Inhibitory Opsin Research Articles

Reward-driven cerebellar climbing fiber activity influences both neural and behavioral learning.

The cerebellum plays a key role in motor coordination and learning. In contrast with classical supervised learning models, recent work has revealed that CFs can signal reward-predictive information in some behaviors. This raises the question of whether CFs may also operate according to principles similar to those described by reinforcement learning models. To test how CFs operate during reward-guided behavior, and evaluate the role of reward-related CF activity in learning, we have measured CF responses in Purkinje cells of the lateral cerebellum during a Pavlovian task using 2-photon calcium imaging. Specifically, we have performed multi-stimulus experiments to determine whether CF activity meets the requirements of a reward prediction error (rPE) signal for transfer from an unexpected reward to a reward-predictive cue. We find that once CF activity is transferred to a conditioned stimulus, and there is no longer a response to reward, CFs cannot generate learned responses to a second conditioned stimulus that carries the same reward prediction. In addition, by expressing the inhibitory opsin GtACR2 in neurons of the inferior olive, and optically inhibiting these neurons across behavioral training at the time of unexpected reward, we find that the transfer of CF signals to the conditioned stimulus is impaired. Moreover, this optogenetic inhibition also impairs learning, resulting in a deficit in anticipatory lick timing. Together, these results indicate that CF signals can exhibit several characteristics in common with rPEs during reinforcement learning, and that the cerebellum can harness these learning signals to generate accurately timed motor behavior.

A brief history of somatostatin interneuron taxonomy or: how many somatostatin subtypes are there, really?

We provide a brief (and unabashedly biased) overview of the pre-transcriptomic history of somatostatin interneuron taxonomy, followed by a chronological summary of the large-scale, NIH-supported effort over the last ten years to generate a comprehensive, single-cell RNA-seq-based taxonomy of cortical neurons. Focusing on somatostatin interneurons, we present the perspective of experimental neuroscientists trying to incorporate the new classification schemes into their own research while struggling to keep up with the ever-increasing number of proposed cell types, which seems to double every two years. We suggest that for experimental analysis, the most useful taxonomic level is the subdivision of somatostatin interneurons into ten or so "supertypes," which closely agrees with their more traditional classification by morphological, electrophysiological and neurochemical features. We argue that finer subdivisions ("t-types" or "clusters"), based on slight variations in gene expression profiles but lacking clear phenotypic differences, are less useful to researchers and may actually defeat the purpose of classifying neurons to begin with. We end by stressing the need for generating novel tools (mouse lines, viral vectors) for genetically targeting distinct supertypes for expression of fluorescent reporters, calcium sensors and excitatory or inhibitory opsins, allowing neuroscientists to chart the input and output synaptic connections of each proposed subtype, reveal the position they occupy in the cortical network and examine experimentally their roles in sensorimotor behaviors and cognitive brain functions.

The role of the parafascicular thalamic nucleus in action initiation and steering.

The parafascicular (Pf) nucleus of the thalamus has been implicated in arousal and attention, but its contributions to behavior remain poorly characterized. Here, using invivo and invitro electrophysiology, optogenetics, and 3D motion capture, we studied the role of the Pf nucleus in behavior using a continuous reward-tracking task in freely moving mice. We found that many Pf neurons precisely represent vector components of velocity, with a strong preference for ipsiversive movements. Their activity usually leads velocity, suggesting that Pf output is critical for self-initiated orienting behavior. To test this hypothesis, we expressed excitatory or inhibitory opsins in VGlut2+ Pf neurons to manipulate neural activity bidirectionally. We found that selective optogenetic stimulation of these neurons consistently produced ipsiversive head turning, whereas inhibition stopped turning and produced downward movements. Taken together, our results suggest that the Pf nucleus can send continuous top-down commands that specify detailed action parameters (e.g., direction and speed of the head), thus providing guidance for orienting and steering during behavior.

Hypothalamic PVN CRH Neurons Signal Through PVN GABA Neurons to Suppress GnRH Pulse Generator Frequency in Female Mice.

Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) are central to the stress response. Chemogenetic activation of PVN CRH neurons decreases LH pulse frequency but the mechanism is unknown. In the present study, optogenetic stimulation of PVN CRH neurons suppressed LH pulse frequency in estradiol-replaced ovariectomized CRH-cre mice, and this effect was augmented or attenuated by intra-PVN GABAA or GABAB receptor antagonism, respectively. PVN CRH neurons signal to local GABA neurons, which may provide a possible indirect mechanism by which PVN CRH neurons suppress LH pulse frequency. Optogenetic stimulation of potential PVN GABAergic projection terminals in the hypothalamic arcuate nucleus in ovariectomized estradiol-replaced Vgat-cre-tdTomato mice via an optic fiber implanted in the arcuate nucleus suppressed LH pulse frequency. To further determine whether PVN CRH neurons signal through PVN GABA neurons to suppress LH pulsatility, we combined recombinase mice with intersectional vectors to selectively target these neurons. CRH-cre::Vgat-FlpO mice expressing the stimulatory opsin ChRmine in non-GABAergic CRH neurons alone or in combination with the inhibitory opsin NpHR3.3 in non-CRH-expressing GABA neurons in the PVN were used. Optogenetic stimulation of non-GABAergic CRH neurons suppressed pulsatile LH secretion; however, LH pulse frequency was not affected when CRH neurons were stimulated and PVN GABA neurons were simultaneously inhibited. Together, these studies demonstrate that suppression of LH pulse frequency in response to PVN CRH neuronal activation is mediated by GABAergic signalling intrinsic to the PVN and may incorporate PVN GABAergic projection to the hypothalamic GnRH pulse generator.

Temporal Dynamics Underlying Prelimbic Prefrontal Cortical Regulation of Action Selection and Outcome Evaluation during Risk/Reward Decision-Making.

Risk/reward decision-making is a dynamic process that includes periods of deliberation before action selection and evaluation of the action outcomes that bias subsequent choices. Inactivation of the prelimbic (PL) cortex has revealed its integral role in updating decision biases in the face of changes in probabilistic reward contingencies, yet how phasic PL signals during different phases of the decision process influence choice remains unclear. We used temporally specific optogenetic inhibition to selectively disrupt PL activity coinciding with action selection and outcome phases to examine how these signals influence choice. Male rats expressing the inhibitory opsin eArchT within PL excitatory neurons were well trained on a probabilistic discounting task, entailing choice between small/certain versus large/risky rewards, the probability of which varied over a session (50-12.5%). During testing, brief light pulses suppressed PL activity before choice or after different outcomes. Prechoice suppression reduced bias toward more preferred/higher utility options and disrupted how recent outcomes influenced subsequent choice. Inhibition during risky losses induced a similar profile, but here, the impact of reward omissions were either amplified or diminished, relative to the context of the estimated profitability of the risky option. Inhibition during large or small reward receipt reduced risky choice when this option was more profitable, suggesting these signals can both reinforce rewarded risky choices and also act as a relative value comparator signal that augments incentive for larger rewards. These findings reveal multifaceted contributions by the PL in implementing decisions and integrating action-outcome feedback to assign context to the decision space.SIGNIFICANCE STATEMENT The PL prefrontal cortex plays an integral role in guiding risk/reward decisions, but how activity in this region during different phases of the decision process influences choice is unclear. By using temporally specific optogenetic manipulations of this activity, the present study unveiled previously uncharacterized and differential contributions by PL in implementing decision policies and how evaluation of decision outcomes shape subsequent choice. These findings provide novel insight into the dynamic processes engaged by the PL that underlie action selection in situations involving reward uncertainty that may aid in understanding the mechanism underlying normal and aberrant decision-making processes.

Hippocampal fear engrams modulate ethanol-induced maladaptive contextual generalization in mice.

The compounding symptomatology of alcohol use disorder (AUD) and co-occurring mental health disorders gives rise to interactions of maladaptive neurobiological processes, the etiology of which are elusive. Here, we devised an optogenetic strategy aimed at rescuing maladaptive fear processing in male c57BL/6 mice that underwent a chronic ethanol administration and forced abstinence paradigm. In the first experiment, we confirmed that fear acquisition and maladaptive contextual generalization was potentiated in ethanol-exposed mice during fear conditioning and exposure to a novel environment, respectively. In the second experiment, using an activity-dependent tet-tag system, we labeled the neural ensemble selectively activated by contextual fear conditioning in the dorsal hippocampus with an inhibitory opsin to attenuate behavioral dysfunctions resulting from ethanol exposure. We found that acute optogenetic perturbations during exposure to a novel environment suppressed maladaptive generalization in ethanol-exposed mice. These results provide further evidence for a crucial link between ethanol exposure and impaired fear memory processing by providing cellular and behavioral insights into the neural circuitry underlying AUD and maladaptive fear processing.

Unblock the Block! Preventing Inhibitory Failure to Maintain Inhibitory Restraint.

