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New optical tools for controlling neuronal activity
Stefan Herlitze and Lynn T Landmesser
Corresponding author: Herlitze, Stefan ([email protected])
This review comes from a themed issue on
Development
Edited by Ben Barres and Mu-Ming Poo
Available online 15th December 2006
or
's
0959-4388/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.conb.2006.12.002
Introduction
Au
th
In cells, light is used for two main purposes: first, to
produce energy through photosynthesis; and second, to
couple extracellular stimuli to intracellular signaling pathways, such as enzymes, ion transporters or ion channels.
The second use is particularly interesting to us because
switching on and off ion transporters, ion channels or
second messenger pathways by light provides an opportunity to control non-invasively the membrane potentials
and second messenger cascades in a living animal.
Recently, it has been demonstrated that neuronal circuits
can be manipulated through the exogenous expression of
mutated ion channels and G-protein-coupled receptors
(GPCRs). Here we review recent progress made in this
area and discuss the specific shortcomings of some of the
methods proposed.
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Invertebrate rhodopsins
During activation of the invertebrate visual system, the
excited GPCR (rhodopsin) stimulates a Gq/11 protein,
which then activates phospholipase C, leading to the
production of inositol (1,4,5)-trisphosphate and diacylglyercol. These second messengers activate non-specific
cation channels, which depolarize the cell. The G protein
signal is turned off when arrestin binds to the GPCR to
initiate regeneration of 11-cis retinal, the ligand of the
photoreceptor.
al
pe
Current Opinion in Neurobiology 2007, 17:87–94
In the visual system of invertebrates and vertebrates, light
of different frequencies can activate a molecular cascade
leading to the depolarization of a nerve cell in invertebrates
or the hyperpolarization of a nerve cell in vertebrates.
The potential for the invertebrate light cascade to depolarize cells outside their normal environment was first
recognized by Miesenbock and colleagues [1]. By expressing ten different proteins of the invertebrate light cascade, they identified the three minimal structural
components of an invertebrate GPCR light-induced
cationic current. The three proteins that were necessary
and sufficient were the GPCR (NinaE), the G protein aq
subunit and arrestin-2. Exogenous expression of these
three proteins, collectively called chARGe, in hippocampal neurons was found to induce action potential firing
(spiking) after light activation. The technique, however,
had some limitations.
rs
Addresses
Department of Neurosciences, Case Western Reserve University,
10900 Euclid Avenue, Cleveland, Ohio 44106-4975, USA
Visual system GPCRs for slow control of
nerve cell activity
on
A major challenge in understanding the relationship between
neural activity and development, and ultimately behavior, is to
control simultaneously the activity of either many neurons
belonging to specific subsets or specific regions within
individual neurons. Optimally, such a technique should be
capable of both switching nerve cells on and off within
milliseconds in a non-invasive manner, and inducing
depolarizations or hyperpolarizations for periods lasting
from milliseconds to many seconds. Specific ion
conductances in subcellular compartments must also be
controlled to bypass signaling cascades in order to regulate
precisely cellular events such as synaptic transmission.
Light-activated G-protein-coupled receptors and ion channels,
which can be genetically manipulated and targeted to neuronal
circuits, have the greatest potential to fulfill these
requirements.
First, the slow activation and deactivation of neuronal
firing made it difficult to control precisely the firing
pattern of the neuron. In addition, the precise cellular
mechanism by which these constructs regulated neuronal
firing was not investigated. The induction of neuronal
firing might be mediated through a decrease in K+ conductance, for example, by KCNQ channels, which are
inhibited by activation of Gq-coupled receptors. If this is
the case, then chARGe would have to be expressed in
neurons with sufficient M-currents to be able to affect
neuronal firing. Second, the construct is very complex,
making it difficult to express as a transgene in other
animals [1]. As an alternative to invertebrate rhodopsin,
melanopsin, a photopigment of retinal ganglion cells,
could be used. Three independent studies have recently
shown that vertebrate melanopsins couple to the Gq
pathway and can, for example, activate transient receptor
potential (TRP) channels [2–4]. Whether or not this
approach will work in neurons remains to be demonstrated.
