Gradient descent decomposition of force-field motor primitives optogenetically elicited for motor mapping of the murine lumbosacral spinal cord

Generating diverse motor behaviors critical for survival is a challenge that confronts the central nervous system(CNS)of all animals.During movement execution,the CNS performs complex calculations to control a large number of neuromusculoskeletal elements.The theory of modular motor control proposes that spinal interneurons are organized in discrete modules that can be linearly combined to generate a variety of behavioral patterns.These modules have been previously represented as stimulus-evoked force fields(FFs)comprising isometric limb-endpoint forces across workspace locations.Here,we ask whether FFs elicited by different stimulations indeed represent the most elementary units of motor control or are themselves the combination of a limited number of even more fundamental motor modules.To probe for potentially more elementary modules,we optogenetically stimulated the lumbosacral spinal cord of intact and spinalized Thy1-ChR2 transgenic mice(n=21),eliciting FFs from as many single stimulation loci as possible(20-70 loci per mouse)at minimally necessary power.We found that the resulting varieties of FFs defied simple categorization with just a few clusters.We used gradient descent to further decompose the FFs into their underlying basic force fields(BFFs),whose linear combination explained FF variability.Across mice,we identified 4-5 BFFs with partially localizable but overlapping representations along the spinal cord.The BFFs were structured and topographically distributed in such a way that a rostral-to-caudal traveling wave of activity across the lumbosacral spinal cord may generate a swing-to-stance gait cycle.These BFFs may represent more rudimentary submodules that can be flexibly merged to produce a library of motor modules for building different motor behaviors.

In vivo fiber photometry of neural activity in response to optogenetically manipulated inputs in freely moving mice

In vito fber photometry is a powerful technique to analyze the dy namics of population neurons during fiunctional study of neuroscience.Here,we introduced a detailed protocol for fiber photometry-based calciun reording in freely moving mice,covering from virus injection,fiber stub insertion,optogenetical stimulation to data procurement and analysis.Furthemnore,we applied this protocol to explore neuronal activity of mice latenal-posterior(LP)thalaric nucleus in response to optogenetical stimulation of primary visual cortex(V1)neurons,and explore axon clusters activity of optogenetically evoked V1 neurons.Final confirmation of virus-based protein expression in V1 and precise fber insertion indicated that the surgery procedure of this protocol is reliable for functional calcium recording.The scripts for data analysis and some tips in our protocol are provided in details.Together,this protocol is simple,low-cost,and effective for neuronal activity detection by fiber photometry,which will hep neuroscience researchers to carry out fiunctional and behavioral study in vivo.

Activation of medial septum cholinergic neurons restores cognitive function in temporal lobe epilepsy

Cognitive impairment is the most common complication in patients with temporal lobe epilepsy with hippocampal scle rosis.There is no effective treatment for cognitive impairment.Medial septum cholinergic neurons have been reported to be a potential target for controlling epileptic seizures in tempo ral lobe epile psy.However,their role in the cognitive impairment of temporal lobe epilepsy remains unclear.In this study,we found that patients with temporal lobe epile psy with hippocampal sclerosis had a low memory quotient and severe impairment in verbal memory,but had no impairment in nonverbal memory.The cognitive impairment was slightly correlated with reduced medial septum volume and medial septum-hippocampus tra cts measured by diffusion tensor imaging.In a mouse model of chronic temporal lobe epilepsy induced by kainic acid,the number of medial septum choline rgic neurons was reduced and acetylcholine release was reduced in the hippocampus.Furthermore,selective apoptosis of medial septum cholinergic neurons mimicked the cognitive deficits in epileptic mice,and activation of medial septum cholinergic neurons enhanced hippocampal acetylcholine release and restored cognitive function in both kainic acid-and kindling-induced epile psy models.These res ults suggest that activation of medial septum cholinergic neurons reduces cognitive deficits in temporal lobe epilepsy by increasing acetylcholine release via projections to the hippocampus.

Application of optogenetics in the study of gastrointestinal motility:A mini review

Disorders of gastrointestinal(GI)motility are associated with various symptoms such as nausea,vomiting,and constipation.However,the underlying causes of impaired GI motility remainunclear,which has led to variation in the efficacy of therapies to treat GI dysfunction.Optoge-netics is a novel approach through which target cells can be precisely controlled by light and hasshown great potential in GI motility research.Here,we summarized recent studies of GI motilitypatterns utilizing optogenetic devices and focused on the ability of opsins,which are geneticallyexpressed in different types of cells in the gut,to regulate the excitability of target cells.We hopethat our review of recent findings regarding optogenetic control of GI cells broadens the scope ofapplication for optogenetics in GI motility studies.

