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Calcium Imaging and Voltage-sensitive dyes. Membrane Biophysics 10/26/2007. - Joseph M. Breza -. Outline. 1) Backgroud Calcium-sensitive dyes, Calcium imaging.

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slide1

Calcium Imaging and Voltage-sensitive dyes

Membrane Biophysics 10/26/2007

- Joseph M. Breza -

slide2

Outline

1) Backgroud Calcium-sensitive dyes, Calcium imaging

2) Riera CE, Vogel H, Simon SA, le Coutre J. Artificial sweeteners and salts producing a metallic taste sensation activate TRPV1 receptors, Am J Physiol Regulatory Integrative Comp Physiol 293:626-634, 2007.

3) Background Voltage-sensitive dyes, imaging

slide3

Calcium

The ion of the nervous system

Neurotransmitter release into synapse via vesicle release proteins

Signaling cascades

Cell division

Blood clotting

Fertilization

Amount of Calcium needed for effect is relatively small

slide4

Fluorescence:

Is the phenomenon in which the molecular absorption of a

photon triggers the emission of another photon with a longer

wavelength.

(named after calcium fluoride “Fluorite”)

* Stimulate in the ultraviolet range, and the emitted light is in the visible range.*

slide5

Agar Plate of Fluorescent Bacteria Colonies

QUIN 2 was a member of the first generation of Ca2+ chelators introduced by Tsien in 1980

slide6

Calcium Imaging

Fluorescence detection system

1) Fluorophore- fluorescent molecule (photo bleaching)

2) Wavelength filters

3) Excitation source (light source UV)

4) Detector

Fluorescence instruments

1) Spectrofluorometer – analyzes fluorescence (before – after)

2) Flow cytometer- analyzes, sorts, counts microscopic particles in stream of fluid-doesn’t produce and image of the cell

3) Fluorescence microscope - filters weak fluorescent light from other lights

slide7

Calcium sensitive dyes are Chelators

Many different types of calcium fluorescent dyes

Type used depends on the cell type - experience

Research goal (salt/lipophilic forms, low/intermediate/high affinity, fast calcium spikes,

fluorescence, method of delivery, etc)

Low-affinity calcium indicators

Fura-FF

BTC

Fura-2

Fura-5,

Indo-1)

Excited by UV light

Intermediate-affinity calcium

Fura-4F

Fura-5F

Fura-6F

Excited by UV light

Excited by visible light under

scanning laser confocal microscopy

High-affinity and selectivity (BAPTA)

Calcium Green, Calcium Orange – Tomchik et al 2007

slide8

Fluorescent microscope

Protects eyes from

UV light

slide9

Figure 2

PLCβ2 negative

G-proteins

CaGD-

Calcium

Fluorescence

slide10

Figure 4

PLCβ2-GFP Mouse

(Receptor cell)

GAD-GFP Mouse

(Presynaptic cell)

slide11

Artificial sweeteners and salts producing a metallic taste sensation activate TRPV1 receptors

Am J Physiol Regulatory Integrative Comp Physiol 293:626-634, 2007.

Authors: Riera CE, Vogel H, Simon SA, le Coutre J.

slide19

Voltage-sensitive dyes

A plethora of them to choose from…depends on tissue (experience)

Vary considerably in active duration, loading, etc.

“Sticks” to the plasma membrane

Changes in membrane potential are recorded with a photo diode (464 pixels 1010 photons per frame)

~1nA photocurrent and dye sensitivity of ~1%/100mV

Can calculate membrane potential within 3 mV

Change in Fluorescence can be between ~1-21% / 100mV change (RH421).

optical recording of synaptic release
Optical recording of synaptic release

Digital Camera

Pseudocolor output of camera

Transgenic olfactory bulb

the ph sensitive protein synaptophluorin
The pH-sensitive protein: synaptopHluorin

Mutagenized Green Fluorescence Protein

Neuron. 2005 Dec 22;48(6):1039-53.

can accept a proton to change fluorescence

fused to VAMP2 expressed in mice

targets to synaptic vessicles in vivo

When synaptic vessicle fuses with membrane

pH increase almost instantly

fluorescence increases almost instantly

slide26

pH of: vesicle extracellular

Gero Miesenböck, Dino A. De Angelis and James E. Rothman

Nature 394, 192-195(9 July 1998)

slide27
Odorant Representations Are Modulated by Intra- but Not Interglomerular Presynaptic Inhibition of Olfactory Sensory Neurons

McGann JP, Pírez N, Gainey MA, Muratore C, Elias AS, Wachowiak M.

