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Written by - Pivotal Secret
Responsible editor—Wang Sizhen, Fang Yiyi
Editor—Summer Leaf
Optogenetics expresses light-sensitive proteins in specific cell populations and manipulates cellular activity
at the subcellular, cellular, circuit, and behavioral levels through light.
Because of its specificity to target targets, the speed and precision of manipulation time, it is more and more widely
used in the field of neuroscience.
Although the application of optogenetics in neuroscience has traditionally focused on controlling spike activity at the level of cell dendrites, the scope of optogenetics that directly manipulates presynaptic function is expanding
.
Presynaptic opsin localization is combined with photostimulation at the end, allowing light-mediated neurotransmitter release, presynaptic inhibition, induction of synaptic plasticity, etc
.
Optogenetics has unique advantages
in neuronal presynaptic regulation.
First, specific optogenetic tools allow for time-accurate manipulation
of presynaptic function.
Secondly, the use of optogenetics to activate axon ends can reveal the synaptic connection between two neuronal populations, and the use of optogenetics to inhibit synapses can reveal the role of
synapses in signal transduction, network oscillation, and behavioral function.
Finally, presynaptic optogenetics can hierarchically regulate the release efficiency of neurotransmitters, and this dynamic regulation of transmitter release can help study the role
of synaptic activity in information processing.
Recently, Benjamin R.
Rost of the German Center for Neurodegenerative Diseases (DZNE) collaborated with the Ofer Yizhar team of the Weizmann Institute of Science in Israel at Nature Neuroscience Published a review article entitled "Optogenetics at the presynapse", which summarizes the application of optogenetic tools to presynapses (species and application strategies) in combination with the unique cell biology of axonal terminals.
and outline future directions
within the field of study.
Biophysical characteristics of optogenetic tools
First, rhodopsin
Rhodopsin is divided into type I and II, and type I rhodopsin is derived from prokaryotic and eukaryotic microorganisms, algae, fungi and even viruses, and its evolved functions can be used to manipulate cell physiology.In most microbial opsins, the covalently bound cofactor retinal isomerizes from the all-trans configuration to the 13-cis configuration after photon absorption
.
This light reaction is cyclic and can be repeatedly activated
according to the time required to complete the light cycle.
Rhodopsin channel proteins (ChRs) are divided into cation-conducted ChRs (CCRs) and anionically conducted ChRs (ACRs)
based on conductivity.
CCRs and ACRs are not selective
for specific cations and anions, respectively.
Type II rhodopsin is derived from special G protein-coupled receptors (GPCRs) in animals, activated by light and using retinal as a chromophore).
Although type II rhodopsin has no sequence homology to type I rhodopsin, they share the same 7 transmembrane α-helical structures and retinal binding sites as microbial rhodopsin
。 In visual type II rhodopsin (e.
g.
, rod and cone), 11-cis-retinal isomerizes to all-trans after photon absorption, which is triggered by conformational changes in the receptor G protein signaling
.
However, in contrast to microbial rhodopsin, light breaks the covalent binding between retinal and opsin, and all trans-retinal must be re-isomerized, and functional visual rhodopsin cannot be regenerated
if the enzymes required for re-isomerization are deficient.
This means that they are bleached and can no longer transmit signals
.
In contrast, non-visual type II rhodopsin present in both vertebrates and invertebrates can reisomerize the bound chromophores to achieve a bistable state
.
Therefore, these bistable, nonbleached type II rhodopsin have a major advantage
when expressed ectopic in neurons or other excitable cells.
Fig.
1 Classification, structure and function of rhodopsin protein
(Source: Rost B R, et al.
) , Nat Neurosci, 2022）
Second, blue light receptors
Most photoreceptors used in optogenetics use flavin as a chromophore protein, namely flavin single nucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin, all of which are well availablein mammalian tissues.
The absorption spectrum of the blue light receptor is up to 500 nm, so it can be complementary to
optogenetic tools that are sensitive to green and red light.
While the blue spectrum limits penetration into deep tissues, it can be done
by two-photon excitation.
Unlike rhodopsin, blue light receptors (BLRs) are soluble proteins
that control the function of various effector proteins.
Importantly, since most BLRs exhibit residual dark activity, they should be considered as light-dependent modulators
of simulated activity.
