In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ChR2is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice.
Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice.
Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram EEG and electromyogram EMG monitoring to evaluate the sleep stage of mice. Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Sleep is essential for optimal cognitive function.
Recent findings also suggest that disturbances in sleep are associated with a wide range of diseases 123. Wakefulness is characterized by fast EEG oscillations Hz of low amplitude with purposeful and sustained motor activity. NREM sleep is defined by slow oscillations Hz of high amplitude delta waveswith lack of consciousness and purposeful motor activity. REM sleep is characterized by relatively fast oscillations Hz of low amplitude and almost complete bilateral muscle atonia 5.
Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans.
Borbely proposed a theory of sleep-wakefulness regulation known as the two process model 67. A homeostatic process, also referred to as process S, represents sleep pressure that accumulates during wakefulness and dissipates during sleep.
Another process, referred to as process C, is a circadian process, which explains why vigilance levels fluctuate in the 24 h cycle. Allostatic factors include nutritional states and emotion. Fear and anxiety are usually accompanied by an increase in arousal along with autonomic and neuroendocrine responses 1011 In particular, recent advances in optogenetics have allowed us to stimulate or inhibit specific neural circuits i n vivo with high spatial and temporal resolutions.
The BNST is thought to play an essential role in anxiety and fear. One of the greatest advances in neuroscience in recent years has been methods that enable manipulation of neurons with particular chemical identities in vivo, with high spatial and temporal resolutions.
Optogenetics is highly useful for demonstrating causal links between neural activity and specific behavioral responses Further, focal expression of photo-sensitive opsins such as channelrhodopsin 2 ChR2 20 or archaerhodopsin ArchT 21 combined with a Cre-loxP or Flp-FRT system allows us to manipulate a selective neuronal population and specific neural pathway To express opsins in a designated neuronal population, appropriate Cre driver mice and Cre-dependent virus vectors are most frequently used.
Transgenic or knock-in lines in which opsins are expressed in particular neuronal populations are also useful. Subscription Required. Please recommend JoVE to your librarian. AAV should be used in an isolated P1A graded room for injection, and the tube carrying AAV must be sterilized with an autoclave after all the volume is used up.Most investigations of behaviour and development have relied on mapping the neural circuits within the brain and CNS.
Understanding the connectivity of these circuits allow neuroscientists to understand behaviour and pathologies Figure 1. In the past, this has meant lesioning or destroying parts of these circuits, or electrophysiologically stimulating these circuits and examining the resulting behaviour.
The vertebrate brain mice, rats, primates and humanscontains many different cell types with distinct molecular expression patterns, physiological activity, and topological connectivity, which are intermingled in a highly heterogeneous network.
3.2 Molecular toolbox – Neural Circuits: The Basics
Studying specific groups of neurons in this milieu becomes very challenging and scientists using lesion, stimulation and tracing studies were never sure about the spatial i. The questions around specificity and temporal reversibility changed with the introduction of optogenetics.
Since the late s, optogenetics has ushered in a new era of potent and targeted control over multiple aspects of neural function. Genetic and optical methods applied together allow tight spatial and temporal control of the activity of specific kinds of neurons in the living brain, a revolutionary advance that will allow us to achieve an unprecedented understanding of neural circuit function in behaving animals.
Using this technique, neurons are first genetically engineered using a variety of mechanisms, described later to express light-sensitive proteins opsins. When these neurons are then illuminated with light of the correct frequency they will be transiently activated or inhibited or their signaling pathways will be modulated, depending on the particular kind of opsin that was chosen for expression. Cell type—specific expression is typically achieved with transgenic animals, viral vectors, or a combination, and spatially restricted light application allows for further refinement in targeting to specific brain regions.
Light can be applied in a variety of temporal patterns in order to optimally influence neuronal function permitting experimental control of spike frequency and burstiness, among other parametersand may be restricted to specific short behavioral periods of examination.
Optogenetic actuators are proteins that modify the activity of the cell in which they are expressed when that cell is exposed to light Figure 3. These actuators can be used to induce single or multiple action potentials which can be organized into regular spike trains or which can be pseudo-random at a user-controlled ratesuppress neural activity, or modify biochemical signaling pathways, with millisecond control over the timing of events.
The most powerful and widely used actuators are opsins—naturally occurring light-sensitive transmembrane proteins—that are found in a variety of organisms ranging from microbes to primates, and that can be used as found in nature or engineered to optimize functioning. Naturally occurring opsins can be broadly categorized into two major classes: microbial opsins Type I and vertebrate opsins Type II. Type I opsins are found in prokaryotic and eukaryotic microbial organisms, including bacteria, archaea, and algae, and are composed of a single membrane—bound protein component that functions as a pump or channel.
