David Ogden

Interaction of the actin cytoskeleton with microtubules regulates secretory organelle movement near the plasma membrane in human endothelial cells

The role of cytoskeletal elements in regulating transport and docking steps that precede exocytosis of secretory organelles is not well understood. We have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the three-dimensional motions of secretory organelles near the plasma membrane in living endothelial cells. Weibel-Palade bodies (WPb), the large tubular storage organelles for von Willebrand factor, were labelled with Rab27a-GFP. By contrast, green fluorescent protein (GFP)-tagged tissue-type plasminogen activator (tPA-GFP) labelled submicron vesicular organelles. Both populations of GFP-labelled organelles underwent stimulated exocytosis. The movement of these morphologically distinct organelles was measured within the evanescent field that penetrated the first 200 nm above the plasma membrane. WPb and tPA-GFP vesicles displayed long-range bidirectional motions and short-range diffusive-like motions. Rotating and oscillating WPb were also observed. TIRF microscopy enabled us to quantify the contribution of actin and microtubules and their associated motors to the organelle motions close to the plasma membrane. Long-range motions, as well as WPb rotations and oscillations, were microtubule-and kinesin-dependent. Disruption of the actin cytoskeleton and inhibition of myosin motors increased the number of long-range motions and, in the case of WPb, their velocity. The actin and microtubules had opposite effects on the mobility of organelles undergoing short-range motions. Actin reduced the mobility and range of motion of both WPb and tPA vesicles, whereas microtubules and kinesin motors increased the mobility of WPb. The results show that the dynamics of endothelial secretory organelles close to the plasma membrane are controlled by the opposing roles of the microtubule and actin cytoskeletal transport systems.

Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning.

Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na+ channels.

Laser photolysis of caged compounds at 405 nm: Photochemical advantages, localisation, phototoxicity and methods for calibration

Rapid, localised photolytic release of neurotransmitters from caged precursors at synaptic regions in the extracellular space is greatly hampered at irradiation wavelengths in the near-UV, close to the wavelength of maximum absorption of the caged precursor, because of inner-filtering by strong absorption of light in the cage solution between the objective and cell. For this reason two-photon excitation is commonly used for photolysis, particularly at multiple points distributed over large fields; or, with near-UV, if combined with local perfusion of the cage. These methods each have problems: the small cross-sections of common cages with two-photon excitation require high cage concentrations and light intensities near the phototoxic limit, while local perfusion gives non-uniform cage concentrations over the field of view. Single-photon photolysis at 405 nm, although less efficient than at 330-350 nm, with present cages is more efficient than two-photon photolysis. The reduced light absorption in the bulk cage solution permits efficient wide-field uncaging at non-toxic intensities with uniform cage concentration. Full photolysis of MNI-glutamate with 100 micros pulses required intensities of 2 mW microm(-2) at the preparation, shown to be non-toxic with repeated exposures. Light scattering at 405 nm was estimated as 50% at 18 microm depth in 21-day rat cerebellum. Methods are described for: (1) varying the laser spot size; (2) photolysis calibration in the microscope with the caged fluorophore NPE-HPTS over the wavelength range 347-405 nm; and (3) determining the point-spread function of excitation. Furthermore, DM-Nitrophen photolysis at 405 nm was efficient for intracellular investigations of Ca2+-dependent processes.

Ca2+ Ion Permeability and Single-Channel Properties of the Metabotropic Slow EPSC of Rat Purkinje Neurons