Disrupting Epileptiform Activity by Preventing Parvalbumin Interneuron Depolarization Block Călin A, Ilie AS, Akerman CJ. J Neurosci. 2021;41(45):9452-9465. doi:10.1523/JNEUROSCI.1002-20.2021. Epub 2021 Oct 5.Inhibitory synaptic mechanisms oppose epileptic network activity in the brain. The breakdown in this inhibitory restraint and propagation of seizure activity has been linked to the overwhelming of feedforward inhibition, which is provided in large part by parvalbumin-expressing (PV) interneurons in the cortex. The underlying cellular processes therefore represent potential targets for understanding and preventing the propagation of seizure activity. Here we use an optogenetic strategy to test the hypothesis that depolarization block in PV interneurons is a significant factor during the loss of inhibitory restraint. Depolarization block results from the inactivation of voltage-gated sodium channels and leads to impaired action potential firing. We used focal NMDA stimulation to elicit reproducible epileptiform discharges in hippocampal organotypic brain slices from male and female mice and combined this with targeted recordings from defined neuronal populations. Simultaneous patch-clamp recordings from PV interneurons and pyramidal neurons revealed epileptiform activity that was associated with an overwhelming of inhibitory synaptic mechanisms and the emergence of a partial, and then complete, depolarization block in PV interneurons. To counteract this depolarization block, we developed protocols for eliciting pulsed membrane hyperpolarization via the inhibitory opsin, archaerhodopsin. This optical approach was effective in counteracting cumulative inactivation of voltage-gated channels, maintaining PV interneuron action potential firing properties during the inhibitory restraint period, and reducing the probability of initiating epileptiform activity. These experiments support the idea that depolarization block is a point of weakness in feedforward inhibitory synaptic mechanisms and represents a target for preventing the initiation and spread of seizure activity.

Investigation of selective vagal afferents subserving baroreflex function and blood pressure control: implications for understanding and alleviating hypertension

Cardiovascular homeostasis is maintained, in part, by neural signals arising from arterial baroreceptors (BRs) that apprise the brain of blood volume and pressure within the carotid sinus and aortic arch. Here, we test the overall hypothesis that neurons within the nodose ganglia that express the angiotensin type 1a receptor (referred to as NDGAT1aR) serve as BR that undergo plasticity during chronic elevations in blood pressure (BP). Towards this end, mice that express Cre‐recombinase at the AT1aR locus were delivered a Cre‐dependent AAV to express tdTomato within NDGAT1aR. These studies revealed that NDGAT1aR receive input from the aortic arch and carotid sinus, project to the caudal nucleus of the solitary tract (cNTS), and synthesize mechanosensitive ion channels, Piezo‐1/‐2. To determine whether NDGAT1aR couple their firing to elevated BP, we generated mice with the expression of the fluorescent Ca2+ indicator, GCaMP6s, directed to the AT1aR locus. Two‐photon (2P) intravital imaging revealed that phenylephrine (PE; i.v.) significantly increased GCaMP6s fluorescence within NDGAT1aR. To determine if optogenetic perturbation of NDGAT1aR at their soma or axon terminals within the cNTS affect the baroreflex, we directed the expression of excitatory (channelrhodpsin2; ChR2) or inhibitory (halorhodopsin; Halo) opsins to the AT1aR locus. Optical excitation of NDGAT1aR at their soma or axons terminals in the cNTS, elicited robust and frequency‐dependent decreases in BP and heart rate. Conversely, optical inhibition of afferents in the cNTS originating from NDGAT1aR blunted pressor responses to PE. The implication is that NDGAT1aR are sufficient and necessary to elicit appropriate compensatory responses to vascular mechanosensation induced by vasoconstriction. Opsin‐ or GCaMP6s‐expressing mice were then subjected to the DOCA‐salt model of hypertension. Optical excitation elicited hypotensive and bradycardic responses; however, the duration of these effects was altered, consistent with hypertension‐induced impairment of BR. Similarly, the increased GCaMP6s fluorescence observed after administration of PE was significantly delayed in mice that were subjected to DOCA‐salt or chronic delivery of angiotensin II. Finally, AT1aR‐flox mice were delivered AAV expressing Cre and/or tdTomato into the nodose ganglia to probe the function of AT1aR(s) themselves, in mediating the baroreflex. Preliminary results indicate deleting AT1aR(s) from the nodose ganglia alters pressor responses to vasoactive drugs. Collectively, these results reveal the structure and function of NDGAT1aR and suggest that such neurons may be exploited to discern and relieve hypertension.

