Hollis Cline, CSHL – Ultrastructural analysis of synaptic convergence during circuit development
The synaptotrophic hypothesis: synaptic inputs drive dendrite development.
Immature synapses have NMDARs some AMPARs, mature get more AMPA.
Degenration of synapses promote branching of dendritic arbor.
Single cell in vivo electroporation of GFP into optic tectal neurons of zebrafish.
Cisual stimulation increases retinotectal synaptogenesis and increases strength of per-existing synapes. Try to block development of GluR synapses.
From Malinow tech. Whole brain electroporation of AMPAR C-tails. GluR1Ct mini amplitude 11->5, GluR2Ct 11->7. Neurons expressing either make arbors that are longer and less dense. Dynamic (2hr timepoints) imaging of arbor growth and retraction in tectal neurons. Hypotheses : Ct neurons have shorter branch durations.
Correlated in vivo 2p and serial em reconstructions, map all synapses.
1. All dynamic branches have synapses.
2. Retracting branches have very low synaptic density.
3. Synapses on stable branches are more mature
4. Divergence of axon terminals decreases with stable synapses.
Synapse Maturation metric – Area of clustered synaptic vesicles to terminal area
David Kleinfeld, UCSD – Resilience and control of neocortical blood flow revealed by Optical Imaging and Manipulation.
Motivation: Connection between network topology, flow dynamics and control
1. Particle tracking w/ 2p imaging to meauser RBC flow
2. Localized occlusion to perturb flow
3. Global occlusion to regulate source flow
Prep: Lightly Anethistized Rodents
Are the penetrating arterioles (PA) the trunk lines that plunge from top of cortex and have a limited specific range?
Sometimes make clot in PAs, still get flow in neighbors, but usually get total stop in neighbors. Need to move 200-300um away to restore flow. PA makes a cylinder of flow. A-hypoxyprobe labeling (mitochondrial stress) shows 400um diameter stain when PA blocked. Microstrokes of PAs cause localized cortical “burn out”.
Surface Vasculature – Many loops in the surface communicating arteiole network, thus many routes to route around blocks. Block between intersections, flow reverses in some places. 1 vessle away, 60% flow rate, OK. 2 vessles away, 100% flow.
Effect of strong forepaw somatotopic stim on vasodynamics in the surface communicating arterioles. Stimulation leads to both vasodialtion and delayed vasoconstriction in SCAs. Reminiscent of +/-BOLD.
Subsurface microvascular – Gated communities in SoCal. 2p imaging, then zap the top off and image the next level. Make 1mm deep map. Making 3d reconstruction of vessles, cell somata. Density of microvessles is flat. Does not increase in layer 4. Somata does pop up in layer 4. When you block flow in deep microvasculature, 1 vessle distance, total blocked flow, 2-3 little blocked flow. Astros are easy to image Ca, so people are looking at them for vasoregulation. But inhibitory cells release peptides too. Tn-XXL Ca indicator expressed w/ exogenous achr gpcr inject HEK cells into animals. See Ca transients due to Ach release.
Questions : Timecourse of vessel changes? Dilation delay of 400ms, 500ms for constriction. How does vasoregulation occurs at distances of mm? Speculation that inhibitory neurons are part of a broad network whose activity spreads widely. Working on imaging spread with the hybrid imaging probes.
Rusty Landsford, Caltech – Multi-modal imaging of avian development.
Transgenic Japanese quail – avian eq. of mouse, small, related to chicken, fast lifecycle 5-6wk sex maturity. Synapsin.H2B-GFP. Continues 5 day imaging of embryo development. Quail atlas with 11.7T MRI with 3d website.
Yukako Yokota, UNC – Novel aspects of interneuronal migration in the developing cerebral cortex. How do neurons decide where to end up during cerebral cortex development? In vitro electroportation. Look at interneuron-radial glia interaction with live 2pimaging. Example, interneuron changes direction when it hits the endfeet of glia. Pass through ,Turn back, turn towards, attach. Only looking at a small subset of radial glia due to sparse labeling.
Tim Murphy, UBC – Two-photon imaging of acute stroke indicates that a propagating ischemic depolarization produces profound but reversible alteration in the structure and function of spiny dendrites in vivo.
Originally used Kleinfeld technique of photothrombosis, but it’s very local and clots are hard to break up. Wanted reversible ischemia. Which ionic events trigger loss of dendritic structure?
7min bilateral CCA ligation to do global strokes. Structure with YFP.
After 4 minutes, dendrite is a “blebbed ugly mess” of swelling and restrictions. Reperfuse, 10 minutes getting better, 90minutes, structure is almost unchanged from original. 30% spine loss during ischemia, 8% after 90min reperfuse.
Very sharp threshold around 1-2 minutes the brain area turns to mush in about 10 sec. During occulusion quiet EEG, then an AC ripple @ 2min, drop in DC potential. Ischemic depolarization wave increases cortical light scattering. Slow drop then dramatic wave of increased scatter 3mm/sec. Intracellular calcium jumps during the wave. MK-801 had no effect on the ischemia wave structural change, but block NMDA evoked signal. Thinks wiring stays attached during the ballon, though some spine loss and AMPAR internalization.
Adi Mizrahi, Hebrew Univeristy – Imaging synaptic development and plasticity of adult born neurons in the mouse olfactory bulb.
In vivo 2p imaging of lenti-GFP migration of neurons from SVZ to olfactory bulb. Highly dynamic dendritic development. PSD95-GFP_IRES_DsRed. About 65% of PSD punta are synapses by EM. Synaptic puncta do not accumulate during migration. Distributions of synaptic puncta along dendrites sharpen as neurons mature from 14 to 51 DPI. PSD95 puncta are dynamic only when mice are awake. Doing bulk loading of Oregon Green BAPTA – AM and look at response in a single Lenti-RFP neuron. Get fractional percent dF from electrical stim. Primary and secondary dendrites are stable at 3 months, but somas still move around.
Mark Schintzer, Stanford – Chronic microendoscopy.
Insert cannula with a microcoverslip at tip. Long term imaging of cells in hippocampus. Can find same cells DIV 239 v. 241. 1p bloodflow imaging at 100Hz. 423um/sec mean velocity. Micromechanical 2p scanner about the size of a dime, carbon fiber frame for weight. Full frame imaging at video rate. Not on freely moving mice, yet. 1p imaging of blood flow in freely moving mice is working, even 1 month after implantation. 1.8g mount. Freely rotating camera to relieve torsional strain. When will we get a commercial solution??