The slow EPSC (sEPSC) of cerebellar parallel fiber → Purkinje neuron synapses is mediated by metabotropic glutamate receptor type 1 (mGluR1) activation of nonselective cation channels. Here, the channel properties were studied with uniform calibrated photorelease of l-glutamate with ionotropic receptors blocked, allowing isolation of postsynaptic processes, or with parallel fiber stimulation or mGluR1 agonist application. Evoked current and fluorescence from Ca2+ indicators were recorded. Noise analysis of the mGluR1 current gave a single-channel conductance of 0.6 pS and showed low open probability at maximal mGluR1 activation. Similar small single-channel conductances were obtained with the mGluR1 agonist (S)-dihydroxyphenylglycine, with parallel fiber or climbing fiber stimulation. The mGluR1 current fluctuations were unaffected by potassium channel blockers. Photoreleased l-glutamate triggered a Ca2+ concentration increase in the distal dendrites with a time course similar to that of the mGluR1 current. The proximal dendritic and somatic Ca2+ changes were delayed with respect to the current. Ca2+ channel blockers and the phospholipase Cδ inhibitor 1-[6-[((17δ)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione, which inhibits mGluR1-activated intracellular Ca2+ release, did not prevent the dendritic Ca2+ concentration increase. Polyamine naphthylacetyl spermine and cationic adamantanes that block the pore of the channel were used to vary the mGluR1 current over a wide range in each cell but still at maximal mGluR1 activation. The Ca2+ influx was inhibited in parallel with the current. The results show that the mGluR1-activated current and the sEPSC are attributable to small-conductance, low-open probability Ca2+-permeable cation channels that will mediate spine-specific Ca2+ influx during the parallel fiber sEPSP.

Laser photolysis of DPNI-GABA, a tool for investigating the properties and distribution of GABA receptors and for silencing neurons in situ

Laser photolysis to release GABA at precisely defined times and locations permits investigation of the distribution of functional GABA(A) receptors in neuronal compartments, the activation kinetics and pharmacology of GABA(A) receptors in situ, and the role of individual neurons in neural circuits by selective silencing with low GABA concentrations. We describe the experimental evaluation and applications of a new nitroindoline-caged GABA, DPNI-GABA, modified to minimize the pharmacological interference commonly found with caged GABA reagents, but retaining the advantages of nitroindoline cages. Unlike the 5-methoxycarbonylmethyl-7-nitroindolinyl-GABA tested previously, DPNI-GABA inhibited GABA(A) receptors with much lower affinity, reducing peak GABA-evoked responses with an IC(50) of approximately 0.5 mM. Most importantly, the kinetics of receptor activation, determined as 10-90% rise-times, were comparable to synaptic events and were little affected by DPNI-GABA present at 1mM concentration, permitting photolysis of DPNI-GABA to mimic synaptic activation of GABA(A) receptors. With a laser spot of 1 microm applied to cerebellar molecular layer interneurons, the spatial resolution of uncaging DPNI-GABA in dendrites was estimated as 2 microm laterally and 7.5 microm focally. Finally, at low DPNI-GABA concentration, photorelease restricted to the area of the soma suppressed spiking in single Purkinje neurons or molecular layer interneurons for periods controlled by the flash intensity and duration. DPNI-GABA has properties better adapted for fast kinetic studies with laser photolysis at GABA(A) receptors than previously reported caged GABA reagents, and can be used in experiments where spatial resolution is determined by the dimensions of the laser light spot.

Photolabile donors of nitric oxide: Ruthenium nitrosyl chlorides as caged nitric oxide

Abstract RuNOCl3 and K2RuNOCl5 are water-soluble, ionic caged NO compounds. On irradiation in the range 300–350 nm they liberate NO rapidly at a rate greater than 105 sec−1, with product quantum yields, Qp, of 0.012 and 0.06, respectively. The NO released on photolysis can be measured from an absorbance change associated with conversion of deoxyHb (FeII) to nitrosylHb (FeII). An alternative assay is to measure the absorbance change associated with conversion of oxyHb (FeIII) to metHb (FeIII) by photoreleased NO. When NO gas from a cylinder is dissolved in distilled water, the resulting solution also contains nitrous acid, which can be identified by its characteristic near-UV spectrum. A commonly used assay for NO dissolved in aqueous solvents involves diazotization of sulfanilic acid. This diazotization arises because of the action of the contaminating nitrite, not NO. Finally, the efficacy of the caged NO compounds in biological systems is shown by the relaxation of contracted aortic rings by photoreleased NO, just as occurs with endothelium-derived relaxing factor, which is known to be NO.