Current Opinion in Neurobiology 2007, 17:87–94
88 Development
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Indirect photoactivation: ligands and channel blockers
In the first approach, chemical compounds were synthesized for the light activation of two ligand-gated ion
channels: the capsaicin receptor TRPV1 and the purinergic receptor P2X2 [12]. Mammalian TRPV1 had previously been used to confer capsaicin avoidance
responses in Caenorhabditis elegans, which is normally
not responsive to capsaicin [13]. Ester linkage of an
inactive, photosensitive molecule to the active ligand
facilitated activation of the ligand and channel by 1 s
pulses of short wavelength (<400 nm) light in neurons to
trigger action potential firing with some delay. According
to the dose–response relationship of the current response,
this method enabled the firing frequencies to be adjusted
by the application of different concentrations of ligands.
This approach would thus be very useful for inducing
different firing patterns during constant application of
light and/or ligand.
rs
Indeed, other Gi/o-coupled receptors have been recently
used to silence neuronal firing. The silencing of cortical
and thalamic neurons from various species, including rats,
ferrets and monkeys, was achieved by exogenously
expressing the insect GPCR AlstR (the Drosophila allatostatin receptor). Application of its peptide ligand allatostatin in neurons expressing the AlstR was found to
inactivate the neurons reversibly within minutes [8,9!].
the tool of choice for rapid control of nerve cell activity.
The regional expression of a genetically modified K+
channel in Drosophila provided the first example of using
an ion channel to control the excitability of the targeted
cell (i.e. muscle, neurons, photoreceptors). Unfortunately, at higher transgene dosages the expression of
these channels led to the loss of neuronal function; thus,
the extent of excitability of the targeted cell could not be
controlled during the experiment [11]. This problem has
been overcome by the development of photoactivated
(caged) chemicals, which are used as ligands or blockers to
increase or to decrease ion conductances once activated
by light.
al
In vertebrates, light activation of the GPCR rhodopsin
leads to dissociation of the G protein and subsequent
activation of a phosphodiesterase, which hydrolyzes cyclic guanosine monophosphate (cGMP) to 50 GMP. The
reduction in cGMP induces the closure of cGMP-gated
cation channels, resulting in hyperpolarization owing to a
reduction in Na+ and Ca2+ influx [5,6]. Vertebrate rhodopsin was found to couple to the G protein transducin.
Because the a subunit of transducin belongs to the Gi/o
subfamily [7], it seemed possible that the mammalian
rhodopsins might couple to other members of this subfamily. Activation of the Gi/o pathway in neurons usually
leads to a reduction in the firing rate through activation of
a G-protein-coupled inward rectifier K+ channel (GIRK)
and a reduction in synaptic transmitter release through
inhibition of presynaptic Ca2+ channels.
on
Vertebrate rhodpsins
Au
th
or
's
pe
We and our co-workers [10!!] also used this approach
when we expressed the vertebrate rhodopsin RO4
together with GIRK or Ca2+ channels in HEK293 cells,
which enabled us to demonstrate that the GPCR can
activate K+ currents and inhibit Ca2+ currents expressed
through recombinant constructs. Expression and light
activation of RO4 in cultured hippocampal neurons
hyperpolarized the cell membrane within a second, leading to a reduction in the firing rate of the neurons and
suggesting that, in neurons, RO4 activates GIRK currents
on somatodendritic areas. Analysis of the presynaptic
transmission parameter ‘paired pulse facilitation’
revealed that, once RO4 was activated, an increase in
paired pulse facilitation in hippocampal autapses was
induced, suggesting that RO4 was also transported and
was active at presynaptic sites. This result has interesting
implications for controlling Gi/o pathways in defined
subcellular structures by targeting the light-activated
rhodopsin in the cell area of choice. Thus, light-activated
GPCRs of the rhodopsin family can be used to increase
neuronal activity when coupled to the Gq pathway and to
decrease activity when coupled to the Gi/o pathway.