Question: How does presynaptic inhibition really work in the olfactory bulb glomerulus?

slide29

Single glomerulus:

2 bundles of OSNs

Single bundle are

cumulative

Paired-pulse results

in depression

Depression alleviated

by: GLU blockers

GABA blockers

optical recording of voltage changes in vivo
Optical recording of voltage changes in vivo

photodiode array

Pseudocolor output of the array

turtle

http://www.redshirtimaging.com

slide31

Voltage-sensitive dyes

A plethora of them to choose from…depends on tissue (experience)

Vary considerably in active duration, loading, etc.

“Sticks” to the plasma membrane

Changes in membrane potential are recorded with a photo diode (464 pixels 1010 photons per frame)

~1nA photocurrent and dye sensitivity of ~1%/100mV can calculate membrane

Potential within 3 mV

Change in Fluorescence can be between ~1-21% / 100mV change (RH421).

binaral interaction and centrifugal input enhances spatial contrast in olfactory bulb activation
Binaral interaction and centrifugal input enhances spatial contrast in olfactory bulb activation.

Singer, Benjamin H., Kim, Soyoun & Zochowski, Michal

European Journal of Neuroscience 25 (2), 576-586.

Question: What role do centrifugal inputs play in OB activation?

slide33

Schematic of olfactory

bulb neurotransmitters

and cellular connections

Note:

All of these different

neurotransmitters are

coming from BOTH

sides of brain

Doty RL (1995), Handbook of Olfaction and Gustation, Marcel Dekker.

slide34

Experimental design and controls

Replicates with one odorant

slide35

Paired-pulse with different

odorants results in increased

inhibition

This would have been difficult

to see with standard

electrophysiology or behavior

slide37
Flash-activation of compounds won the 1967 prize in Noble Prize in Chemistry

Light-induced isomerization acetylcholine agonists by Wasserman and Erlanger 1971 only trans isomer biologically active (Bis-Q)

Many kinds today

used on: Ca++

cNMP

glutamate

GABA

peptides

photolysis of caged glutamate
Photolysis of Caged Glutamate

UV photon

Caging compound

Glutamate

Requirements of caging compounds/ systems:

Masks the biomolecule completely

Releases the biomolecule completely

No deleterious by-products

No biomolecule breakdown

http://flavor.monell.org/%7Eloweg/Techniques.htm

photolysis and recording set up
Photolysis and Recording Set-up

Beam can be moved in

X-Y plane and in

Z-plane for discrete anatomical

placement

Or discrete channels can be placed

with the compound

http://flavor.monell.org/%7Eloweg/Techniques.htm

slide40

Discrete Release of Caged Compound

Lowe, G. J Neurophysiol 88: 64-85 2002

slide41
Flash photolysis reveals a diversity of ionotropic glutamate receptors on the mitral cell somatodendritic membrane.

Lowe G.

J Neurophysiol. 2003 90(3):1737-46.

Question: Are glutamate rececptors located on the mitral cell soma/ proximal dendrites?

slide42

Glutamate elicts fast and slow currents

Photolysis at arrow on soma/

proximal dendrite

NBQX= AMPA antagonist

APV= NMDA antagonist (comp)

dCK= NMDA glycine site

antagonist (comp)

Mg++ -free bath

Lowe, G. J Neurophysiol 90: 1737-1746 2003;

doi:10.1152/jn.00180.2003

slide43

Competitive inhibition of NMDA current

Lowe, G. J Neurophysiol 90: 1737-1746 2003;

doi:10.1152/jn.00180.2003

slide44

Fast Glutamate Current = AMPA?

All slow glutamate

currents blocked

Fast current remains

CTZ= AMPA postitve

allosteric

modulator

Lowe, G. J Neurophysiol 90: 1737-1746 2003;

doi:10.1152/jn.00180.2003

slide45

Fast Glutamate Current = a little Kainate?

All slow glutamate

currents blocked

Fast current remains

SYM2206= Kainate

blocker