporting the involvement of nanodomain calcium signals in triggering of neurotransmitter release. At the squid synapse, there are three main lines of experimental evidence for nanodomain signaling. First, changing the size of the unitary calcium channel current by altering external calcium concentration causes a non-linear change in transmitter release, while changing the number of open channels by broadening the presynaptic action potential causes a linear change in release. Second, low-affinity calcium indicators, calcium chelators, and uncaging of calcium all suggest that presynaptic calcium concentrations are as high as hundreds of micromolar, which is more compatible with a nanodomain type of calcium signal. Finally, neurotransmitter release is much less affected by the slow calcium chelator, ethylene glycol tetraacetic acid (EGTA), in comparison to the rapid chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Similarly, as the calyx of Held synapse matures, EGTA becomes less effective in attenuating transmitter release while the number of calcium channels required to trigger a single fusion event declines. This suggests a developmental transformation of microdomain to nanodomain coupling between calcium channels and transmitter release. Calcium imaging and uncaging experiments, in combination with simulations of calcium diffusion, indicate the peak calcium concentration seen by presynaptic calcium sensors reaches at least tens of micromolar at the calyx of Held. Taken together, data from these provide a compelling argument that nanodomain calcium signaling gates very rapid transmitter release.
  • Article
    Optogenetic visualization of presynaptic tonic inhibition of cerebellar parallel fibers
    (Society for Neuroscience, 2016-05-25) Berglund, Ken ; Wen, Lei ; Dunbar, Robert L. ; Feng, Guoping ; Augustine, George J.
    Tonic inhibition was imaged in cerebellar granule cells of transgenic mice expressing the optogenetic chloride indicator, Clomeleon. Blockade of GABAA receptors substantially reduced chloride concentration in granule cells due to block of tonic inhibition. This indicates that tonic inhibition is a significant contributor to the resting chloride concentration of these cells. Tonic inhibition was observed not only in granule cell bodies, but also in their axons, the parallel fibers (PFs). This presynaptic tonic inhibition could be observed in slices both at room and physiological temperatures, as well as in vivo, and has many of the same properties as tonic inhibition measured in granule cell bodies. GABA application revealed that PFs possess at least two types of GABAA receptor: one high-affinity receptor that is activated by ambient GABA and causes a chloride influx that mediates tonic inhibition, and a second with a low affinity for GABA that causes a chloride efflux that excites PFs. Presynaptic tonic inhibition regulates glutamate release from PFs because GABAA receptor blockade enhanced both the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs at the PF-Purkinje cell synapse. We conclude that tonic inhibition of PFs could play an important role in regulating information flow though cerebellar synaptic circuits. Such cross talk between phasic and tonic signaling could be a general mechanism for fine tuning of synaptic circuits.
  • Article
    All-optical mapping of barrel cortex circuits based on simultaneous voltage-sensitive dye imaging and channelrhodopsin-mediated photostimulation
    (SPIE, 2015-03-31) Lo, Shun Qiang ; Koh, Dawn X. P. ; Sng, Judy C. G. ; Augustine, George J.
    We describe an experimental approach that uses light to both control and detect neuronal activity in mouse barrel cortex slices: blue light patterned by a digital micromirror array system allowed us to photostimulate specific layers and columns, while a red-shifted voltage-sensitive dye was used to map out large-scale circuit activity. We demonstrate that such all-optical mapping can interrogate various circuits in somatosensory cortex by sequentially activating different layers and columns. Further, mapping in slices from whisker-deprived mice demonstrated that chronic sensory deprivation did not significantly alter feedforward inhibition driven by layer 5 pyramidal neurons. Further development of voltage-sensitive optical probes should allow this all-optical mapping approach to become an important and high-throughput tool for mapping circuit interactions in the brain.
  • Article
    Optogenetic mapping of cerebellar inhibitory circuitry reveals spatially biased coordination of interneurons via electrical synapses
    (Cell Press, 2014-05-22) Kim, Jinsook ; Lee, Soojung ; Tsuda, Sachiko ; Zhang, Xuying ; Asrican, Brent ; Gloss, Bernd ; Feng, Guoping ; Augustine, George J.
    We used high-speed optogenetic mapping technology to examine the spatial organization of local inhibitory circuits formed by cerebellar interneurons. Transgenic mice expressing channelrhodopsin-2 exclusively in molecular layer interneurons allowed us to focally photostimulate these neurons, while measuring resulting responses in postsynaptic Purkinje cells. This approach revealed that interneurons converge upon Purkinje cells over a broad area and that at least seven interneurons form functional synapses with a single Purkinje cell. The number of converging interneurons was reduced by treatment with gap junction blockers, revealing that electrical synapses between interneurons contribute substantially to the spatial convergence. Remarkably, gap junction blockers affected convergence in sagittal slices, but not in coronal slices, indicating a sagittal bias in electrical coupling between interneurons. We conclude that electrical synapse networks spatially coordinate interneurons in the cerebellum and may also serve this function in other brain regions.
  • Article
    Next-generation transgenic mice for optogenetic analysis of neural circuits
    (Frontiers Media, 2013-11-26) Asrican, Brent ; Augustine, George J. ; Berglund, Ken ; Chen, Susu ; Chow, Nick ; Deisseroth, Karl ; Feng, Guoping ; Gloss, Bernd ; Hira, Riichiro ; Hoffmann, Carolin ; Kasai, Haruo ; Katarya, Malvika ; Kim, Jinsook ; Kudolo, John ; Lee, Li Ming ; Lo, Shun Qiang ; Mancuso, James ; Matsuzaki, Masanori ; Nakajima, Ryuichi ; Qiu, Li ; Tan, Gregory ; Tang, Yanxia ; Ting, Jonathan T. ; Tsuda, Sachiko ; Wen, Lei ; Zhang, Xuying ; Zhao, Shengli
    Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of channelrhodopsins, behind various neuron-specific promoters. These mice permit photoinhibition or photostimulation both in vitro and in vivo. Our results also reveal the important influence of fluorescent tags on optogenetic probe expression and function in transgenic mice.