Precise Inhibition of Individual Neurons
Scientists can learn about the functions of specific neurons in networks, like the
brain using proteins that bind natural or synthetic photoswitches. Light can turn these
photoswitches on and off, allowing the control of activity in specific cells and thus
observation of how processing and behavior are altered by defined neuronal populations.
Processes happen fast in neurons, so speed can be critical for some
studies. The bacteriorhodopsin and HaloR families are the only lightactivated
inhibitory proteins with millisecond resolution. However,
they aren't without room for improvement. Both require continuous
illumination to stay activated, and continuous light can cause problems
such as rebound excitation or eventual partial inactivation. Also,
HaloR can require high light intensities because the magnitude of the
current is driven by the constant activation of the pumping cycle.
Thus researchers led by Ehud Y. Isacoff from the University of
California, Berkeley and the Lawrence Berkeley National Laboratory
designed a ligand-gated ion channel that is K+ selective and
controlled with light. Called HyLighter, the ion-channel isn’t active
until exposed to light, is more sensitive to light than HaloR, and can
reach its maximum current over a wide range of light intensities.
The light-activated ion channel can convert a light pulse into a stable
hyperpolarizing current that stays on with no illumination until it
is turned off using a complementary wavelength. It is based on an
ionotropic glutamate receptor (iGluR). These cation channels mediate
excitatory neurotransmission in higher organisms and are permeant
to Na+ and K+.
To create HyLighter, the researchers first engineered chimeric iGluRs
with the ligand-binding domain of iGluR6, the photoswitched tethered
ligand attachment site that encodes light sensitivity in the excitatory
LiGluR channel, and the pore of sGluR0, an exotic bacterial iGluR
homologue. They experimented with the various chimeras they
created and found that one had all the characteristics they needed. It
functioned as a light-gated K+ channel, was maximally activated by
low light intensity at wavelengths ~390 nm, maintained activity in the
dark, and could be turned off with 500 nm light.
They tested HyLighter by transfecting cultured hippocampal slices
from early postnatal rats with HyLighter-GFP using biolistic gene
transfer. It was expressed well in all regions of the hippocampus and
homogenously distributed in all parts of the neuron. They activated
HyLighter with 390 nm illumination from a Lambda DG-4 light
source coupled to the microscope and projected onto the sample
through the Mosaic DMD through a 40X objective.
"Among the existing microscopy solutions, only the Mosaic allows
you to continuously illuminate an arbitrary mask in the imaging field.
You simply can't do this using laser-scanning microscopes, which
never provide true simultaneous illumination. This important feature
combined with ease of use and direct interfacing with software makes
the Mosaic a unique and valuable tool for the optogenetic community,"
said Dr. Harald Janovjak, who was part of the research team and is
now at the Institute of Science and Technology Austria (IST Austria).
In the hippocampal slices, HyLighter induced strong hyperpolarization
that could silence neuron firing until deactivated by 500 nm light. At
the sample, the light intensity was approximately 20 mW mm-2 at 390
nm and 40 mW mm-2 at 500 nm.
The researchers also generated transgenic zebrafish expressing
HyLighter. When the tails of these fish received 390 nm, the probability
of an escape response after a mechanical stimulus was reduced. This
was reversed by 500 nm illumination. The illumination had no effect
on escape responses in zebrafish not expressing HyLighter.
The researchers look forward to HyLighter emerging as an important
tool for the optogenetic control of neuronal activity. "When it comes
to silencing of nerves cells, bi-stability and the requirement for small
amounts of light make HyLighter unique," said Dr. Janovjak.
- Olympus IX70 for electrophysiology
- Zeiss AxioExaminer for imaging
- Axopatch and P-Clamp (Molecular Devices Inc.)
was used for patch-clamp voltage recording
- Mosaic DMD and software for targeted inhibition
of neuronal firing
- Sutter DG4P light source for Mosaic illumination
- CSU laser spinning disk used to acquire confocal
images of neurons