Genetically engineered systems revealed the roles of basal ganglia in sleep-wake regulation

The basal ganglia(BG) act as a cohesive functional unit that regulates motor function,habit formation,and reward/addictive behaviors. However,it is still not well understood how the BG maintains wakefulness and suppresses sleep to achieve al these fundamental functions until genetical y engineered systems developed these years. Significant research efforts have recently been directed at developing genetic-molecular tools to achieve reversible and cell-type specific in vivo silencing or activation of neurons in behaving animals. Optogenetic tools can be used both to specifically activate or inhibit neurons of interest and identify functional synaptic connectivity between specific neuronal populations,both in vivo and in brain slices. Another recently developed system by Roth and colleagues permits the selective and ″remote″ manipulation(activation and silencing) of neuronal activity via all 3 major GPCR signaling pathways(G_i,G_s and G_q). These so-called ″ designer receptors exclusively activated by designer drugs″(DREADD) involve mutant GPCRs that do not respond to their endogenous ligands but are responsive to otherwise inert biological compounds. Recently,we demonstrated the essential roles and the neural pathways of the neurons expressing adenosine A_(2A) receptors or dopamine D_1 receptors in the BG for sleep-wake regulation using the genetically engineered systems including optogenetics and DREADD. We proposed a plausible model in which the caudate-putamen and the nucleus accumbens integrates behavioral processes with sleep/wakefulness through adenosine and dopamine receptors.

A Micro Peristaltic Pump Using an Optically Controllable Bioactuator

Peristalsis is widely seen in nature, as this pumping action is important in digestive systems for conveying sustenance to every corner of the body. In this paper, we propose a muscle-powered tubular micro pump that provides peristaltic transport. We utilized Drosophila melanogaster larvae that express channelrhodopsin-2 (ChR2) on the cell membrane of skeletal muscles to obtain light-responsive muscle tissues. The larvae were forced to contract with blue light stimulation. While changing the speed of the propagating light stimulation, we observed displacement on the surface of the contractile muscle tissues. We obtained peristaltic pumps from the larvae by dissecting them into tubular structures. The average inner diameter of the tubular structures was about 400 lm and the average outer diameter was about 750 lm. Contractions of this tubular structure could be controlled with the same blue light stimulation. To make the inner flow visible, we placed microbeads into the peristaltic pump, and thus determined that the pump could transport microbeads at a speed of 120 lm-s1.

Optogenetics:a novel optical manipulation tool for medical investigation

Optogenetics is a new and rapidly evolving gene and neuroengineering technology that allows optical control of specific populations of neurons without affecting other neurons in the brain at high temporal and spatial resolution. By heterologous expression of the light-sensitive membrane proteins, cell type-specific depolarization or hyperpolarization can be optically induced on a millisecond time scale. Optogenetics has the higher selectivity and specificity compared to traditional electrophysiological techniques and pharmaceutical methods. It has been a novel promising tool for medical research. Because of easy handling, high temporal and spatial precision, optogenetics has been applied to many aspects of nervous system research, such as tactual neural circuit, visual neural circuit, auditory neural circuit and olfactory neural circuit, as well as research of some neurological diseases. The review highlights the recent advances of optogenetics in medical study.

Therapeutic potential of neuromodulation for demyelinating diseases

Neuromodulation represents a cutting edge class of both invasive and non-invasive therapeutic methods which alter the activity of neurons.Currently,several different techniques have been developed-or are currently being investigated–to treat a wide variety of neurological and neuropsychiatric disorders.Recently,in vivo and in vitro studies have revealed that neuromodulation can also induce myelination,meaning that it could hold potential as a therapy for various demyelinating diseases including multiple sclerosis and progressive multifocal leukencepalopathy.These findings come on the heels of a paradigm shift in the view of myelin's role within the nervous system from a static structure to an active co-regulator of central nervous system plasticity and participant in neuron-mediated modulation.In the present review,we highlight several of the recent findings regarding the role of neural activity in altering myelination including several soluble and contact-dependent factors that seem to mediate neural activitydependent myelination.We also highlight several considerations for neuromodulatory techniques,including the need for further research into spatiotemporal precision,dosage,and the safety and efficacy of transcranial focused ultrasound stimulation,an emerging neuromodulation technology.As the field of neuromodulation continues to evolve,it could potentially bring forth methods for the treatment of demyelinating diseases,and as such,further investigation into the mechanisms of neuron-dependent myelination as well as neuro-imaging modalities that can monitor myelination activity is warranted.

