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Optogenetic TrkB activation in mouse hippocampal DG GCs. (A) Upper images: Structure of the AAVDJ/8-CaMKIIa::Opto-cytTrkB(E281A)-HA construct. Lower images: Virus was injected into the DG, after which 3 weeks were allowed for recovery from surgery and vector expression. (B) Injected mouse brains were sectioned and immunostained for brain cell-specific markers. White arrows show GAD67-, Iba1-and GFAP-positive cell bodies. (C) Upper image: AAV injection with implantation of a fiber optic cannula on the DG and induction of pERK1/2 in DG GCs by 473-nm blue laser light stimulation. Lower images: Activation of Opto-cytTrkB(E281A)-HA by light stimulation. (D) Light sensitivity of OptocytTkrB(E281A)-HA. Light was administered at intensities of 50, 100, 500 mW/mm 2 , and 1 mW/mm 2 , and the percentage of pERK1/2-positive neurons was calculated as described in Methods. Data are presented as means ± s.e.m. (error bars); F ¼ 0.2957, p ¼ 0.8279 versus the light-stimulated Opto-cytTrkB(E281A)-HA group (one-way ANOVA). n.s., no significant difference. The number of filled circles corresponds to the number of mice used in the experiment. Slice images were obtained using 20Â and 60Â lenses. All scale bars: 50 mm.
Source publication
Optogenetic activation of receptors has advantages compared with chemical or ligand treatment because of its high spatial and temporal precision. Especially in the brain, the use of a genetically encoded light-tunable receptor is superior to direct infusion or systemic drug treatment. We applied light activatable TrkB receptor in mouse brain with r...
Contexts in source publication
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... fulllength TrkB. A myristoylation sequence (Lyn signal peptide) was added at the N-terminus of cytTrkB to promote localization of cytTrkB to the plasma membrane, and a Cry2PHR domain was conjugated to the end of cytTrkB. Finally, an HA-tag sequence was added to the C-terminus to facilitate the detection of its expression in immunohistochemistry (Fig. 1A). Because genes transduced via AAVmediated delivery require much longer to achieve full expression (>2e3 weeks) than genes transiently expressed in cultured neurons, it is necessary that tools show reduced basal activity in the brain. Our group recently developed a Cry2PHR mutant, Cry2PHR(E281A), by structure prediction and showed that ...
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... evaluate the basal activity, we infected hippocampal excitatory neurons with AAVs expressing Cry2PHR (OptocytTrkB-HA), the Cry2PHR(E281A) mutant (OptocytTrkB(E281A)-HA), or virus without the cytTrkB domain (Lyn-Cry2PHR(E281A)-HA). Three weeks after injection, we measured the level of phosphorylated ERK1/2 (pERK1/2) in hippocampal lysates in the absence of light stimulation by Western blotting (Supplementary Fig. 1). Mice expressing OptocytTrkB containing Cry2PHR showed an increase in basal pERK1/2 compared with Lyn-Cry2-PHR(E281A)-HA, but no significant change in basal pERK1/2 was detected in mice expressing Opto-cytTrkB containing the E281A mutant. ...
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... in the hippocampal DG. To this end, 0.3e0.4 ml of the Opto-cytTrkB(E281A)-HA viral construct under the control of the CaMKIIa(0.4) promoter was injected into the DG. After 3 weeks from the surgery, Immunohistochemical experiments were performed. The images showed that Opto-cytTrkB(E281A)-HA was expressed in DG granule cells (GCs) (Fig. 1A). To determine whether the construct was expressed in other cell types in the DG, hippocampal sections were immunostained for CaMKII (calcium/calmodulin-dependent protein kinase II), GAD67 (glutamate decarboxylase 67 kD), Iba1 (ionized calcium-binding adaptor molecule 1) and GFAP (glial fibrillary acidic protein)dspecific markers for ...
