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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
  • 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
  • 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
  • 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
Methods
  • 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
  • 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
  • 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.
slide20
Uncaging at 4ms pulses
  • 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.

slide21
Subthreshold protocol: similar to original protocol but with a shorter uncaging duration (1ms).
  • Result: No change in uEPSC amplitude or spine volume in both specified and neighboring spines.
slide22
LTP induced at spine, with a subthreshold induction delivered to a neighboring sprine 90s later
  • Result: Subthreshold induction now triggers LTP and long-lasting spine enlargetment.
crosstalk between plasticity at nearby synapses1
Crosstalk between plasticity at nearby synapses
  • 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.

slide24
Unperturbed Neurons

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
  • 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
  • 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
  • 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
  • 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.

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