Ion channels for rapid control of nerve cell
activity
Activation and deactivation of GIRK channels by pertussis toxin (PTX)-sensitive G proteins in neurons occur
within one to several seconds. By contrast, ligand-gated
and voltage-gated ion channels can be activated within
microseconds, making the direct gating of ion channels
Current Opinion in Neurobiology 2007, 17:87–94
The second approach was based on the photoconversion
of a K+ channel blocker [14]. This blocker is in the
stretched trans configuration at longer wavelengths
(500 nm) and is converted to a more bulky cis form at
shorter wavelength (380 nm). The stretched form can
enter the open pore and reduce the K+ conductance,
whereas the bulky form cannot. The experiments were
done with the shaker K+ channel, a voltage-gated channel, and were based on the idea that molecules containing
a quaternary ammonium, such as tetraethylammonium,
block the channel when entering from the extracellular
site. A glutamate to cysteine substitution in proximity of
the tetraethylammonium-binding site facilitated tethering of a photoactivated compound containing the quaternary ammonium ion MAL-AZO-QA (MAL is
malemide for attaching the molecule to the cysteine near
the channel pore, AZO is a photoisomerizable group, and
QA is the quaternary ammonium). By expressing the
modified shaker K+ channel in hippocampal pyramidal
neurons and loading the neurons with MAL-AZO-QA
15 min before recording, it was shown that switching the
wavelength between 500 and 390 nm could control action
potential firing in these neurons. At 500 nm, neuronal
activity was induced owing to blockage of the shaker K+
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Controlling neuronal activity Herlitze and Landmesser 89
channel, whereas at 390 nm neurons were silenced
because the blocker was released from the channel.
acids, has weak sequence similarity to bacteriorhodopsin
and halorhodopsin, and comprises seven putative transmembrane segments. Most importantly, expression of
ChR1 or carboxy (C)-terminally truncated ChR1 mRNAs
in the Xenopus oocyte expression system results in a robust
photocurrent with fast activation and deactivation
kinetics during light pulses. The current carrier is H+
and current activation is wavelength dependent. The
inward photocurrent in Xenopus oocytes is maximal at
"500 nm, and there is no detectable activation of ChR1 at
either 400 nm or 600 nm, raising the possibility of using
blue- and red-shifted variants of the photoreceptor as
probes for coactivation and/or colocalization. Furthermore, during light activation of the channel, the H+
current does not attenuate. Whether H+ conductances
can be used to depolarize neurons sufficiently to trigger
action potential firing remains to be demonstrated but
ChR1, with its fast activation and deactivation kinetics,
set the stage for using a light-activated channel to control
membrane potential.
co
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on
A breakthrough came with the cloning and electrophysiological characterization of channelrhodopsin-2 (ChR2)
[21]. ChR2 is also a proton channel, but it is non-selective
and permeable to various cations including Na+, K+ and
Ca2+. The ChR2 current has two components: a large
inactivating or desensitizing current, followed by a
steady-state current. The activation and deactivation
kinetics, the current size, and the ratio between the
inactivation current and the steady-state current are
dependent on the duration and intensity of the light
[10!!,22!]. This channel therefore provides the potential
to control precisely the amount of depolarization by
adjusting the light stimulation protocol. The ChR2 current is maximally activated around 480 nm and shows no
activation at wavelengths >575 nm [21].
or
's
pe
rs
In a similar approach, the ligand-binding domain of the
ionotropic glutamate receptor (iGluR-L439C mutation)
was modified to bind a photoactivated-glutamate-containing compound called MAG (M is the cysteine-reactive maleimide, A is the azobenzene photoswitch, and G
is the glutamate head group) [16!]. This mutated channel,
termed LiGluR, can be gated by both light and ligand.
The covalent attachment of MAG to the cysteine located
at the lip of the clam shell binding pocket of the GluR
facilitates stabilization of the closed and open state of the
channel, depending on whether the compound is in the
trans (closed channel) or cis (open channel) configuration.
Short wavelengths of light (380 nm) stabilize the cis
conformation, whereas 500 nm light stabilizes the trans
form. Although the biophysical designs of these experiments are very elegant, the modified channels must be
loaded with the light-activated blocker before the experiment for a lengthy amount of time. This drawback might
limit the application of this approach in terms of neurons,
but it has potential to be very useful for controlling cell
activity in other organs such as heart, where the compound can be applied through the blood stream at sufficient concentration.
y
Introduction of the point mutation V443Q in the selectivity filter converted the K+-selective shaker channel
into a non-selective cation channel [15!]. When expressed
in primary neuronal cultures, this channel could conduct
Na+ and thus depolarize the cell membrane. Action
potential firing was elicited once QA-mediated channel
block was released at 390 nm. These light-controllable K+
channels are now called H-SPARK and D-SPARK for
‘synthetic photoisomerizable asobenzene-regulated K+
channels and will, respectively, hyperpolarize and depolarize the cell membrane.