On the development of optical peripheral nerve interfaces

Limb loss and spinal cord injury are two debilitating conditions that continue to grow in prevalence. Prosthetic limbs and limb reanimation present two ways of providing affected individuals with means to interact in the world. These techniques are both dependent on a robust interface with the peripheral nerve. Current methods for interfacing with the peripheral nerve tend to suffer from low specificity, high latency and insufficient robustness for a chronic implant. An optical peripheral nerve interface may solve some of these problems by decreasing invasiveness and providing single axon specificity. In order to implement such an interface three elements are required:(1) a transducer capable of translating light into a neural stimulus or translating neural activity into changes in fluorescence,(2) a means for delivering said transducer and(3) a microscope for providing the stimulus light and detecting the fluorescence change. There are continued improvements in both genetically encoded calcium and voltage indicators as well as new optogenetic actuators for stimulation. Similarly, improvements in specificity of viral vectors continue to improve expression in the axons of the peripheral nerve. Our work has recently shown that it is possible to virally transduce axons of the peripheral nerve for recording from small fibers. The improvements of these components make an optical peripheral nerve interface a rapidly approaching alternative to current methods.

Optogenetic dissection of neuronal circuit underlying temporal lobe epilepsy

Temporal lobe epilepsy(TLE) is a common type of epilepsy and is not well controlled by current treatments.The frequent failure to treat TLE may be due to our lack of precise cellular/circuit mechanisms underlying TLE.The early series of our studies have proved the success of low-frequency stimulation treatment for epilepsy,which was mainly depending on the stimulation target,the stimulation frequency and stimulation time(the therapeutic-window phenomenon).Now,by using optogenetics,viral tracing,multiple-channel EEG analysis,imaging,electrophysiology and pharmacology strategies,we are continued to investigate the circuit mechanism of therapeutic deep brain stimulation,and found that entorhinal principal neurons mediate antiepileptic ″ glutamatergic-GABAergic″ neuronal circuit for brain stimulation treatments of epilepsy.Meanwhile,we are currently focusing on the interplay of inhibitory and excitatory network in the key input/output regions of the hippocampus that related to the generation of in TLE.Specially,we found that depolarized GABAergic signaling in subicular microcircuit mediates generalized seizures in TLE and a direct septal cholinergic circuit attenuates TLE through driving hippocampal somatostatin inhibition.These findings may be of therapeutic interest in understanding the pathological neuronal circuitry in TLE and further the development of novel therapeutic approaches or drug targets.

Modulation of abnormal neuronal circuit in temporal lobe epilepsy

OBJECTIVE Temporal lobe epilepsy(TLE)is one of the most common types of human epilepsy,and they are often resistant to current treatments.METHODS By using optogenetic,electrophysiological,imaging and pharmacology strategies,we aimed toinvestigate the underlying circuit mechanism of TLE and tried to developthe novel and efficient approach to control epilepsy.RESULTS(1)Using micro PET and multichannel EEG recording,we found an abnormal neural network,characterized by early hypometabolism and after discharge spread,during the epileptogenensis of TLE.(2)Deep brain stimulation,especially low frequency stimulation,targeted the epileptic focus and the areas outside of the focus(critical regions for seizure spread),such as the piriform cortex,cerebellum,entorhinal cortex or subiculum,reduced seizure severity in TLE.Its anti-epileptic effect is time-window dependent and polarity dependent,which shows a promising strategy for treating epileptic seizures.(3)Using an optogenetic strategy,we demonstrated that excitatory projection from entorhinal cortex to hippocampus instructs the brain-stimulation treatments of epilepsy.(4)Our data from both the clinical and experimental studies further demonstrated that a disinhibitory GABAergic neuronmediated microcircuit in the subiculum contributes to secondary generalized seizures in TLE.(5)Finally,based on abnormal synchronization of the electrical activity in epileptic circuit,we developed electroresponsive hydrogel nanoparticles modified with angiopep-2 to facilitate the delivery of the antiepileptic drug phenytoin sodium,which greatly improves the therapeutic index.CONCLUSION Our findings may update the current view of epileptic neuronal networks and suggest possible promising ways for epilepsy treatment.

