Presentation on theme: "Uncaging Compunds: Stimulating Neurons with Light & Electrophysiology."— Presentation transcript:
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: 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) ATP GABA NMDA Glutamate IP 3 Ca 2+ Nitric Oxide
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: Q-switched Attenuation of high-energy pulses: Pockels cell nitrogen frequency-doubled ruby argon neodymium-doped yttrium
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: 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; phototoxicity photobleaching Difficulty detecting single photon from excitation events
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 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 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 Ca 2+ 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 [Ca 2+ ] accumulation similar to other protocols of LTP induction (tetanic). Glutamte activation was restricted to specified spines as indicated by the absence of spreading [Ca 2+ ]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 Ca 2+, 1 mM Mg 2+, 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 0mMg 2+ ) 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 Ca 2+, 0 mM Mg 2+, 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 Mg 2+, 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.