In contrast, rhodopsin is generally inactive
in the absence of light.
In addition, because the termination of the BLR signal depends on the thermal relaxation effect, precise time control
of this photoreceptor is not possible.
The BLR family can be divided into optical-oxygen-voltage domain (LOV), which utilizes FAD blue light sensors (BLUF) domains and cryptoanthocyanidins (CRYs).
The LOV domain is generally derived from phototaxis of higher plants and microalgae, which bind FMN
in a non-covalent manner.
In the BLUF domain, the flavin chromophores are not covalently bound, but the activation mechanism is different
from that of the LOV domain.
The activation of the BLUF domain causes only changes in non-covalent electron bonds, not changes in oxidation states and the formation
of adducts.
Although these subtle protein alterations have not yet been fully elucidated, the BLOF domain has a persistent signaling state (seconds to minutes).
CRYs are present in plants and animals, and although highly homologous to photolytic enzyme proteins, they cannot interact
with DNA.
To date, there is no consensus
on the activation mechanism of CRI.
Fig.
2 Classification, structure and function of blue light receptors
(Source: Rost B R, et al.
) , Nat Neurosci, 2022）
Third, photosensitizer
The genetically encoded photosensitizers are all derived from the green fluorescent protein (GFP) or LOV2 domainof Arabidopsis phototropic 2.
Under light, they produce reactive oxygen species (ROS) instead of fluorescence
.
At 20-150 nm light, singlet oxygen (O2) oxidizes cysteine, histidine, methionine, tryptophan, and tyrosine side chains, disrupting protein function
.
Thus, protein-fused photosensitizers enable in situ spatiotemporal precision chromophore-assisted photoinactivation (CALI).
However, photosensitizers are difficult to use
compared to other optogenetic tools.
Because it is necessary to carefully assess the degree of
inactivation of specific proteins associated with nonspecific tissue damage.
Fig.
3 Classification, structure and function of photosensitizers
(Source: Rost B R, et al.
) , Nat Neurosci, 2022）
Optogenetic drives targeting axons and presynapses
Since most optogenetic tools originate from species with distant germlines, transport signals
are lacking in mammalian cells.
At high expression levels, it often results in ineffective membrane localization, intracellular aggregation, and cytotoxicity
.
Presynaptic proteins are synthesized within cells and transported
over considerable time and distance.
There are different transport mechanisms for synaptic vesicle proteins, active region components, and presynaptic membrane proteins, which can be used to target optogenetic tools to axon endings
.
Synaptophysin is the most abundant protein in both glutamate and GABA vesicles, and is particularly suitable for targeting optogenetic drives and fluorescent proteins to synaptic vesicles
.
Importantly, overexpression of synaptophysin does not appear to affect synaptic transmission
in rodent neurons.
Studies have shown that it is much
more difficult to effectively target optogenetic tools to the axon plasma membrane.
Because axonal membrane proteins must pass through the axon compartment and axon initiation at the cell and dendritic boundaries, and the universal signal sequence of membrane protein axon transport or presynaptic localization is not currently well defined
.
Nevertheless, two mechanisms are known to facilitate axon localization: one is unidirectional membrane insertion through preferential endocytosis of the dendritic compartment of the cell body; The second is specific vesicle-mediated axon-guided transport
.
Fig.
4 Axon transport and presynaptic localization of optogenetic drives
(Source: Rost B R, et al.
) , Nat Neurosci, 2022）
Presynaptic application of optogenetic tools
First, light-induced neurotransmitter release
For rhodopsin channelin-assisted loop mapping (CRACM), CCRs are expressed in one brain region, and functional connectionsare then assessed by local illumination of the target (targeted) brain region.
Mechanically, photocurrent depolarizes axons and induces action potentials (APs).
At the end of the axon, APs cause Ca2+ influx through voltage-gated channels, triggering neurotransmitter release
.
Light-induced postsynaptic currents indicate that there are functional synapses between CCR-expressed neuronal populations and postsynaptic
neuronal populations.
Widely used to study the functional connections of gene-defined neurons, combined with cell-specific gene knockout or knockdown strategies, CRACM can also study the effect
of protein loss on transmitter release.
Fig.