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... kinase II), GAD67 (glutamate decarboxylase 67 kD), Iba1 (ionized calcium-binding adaptor molecule 1) and GFAP (glial fibrillary acidic protein)dspecific markers for excitatory neurons, GABAergic neurons, microglia, and astrocytes, respectively. HA-tag signals were colocalized with CaMKII-positive DG GCs, but not with other cellspecific markers (Fig. 1B). To verify the potential for activating TrkB signaling in DG GCs with blue light stimulation, we implanted a cannula optic fiber (200 mm diameter) into the outer molecular layer (DV: 1.5 mm) followed by stimulation with a 473-nm laser. Because continuous, prolonged light stimulation with strong intensity light has the potential to ...
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... laser. Because continuous, prolonged light stimulation with strong intensity light has the potential to cause phototoxicity [37], we utilized a pulse-type light stimulation strategy rather than a continuous stimulation paradigm. Stimulation with a 1 mW/mm 2 laser for 1 s per 5 s pulse was sufficient to activate the MAPK/ERK pathway in DG GCs (Fig. 1C). Next, we tested light stimulation at powers less than 1 mW/ mm 2 . Interestingly, Opto-cytTrkB(E281A)-HA could be activated at powers as low as 50 mW/mm 2 with no noticeable change in the percentage of pERK1/2-positive neurons (Fig. 1D). No induction of pERK1/2 was observed in mice injected with Lyn-PHR(E281A)-HA virus and stimulated ...
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... with a 1 mW/mm 2 laser for 1 s per 5 s pulse was sufficient to activate the MAPK/ERK pathway in DG GCs (Fig. 1C). Next, we tested light stimulation at powers less than 1 mW/ mm 2 . Interestingly, Opto-cytTrkB(E281A)-HA could be activated at powers as low as 50 mW/mm 2 with no noticeable change in the percentage of pERK1/2-positive neurons (Fig. 1D). No induction of pERK1/2 was observed in mice injected with Lyn-PHR(E281A)-HA virus and stimulated with 1 mW/ mm 2 light, indicating that pERK1/2 induction was not caused by light stimulation itself. Given that this construct could be activated by very low light intensity, we speculated that it might be activated by noninvasive blue ...
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... Cry2PHR-based optogenetic tool does not have a fast turn OFF feature (~15min for dissociation from dimer or oligomer of Cry2PHR) [39]. Also, its characteristics, as shown Fig. 1D, activating OptocytTrkB(E281A)-HA cannot be adjustable with manipulating light intensity. Thus, a new strategy for signal turn ON and OFF is needed for the PHR based RTKs systems. To overcome this limitation and establish new signal-modulating conditions, we tested various light-pulse types and ultimately selected two types of pulses ...
Citations
... construct [27] using the In-Fusion cloning system. To minimize basal activity, we performed site-directed mutagenesis to mutate residue E281 to alanine (PHR E281A ) [30,31]. This final form of the construct-pcDNA3.1-CMV-Lyn-RET ...
... and pAAV-hSyn1-DIO-optoRET(HA) were constructed from the respective constructs, pAAV-CamKIIα(0.4)-DIO-Opto-cytTrkB(E281A)-HA (Addgene #180588) and pAAV-hSyn1-DIO-Opto-cytTrkB(E281A)-HA (Addgene #180590), by replacing Lyn-cyTrkB-PHR sequences with the Lyn-RET aa658−1062 -PHR E281A sequences of optoRET using the NheI/AgeI restriction enzyme sites [31]. pAAV-CamKIIa(0.4)-ERK-KTR-Clover ...
RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light–inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.
... construct [27] using the In-Fusion cloning system. To minimize basal activity, we performed site-directed mutagenesis to mutate residue E281 to alanine (PHR E281A ) [30,31]. This nal form of the construct-pcDNA3.1-CMV-Lyn-RET ...
... and pAAV-hSyn1-DIO-optoRET(HA) were constructed from the respective constructs, pAAV-CamKIIα(0.4)-DIO-Opto-cytTrkB(E281A)-HA (Addgene #180588) and pAAV-hSyn1-DIO-Opto-cytTrkB(E281A)-HA (Addgene #180590), by replacing Lyn-cyTrkB-PHR sequences with the Lyn-RET aa658-1062 -PHR E281A sequences of optoRET using the NheI/AgeI restriction enzyme sites [31]. pAAV-CamKIIa(0.4)-ERK-KTR-Clover ...
RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light–inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, our findings suggest that optoRET has potential for treating neurological disorders such as Parkinson’s disease by promoting the ramification of auxiliary fibers on axon terminals.
... Similarly, a photoactivable domain, inspired by Arabidopsis thaliana cryptochrome 2, was engineered to allow blue-light activation of Trk signaling in a cell-specific and spatially directed manner [109,110]. OptoTrkB was also used to activate receptor signaling in the mouse brain [111]. An Optogenetically activatable Fas receptor (optoFAS) was developed using the blue light-induced homo-oligomerizing property of cryptochrome 2 (CRY2). ...
Systems neuroscience is focused on how ensemble properties in the brain, such as the activity of neuronal circuits, gives rise to internal brain states and behavior. Many of the studies in this field have traditionally involved electrophysiological recordings and computational approaches that attempt to decode how the brain transforms inputs into functional outputs. More recently, systems neuroscience has received an infusion of approaches and techniques that allow the manipulation (e.g., optogenetics, chemogenetics) and imaging (e.g., two-photon imaging, head mounted fluorescent microscopes) of neurons, neurocircuits, their inputs and outputs. Here, we will review novel approaches that allow the manipulation and imaging of specific molecular mechanisms in specific cells (not just neurons), cell ensembles and brain regions. These molecular approaches, with the specificity and temporal resolution appropriate for systems studies, promise to infuse the field with novel ideas, emphases and directions, and are motivating the emergence of a molecularly oriented systems neuroscience, a new discipline that studies how the spatial and temporal patterns of molecular systems modulate circuits and brain networks, and consequently shape the properties of brain states and behavior.
... OptoTrkB is preferable to other approaches to manipulate TrkB signalling, since optoTrkB can be activated in specific cells and brain areas at specific times. This allows us to establish causal relationships between TrkB signalling, neural network changes, and behavioural effects with temporal and spatial precision [13]. Recently, a new type of optoTrkB (E281A) was developed and successfully activated by blue light stimulation through the intact skull and fur of mice [13]. ...
... This allows us to establish causal relationships between TrkB signalling, neural network changes, and behavioural effects with temporal and spatial precision [13]. Recently, a new type of optoTrkB (E281A) was developed and successfully activated by blue light stimulation through the intact skull and fur of mice [13]. OptoTrkB (E281A) also showed a low level of basal activity, allowing for more experimental control as spontaneous optoTrkB signalling was reduced. ...
... The analyses of pCREB and pERK intensity show that LED light stimulation results in an increase in downstream signalling of optoTrkB (E281A), both in inhibitory PV + interneurons, as well as in the excitatory CKII + neurons, as shown by a previous study [13]. Interestingly, the higher the expression of optoTrkB (E281A) in PV + interneurons, the more downstream signalling was promoted by light stimulation, while LED light stimulation failed to activate neurons with low levels of optoTrkB (E281A). ...
The activation of tropomyosin receptor kinase B (TrkB), the receptor of brain-derived neurotrophic factor (BDNF), plays a key role in induced juvenile-like plasticity (iPlasticity), which allows restructuring of neural networks in adulthood. Optically activatable TrkB (optoTrkB) can temporarily and spatially evoke iPlasticity, and recently, optoTrkB (E281A) was developed as a variant that is highly sensitive to light stimulation while having lower basal activity compared to the original optoTrkB. In this study, we validate optoTrkB (E281A) activated in alpha calcium/calmodulin-dependent protein kinase type II positive (CKII+) pyramidal neurons or parvalbumin-positive (PV+) interneurons in the mouse visual cortex by immunohistochemistry. OptoTrkB (E281A) was activated in PV+ interneurons and CKII+ pyramidal neurons with blue light (488 nm) through the intact skull and fur, and through a transparent skull, respectively. LED light stimulation significantly increased the intensity of phosphorylated ERK and CREB even through intact skull and fur. These findings indicate that the highly sensitive optoTrkB (E281A) can be used in iPlasticity studies of both inhibitory and excitatory neurons, with flexible stimulation protocols in behavioural studies.