Au
th
In general, for ion channels and GPCRs activated by
chemical stimuli, the need for ligand application and
especially the time for wash out will limit the use of
these constructs in large tissues such as brain slices or
whole brain. The above examples demonstrate, however,
that expression of foreign genes in neurons or other cells
is a suitable method to non-invasively modulate the state
of a cell and thus to elucidate mechanisms underlying
neuronal development, neuronal circuit function, and
behavior.
Direct photoactivation: channelrhodopsin
In phototactic green algae, photoreceptor currents
induced by light are carried by Ca2+ or H+ [17] and are
activated within 30 ms [18,19]. One of the rhodopsinmediated photoresponses from Chlamydomonas reinhardtii
is mediated by a rhodopsin-like protein, the channelrhodopsin-1 (ChR1) [20]. This protein consists of 712 amino
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Three groups have independently tested the potential of
ChR2 to control nerve cell firing [10!!,22!,23!] and
demonstrated that, in both cultured hippocampal neurons
and hippocampal slices, short light pulses (5–10 ms) are
sufficient to trigger action potentials; in addition, neurons
can follow light stimulation protocols with action potential firing up to 30 Hz. These observations correlate well
with the deactivation of ChR2 within 20–40 ms [10!!,22!].
Thus, ChR2 is currently the molecular probe of choice to
control action potential firing in neurons. One problem
might be the small single channel conductance of ChR2,
which has been estimated at "50 fS. As a result, high
expression of ChR2 in the target cell might be necessary
to activate sufficient inward current to depolarize the cell
membrane.
Chloride conductance
As mentioned above, hyperpolarization within a cell can
be caused by increasing not only the K+ conductance but
also the Cl# conductance. Thus, another way to silence
Current Opinion in Neurobiology 2007, 17:87–94
90 Development
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RO4 and ChR2 in chicken spinal cord
al
We and our co-workers [10!!] have tested two constructs
for their capability to silence or to activate neuronal
circuits. Embryonic chicken spinal cord was electroporated with either vertebrate rhodopsin (RO4) or ChR2 to
try and control precisely the spontaneous firing of the
spinal cord motor neurons [10!!]. Embryonic chicken
spinal cords show rhythmic episodes of spontaneous
bursting activity, the frequency of which is important
for motor axon pathfinding. Spinal cord preparations were
incubated with all-trans retinal 2 min before the recording
to supply a sufficient amount of chromophore for either
RO4 or ChR2.
rs
The concept that controlling the Cl# conductance in
cultured hippocampal neurons could silence neuronal
activity was first demonstrated with an invertebrate
ligand-gated Cl# channel [30]. Coexpression of a and
b subunits of the glutamate-gated Cl# channel from C.
elegans in neurons was necessary to activate the channels
by application of the chemical compound ivermectin.
Light-activated Cl# transport also could be achieved with
halorhodopsin, which was first detected in the purple
membranes of Halobacterium salinarum. Halorhodopsin
is activated by light and is a Cl# transporter that carries
Cl# ions inward, thereby resulting in hyperpolarization
of the cell membrane. When halorhodopsin is excited,
Cl# binds to the extracellular site of the receptor [31].
Light activation leads to several conformational changes
in the protein, accompanied by an alteration in the
accessibility and affinity of the binding site, resulting
in release of Cl# at the intracellular surface of the cell
and subsequent restoration of the unexcited state of the
receptor [32]. Thus, light-activated Cl# transport provides another mechanism for hyperpolarizing cells and
could be of particular interest for developmental neuroscientists.
In the second application [33!!], P2X2 receptors were
expressed in Drosophila dopaminergic neurons to elucidate the modulatory function of dopamine on movement.