Current and Calcium Responses to Local Activation of Axonal NMDA Receptors in Developing Cerebellar Molecular Layer Interneurons

In developing cerebellar molecular layer interneurons (MLIs), NMDA increases spontaneous GABA release. This effect had been attributed to either direct activation of presynaptic NMDA receptors (preNMDARs) or an indirect pathway involving activation of somato-dendritic NMDARs followed by passive spread of somatic depolarization along the axon and activation of axonal voltage dependent Ca2+ channels (VDCCs). Using Ca2+ imaging and electrophysiology, we searched for preNMDARs by uncaging NMDAR agonists either broadly throughout the whole field or locally at specific axonal locations. Releasing either NMDA or glutamate in the presence of NBQX using short laser pulses elicited current transients that were highly sensitive to the location of the spot and restricted to a small number of varicosities. The signal was abolished in the presence of high Mg2+ or by the addition of APV. Similar paradigms yielded restricted Ca2+ transients in interneurons loaded with a Ca2+ indicator. We found that the synaptic effects of NMDA were not inhibited by blocking VDCCs but were impaired in the presence of the ryanodine receptor antagonist dantrolene. Furthermore, in voltage clamped cells, bath applied NMDA triggers Ca2+ elevations and induces neurotransmitter release in the axonal compartment. Our results suggest the existence of preNMDARs in developing MLIs and propose their involvement in the NMDA-evoked increase in GABA release by triggering a Ca2+-induced Ca2+ release process mediated by presynaptic Ca2+ stores. Such a mechanism is likely to exert a crucial role in various forms of Ca2+-mediated synaptic plasticity.

Readily releasable pool of synaptic vesicles measured at single synaptic contacts

To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.

New caged neurotransmitter analogs selective for glutamate receptor sub-types based on methoxynitroindoline and nitrophenylethoxycarbonyl caging groups

Photolysis is widely used in experimental neuroscience to isolate post-synaptic receptor activation from presynaptic processes, to determine receptor mechanisms in situ, for pharmacological dissection of signaling pathways, or for photostimulation/inhibition in neural networks. We have evaluated new caged neuroactive amino acids that use 4-methoxy-7-nitroindolinyl- (MNI) or 1-(2-nitrophenyl)ethoxycarbonyl (NPEC) photoprotecting groups to make caged ligands specific for glutamate receptor sub-types. Each was tested for interference with synaptic transmission and excitability and for receptor-specific actions in slice preparations. No adverse effects were found at glutamate receptors. At high concentration, MNI-caged, but not NPEC-caged ligands, interfered with GABA-ergic transmission. MNI-caged amino acids have sub-microsecond release times suitable for investigating mechanisms at fast synaptic receptors in situ. MNI-NMDA and MNI-kainate were synthesized and tested. MNI-NMDA showed stoichiometric release of chirally pure NMDA. Wide-field photolysis in cerebellar interneurons produced a fast-rising sustained activation of NMDA receptors, and localized laser photolysis gave a fast, transient response. Photolysis of MNI-kainate to release up to 4 μM kainate generated large inward currents at resting membrane potential in Purkinje neurons. Application of GYKI 53655 indicated that 40% of the current was due to AMPA receptor activation by kainate. Signaling via metabotropic glutamate receptors (mGluR) does not require fast release rates. NPEC cages are simpler to prepare but have slower photorelease. Photolysis of NPEC-ACPD or NPEC-DHPG in Purkinje neurons generated slow inward currents blocked by the mGluR type 1 antagonist CPCCOEt similar to the slow sEPSC seen with parallel fiber burst stimulation. NPEC-AMPA was also tested in Purkinje neurons and showed large sustained inward currents selective for AMPA receptors with little activation of kainate receptors. MNI-caged l-glutamate, NMDA and kainate inhibit GABA-A receptors with IC₅₀ concentrations close to the maximum concentrations useful in receptor signaling experiments.

Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics

Faithful reporting of temporal patterns of intracellular Ca2+ dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca2+ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca2+ affinities and accelerated kinetics by weakening the Ca2+-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca2+ decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca2+ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca2+ at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca2+ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.