These opsins are used by their host microorganisms for a variety of functions, including navigation towards sources of energy and away from hazardous environments, and control the intracellular concentrations of a variety of ions and the beating of flagella. Type I opsins were used in the first optogenetics experiments to control neuronal function, both because of the ease of genetic engineering using a single component protein and because of their faster kinetics, and remain the primary but not exclusive source for new natural and engineered opsins.
Systems neuroscience aims to understand how neural dynamics create behavior. Optogenetics has accelerated progress in this area by making it possible to stimulate or inhibit neurons that express light-activated proteins — e.
The nematode C. Being able to deliver light to one cell with spatial selectivity is essential for targeted optogenetic perturbation in the many cases in C. In the worm motor circuit, for example, promoters are not available to drive expression of light-activated proteins in only one or a few neurons of the ventral nerve cord. Optogenetics has been applied to the mechanosensory circuit in C. Laser killing allows one to study the contribution of single touch receptor neurons to overall behavior by removing neurons, but it is often preferable to work with intact circuits 13 — Recently it has been shown that a digital micromirror device DMD can be used to deliver light with high spatial selectivity in immobilized C.
In many cases, however, the normal operation of neural circuits can only be studied in freely behaving animals, requiring a more sophisticated instrument. Here, we describe an optogenetic illumination system that allows perturbations of neural activity with high spatial and temporal resolution in an unrestrained animal, enabling us to co ntrol l ocomotion and be havior in r eal t ime CoLBeRT in C.
Machine-vision algorithms estimate the coordinates of targeted cells within the worm body and generate an illumination pattern that is projected onto the worm by a DMD with laser light, and the cycle repeats itself for the next frame.
Because the worm is a moving target, the faster an image can be captured and translated into DMD directives, the more accurately an individual cell can be targeted.
Here, we carry out studies of the motor circuit and mechanosensory circuit of unrestrained animals that illustrate the performance of the CoLBeRT system, a new tool for C. Either laser was incident onto a DMD with x elements. Filter cubes reflected the wavelengths for optogenetic illumination from the DMD onto the sample, while passing longer wavelengths for dark-field illumination to a camera.
A motorized stage keeps the specimen in the field of view. High resolution optogenetic control of freely moving C. A high-speed camera images the worm. Custom software instructs a DMD to reflect laser light onto targeted cells. Head and tail are located as maxima of boundary curvature red arrows. Centerline is calculated from the midpoint of line segments connecting dorsal and ventral boundaries blue barand is resampled to contain equally spaced points.
The worm is partitioned into segments by finding vectors green arrows from centerline to boundary, and selecting one that is most perpendicular to the centerline orange arrow.
Targets defined in worm coordinates are transformed into image coordinates and sent to the DMD for illumination green bar. Anterior is to the left and dorsal is to the top. Once an egg was laid, the worm was discarded. To accelerate real-time image analysis of worm posture, we developed the MindControl software package using the open-source OpenCV computer vision library An intuitive graphical user interface GUI enables the user to dynamically target specific regions of freely moving animals.
The MindControl software performs a sequence of image analysis operations on each frame received from the camera Fig.Early brain activity in health and disease View all 12 Articles. Coordinated patterns of electrical activity are critical for the functional maturation of neuronal networks, yet their interrogation has proven difficult in the developing brain. Optogenetic manipulations strongly contributed to the mechanistic understanding of network activation in the adult brain, but difficulties to specifically and reliably express opsins at neonatal age hampered similar interrogation of developing circuits.
Here, we introduce a protocol that enables to control the activity of specific neuronal populations by light, starting from early postnatal development. We show that brain area- layer- and cell type-specific expression of opsins by in utero electroporation IUEas exemplified for the medial prefrontal cortex PFC and hippocampus HPpermits the manipulation of neuronal activity in vitro and in vivo.
Both individual and population responses to different patterns of light stimulation are monitored by extracellular multi-site recordings in the medial PFC of neonatal mice. The expression of opsins via IUE provides a flexible approach to disentangle the cellular mechanism underlying early rhythmic network activity, and to elucidate the role of early neuronal activity for brain maturation, as well as its contribution to neurodevelopmental disorders.
Specific patterns of rhythmic network activity synchronize neuronal networks during early brain development Hanganu et al. Together with genetic programs this electrical activity controls the maturation of neuronal networks Khazipov and Luhmann, ; Hanganu-Opatz, However, subsequently diverse developmental processes such as neuronal migration, differentiation, axon growth, synapse formation, programmed cell death, and myelination are influenced by neuronal activity and mediate the activity-dependent maturation of neuronal networks Spitzer, ; Heck et al.
