Transcript Uncaging Compunds: - Florida State University

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.
• 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 high spatial and
temporal resolution!
Caged Compounds
• Caging groups can be synthetically added onto neurotransmitter, second
messengers, and peptides.
• Commercially caged compounds are available for:
•ATP
•GABA
•NMDA
•Glutamate
•IP3
•Ca2+
•Nitric Oxide
• Good caged compounds must possess several properties:
1. Minimal interaction with biological system of interest in inactive state
2. Product of photolysis reaction should not affect the system
3. A caged compound must release ligand efficiently and quickly in response to UV
illumination (and not other times)
•
Uncaging index (next slide)
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
• 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 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:
•nitrogen
•frequency-doubled ruby
•argon
•neodymium-doped yttrium
• Q-switched
• Attenuation of high-energy pulses: Pockels cell
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
• 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:
Wide-Field Fluorescence
• Strong scattering
Scattering: bending of light in random ways when in complex tissue.
Confocal
• Scanning damages specimen
• Deep tissue; phototoxicity photobleaching
• Difficulty detecting single photon from excitation events
2PE allows high-resolution and high-contrast fluorescence
microscopy deep in the brain & minimizes photodamage.
When is uncaging useful?
• Electrically stimulating neurons to manipulate neuronal
activity is typically done with electrodes
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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
Christopher D. Harvey & Karel
Svoboda. Nature, December 2007.
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
• 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.
– Heterosynaptic metaplasticity: 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
• 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
• 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
• 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 twophoton 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
• Pair train of 30 stimuli (0.5hz, 4 ms) with postsynaptic depolarization to
~0mV.
Uncaging stimulus 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
• 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.
• 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.
• 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 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.
Unperturbed Neurons
Remove sustained postsynaptic
depolarization (at 0mMg2+)
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•
•
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
• 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 subthreshold uncaging
LTP of a neighboring spine.
Result: The combination of synaptic stimulation with
subthreshold LTP uncaging protocol brought upon a persistant
spine enlargement.
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
• 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 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.