... For example, by using CRY2-based membrane-bound photoactivatable optoTrkB, Woo et al. (2019) have found that local activation of TrkB signaling generates actin waves and can recruit key proteins to stimulated axonal areas during neuron polarization in cultured neurons. They further proved TrkB signaling can be activated by non-invasive blue light LEDs on the hairremoved mouse head (Hong and Heo, 2020). Letellier et al. (2020) recently showed that activation of optoFGFR1 was able to phosphorylate endogenous neuroligin 1 protein that can selectively increase dendritic spine density in mouse hippocampal neurons. ...
Dynamic protein-protein interactions are essential for proper cell functioning. Homo-interaction events-physical interactions between the same type of proteins-represent a pivotal subset of protein-protein interactions that are widely exploited in activating intracellular signaling pathways. Capacities of modulating protein-protein interactions with spatial and temporal resolution are greatly desired to decipher the dynamic nature of signal transduction mechanisms. The emerging optogenetic technology, based on genetically encoded light-sensitive proteins, provides promising opportunities to dissect the highly complex signaling networks with unmatched specificity and spatiotemporal precision. Here we review recent achievements in the development of optogenetic tools enabling light-inducible protein-protein homo-interactions and their applications in optical activation of signaling pathways.
... After the earlier design of optogenetic control of RTKs [5], a series of works in this collection further demonstrated the versatility of optogenetic regulation of RTK signaling. For example, RTKs such as EGFR, FGFR1, and tropomyosin receptor kinase (Trks) can be controlled by the dimerization of receptor [6,7] or a membrane translocation of their intracellular domain [8][9][10]. These strategies offer dynamic control of the RTK activity in live cells. ...
... These strategies offer dynamic control of the RTK activity in live cells. By optimizing the light-sensitive protein CRY2, Hong et al. successfully regulated TrkB signaling in the mouse brain with blue light [7]. The ability to use near-infrared light to control the RTK signaling, as shown by Leopold et al. in this collection, promises to further accelerate the optogenetic application in deep tissues and intact organisms [6]. ...
... In a demonstration of subcellular spatial control, Li et al. used optogenetics to deplete the level of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), from glucose transporter 4 (GLUT4) vesicle docking sites and identified an essential role for PI(4,5)P 2 in GLUT4 vesicle docking [13]. The integration of optogenetics with live-cell TIRFM imaging allowed the authors to reveal versatile functions of PI(4,5)P 2 in the regulation of insulin signaling and GLUT4 vesicle trafficking in adipocytes [7]. Li and coworkers further proposed that synaptic vesicle docking and membrane fusion could be fine-tuned by optogenetic manipulation of syntaxin, a transmembrane protein that forms the core SNARE complex with synaptobrevin and SNAP-25 [25]. ...
... In NHP opto-and chemogenetics studies, AAVs primarily have been used to manipulate neural activity local to the injection site (Acker et al., 2016;Afraz et al., 2015;Cavanaugh et al., 2012;Dai et al., 2014;El-Shamayleh et al., 2017;Fetsch et al., 2018;Grayson et al., 2016;Ju et al., 2018;Klein et al., 2016;Lu et al., 2015;May et al., 2014;Nagai et al., 2016;Nakamichi et al., 2019;Raper et al., 2019;Stauffer et al., 2016;Tamura et al., 2017;Upright et al., 2018;Yazdan-Shahmorad et al., 2016). However, a new rAAV variant-rAAV2-retro-developed through in vivo directed evolution (Tervo et al., 2016) has gained recent popularity because of its highly efficient retrograde transport in rodents (Birdsong et al., 2019;Hong and Do Heo, 2020). ...
Background
Recent genetic technologies such as opto- and chemogenetics allow for the manipulation of brain circuits with unprecedented precision. Most studies employing these techniques have been undertaken in rodents, but a more human-homologous model for studying the brain is the nonhuman primate (NHP). Optimizing viral delivery of transgenes encoding actuator proteins could revolutionize the way we study neuronal circuits in NHPs.
New Method
rAAV2-retro, a popular new capsid variant, produces robust retrograde labeling in rodents. Whether rAAV2-retro’s highly efficient retrograde transport would translate to NHPs was unknown. Here, we characterized the anatomical distribution of labeling following injections of rAAV2-retro encoding opsins or DREADDs in the cortico-basal ganglia and oculomotor circuits of rhesus macaques.