Regulation of dark period sleep by the Amygdala: A microinjection and optogenetics study

The central nucleus of the amygdala (CNA) projects to brainstem regions that generate and regulate rapid eye movement sleep (REM). We used optogenetics to assess the influence of CNA inputs into reticularis pontis oralis (RPO), pedunculopontine tegmentum (PPT) and nucleus subcoeruleus (SubC) on dark period sleep. We compared these results to effects of microinjections into CNA of the GABAA agonist, muscimol (MUS, inhibition of cell bodies) and tetrodotoxin (TTX, inhibition of cell bodies and fibers of passage). For optogenetics, male Wistar rats received excitatory (AAV5-EF1a-DIO -hChR2(H134R)-EYFP) or inhibitory (AAV-EF1a-DIO-eNpHR3.0-EYFP; DIO-eNpHR3.0) opsins into CNA and AAV5-EF1a-mCherry-IRES-WGA-Cre into RPO, PPT, or SubC. This enabled only CNA neurons synaptically connected to each region to express opsin. Optic cannulae for light delivery into CNA and electrodes for determining sleep were implanted. Sleep was recorded with and without blue or amber light stimulation of CNA. Separate rats received MUS or TTX into CNA prior to recording sleep. Optogenetic activation of CNA neurons projecting to RPO enhanced REM and did not alter non-REM (NREM) whereas activation of CNA neurons projecting to PPT or SubC did not significantly affect sleep. Inhibition of CNA neurons projecting to any region did not significantly alter sleep. TTX inactivation of CNA decreased REM and increased NREM whereas muscimol inactivation did not significantly alter sleep. Thus, the amygdala can regulate decreases and increases in REM, and RPO is important for CNA promotion of REM. Fibers passing through CNA, likely from the basolateral nucleus of the amygdala, also play a role in regulating sleep.

Dysfunction of parvalbumin-expressing cells in the thalamic reticular nucleus induces cortical spike-and-wave discharges and an unconscious state.

Spike-and-wave discharges and an accompanying loss of consciousness are hallmarks of absence seizure, which is a childhood generalized epilepsy disorder. In absence seizure, dysfunction of the cortico-thalamo-cortico circuitry is thought to engage in abnormal cortical rhythms. Previous studies demonstrated that the thalamic reticular nucleus has a critical role in the formation of normal cortical rhythms; however, whether thalamic reticular nucleus dysfunction leads directly to abnormal rhythms, such as epilepsy, is largely unknown. We found that expressing the inhibitory opsin, archaerhodopsin, including in the thalamic reticular nucleus, caused abnormal cortical rhythms in Pvalb-tetracycline transactivator::tetO-ArchT (PV-ArchT) double transgenic mice. We validated the PV-ArchT line as a new mouse model of absence seizure through physiological and pharmacological analyses, as well as through examining their behavioural features. We then discovered that archaerhodopsin expression exclusively in thalamic reticular nucleus parvalbumin-positive neurons was sufficient to induce cortical spike-and-wave discharges using adeno-associated virus-mediated thalamic reticular nucleus targeting. Furthermore, we found that archaerhodopsin expression impaired rebound burst firing and T-current in thalamic reticular nucleus parvalbumin-positive cells by slice physiology. Although T-current in the thalamic reticular nucleus was impaired, the T-current blocker ethosuximide still had a therapeutic effect in PV-ArchT mice, suggesting a gain of function of T-type calcium channels in this absence seizure model. However, we did not find any over- or misexpression of T-type calcium channel genes in the thalamus or the cortex. Thus, we demonstrated that thalamic reticular nucleus dysfunction led to an absence seizure-like phenotype in mice. In a final set of experiments, we showed that the archaerhodopsin-mediated absence seizure-like phenotype disappeared after the removal of archaerhodopsin by using a time-controllable transgenic system. These data may provide a hint as to why many absence seizures naturally regress.

