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 – – – – 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+) • • • 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.