Results
rAAV2-retro injections in striatum, frontal eye field, and superior colliculus produced local labeling at injection sites and robust retrograde labeling in many afferent regions. In every case, however, a few brain regions with well-established projections to the injected structure lacked retrogradely labeled cells. We also observed robust terminal field labeling in downstream structures.
Comparison with existing method(s)
Patterns of labeling were similar to those obtained with traditional tract-tracers, except for some afferent labeling that was noticeably absent.
Conclusions
rAAV2-retro promises to be useful for circuit manipulation via retrograde transduction in NHPs, but caveats were revealed by our findings. Some afferently connected regions lacked retrogradely labeled cells, showed robust axon terminal labeling, or both. This highlights the importance of anatomically characterizing rAAV2-retro’s expression in target circuits in NHPs before moving to manipulation studies.
The structural plasticity of synapses is crucial for regulating brain functions. However, currently available methods for studying synapse organization based on split fluorescent proteins (FPs) have been limited in assessing synaptic dynamics in vivo due to the irreversible binding of split FPs. Here, we develop ‘SynapShot’, a method for visualizing the structural dynamics of intact synapses by combining dimerization-dependent FPs (ddFPs) with engineered synaptic adhesion molecules. SynapShot allows real-time monitoring of reversible and bidirectional changes of synaptic contacts under physiological stimulation. The application of green and red ddFPs in SynapShot enables simultaneous visualization of two distinct populations of synapses. Notably, the red-shifted SynapShot is highly compatible with blue light-based optogenetic techniques, allowing for visualization of synaptic dynamics while precisely controlling specific signaling pathways. Furthermore, we demonstrate that SynapShot enables real-time monitoring of structural changes in synaptic contacts in the mouse brain during both primitive and higher-order behaviors.
The effects of microalgae Chlorella pyrenoidosa on the hybrid groupers’ ( Epinephelus lanceolatus ♂× E. fuscoguttatus ♀) growth, gut microbiome and transcriptome were examined in this study. Feeding trials with duration of 15-days (15d) and 60-days (60d) were conducted on three experimental groups (n=3) as follow; (T1) grouper fed with basal diet and reared in filtered seawater (control treatment), (T2) grouper fed with basal diet and reared in C. pyrenoidosa monoculture water and (T3) grouper fed with basal diet partially replaced with 15% of dried C. pyrenoidosa (CRM) and reared in filtered seawater. The findings revealed that groupers reared 15d in T2 treatment (T2-15d) had a higher fat content, with apparent shift of microbial composition and functional pathways in the gut. Groupers reared 60d in T2 treatment (T2-60d) displayed an increased NADH dehydrogenases and cytochrome c oxidases gene expression, indicating more robust oxidative phosphorylation activity and ATP production crucial for the metabolic homeostasis. The reduction in the total amino acid content was also detected in groupers raised in T2-60d. Gastritis, enteritis and lipid malabsorption syndrome were observed in groupers raised 60d in T3 treatment (T3-60d), with the condition likely due to a higher intake of omega-6 to omega-3 fatty acids ratio in grouper raised 15 days in T3 (T3-15d) and T3-60d. This result indicated that grouper intestinal inflammation could arise from the inclusion of dried C. pyrenoidosa . Overall, the study outcomes demonstrated that introducing live C. pyrenoidosa to the culture water is advantageous in the digestion and enhance the energy metabolism of juvenile groupers.
Optogenetics combines genetics and biophotonics to enable noninvasive control of biological processes with high spatiotemporal precision. When engineered into protein machineries that govern the cellular information flow as depicted in the central dogma, multiple genetically encoded non-opsin photosensory modules have been harnessed to modulate gene transcription, DNA or RNA modifications, DNA recombination, and genome engineering by utilizing photons emitting in the wide range of 200–1000 nm. We present herein generally applicable modular strategies for optogenetic engineering and highlight latest advances in the broad applications of opsin-free optogenetics to program transcriptional outputs and precisely manipulate the mammalian genome, epigenome, and epitranscriptome. We also discuss current challenges and future trends in opsin-free optogenetics, which has been rapidly evolving to meet the growing needs in synthetic biology and genetics research.