Spontaneous and sensory-triggered discontinuous patterns of oscillatory activity e. However, the cellular mechanisms generating the different patterns of early network activity are still largely unknown. Furthermore, direct evidence for the impact of early activity on the maturation of neuronal networks is still missing. Specific contributions of distinct neuronal populations to rhythmic network activity in the adult brain and their influence on behavior have been resolved during the last decade using optogenetics approaches Cardin et al.
Selective expression of light sensitive membrane channels and pumps in defined neuronal populations allow for precisely timed control of the activity of these neurons in intact networks Fenno et al.
The optogenetic approach helped to interrogate a large diversity of neural circuits in the adult brain involved in sensory processing Lepousez and Lledo, ; Olcese et al. Similar application of optogenetics in the developing brain has been hampered by the lack of flexible methods to selectively and effectively target neurons at early age.
The most common strategies to express light-sensitive proteins in the adult rodent brain are viral transduction and genetically modified mouse lines Zhang et al. Both techniques require cell-type specific promoters to restrict the expression to a neuronal subpopulation. While recently synapsin has been successfully used as promoter for viral injections in neonatal rats and led to reliable activation of neurons in the visual cortex Murata and Colonnese,most promoters that specifically label neuronal subpopulations yield only little expression during development.
Thereby, viral transduction is only of limited usability to investigate local network interactions during development. Furthermore, most viruses require 10—14 days until reliable and sufficient expression is achieved, too long for the interrogation of neonatal networks.
On the other hand, recently engineered viruses yielding fast expression are often toxic to the expressing cells even after short time periods Klein et al.
Optogenetic manipulation of neural activity in C. elegans: from synapse to circuits and behaviour
Another strategy for controlling the activity of developing circuits relies on genetically modified mouse lines. By these means the activity of gamma-aminobutyric acid GABA ergic interneurons was controlled by light during early postnatal development using the glutamic acid decarboxylase promoter Valeeva et al. However, the major drawback of this approach is the lack of area specificity, the light-sensitive opsins being expressed in the entire brain.
Attempts to spatially confine the illumination are useful, but cannot avoid that long range projections are co-activated and interfere with the investigation of the area of interest.Georgia Institute of Technology. Atlanta, GA. Although optogenetic techniques have proven to be invaluable for manipulating and understanding complex neural dynamics over the past decade, they still face practical and translational challenges in targeting networks involving multiple, large, or difficult-to-illuminate areas of the brain.
We utilized inhibitory luminopsins to simultaneously inhibit multiple limbic structures of the rat brain in a hardware-independent and cell-type specific manner to suppress seizure activity in a rat model of epilepsy. In addition to elucidating mechanisms of seizure suppression never directly demonstrated before, this work also illustrates how precise multi-focal control of pathological circuits can be advantageous for the treatment of epilepsy, which may also be applicable to the treatment and understanding of other neurological disorders involving broad neural circuits.
Optogenetic tools have provided scientists with an unprecedented ability to manipulate neuronal activity in a temporally and spatially precise manner that has proven to be invaluable for understanding and controlling complex neural dynamics in various animal models of neurological disease 1. Epilepsy is one such disease in which much progress has been made in identifying the crucial cell types and structures that are involved in generating and propagating seizure activity, and many of these targets have been effectively manipulated with optogenetics as a direct neuromodulatory therapy to suppress seizure activity see reviews by Krook-Magnuson et.
Despite these promising results, there still exists a translational challenge in controlling broad circuits, multiple targets, or large structures in the brain due to the practical limitations of light delivery. Although progress has been made towards developing devices that can illuminate multiple sites or larger tissue volumes e.
We previously demonstrated that optogenetic inhibition of neural activity can be achieved in a hardware-independent and scalable manner with inhibitory luminopsins iLMO 14optogenetic probes with their own genetically encoded light source that can be activated remotely by a chemical substrate coelenterazine, CTZ.
These optogenetic probes achieve the same cell-type specific inhibition as conventional opsins without the need for external hardware or chronic implants for light delivery. We were able to demonstrate the scalability of this approach by targeting expression to multiple structures of the brain covering an estimated volume of greater than 20 mm 3 These probes are therefore uniquely suited to address the hardware limitations and translatability of traditional optogenetic approaches. To evaluate the utility of inhibitory luminopsins for interrogating and manipulating circuit-level activity across multiple brain regions, we expressed the inhibitory luminopsin, iLMO2, in various limbic structures of the rat and evaluated its efficacy at suppressing seizure activity.