Noninvasive Manipulation of Ion Channels for Neuromodulation and Theranostics

ConspectusFunctional and structural studies of ion channels have deepened our understanding of their mechanisms and important role in regulating neuronal activity and treating disease. Manipulation of ion channels can directly control electrochemical signals in the nervous and cardiovascular systems for advanced neuromodulation and theranostics. Manipulation tools based on ion channel control, such as optogenetics, electrical stimulation, chemogenetics, and gene editing, have advanced basic biomedical research and enabled unprecedented treatment strategies. Nevertheless, conventional approaches are associated with limitations such as invasiveness, irreversibility, or low spatiotemporal resolution, which limit their clinical application. Therefore, targeted noninvasive or minimally invasive modulation of various ion channel activities is highly desirable.In recent years, nanomaterials have enabled noninvasive and precise modulation of ion channels due to their tunable morphology and physicochemical properties. Based on activation modes, current nanomaterial-based methods for ion channel control include light stimulation, thermal regulation, and mechanical manipulation. Nanomaterials can serve as energy transducers to convert macroscopic signals that penetrate deep into tissue, such as near-infrared light or magnetic fields, into local stimuli for ion channel manipulation. For photomodulation, lanthanide-doped nanoparticles convert near-infrared light into visible energy by upconversion, enabling bidirectional control of excitatory and inhibitory opsins in deep tissues. For thermal modulation, photothermal nanoparticles transform absorbed light, such as near-infrared, into heat to activate thermosensitive channels in deep targets. For mechanical manipulation, magnetic nanoparticles convert external magnetic fields into local attractive and torque forces to regulate the functionality of mechanosensitive channels without concern for penetration depth. Moreover, nanomaterial-based strategies can realize targeted and minimally invasive control over the activities of various ion channels even at the single-cell level, maximizing their feasibility and application potential.In this Account, we focus on the manipulation of ion channels using nanomaterials and categorize current approaches into photoconversion optogenetics, thermogenetics, and magnetogenetics. We first introduce the biological principles of various opsins, discuss thermo- and mechanosensitive ion channels and describe their molecular mechanisms in the context of channel structures. We also outline the general principles of emerging nanomaterials in terms of energy conversion capability. We highlight the major advances in nanoprobe-enabled systems and their modern applications in neuromodulation and theranostics. We conclude the Account with a discussion of existing challenges and future prospects.

In vivo blue light illumination for optogenetic inhibition: effect on local temperature and excitability of the rat hippocampus

Objective. The blue light-activated inhibitory opsin, stGtACR2, is gaining prominence as a neuromodulatory tool due its ability to shunt-inhibit neurons and is being frequently used in in vivo experimentation. However, experiments involving stGtACR2 use longer durations of blue light pulses, which inadvertently heat up the local brain tissue and confound experimental results. Therefore, the heating effects of illumination parameters used for in vivo optogenetic inhibition must be evaluated. Approach. To assess blue light (473 nm)-induced heating of the brain, we used a computational model as well as direct temperature measurements using a fiber Bragg grating (FBG). The effects of different light power densities (LPDs) and pulse durations on evoked potentials (EP) recorded from dentate gyrus were assessed. For opsin-negative rats, LPDs between 127 and 636 mW mm−2 and pulse durations between 20 and 5120 ms were tested while for stGtACR2 expressing rats, LPD of 127 mW mm−2 and pulse durations between 20 and 640 ms were tested. Main results. Increasing LPDs and pulse durations logarithmically increased the peak temperature and significantly decreased the population spike (PS) amplitude and latencies of EPs. For a pulse duration of 5120 ms, the tissue temperature increased by 0.6 °C–3.4 °C. All tested LPDs decreased the PS amplitude in opsin-negative rats, but 127 mW mm−2 had comparatively minimal effects and a significant effect of increasing light pulse duration was seen from 320 ms and beyond. This corresponded with an average temperature increase of 0.2 °C–1.1 °C at the recorded site. Compared to opsin-negative rats, illumination in stGtACR2-expressing rats resulted in much greater inhibition of EPs. Significance. Our study demonstrates that light-induced heating of the brain can be accurately measured in vivo using FBG sensors. Such light-induced heating alone can affect neuronal excitability. Useful neuromodulation by the activation of stGtACR2 is still possible while minimizing thermal effects.

Corticothalamic projections deliver enhanced responses to medial geniculate body as a function of the temporal reliability of the stimulus.

