nm wavelength and an average power
of 30 m W for each spot. The neuron
depolarizes in response to uncaging; the
amplitude depends on the total uncaging
laser energy and the number of uncaging
sites chosen. With sufficient depolarization, the neuron may generate a series of
outputs called action potentials.
Our method opens up new prospects for studying neuronal synaptic
integration in 3-D. Future work
on the dynamics and principles of
neuronal circuitry will rely heavily
on optical methods to manipulate
neuronal activity and to selectively
excite subsets of neurons.
Information processing in brain circuits is achieved via synaptic transmission
of neurotransmitters between neurons.
Synaptic inputs to a neuron are distributed
along its entire dendritic tree, which
extends spatially in 3-D. As the receiving
neuron processes its inputs, it sends an
output by firing an action potential, thereby
propagating its processed information to
other neurons. How a particular neuron
processes its inputs to arrive at an output
remains a complex process that is not yet
fully understood. Technological advancements in laser optics have greatly improved
the methods for probing this process
and overcoming technical limitations of
standard electrophysiological systems.
Photochemical compounds facilitated
the development of optical methods that
emulate synaptic inputs to the neuron. 1–3
Our approach incorporates a 3-D
holographic laser projector into a two-photon microscope. 4 The projector generates multiple laser foci used to stimulate
the synaptic inputs. The accompanying diagram shows three projected foci at different
depths across the neuron’s dendritic arbor.
At each site, two-photon photolysis releases
the caged neurotransmitters (glutamate),
which bind to ionotropic ligand-gated ion
channels causing an excitatory postsynaptic potential in the neuron.
Prior to uncaging, we render the 3-D
morphology of a neuron in a 300-µm-thick
slice of rat somatosensory cortex. The
cell is loaded with Alexafluor-488 via a
patch pipette. From the neuron image, we
determine the uncaging locations along the
dendritic tree and apply an MNI-caged glutamate bath ( 3 mM effective concentration).
Uncaging is performed using short pulses
( 2 ms) of the incident laser operating at 720
(a) Holographic projection generates multiple foci. (b) Multi-foci uncaging of MNI-caged glutamate. Glutamate is released and binds to ionotropic ligand-gated ion
channels. (c) 3-D projection of layer II/III neuron using two-photon microscope. (d)
Magnified view. (e) Uncaging-evoked voltage responses to foci locations indicated (d).
Mary Ann Go,
and Vincent R. Daria
Australian National University,
1. E. Callaway et al. Proc.
Natl. Acad. Sci. U.S.A.
90, 7661 (1993).
2. S. Shoham et al. Nat.
Methods 2, 837 (2005).
3. C. Lutz et al. Nat.
Methods 5, 821 (2008).
4. M.A. Go et al. J. Biophotonics 5, 745 (2012).
3-D Light Patterns for Spine-Targeted
Probing of Neuronal Function
3-D light fields
Laser (a) (b)
(d) (e) (c)