Four 150 ms UV light pulses, separated by 1.5 s, increased
locomotor activity in flies, as evaluated by travel speed
and pause duration. Interestingly, light-induced dopamine release also seemed to influence the flight routes,
suggesting that dopamine might be involved in controlling motivational behavior in flies. This study shows that
caged compounds, which activate ion channels, can be
used to control neuronal circuits in invertebrate systems.
on
neurons would be through an increase in Cl# conductance. The equilibrium potential of Cl# changes, however, during development. Although Cl#-based g-amino
butyric acid (GABA)-mediated currents in the hippocampus, neocortex, hypothalamus and spinal cord are depolarizing early in development, they are hyperpolarizing in
the adult animal [24–29]. Thus, an increase in Cl# conductance in adult mice will cause hyperpolarization rather
than depolarization.
pe
Light application increased the intervals between bursting episodes and decreased asynchronous firing of motor
units between episodes in spinal cords expressing RO4, as
would be expected if RO4 were causing hyperpolarization
of the cell membrane. Notably, switching off the light
often induced immediate bursting of the neuronal network, which suggests that the RO4 induced hyperpolarization acts to synchronize motor neurons most probably
by relieving the Na+ channels from inactivation. By
contrast, exposing cords expressing ChR2 to continuous
light (485 nm) increased the frequency of bursting of the
neuronal circuit and decreased the intervals between
episodes. The precise pattern of frequency bursts could
be controlled by brief, 3 s applications of light, each of
which elicited immediate bursts.
's
Light-activated switches in physiologically
relevant neuronal networks
or
So far three different light-activated proteins have been
used to control neuronal circuits in intact tissue and live
animals.
P2X2 receptors in Drosophila
Au
th
The first approach, involving the expression of the ATPgated P2X2 receptors in the Drosophila nervous system,
has been used to trigger action potentials for controlling
several escape-related behaviors and to investigate the
role of dopaminergic input for motor control [33!!]. Specific expression of the P2X2 receptors in the Drosophila
giant fiber system — a well-defined and well-characterized neuronal circuit responsible for the escape movements of jumping and initiation of flight — revealed that
short (150–250 ms) UV laser pulses were sufficient to
trigger flight behavior. Flight and jumping could be
induced in both blind and decapitated flies, supporting
the notion that the light-induced behavior was due to the
exogenous receptors. Flies had to be microinjected with
caged-ATP 10 min before the experiments and the efficacy of the response behavior had a half-life of "75 min.
Current Opinion in Neurobiology 2007, 17:87–94
To examine the possibility that ChR2 could be used to
control neuronal activity in intact animals, we also
assessed whether light could induce axial movement of
intact embryos in ovo. Depending on the developmental
stage, bursts of cord activity cause axial movements.
Light application through an egg shell window was sufficient to activate ChR2 and to induce movement of the
embryo. This observation indicates that a retinal derivative sufficient to act as a light-activated ligand is supplied
by the embryo or egg. This approach has potential applications. First, expression of RO4 and ChR2 in specific
subsets of spinal cord motor neurons and interneurons
could be used to understand the circuitry in the spinal
cord and the influence that rhythmic activity has on spinal
cord development. Second, in cases of spinal cord injury,
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Controlling neuronal activity Herlitze and Landmesser 91
light could be used either to non-invasively activate
regenerating descending input to determine the effect
of activity on regeneration, or to directly activate denervated neurons to prevent inactivity-induced changes.
co
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In addition, ChR2(H134R) was expressed in mechanosensitive neurons in C. elegans, with the result that light
activation of these neurons led to withdrawal responses
that are normally observed when the worms are tapped.
Interestingly, the Na+ and Ca2+ influx through
ChR2(H134R) could substitute for mechanosensitive
ion channels: worms carrying mutations in MEC-4 and
MEC-10 mechanosensitive ion channels are insensitive to
touch, but touch responses could be restored by light
activation of ChR2. Ultimately, the withdrawal response
habituated during the light stimulation as do touch
responses. The mechanism underlying habituation has
not been addressed in detail. In summary, this study shows
that ChR2 can be used not only to induce action potentials
but also to trigger signaling events, which depend on both
depolarization and ion specificity such as Ca2+. Lastly, the
transparency of C. elegans makes it the optimal biological
system for using light-activated switches to understand
neuronal circuitry underlying behavior.
