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Uncaging Compunds:. Stimulating Neurons with Light & Electrophysiology. What is uncaging?. Caged compounds are biologically active molecules that are made inactive by the addition of light sensitive caging groups.

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Uncaging compunds

Uncaging Compunds:

Stimulating Neurons with Light & Electrophysiology

What is uncaging
What is uncaging?

  • Caged compounds are biologically active molecules that are made inactive by the addition of light sensitive caging groups.

  • When illuminated by UV light (photolysis), the caging group absorbs a photon, resulting in a breakage of a covalent bond linking it to the rest of the molecule.

    • END RESULT: Activation of cells with highspatial and temporal resolution!

Caged compounds
Caged Compounds

  • Caging groups can be synthetically added onto neurotransmitter, second messengers, and peptides.

  • Commercially caged compounds are available for:

  • Good caged compounds must possess several properties:

    • Minimal interaction with biological system of interest in inactive state

    • Product of photolysis reaction should not affect the system

    • A caged compound must release ligand efficiently and quickly in response to UV illumination (and not other times)

      • Uncaging index (next slide)

  • ATP

  • GABA

  • NMDA

  • Glutamate

  • IP3

  • Ca2+

  • Nitric Oxide

Application of compound
Application of Compound

  • High concentrations of the uncaging compound are applied to the preparation for long periods of time (recirculating bath with peristaltic pump)

    • To avoid spontaneous uncaging reduce exposure to ambient light, and keep in ice

    • Reduce uncaging by visualizing specimen with infrared differential interference contrast (IR-DIC) imaging

    • Double-caging compounds also minimizes accidental uncaging

Uncaging setup
Uncaging Setup

  • Brief pulse of UV light (whole field uncaging)

    • UV flashlamp mounted to optical port of microscope

    • Advantages: temporal resolution, low cost, simplicity

    • Disadvantages: low spatial resolution (>50μm), lamp generates electrical discharge which interferes with electrophysiological recording

Uncaging setup1
Uncaging Setup

  • Focal uncaging system using laser

    • Uses system of mirrors to focus laser beam through objective

    • Advantages: temporal & spatial resolution (diffraction limit of light)

    • Disadvantage: expensive, complicated

      • Types of lasers:

      • Q-switched

      • Attenuation of high-energy pulses: Pockels cell

  • nitrogen

  • frequency-doubled ruby

  • argon

  • neodymium-doped yttrium

Recent developments
Recent Developments

  • Optical two-photon uncaging

    • Cage group absorbs two photons of IR light of similar energy to one UV uncaging photon

    • Pulsed IR laser on two-photon microscope

      • Imaging beam used for uncaging

    • Advantage: IR light scatters less than UV light, minimal phototoxicity, allows imaging deep in living tissue, suppression of background signal

    • Disadvantage: high cost

  • Chemical two-photon uncaging

    • Addition of a second inactivating cage group to molecule of interest

      • Requires absorption of two UV photons

      • Very focalized, reduces uncaging of compounds above or below focal point.

    • More dissimilar to native compounds, and easier to handle.

Two photon microscopy
Two-photon microscopy

  • Objects can be selectively visualized and activated in slice or in vivo

  • Genetically encoded fluorescent protein tagging elucidates the spatial distribution and dynamics of numerous proteins of interest

    • Allowing the labeling of specific cell populations

  • Optical Microscopy can resolve single synapses

  • Downfalls of microscopic methods which 2P microscopy avoids:

    2PE allows high-resolution and high-contrast fluorescence microscopy deep in the brain & minimizes photodamage.

  • Wide-Field Fluorescence

  • Strong scattering

    • Scattering: bending of light in random ways when in complex tissue.

  • Confocal

  • Scanning damages specimen

  • Deep tissue; phototoxicityphotobleaching

  • Difficulty detecting single photon from excitation events

When is uncaging useful
When is uncaging useful?

  • Electrically stimulating neurons to manipulate neuronal activity is typically done with electrodes

    • Mechanical damage to tissue

    • Poor spatial resolution

    • Stimulation at multiple sites requires multiple electrodes

    • Difficult to stimulate isolated somata/cell

  • Uncaging is useful in slice physiology involving…

    • Multisite activation of neural circuitry

    • Intracellular signaling

    • Dendritic spine physiology…

Locally dynamic synaptic learning rules in pyramidal neuron dendrites

Locally dynamic synaptic learning rules in pyramidal neuron dendrites

Christopher D. Harvey & KarelSvoboda. Nature, December 2007.

Synaptic transmission plasticity
Synaptic Transmission & Plasticity dendrites

  • Synapses: “The tiny junctions between neurons that underlie your perception of the world, as well as the places where memories are stored in the brain.”*

  • Structure in the neuropil consisting of presynaptic terminal opposed to a dendritic spine, which is a hair-like structure coming off the postsynaptic dendrite.

    • Action potentials (Aps) propegate though the axonal arbor and where axons and dendrites overlap in the neuropil a synapse sometimes forms, and synaptic transmission occurs when APs reaches the synapse.

    • Action potentials invade the presynaptic terminal causing glutamate to be released and then to bind onto receptors on the postsynaptic spine.

    • 1:1 correspondence between spines and presynaptic terminals

    • Neurons have about 10,000 inputs and outputs

Karel Svoboda

Input specificity in ltp
Input Specificity in LTP dendrites

  • Long-term potentiation (LTP) is believed to be critical for learning and memory.

  • May be input specific, so synapses may function as independent units of plasticity.

  • Spine size is believed to be correlated with synaptic strength

  • Potential for co-regulation by neighboring synapses as LTP spreads.

    • Heterosynapticmetaplasticity: LTP at one synapse may increase threshold for potentiation at other synapses.