颞叶癫痫异常神经环路分析(英文)

OBJECTIVE Temporal lobe epilepsy(TLE) is one of the most common types of human epilepsy,and they are often resistant to current treatments. METHODS By using optogenetic,electrophysiological,imaging and pharmacology strategies,we aimed toinvestigate the underlying circuit mechanism of TLE and tried to developthe novel and efficient approach to control epilepsy. RESULTS(1) Deep brain stimulation,especially low frequency stimulation,targeted the epileptic focus and the areas outside of the focus(critical regions for seizure spread),such as entorhinal cortex or subiculum,reduced seizure severity in TLE. Its anti-epileptic effect is time-window dependent and polarity dependent,which shows a promising strategy for treating epileptic seizures.(2) Using an optogenetic strategy,we demonstrated that excitatory projection from entorhinal cortex to hippocampus instructs the brain-stimulation treatments of epilepsy.(3) Our data from both the clinical and experimental studies further demonstrated that a disinhibitory GABAergic neuron-mediated microcircuit in the subiculum contributes to secondary generalized seizures in TLE.(4) Finally,based on abnormal synchronization of the electrical activity in epileptic circuit,we developed electro-responsive hydrogel nanoparticles modified with angiopep-2to facilitate the delivery of the antiepileptic drug phenytoin sodium,which greatly improves the therapeutic index. CONCLUSION Our findings may update the current view of epileptic neuronal networks and suggest possible promising ways for epilepsy treatment.

Optical read-out and modulation of peripheral nerve activity

Numerous clinical and research applications necessitate the ability to interface with peripheral nerve fibers to read and control relevant neural pathways. Visceral organ modulation and rehabilitative prosthesis are two areas which could benefit greatly from improved neural interfacing approaches. Therapeutic neural interfacing, or ‘bioelectronic medicine’, has potential to affect a broad range of disorders given that all the major organs of the viscera are neurally innervated. However, a better understanding of the neural pathways that underlie function and a means to precisely interface with these fibers are required. Existing peripheral nerve interfaces, consisting primarily of electrode-based designs, are unsuited for highly specific （individual axon） communication and/or are invasive to the tissue. Our laboratory has explored an optogenetic approach by which optically sensitive reporters and actuators are targeted to specific cell （axon） types. The nature of such an approach is laid out in this short perspective, along with associated technologies and challenges.

Optogenetic activation of intracellular signaling based on light-inducible protein-protein homo-interactions

Dynamic protein-protein interactions are essential for proper cell functioning.Homointeraction 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.

All-fiber-transmission photometry for simultaneous optogenetic stimulation and multi-color neuronal activity recording

Manipulating and real-time monitoring of neuronal activities with cell-type specificity and precise spatiotemporal resolution during animal behavior are fundamental technologies for exploring the functional connectivity, information transmission, and physiological functions of neural circuits in vivo. However, current techniques for optogenetic stimulation and neuronal activity recording mostly operate independently. Here, we report an all-fiber-transmission photometry system for simultaneous optogenetic manipulation and multi-color recording of neuronal activities and the neurotransmitter release in a freely moving animal. We have designed and manufactured a wavelength-independent multi-branch fiber bundle to enable simultaneous optogenetic manipulation and multi-color recording at different wavelengths. Further, we combine a laser of narrow linewidth with the lock-in amplification method to suppress the optogenetic stimulation-induced artifacts and channel crosstalk. We show that the collection efficiency of our system outperforms a traditional epi-fluorescence system. Further, we demonstrate successful recording of dynamic dopamine(DA) responses to unexpected rewards in the nucleus accumbens(NAc) in a freely moving mouse. We also show simultaneous dual-color recording of neuronal Ca2+ signals and DA dynamics in the NAc upon delivering an unexpected reward and the simultaneous optogenetic activating at dopaminergic terminals in the same location. Thus, our multi-function fiber photometry system provides a compatible, efficient, and flexible solution for neuroscientists to study neural circuits and neurological diseases.

Optogenetics stimulates nerve reorganization in the contralesional anterolateral primary motor cortex in a mouse model of ischemic stroke

The anterolateral motor cortex of rodents is an important motor auxiliary area,and its function is similar to that of the premotor area in humans.Activation and inhibition of the contralesional anterolateral motor cortex(cALM)have been shown to have direct effects on motor behavior.However,the significance of cALM activation and inhibition in the treatment of stroke remains unclear.This study investigated the role of optogenetic cALM stimulation in a mouse model of cerebral stroke.The results showed that 21-day optogenetic cALM inhibition,but not activation,improved neurological function.In addition,optogenetic cALM stimulation substantially altered dendritic structural reorganization and dendritic spine plasticity,as optogenetic cALM inhibition resulted in increased dendritic length,number of dendritic spines,and number of perforated synapses,whereas optogenetic activation led to an increase in the number of multiple synapse boutons and the number of dendritic intersections.Furthermore,RNA-seq analysis showed that multiple biological processes regulated by the cALM were upregulated immediately after optogenetic cALM inhibition,and that several immediate-early genes(including cFOS,Erg1,and Sema3f)were expressed at higher levels after optogenetic inhibition than after optogenetic activation.These results were confirmed by quantitative reverse transcription-polymerase chain reaction.Finally,immunofluorescence analysis showed that the c-FOS signal in layer V of the primary motor cortex in the ischemic hemisphere was higher after optogenetic cALM activation than it was after optogenetic cALM inhibition.Taken together,these findings suggest that optogenetic cALM stimulation promotes neural reorganization in the primary motor cortex of the ischemic hemisphere,and that optogenetic cALM inhibition and activation have different effects on neural plasticity.The study was approved by the Experimental Animal Ethics Committee of Fudan University(approval No.201802173 S)on March 3,2018.

