Evening Session – Optical Measures of Neuronal Excitation And Signaling
Rainer Friedrich – Measuring spatio-temporal activity patterns by temporally deconvolved 2-photon calcium imaging
Bulk loading of olfactory cortex with AM Ca dyes. Calcium signal is much slower than action potentials because the signal is a convolution of Ca trace with binary spikes. Deconvolution of the raw Ca signal with a canonical Ca response derives the firing rate. Reconstruction of population activity pattern improves only a little bit with letting tau or spike amplitude vary. This technique requires low noise. Published in nature methods. Application in olfactory bulb of zebrafish. Factor analysis of spatially clustered activity in OB maps regions of activity to amino acid properties (aromatic, long-chain, basic). Canonical trace is average of single APs. Little contribution of intracellular stores or synaptic input due to the localization of those calcium transients. They image Ca flux in nucleus that has slow timeconstant, but that doesn’t have a negative impact with deconvolution.
Wen-hong Li, UT Southwestern – Dynamic gap junctional communication in developing nervous systems
Vivek Jayaraman – Calibrating a genetically encoded optical sensor of neural activity with electrophysiological techniques in intact fruit flies
1. Does the GECI affect the system’s normal development and function?
How are odors in PNs in flys generally represented? Simultaneous loose patch and 2p imaging with GCaMP1.3 in PN neurons. GCaMP+ neurons not significantly different from matching EGFP+ neurons.
2. Is GECI signal a good proxy for spiking activity? If not what is threshold?
It is OK if you have a high number and rate of spikes. Mean spike rate of 30Hz is the minimum to see GCaMP1.3 response.
3. Can we find a GECI -> spike transfer function?
GCaMP singal rises faster in glomeruli than in PNs. However it still doesn’t track the temporal dynamics. So No.
4. How does expression level and compartment imaged effect the GECI signal?
GCaMP singal rises faster in glomeruli than in PNs. Must calibrate in the specific structure that is being imaged.
Venky Murthy, Harvard – Studying neurovascular coupling using multi-photon microscopy of olfactory glomeruli in vivo
What are noninvasive methods for brain imaging? fMRI measures bloodflow. Olfactory bulb as a model system for studying neurovascular coupling. Can isolate post vs. presynaptic activity.
Measure neural activity using the oldskool (2004) synaptopHluorin mouse from Mombaerts. Look at dF/F changes in olfactory glomerulus. Very slow timecourse of response with 15s odor pulse, increases in a dose dependent manner.
Measure bloodflow by injecting dextran conjugated dye into the bloodstream.
Simultaneous imaging of presynatpic activity and blood flow by linescanning along a vessel and beyond on both sides. Odor stimulation increases bloodflow, which is also correlated with vesicle release. Vasoregulation is likely coupled through astrocytes. Coupled via glutamate, ATP or COX-2. Applying MK-801 and others to block postsynaptic activity does not change blood flow. MCPG, a broad mGluR blocker reduces flow increase by 50% with no affect on the presynaptic activity seen with SpH. What is the other 50% of the coupling from? TBOA (general) and DHK (astrocyte specific) glutamate reuptake blockers also reduce flow increase by about 50%. This is very interesting as this might be expected to increase postsynaptic activation due to greater spillover glutamate levels. Therefore direct activation of mGluRs and glutamate reuptake into astrocytes both regulate cerebral blood flow. COX-2 inhibitors occlude the mGluR effect, but not the TBOA effect suggesting the two pathways are independent.
Orie Shafer, Wash. U – Live-imaging of cycle AMP signaling in the mushroom bodies and circadian pacemaker network of the D. melanogaster brain
Trying to find target neurons sensitive to pigment dispersing factor based on camp responses to PDF
EpacCamps: FRET-based genetically encoded cAMP reporter. YFP/CFP goes down upon stimulation. Try to image camp increases in the large ventrolateral neurons of circadian pacemaker network. Small, uneven response to PDF. However small vLNs respond strongly to PDF, with a variable delay. He suspected differences in the delay of FRET change was brain dependent. I suspect it is variation in the pipetting and diffusion when applying the PDF bolus to the dish. KO of PDFR in these cells abolishes the response. Does increased PDFr expression result in an increased PDF response in the large vLNs? Yes. Now express EpacCamps across all neurons in pacemaker network and screen for response to PDF.
Leon Reijmers, TSRI – The TetTag mouse—A transgenic mouse that enables long-term tagging of activated neurons
Dual transcription factor strategy. Fos-promoter for tTA, which is blocked by Doxicycline. Second promoter that is activated by tTA that drives a mutant tTA* transcription element that has a feedback loop and drives the gene of choice. Doxicyclin blocks activation. Removal of Dox turns gene on, with a feedback loop so when Dox is re-fed to mouse, gene stays on.