    • Clustered plasticity: Neighboring synapses to recently potentiated synapses show a decreased threshold for potentiation.

      Probe for between synapse crosstalk:

  • uEPSC + spine volume using 2 photon glutamate uncaging

  • Measure time-window of STDP protocol

    • Synaptic stimulation + uncaging

  • Elucidate crosstalk characteristics using uncaging

Advantages of optical methods
Advantages of Optical Methods dendrites

  • Classical ways of studying brain slices is with an electrical stimulating electrode.

    • Electrical stimulus evokes synchronous action potentials in the presynaptic axon, and one then records postsynaptic currents.

  • Limitations:

    • These events combine both presynaptic and postsynaptic factors, such as the amount of Glu released, or the number of receptors activated.

    • Synaptic activity is measured at the level of populations (~12 synapses), with synapses acting in chorus. This washes out the single synapse component, which can be mechanistically valuable.

Hestrin et al. 1990

Methods dendrites

  • Thy1 GFP mice (line M; P 14-18)

  • 2PE uncaging: 2.5mM MNI-caged-L-glutamate

  • 2PE microscopy: Two Ti:sapphire lasers

    (910 nm for GFP)

    (720 nm for uncaging)

  • Various LTP protocols

Crosstalk between plasticity at nearby synapses
Crosstalk between plasticity at nearby synapses dendrites

  • Dendritic spines were visualized on apical dendrites of CA1 pyramidal neurons (proximal, secondary and tertiary) in a GFP expressing transgenic mouse.

  • Glutamate receptors on individual spines were stimulated using two-photon glutamate uncaging.

  • Uncaging-evoked excitatory postsynaptic currents (uEPSCs) were measured at the soma using perforated patch-clamp electrophysiology.

  • Postsynaptic cell was held at 0mV (depolarized), to ensure NMDA receptor mediated Ca2+ influx, which needs synchronous depolarization and glutamate binding.

Ltp protocols
LTP Protocols dendrites

  • Pair train of 30 stimuli (0.5hz, 4 ms) with postsynaptic depolarization to ~0mV.

    Uncagingstimulus elicits a NMDA-R mediated spine [Ca2+] accumulation similar to other protocols of LTP induction (tetanic).

  • Glutamte activation was restricted to specified spines as indicated by the absence of spreading [Ca2+]accumulation (sup figures 1a-c)

  • Plasticity was measured by increase in spine size and test stimuli evoked uEPSC.

  • Uncaging at 4ms pulses dendrites

  • Result: Increase in uEPSC amplitude and spine volume at LTP spine, but not nearby spines.

30 uncaging pulses at 0.5 Hz

Depolarization to ~0mV, 2 mM Ca2+ , 1 mM Mg2+ , and 1mM TTX.

Crosstalk between plasticity at nearby synapses1
Crosstalk between plasticity at nearby synapses delivered to a neighboring sprine 90s later

  • Crosstalk did not occur after application of LTP protocol with cell held at -70mV. LTP was not induced in this case, therefore it’s LTP induction that causes crosstalk, not glutamate uncaging.

Therefore, LTP induction at one synapse results in a lowering of LTP threshold for an adjacent spine.

Unperturbed Neurons delivered to a neighboring sprine 90s later

Remove sustained postsynaptic depolarization (at 0mMg2+)

  • B: persistent spine enlargement

  • C: transient spine enlargement

  • D: sustained spine enlargement in neighboring spine

    Persistent postsynaptic depolarization is unnecessary for observing crosstalk in plasticity between synapses.

30 uncaging pulses at 0.5 Hz

4 mM Ca2+ , 0 mM Mg2+ , and 1mM TTX.

Crosstalk with synaptically induced plasticity
Crosstalk with synaptically induced plasticity delivered to a neighboring sprine 90s later

  • Compared to synaptically released glutamate, glutamate released by uncaging might be activating a distinct set of receptors

  • To compare uncaging and synaptically induced crosstalk:

    Schaffer collateral axons were stimulated (120 pulses, 2Hz) in low extracellular Mg2+, 2 min later followed by subthresholduncaging LTP of a neighboring spine.

    Result: The combination of synaptic stimulation with subthreshold LTP uncaging protocol brought upon a persistantspine enlargement.

Modulation of the window for stdp
Modulation of the window for STDP delivered to a neighboring sprine 90s later

  • EPSPs followed by action potentials with a brief time window can trigger LTP

  • Does crosstalk broaden the time window for STDP at neighboring spines?

  • STDP:

    • Uncaging pulses (60, 2Hz), 3 action potentials(50Hz, 5ms)

      = Long lasting increase in uEPSCs and spine volume, but not on neighboring spines

    • As timing between uncaging and action potentials increased, STDP was not observed.

    • First STDP protocol repeated, followed 90s later by uEPSP-action potential interaval of 35ms

      = 35-ms time window now induced STDP in neighboring spines to STDP synapses.

Characterization of crosstalk
Characterization of crosstalk delivered to a neighboring sprine 90s later

  • Volume change experienced by the sub spine was measured as distances and time between the LTP and sub-LTP uncaging protocol was carried out.

Characterization of crosstalk1
Characterization of crosstalk delivered to a neighboring sprine 90s later

  • Is the heterosynaptic spread of LTP due to extracellular or intracellular diffusible factors?

  • Can crosstalk occur between cells that are close within the neurpil but are located on different dendrites (on the same cell)?

  • Induce LTP on one spine, and 90s later induce sub-LTP on a spine <4µm away on a different dendrite from the same cell.

    Result: Failed to induce LTP, therefore intracellular factors are responsible for synaptic crosstalk.