We first demonstrated that iLMO2 is able to suppress focal epileptic discharges induced by bicuculline injection in the hippocampus of anesthetized rats. We then found that simultaneous inhibition of four different nodes in the brain including the dentate gyrus DG and anterior nucleus of the thalamus ANT had an additive effect of suppressing behavioral seizures induced by pentylenetetrazol PTZ compared to inhibition of the individual nodes, demonstrating the utility and need for optogenetic tools capable of multi-focal, scalable, and cell-type specific control of neural activity.
To evaluate the ability of inhibitory luminopsins to suppress seizure activity, iLMO2 was selectively expressed in principal cells of the dorsal hippocampus of rats after induction of seizures by focal injection of bicuculline methiodide BM. We confirmed that CTZ was indeed reaching the principal cells around the recording electrodes by direct visualization of Hoescht dye which has a similar molecular weight to that of CTZ injected through the cannula Supplementary Figure 1A.Employing Electrophysiology and Optogenetics to Measure and Manipulate Neuronal Activity in Laborato
The characteristics of the induced discharges are shown in Supplementary Figure 1B. Bicuculline-induced epileptic discharges were acutely suppressed, with a corresponding decrease in broad band 0—60 Hz power of the local field potential LFPfollowing injections of CTZ Figure 1Atop.Understanding how an organism's nervous system transforms sensory input into behavioral outputs requires recording and manipulating its neural activity during unrestrained behavior.
Here we present an instrument to simultaneously monitor and manipulate neural activity while observing behavior in a freely moving animal, the nematode Caenorhabditis elegans.
Neural activity is recorded optically from cells expressing a calcium indicator, GCaMP3. Neural activity is manipulated optically by illuminating targeted neurons expressing the optogenetic protein Channelrhodopsin. Real-time computer vision software tracks the animal's behavior and identifies the location of targeted neurons in the nematode as it crawls. Patterned illumination from a DMD is used to selectively illuminate subsets of neurons for either calcium imaging or optogenetic stimulation.
Real-time computer vision software constantly updates the illumination pattern in response to the worm's movement and thereby allows for independent optical recording or activation of different neurons in the worm as it moves freely. We use the instrument to directly observe the relationship between sensory neuron activation, interneuron dynamics and locomotion in the worm's mechanosensory circuit.
We record and compare calcium transients in the backward locomotion command interneurons AVA, in response to optical activation of the anterior mechanosensory neurons ALM, AVM or both. Understanding the neural basis of behavior is a fundamental goal of neuroscience. In many cases, however, the normal operation of neural circuits can be studied only in freely behaving animals. Previous neurophysiology experiments in behaving animals employed one of two approaches: Either probes, such as electrodes or optical fibers, were surgically implanted into an animal and recorded via tether or wireless backpack Wilson and McNaughton, ; Lee et al.
The nematode Caenorhabditis elegansdue to its small size and optical transparency allows for a third approach, whereby an external and non-invasive tracking microscope keeps pace with the worm's motion to image the worm brain while allowing the animal to roam freely without restraint. Moreover, the worm's small nervous system of neurons, its genetic tractability and its known connectome make it well suited to an investigation of the neural basis of behavior.
Early recordings of neural activity from freely moving C. Subsequently, automated tracking systems were developed that used computer vision Ben Arous et al. For many of these systems, intracellular calcium transients can be recorded while also observing the worm's behavior.
Similar systems have been employed to measure neural activity in zebrafish larvae, another small optically transparent organism Naumann et al. These systems have provided a valuable means to correlate activity with behavior and in worms they have elucidated neural coding of temperature during thermotaxis Clark et al.
Optogenetics allows for optically stimulating or inhibiting neurons that express light activated proteins, like Channelrhodopsin.
Early experiments relied on genetic specificity for targeting their stimulus.
For example, optogenetics was first used to study the mechanosensory circuit in C. Patterned illumination overcomes this limitation by combining genetic specificity with optical targeting. By delivering light to only targeted cells or tissues, neurons can be illuminated individually provided that there is a sufficiently sparse expression pattern.Meaning that despite a growing market share Bet365 are still trailing behind.
Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans.
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In his judgement, Justice Jonathan Beach noted the companies were large and collectively marketed themselves in Australia as the world's biggest online betting company. Topics: business-economics-and-finance, company-news, gambling, courts-and-trials, australiaIf you have inside knowledge of a topic in the news, contact the ABC.
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