5 Concept and defects of light-induced neurotransmitter release
(Source: Rost B R, et al.
) , Nat Neurosci, 2022）
Second, optogenetics inhibits neurotransmitter release
Optogenetic inhibition of neurotransmitter release is an important complementary pathwayfor optogenetic excitation.
Because it circumvents the problems caused by uncontrolled retrograde AP in the
body.
There are three experimental strategies for presynaptic optogenetic inhibition: the first is through the extroverted Arch3, ArchT97, Jaws98 proton pump, and introverted NpHR The chloride pump causes hyperpolarization of axon terminals to inhibit AP transmission and reduce presynaptic Ca2+ influx
.
This presynaptic inhibition of light-driven ion pumps has been applied to inhibit the propagation of spontaneous network oscillations between different brain regions, and to silencate specific synaptic connections in vivo to study their role in
behavior.
The second is inhibition of transmitter release
through GPCRs.
Presynaptic Gαi/o-coupled receptors are natural inhibitors
of presynaptic transmitter release.
The opening probability of voltage-gated calcium channels (VGCCs) is reduced mainly by the βγ subunit of heterotrimeric Gi/o proteins, thereby inhibiting transmitter release
.
Due to the nonlinear dependence of vesicle fusion on presynaptic Ca2+, a modest reduction in Ca2+ influx significantly reduces neurotransmitter release
。 In addition, the βγ subunit inhibits release by directly interfering with the release
mechanism.
Based on many type II rhodopsin and Gα i/o or related transducin (Gα t Conjugation, studies have shown that exogenously expressed photosensitive GPCRs in neurons may conduct photogated presynaptic inhibition under the premise of effective localization of rhodopsin to axon ends.
Currently, the best GPCRs for presynaptic inhibition are lamprey paraprotease (LcPPO) and mosquito OPN3
.
In neurons, LcPPO and transporter-enhanced eOPN3 [combine ER transport signals (ER) with Golgi transport signals (ts) Together added to OPN3, it produces enhanced OPN3-ts-mScarlet-ER], inhibiting presynaptic Ca by VGCCs 2+ influx, which in turn inhibits neurotransmitter release
.
The third is to disrupt the transmitter release mechanism
.
Optogenetic tools that disrupt the release mechanism can be used to inhibit neurotransmitter release for hours or days
for a long time.
The first tool based on this approach is InSynC (synapse inhibition using CALI).
The photoactivated photosensitizer miniSOG attaches to the cytoplasmic end of the synaptic vesicle and produces ROS under continuous light, which in turn oxidizes the synaptic protein
.
In cultured neurons, a few minutes is enough to impair AP-induced transmitter release while increasing spontaneous transmitter release
.
Studies have shown that the hydrolysis of soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein by botulinum toxin or tetanus neurotoxin can effectively eliminate synaptic transmission
.
Based on the AsLOV2-derived iLID dimer system, a vesicle attached photocontrolled splitting protein complementary system was developed-botulinum neurotoxin B.
( vPA-BoNT), which specifically cleaves VAMP2 (SNARE protein core).
Photoactivation of vPA-BoNT reduces the frequency of spontaneous microexcitatory postsynaptic current (EPSC) in isolated cultures, reducing inducing transmitter release in acute mouse brain slices by 50%.
InSynC or vPA-BoNT can continue to disrupt the release mechanism for a long time to inhibit transmitter release, but because it takes more than ten minutes to take effect and recovery depends on the protein synthesis process, the timing accuracy of the manipulation is poor
.
Fig.
6 Different principles of optogenetic inhibition of neurotransmitter release
(Source: Rost B R, et al.
) , Nat Neurosci, 2022）
Fig.
7 Verification of optogenetic presynaptic inhibition
(Source: Rost B R, et al.
) , Nat Neurosci, 2022）
Third, light-induced presynaptic enhancement effect
Persistent changes in synaptic strength alter neuronal memory traces.
Studies have shown that the structure and functional mechanisms of postsynaptic long-term enhancement (LTP) are closely related to memory-guided behavioral performance, but the role of presynaptic LTP found in various synapses throughout the central nervous system in learning and behavioral output is unclear
.
