demo-lecture-07/excercise

Recording description

InVivo single cell recording in strain N2 Caenorhabditis elegans. Activity shown in oxygen sensitive neuron(s) using Calcium Indicator GCaMP5k.

Oxygen shift paradigm

Experiments used an oxygen concentration shift paradigm:

• 110 seconds 21% O2 concentration; hard oxygen switch
• 360 seconds 04% O2 concentration; hard oxygen switch
• 140 seconds 21% O2 concentration

Recording files

Calcium imaging traces can be found in files with the extension "*.log".

The nine columns contained in these log files are to be interpreted in this order: "current_frame", "time_elapsed", "obj_substracted", "substracted_value", "obj_value", "obj_size", "background_value", "x_old", "y_old"

e.g. [1], [2], [3], [4], [5], [6], [7], [8], [9] 6046, 605491, 10768.6, 12809.4, 23578, 80, 160.118, 264, 65

The columns are interpreted as:

[1] current_frame ... recorded frame [2] time_elapsed ... time in milliseconds [ms] [3] obj_substracted ... object_value - background_value; Fluorescence [AU] [4] substracted_value ... background_value; Fluorescence [AU] [5] obj_value ... average value of region of interest; Fluorescence [AU] [6] obj_size ... whole area of region of interest in pixel [px] [7] background_value ... average of designated background region; Fluorescence [AU] [8] x_old ... previous x position of region of interest [px] [9] y_old ... previous y position of region of interest [px]

Recording files metadata

file name, strain, protocol [%], feeding condition [hour], worm resting time [min], tracked neuron, intensity, Comments, tracking comment, frames, genetic modification, shifting times [s], worm, paralytic agent, EM gain setting, LED I, Gray Filter, exposure

20121202Pflp178GCaMP5kN2shift_210421W1, N2, cs01_shift 21-04-21, 1h50, 7', BAG, 4000/400,'' ,'' 6100, Pflp-8; Pflp-17::GCaMP5.k, 110-360-150, 1, Tetramisol 5mM, 1500, 20, 0.2, 100 ms

20121202Pflp178GCaMP5kN2shift_210421W2, N2, cs01_shift 21-04-21, 2h17, 5'30'', BAG (+URX), 4400/500, process visible, '', 6100, Pflp-8; Pflp-17::GCaMP5.k, 110-360-150, 1, Tetramisol 5mM, 1500, 20, 0.2, 100 ms

20121205Pflp178GCaMP5kN2shift_210421W4, N2, cs01_shift, 21-04-21, 2h20, 5', BAG, 6200/500, process visible, '', 6100, Pflp-8; Pflp-17::GCaMP5.k, 110-360-150, 1, Tetramisol 5mM, 1500, 20, 0.2, 100 ms

Recording equipment and settings

Zeiss epifluorescence microscope equipped with a CoolLED pE-100 excitation system. Images were acquired with an Andor iXon 397 EMCCD camera and MetaMorph software (Universal Imaging) Objective: Pln Apo 40x/1.3 oil DIC II

Zeiss recording settings: pE LED intensity 2 ... 20 Zeiss 6x reflector changer ... none Zeiss 3x Optovar turret ... 1x Tubelens Zeiss 3x Beam Path Switching Baseport ... 100% Baseport Zeiss 3x Sideport Baseport ... 100% L Zeiss RL 6x FL Attenuator ... 20%

Digitizer ... 14 bit (10MHz) Exposure Time ... 100ms EM Gain ... 1500

Background

The Caenorabdhitis elegans neurons labeled BAG and URX have been reported to be tonic signaling neurons. The constant high concentration of increased intracellular Ca 2+ is thought to be mediated by L-type voltage gated Ca 2+ channels (L-VGCC), IP3 and ryanodine receptor Ca 2+ channels. (Busch et al, 2011) The activity of the oxygen de- and increase sensory neurons BAG and URX is tested in an N2 strain (ZIM226) background. The strain contains an extra-chromosomal array expressing GCaMP5k under the flp-8 and the flp-17 promoter. Flp-8 is expressed in URX, flp-17 in BAG. GCaMP5k is a genetically encoded calcium indicator (Akerboom et al, 2012) which links calmodulin to an inactive form of GFP. Upon binding of Ca 2+ to calmodulin the conformation of GFP changes and becomes fluorescent. Depolarization of a neuron leads to an intracellular release of Ca 2+. GCaMP therefore is a useful second messenger in visualizing neuronal activity.

Neuronal activity in an oxygen shift paradigm

To study the response of the sensory neurons to a harsh stimulus, animals were exposed to subsequent oxygen shifts from 21-4% and back to 21%, oxygen concentrations are always balanced with nitrogen. The oxygen "down shift" (DS) took place 110 seconds, the "up shift" (US) 470 seconds after the start of the experiment.

Data provenance

All recordings were done at and by: The Research Institute of Molecular Pathology (IMP), Vienna, Austria Research Group Manuel Zimmer