Visual prostheses, optogenetics, stem cell and gene therapies: splitting the cake

The size of the blind population in 2015 was estimated to be approximately 36 million（Bourne et al.,2017）.According to the predictions by Bourne and co-workers,the number of the visually impaired is expected to reach nearly 100 million by 2050.

Studying neurological disorders using induced pluripotent stem cells and optogenetics

Neurological disorders are amongst the most widely studied human aliments.Yet,they are also one of the most poorly understood.Although most of these disorders are polygenic,genotype still plays an important role in their etiologies.For example,in schizophrenia and autism spectrum disorders,there is a 40-60%concordance rate in monozygotic twins,with 60-90%heritability（Burmeister et al.,2008）.However,the mechanisms by which multiple genes and their genomic variations influence the phenotypes of the disorders remain to be understood. The complexities of the disorders are tur- ther compounded by the individual rarity of the genomic variations and their variable penetrance （Cook and Scherer, 2008）. Thus, conventional disease modeling, such as gene knockout in cells or in animals, to attain the desired disease genotype may not be the most suitable platform for tackling most neurological disorders.

Optogenetic activation of glutamatergic neu⁃rons in somatosensory cortex promotes remy⁃elination in ischemic vascular dementia

OBJECTIVE Chronic cerebral hy⁃poperfusion can lead to progressive demyelin⁃ation and ischemic vascular dementia,yet there are no effective treatments.METHODS Magnetic resonance imaging was employed in patients with white matter damage,and optogenetics and skin stroking were exerted to activate glutamater⁃gic neurons in the somatosensory cortex in a clas⁃sical mouse model of ischemia vascular dementia.RESULTS White matter damage was correlated with disrupted cortical structure from MRI results.In a mouse model,activating glutamatergic neu⁃rons in the somatosensory cortex promotes prolif⁃eration of OPCs and remyelination to rescue cog⁃nitive impairment after chronic cerebral hypoper⁃fusion.Such therapeutic action was limited to stimulation with moderate intensity at the upper layers of the cortex,but was achieved over a wide time window after ischemia.Mechanistically,enhanced glutamatergic neuron-OPC functional synaptic connections are required for protection from activation of cortical glutamatergic neurons.Finally,skin stroking activation of the somatosen⁃sory cortex,an easier approach for clinical trans⁃lation,promoted OPC proliferation and remyelin⁃ation as well as cognitive recovery after cerebral hypoperfusion.CONCLUSION Activation of gluta⁃matergic neurons in the somatosensory cortex may serve as novel approaches for treating isch⁃emic vascular dementia through precise modula⁃tion of glutamatergic neuron-OPC circuits.

S6B-4 Roles of the Basal Ganglia in Sleep-Wake Regulation

The basal ganglia(BG)act as a cohesive functional unit that regulates motor function,habit formation,and reward/addictive behaviors.However,it is still not well understood how the BG maintains wakefulness and suppresses sleep to achieve all these fundamental functions until genetically engineered systems developed these years.We focused on the adenosine A2A and dopamine D1 Receptors(R)in the BG and obtained following 4 findings:①Nucleus accumbens(NAc)dopamine D1R-expressing neurons are essential in controlling wakefulness and are involved in physiological arousal via the lateral hypothalamus and midbrain circuits;②The rostromedial tegmental nucleus(RMTg),also called the GABAergic tail of the ventral tegmental area,projects to the midbrain dopaminergic system and other regions.Our findings reveal an essential role of the RMTg in the promotion of non-rapid eye movement(non-REM,NREM)sleep and homeostatic regulation;③Opposite to the D1R in the NAc,A2AR made a prominent contribution to sleep control associated with motivation.④Striatal adenosine A2AR neurons control active-period sleep via parvalbumin neurons in external globus pallidus.Taken together,we proposed a plausible model in which the caudate-putamen and NAc integrate behavioral processes with sleep/wakefulness through adenosine and dopamine receptors.The impacts of the BG in physiological sleep and insomnia will be discussed.