Previous studies have used high-frequency electrical stimulation to increase cAMP levels through protein kinase A and guanine nucleotide exchange factor Epac2 to increase vesicle release and activate new release sites, resulting in a sustained increase in transmitter release and induction of presynaptic LTP
。 However, high-frequency stimulation of axons can lead to unintended off-target effects, making it difficult to assess the effects of
presynaptic LTP on live animals.
Therefore, recent studies have recently been studied to generate presynaptic LTP by directly increasing cAMP levels at axonal ends by optogenetic induction, i.
e.
, attaching the light-activated adenylate cyclase bPAC to the cytosolic C of synaptophystin At the end, the formed blue light activates "synaptoPAC" to rapidly increase synaptic transmission, only enhancing the release
of terminal transmitters with presynaptic plasticity.
SynaptoPAC may be a useful tool for mimicking the presynaptic plasticity of genetically defined synapses in living animals and may help elucidate the behavioral effects
of presynaptic enhancement.
Summary and outlook
The unique physiology and complex transport mechanisms of axons have hindered the application
of optogenetics at presynaptic terminals for many years.
But based on a better understanding of the interactions between the unique intracellular environment of axons and the biophysical properties of optogenetic drives, optogenetic tools
specifically adapted to presynaptic applications have been developed.
The combination of technological advances in better subcellular targeting for exclusive localization of axons, more complex gene expression systems for activity-dependent and connection-dependent control of tool expression improves the spatial and temporal accuracy of
optogenetics.
But there are limitations
to using a specific synapse between two sets of neurons as the sole target for optogenetic manipulation.
Bidirectional regulation of synaptic transmission using optogenetic tools is a promising research direction, such as presynaptic inhibition of optoGPCRs (non-visual type II rhodopsin, coupled to GPCRs).
and excitability The binding of CCRs enables bidirectional regulation
of transmitter release.
These tools activate axons through CCRs in the region of interest on the one hand, and inhibit local transmitter release
through optoGPCR at the same time.
Overall, presynaptic optogenetic tools provide neuroscientists with a variety of experimental methods to perform fine manipulation of neurons with unparalleled spatiotemporal resolution, and understanding their classification and application is critical
to how they are used later and the development of optogenetics.
These tools will support the further development and clinical translation
of neuroscience.
org/10.
1038/s41593-022-01113-6 selection of previous articles
[1] Neural Regen Res Review—The role and potential of microglial polarization and dialogue with other cells in the treatment of Alzheimer's disease
[2] Nat Commun-Chu Jun's research group has developed a new high-performance genetically encoded cAMP fluorescent probe
[3] Mol Neurobiol—Zhu Lixin/Guo Jiasong's team revealed the important role of the basement membrane protein Perlecan in the recovery of blood-spinal cord barrier function after spinal cord injury
[4] Transl Psychiatry—Yang Xun's team revealed the different neural mechanisms of schizophrenia in the reward expectation and reward outcome stages
[5] Schizophrenia—Brain network hub and working memory performance in patients with schizophrenia
[6] Front Cell Neurosci—Using bibliometrics to analyze research trends in astrocytes and stroke
[7] Cereb Cortex-Liu Tao's team revealed the accelerated degeneration pattern of white matter structure in the elderly
[8] Prof.
Biophys J—Professor Xu Guangkui's research group reveals the network dynamics of nonlinear power-law relaxation in the cell cortex
[9] Science—Analysis of neurogenesis and regeneration in Mexican axolotls using single-cellular, multi-omics techniques
[10] J Neuroinflammation Review—Ni Wenfei/Zhou Kailiang team focused on the important role of STING pathway in neuroinflammation and cell death after CNS injury
Recommended high-quality scientific research training courses[1] Symposium on Single Cell Sequencing and Spatial Transcriptomics Data Analysis (October 29-30 Tencent Online Conference) Conference/Forum/Seminar Preview[1] Immune Zoom Seminar—Screening of B cells in the immune and nervous system (Professor Xu Heping)
[2] Academic Conference - 2022 Symposium on Neural Circuit Tracing Technology and the Second Round of the Second Round of the 6th National Training Course on Neural Circuit Tracing Technology
[3] Roundtable – Xu Fuqiang/Jia Yichang/Han Lanqing/Cai Lei et al.
discussed gene therapy innovation for neurodegenerative diseases and ophthalmic diseases
End of this article