Au
th
or
's
pe
rs
Expression of ChR2 was found to be stable for up to 16
months, suggesting that viral gene transfer might be
useful for long-term expression of ChR2 not only in
the eye but also in other tissues [34!!]. Electrophysiological recordings from ChR2-expressing retinal neurons
indicated that light induced cell-type-specific spiking in
these neurons. Spike frequency was dependent on the
light intensity, suggesting that third-order neurons in the
retina (which are normally not responsive to light) can
translate light intensities into spike firing rates. Lightinduced responses could be observed for several hours
without loss of activity, which suggests that the lightsensitive chromophore (the retinal compound) is constantly available and can be restored for ChR2 within
the retinal ganglion cells without external application. By
measuring visually evoked potentials from the visual
cortex — the brain area into which retinal ganglion cells
project — 460 nm light but not 580 nm light evoked
potentials in blind rd1/rd1 mice that expressed ChR2
in the ganglion cells. Whether these mice were now able
to see or to react to light stimuli in a meaningful, behavioral context was not assessed; however, the study shows
that ChR2 has the potential to restore light sensitivity in
the vertebrate eye.
on
Indeed, ChR2 has been recently used to restore visual
responses in mice by converting inner retinal neurons,
which are normally light insensitive, into light-sensitive
neurons [34!!]. In a mouse model deficient in functional
photoreceptor cascades, ChR2 was expressed throughout
the retina, and prominently in retinal ganglion cells, by an
adeno-associated virus vector. Maximal ChR2 currents
were measured, without the addition of all-trans retinal,
with 460 nm light in acutely dissociated inner retinal
neurons from wild-type mice. ChR2 was also expressed
in homozygote rd1 mice through injection of ChR2 into
the eye of newborn or adult mice. rd1 mice carry a
mutation in cGMP phosphodiesterase (PDE6) and therefore their photoreceptor signaling is non-functional.
Because similar mutations are found in humans in some
forms of retinitis pigmentosa, it was proposed that ChR2
might be used as a therapeutic approach to cure blindness.
y
ChR2 in mouse retina
osensory neurons of C. elegans. They used ChR2(H134R),
a mutant with a larger stationary photocurrent in comparison to wild-type protein, for these studies. The transgenic worms were raised in the presence of all-trans
retinal and ChR2 was activated by light of 450–490 nm.
ChR2(H134R) was expressed in the body wall and egglaying muscles and was localized in the muscles in the
endoplasmic reticulum and the cell membranes, as
expected for a transmembrane protein and as previously
observed in hippocampal neurons and HEK293 cells
[10!!,23!]. Light activation of ChR2(H134R) induced
muscle contraction, often accompanied with egg-laying
as a result of the contraction of the vulva muscles. Switching off the light led to muscle relaxation within a second.
Interestingly, ChR2(H134R) might be sufficient to
induce contraction directly by means of Na+ and Ca2+
influx through its own pore, bypassing the nicotinic
nAChR and L-type Ca2+ channels (egl-19). Note that
muscle contraction in C. elegans is mediated by Na+ influx
through nAChR and the depolarization-induced opening
of voltage-gated Ca2+ channels (egl-19), followed by
Ca2+-induced Ca2+ release from the sarcoplasmic reticulum through activating ryanodine receptors.
ChR2 in C. elegans
A limitation of light-activated constructs in, for example,
deep brain areas is that light might not be able to
penetrate to the relevant cellular layers. This problem
should not exist for transparent or thin cell layers, or for
transparent animals such as C. elegans. Nagel et al. [35!]
have therefore expressed ChR2 in muscle and mechanwww.sciencedirect.com
Application of light-activated ligands:
the cell helps itself
Light-activated receptors or channels require the application and regeneration of a light-sensitive ligand to activate
the receptor, which can be a problem for approaches that
use synthesized compounds. The receptors have to be
loaded before the experiment with a compound that has
the potential to induce toxicity depending on the length
of application and the compound itself. Fortunately, by
using retinal-derived ligands, the cellular environment
seems to help itself by providing the receptor with its own
active molecule. The importance of this issue warrants
that we discuss it in more detail.
Current Opinion in Neurobiology 2007, 17:87–94
92 Development
Conclusions
co
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In any case, whether one uses vertebrate, invertebrate,
bacterial or plant-derived light-sensitive proteins, application of all-trans retinal seem to be sufficient to achieve
light responses in mammalian cells. At least in the vertebrate eye and the chicken spinal cord, sufficient retinal
compounds are available in the tissue to drive lightactivated currents without external supply of the chromophore. Whether this is true for every tissue remains to
be investigated.
al
Controlling cellular signaling by light using light-activated switches has great potential for understanding basic
cellular mechanisms and to elucidate how these mechanisms influence and determine system function and animal
behavior. Several studies in Drosophila, C. elegans, chicken
and mice have revealed the feasibility of the approach. In
addition, development of light-activated molecular
machines will have important implications for curing
disease. For example, controlling ion conductance in
the heart through light could be used to regulate the
frequency of the heartbeat in disease conditions such as
atrial fibrillation. In addition, locomotor circuits capable
of producing walking behavior are contained in the lower
spinal cord after cord injury, but the level of excitability is
below the threshold for their activation. Light-controlled
ion conductances could here be used to activate these
circuits after spinal cord injury or to raise the level of
excitation so that spared fibers of the brain are capable of
causing locomotion. Moreover, neuronal function in the
basal ganglia is severely impaired in Parkinson’s disease.
