A new way to select and switch on one cell type in an
organism using light has helped answer a long-standing question about the function
of one class of enigmatic nerve cells in the spinal cord.
Through targeted insertion of light-sensitive switches into
these cells in awake zebrafish larvae, University of California, Berkeley, and
UC San Francisco scientists have found that these mysterious cells trigger
burst swimming – the periodic tail twitching typical of larvae.
A short pulse of ultraviolet light followed by green light triggers burst swimming in a genetically engineered zebrafish larva expressing a light-gated channel in a specific cell type: the KA neuron. This is a still from a video on UC Berkeley’s Web site which proves that these cells, whose function had remained unknown for decades, are responsible of inducing spontaneous swimming activity. (Isacoff lab/UC Berkeley)
While the finding could have implications for humans,
because mammals have similar cells protruding into the spinal fluid, the
discovery highlights the power of new techniques that employ photoswitches –
light-gated ion channels – and gene targeting to non-invasively turn on small
populations of cells as easily as flipping a light switch.
Claire Wyart, post-doctoral fellow at UC Berkeley's
Department of Molecular and Cell Biology, and UCSF post-doctoral fellow Filippo
Del Bene are the joint first authors of a paper describing these results that
appears in the Sept. 17 issue of the journal Nature.
"With these optogenetic tools, we can activate single
neurons in awake behaving animals and directly demonstrate the consequence of
neuron activation on behavior," said Wyart. "This 'optogenetic'
approach enabled us to learn something important about spinal circuits."
Wyart said that the strategy used here could be generalized
to study all types of neurons, such as those in the smell, vision, touch and
hearing centers of the brain.
"Optogenetics opens up a new and extremely exciting
area of study, singling out one type of cell and finding out what it's
doing," she said.
"This is a new way to do neuroscience," said
coauthor Herwig Baier, professor of physiology at UCSF. "Instead of
sticking electrodes into the brain to record and monitor activity in the
nervous system, what we are doing is manipulating the function of neurons
noninvasively with light, the gentlest way to make a manipulation."
"With these optically sensitive channels, it becomes
possible to play back to the nervous system its normal innate activity and see
what behavior results," added co-author Ehud Isacoff, UC Berkeley
professor of molecular and cell biology.
Other coauthors of the Nature paper, in addition to senior
authors Isacoff and Baier, are former UC Berkeley chemist Dirk Trauner, now at
the University of Munich; Erica Warp, a graduate student in Isacoff's UC Berkeley
lab; and Ethan Scott from Baier's UCSF laboratory. Scott is now at the University of Queensland
in Brisbane, Australia.
Trauner, along with Isacoff and Richard Kramer, UC Berkeley
professors of molecular and cell biology, worked for more than six years to
perfect the technique of inserting optical switches into cells, and they formed
the Nanomedicine Development Center for Optical Control of Biological Function
to spearhead applications. One of the long-term goals of the joint UC
Berkeley-Lawrence Berkeley National Laboratory center, which is funded by the
National Institutes of Health, is to insert photoswitches into retinal cells to
restore vision.
So far they have succeeded in engineering light-sensitive
potassium ion channels and glutamate receptors to turn neurons on when zapped
by ultraviolet light and turn the neurons off when zapped by green light, or
vice versa. The researchers achieve this by attaching to the channel a
chemical, called azobenzene, that changes shape when hit by light, opening or
closing the ion channel. When the potassium channel opens, potassium ions flow
through it and inhibit the cell; when the glutamate receptor channel opens,
sodium, potassium and calcium ions flow through it and excite the cell.
Much of the early optogenetic work has confirmed results
suggested by other approaches. Wyart and her UCSF and UC Berkeley colleagues
have now applied the technique to search for a behaviorally relevant cell and
found, to their surprise, a previously unknown function for the Kolmer-Agduhr
(KA) cells in the spinal cord. The KA cells aren't standard relay neurons with
dendrites and axons, but sensory neurons with cilia – small, movable hairs –
that protrude into the spinal fluid, plus long axons extending up the spinal
cord. They evidently sense something, but what, the researchers wondered.
Wyart and Isacoff teamed up with Baier's laboratory, where
Del Bene and Scott produced 10 strains of zebrafish with photoswitches inserted
in specific spinal cord nerve cell populations. When Wyart shined light on the
fish with photoswitches in their KA neurons, the fish waggled their tails in a
manner that exactly mirrored spontaneous slow forward swimming. Placing the
transparent zebrafish larvae under a microscope, Wyart used a Digital Micromirror
Device (DMD) to strongly focus light onto a small number of KA neurons,
successfully switching on only a few KA cells at a time. She found that she had
to switch on about 10 of the KA neurons to trigger swimming.
Knocking out these cells greatly reduced burst swimming, but
did not eliminate it, suggesting that the KA neurons may be lowering the
threshold for triggering reflex swimming.
"It came as a great surprise that these neurons played
a role in locomotion at all," said Isacoff. "There is an apparent
homologue of the KA neuron in mammals, so this may be a general modulatory
principle for vertebrate locomotion, although it may change from positive drive
early in development to negative drive later."
Earlier studies in lampreys by Sten Grillner, a professor in
Stockholm at
the Karolinska Institute's Nobel Institute for Neurophysiology, showed that
nerve cells – including KA neurons – using GABA (gamma aminobutyric acid) as an
inhibitory neurotransmitter were important modulators of swimming. The current study
narrows this down to one kind of GABAergic neuron: the KA neuron.
Wyart continues to explore the role of KA neurons, but hopes
to exploit the new optogenetic and gene targeting techniques to discover the
roles of other types of neurons in the spinal cord.
Other researchers have developed an alternative optogenetic
approach – inserting the gene for a light-sensitive ion channel isolated from
algae – that also shows promise for directly showing the behavior triggered by
activating cells. In fact, it is easier to use, though not as flexible as the
approach developed by Isacoff, Trauner and Kramer, Baier said.
"Optogenetic targeting is a powerful approach, and we
have really only started the work," he added. "We still have to learn
how the KA neurons are connected to drive the muscles. Really, there is no way
we could have done this experiment other than with optogenetics."
The work was supported by the National Institutes of Health.
Original
article, with video
SOURCE: Univ. of California, Berkeley