Deep brain stimulation is currently used in individuals
with Parkinson’s to overcome tremor, rigidity, akinesia
and gait. Light-activated channels could be used for
precise activation or silencing of neuronal circuits of
hyper- or hypoactive neuronal circuits involved in Parkinson’s disease such as the internal segment of the
globus pallidus and subthalamic nucleus.
pe
rs
In contrast to invertebrates, the vertebrate system possesses numerous enzymes and retinoid-binding proteins
in its visual cycle [37]. In vertebrates, the cis–trans and
trans–cis isomerization of retinal are locally separated and
do not occur in the same photoreceptor; thus, the isomerization products must move between different cell
types and distinct subcellular regions within a cell of the
retina by means of retinoid-binding proteins. The photoconversion from trans to cis is mediated by an isomerase.
to all-trans is driven by the energy conversion produced
by light and by conformational changes in the protein
during ion translocation.
on
Phototransduction in many systems involves the isomerization of a photosensitive pigment — namely, an aldehyde of vitamin A retinal. This light-sensitive pigment
binds to a lysine through a protonated Schiff base in all
rhodopsin-type proteins. Vertebrates and invertebrates
use derivatives of 11-cis retinal as chromophores. Light
responses are initially mediated by the photoisomerization of this 11-cis retinal to all-trans retinal in all systems
described so far, but differences exist in the regeneration
of 11-cis chromophores. In invertebrates, the all-trans
isomer remains bound in the receptor pocket in the
thermally stable metarhodopsin state. The advantage
here is that the active photoproduct can be reformed
without deprotonation of the Schiff base by the absorption of a second photon [36]. Thus, the equilibrium
between rhodopsin and metarhodopsin is sufficient to
drive the photocycle and additional retinoid-binding proteins are not necessary.
th
or
's
In contrast to the complicated mechanism in the vertebrate eye, in heterologous expression systems such as
HEK293 cells, activation of human rhodopsin (when
expressed in these cells) can be achieved for longer than
4 h once the cells are loaded with 11-cis retinal. No further
application of 11-cis retinal is necessary, suggesting that at
least HEK293 cells contain the intrinsic capability to
regenerate 11-cis retinal or other analogs such as 9-cis or
13-cis retinal to activate their photoreceptors [38]. Thus,
single application and/or long-time perfusion of 11-cis
retinal or its analogs 9-cis or 13-cis or even all-trans retinal
(see [38]) is sufficient to regenerate the active compounds
necessary for repetitive light activation of vertebrate
photoreceptors.
Au
Bacteria and plants use the all-trans isomer of retinal as
the chromomer of choice for the active receptor state. In
the dark, retinal in its receptor-binding pocket is in an
equilibrium of the all-trans, 15-anti and 13-cis, 15-syn
configurations [39,40]. Only the all-trans configuration
can induce the ion transport mechanism, at least in
bacteriorhodopsins and halorhodopsins [41]. During illumination, all-trans retinal is converted to 13-cis retinal,
accompanied by several conformational changes in the
protein, resulting in net ion transport. Reisomerization to
all-trans retinal after the ion is released restores halorhodopsin in the unexcited state [31,32,42]. Thus, the isomerization of the retinal from all-trans to 13-cis and back
Current Opinion in Neurobiology 2007, 17:87–94
Whatever new applications and designs for light-activated
switches arise, improvement in light sources are needed
for precise triggering of signaling events, such as activation of channels or GPCRs at defined subcellular regions
such as presynaptic terminals. In addition, light sources
must be developed that can be used as implants in living
animals to control cellular activity in vivo and to deliver
light deep into tissue — an issue that will be particularly
important for controlling neuronal activity in the brain.
Chemically controlled methods using compounds that
can cross the blood–brain barrier might provide an alternative to light activation. For example, the chemical
induction of homo- or heterodimerization of modified
synaptic proteins has been recently applied to reversibly
www.sciencedirect.com
Controlling neuronal activity Herlitze and Landmesser 93
4.
Melyan Z, Tarttelin EE, Bellingham J, Lucas RJ, Hankins MW:
Addition of human melanopsin renders mammalian cells
photoresponsive. Nature 2005, 433:741-745.
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Ebrey T, Koutalos Y: Vertebrate photoreceptors. Prog Retin Eye
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Montell C: Visual transduction in Drosophila. Annu Rev Cell Dev
Biol 1999, 15:231-268.
Among the different light switches that have been used,
ChR2 seems to have the greatest potential for fast and
continuous control of membrane depolarization; however,
further improvements of ChR2 regarding its ion selectivity, ion conductance and its spectral sensitivity are
needed. Combination of light-activated GPCRs and ion
channels that differ in their spectral sensitivity would
provide the means for simultaneous control of depolarization and hyperpolarization. We think that we are
looking at a bright future. Let the light shine!
7.
Downes GB, Gautam N: The G protein subunit gene families.
Genomics 1999, 62:544-552.
8.
Lechner HA, Lein ES, Callaway EM: A genetic method for
selective and quickly reversible silencing of Mammalian
neurons. J Neurosci 2002, 22:5287-5290.
co
p
9.
!
Tan EM, Yamaguchi Y, Horwitz GD, Gosgnach S, Lein ES,
Goulding M, Albright TD, Callaway EM: Selective and
quickly reversible inactivation of Mammalian neurons in vivo
using the Drosophila allatostatin receptor. Neuron 2006,
51:157-170.
This paper, together with [8], establishes use of the Drosophila allatostatin
receptor, a Gi/o-coupled receptor, to silence neuronal function. Functionality in several tissues is demonstrated.
10. Li X, Gutierrez D, Hanson MG, Han J, Mark MD, Chiel H,
!! Hegemann P, Landmesser LT, Herlitze S: Fast non-invasive
activation and inhibition of neural and network activity by
vertebrate rhodopsin and green algae channelrhodopsin.
Proc Natl Acad Sci USA 2005, 102:17816-17821.
This study uses the vertebrate rhodopsin and the green algae channelrhodopsin-2 (ChR2) to either hyperpolarize or depolarize the cell membrane of HEK293 cells, hippocampal neurons and chicken spinal cord. It
is the first paper to demonstrate the use of antagonistically acting light
switches in an intact tissue.
on
al
Update
11. White BH, Osterwalder TP, Yoon KS, Joiner WJ, Whim MD,
Kaczmarek LK, Keshishian H: Targeted attenuation of electrical
activity in Drosophila using a genetically modified K+ channel.
Neuron 2001, 31:699-711.
or
's
pe
rs
Recent work has also described the application of ChR2 in
Drosophila larvae to determine whether defined neuronal
circuits can distinguish between appetitive or aversive
learning [44]. Pairing of odor stimuli, which were either
appetitive or aversive, with a second neutral stimuli (fructose) led to an odor specific behavior. Expression and
activation of ChR2 either in octopaminergic or dopaminergic neurons during application of the odors could substitute for fructose, suggesting that octopaminergic
pathways underlie appetitive learning while the dopaminergic pathway determines aversive learning. In another
study, ChR2 was delivered stereotaxically using lentivirus
into the hippocampus to control excitation of ChR2 negative cells by light [45,46]. In a very interesting study a
photoacivated adenylyl cyclase (PAC) from the flagellate
Euglena gracilis was used to control cAMP levels in Xenopus oocytes, HEK293 cells, and Drosophila. PAC is activated by 455 nm and does not need any cofactor. Whether
the basal activity of the PAC used will limit its application
in transgenic animals has to be investigated [47].
y
silence neurons: in cerebellar Purkinje cells, synaptic
transmission was blocked by chemically induced oligomerization of synaptobrevin fused to a variant of FK506binding protein, resulting in impaired motor behavior of
the mice [43!].
Acknowledgements
th
We thank Davina Gutierrez for reading the manuscript. This work was
supported by grants from the National Institutes of Health (NS447752
and NS42623 to SH, and NS19640 and NS23678 to LTL).
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Au
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