Ageing and challenging signal-in-noise conditions are known to engage the use of cortical resources to help maintain speech understanding. Extensive corticothalamic projections are thought to provide attentional, mnemonic and cognitive-related inputs in support of sensory inferior colliculus (IC) inputs to the medial geniculate body (MGB). Here we show that a decrease in modulation depth, a temporally less distinct periodic acoustic signal, leads to a jittered ascending temporal code, changing MGB unit responses from adapting responses to responses showing repetition enhancement, posited to aid identification of important communication and environmental sounds. Young-adult male Fischer Brown Norway rats, injected with the inhibitory opsin archaerhodopsin T (ArchT) into the primary auditory cortex (A1), were subsequently studied using optetrodes to record single-units in MGB. Decreasing the modulation depth of acoustic stimuli significantly increased repetition enhancement. Repetition enhancement was blocked by optical inactivation of corticothalamic terminals in MGB. These data support a role for corticothalamic projections in repetition enhancement, implying that predictive anticipation could be used to improve neural representation of weakly modulated sounds. KEY POINTS: In response to a less temporally distinct repeating sound with low modulation depth, medial geniculate body (MGB) single units show a switch from adaptation towards repetition enhancement. Repetition enhancement was reversed by blockade of MGB inputs from the auditory cortex. Collectively, these data argue that diminished acoustic temporal cues such as weak modulation engage cortical processes to enhance coding of those cues in auditory thalamus.

Disrupting Epileptiform Activity by Preventing Parvalbumin Interneuron Depolarization Block

Inhibitory synaptic mechanisms oppose epileptic network activity in the brain. The breakdown in this inhibitory restraint and propagation of seizure activity has been linked to the overwhelming of feedforward inhibition, which is provided in large part by parvalbumin-expressing (PV) interneurons in the cortex. The underlying cellular processes therefore represent potential targets for understanding and preventing the propagation of seizure activity. Here we use an optogenetic strategy to test the hypothesis that depolarization block in PV interneurons is a significant factor during the loss of inhibitory restraint. Depolarization block results from the inactivation of voltage-gated sodium channels and leads to impaired action potential firing. We used focal NMDA stimulation to elicit reproducible epileptiform discharges in hippocampal organotypic brain slices from male and female mice and combined this with targeted recordings from defined neuronal populations. Simultaneous patch-clamp recordings from PV interneurons and pyramidal neurons revealed epileptiform activity that was associated with an overwhelming of inhibitory synaptic mechanisms and the emergence of a partial, and then complete, depolarization block in PV interneurons. To counteract this depolarization block, we developed protocols for eliciting pulsed membrane hyperpolarization via the inhibitory opsin, archaerhodopsin. This optical approach was effective in counteracting cumulative inactivation of voltage-gated channels, maintaining PV interneuron action potential firing properties during the inhibitory restraint period, and reducing the probability of initiating epileptiform activity. These experiments support the idea that depolarization block is a point of weakness in feedforward inhibitory synaptic mechanisms and represents a target for preventing the initiation and spread of seizure activity.SIGNIFICANCE STATEMENT GABAA receptor-mediated synaptic transmission opposes seizure activity by establishing an inhibitory restraint against spreading excitation. Parvalbumin-expressing (PV) interneurons contribute significantly to this inhibitory restraint, but it has been suggested that these cells are overwhelmed as they enter a state of "depolarization block." Here we test the importance of this process by devising an optogenetic strategy to selectively relieve depolarization block in PV interneurons. By inducing brief membrane hyperpolarization, we show that it is possible to reduce depolarization block in PV interneurons, maintain their action potential firing in the face of strong excitation, and disrupt epileptiform activity in an in vitro model. This represents a proof of principle that targeting rate-limiting processes can strengthen the inhibitory restraint of epileptiform activity.

Brushing-Assisted Two-Color Quantum-Dot Micro-LED Array Towards Bi-Directional Optogenetics

Bi-directional optogenetics at single-cell level requires localized, bright, and multi-color light sources to activate both excitatory and inhibitory opsins. To this end, here we report a simple fabrication method of high-density, two-color, quantum dot (QD) based micron-sized light emitting diode (micro-LED) arrays. In particular, we micro-patterned InP/ZnS QDs on top of GaN-based micro-LED pixels via a simple brushing method, and coated them with a spectral filter. The resulting array featured sub- <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$20~\mu \text{m}$ </tex-math></inline-formula> sized light spots near both 462 nm and 623 nm, with their optical power density being <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">ca.</i> 0.1 – 1.0 mW/mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> . Combined with its low crosstalk and fast response, our two-color QD-LED array may hold promise for bi-directional optogenetics ultimately at the cellular level.

BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.

Remote control of neural function by X-ray-induced scintillation

Scintillators emit visible luminescence when irradiated with X-rays. Given the unlimited tissue penetration of X-rays, the employment of scintillators could enable remote optogenetic control of neural functions at any depth of the brain. Here we show that a yellow-emitting inorganic scintillator, Ce-doped Gd3(Al,Ga)5O12 (Ce:GAGG), can effectively activate red-shifted excitatory and inhibitory opsins, ChRmine and GtACR1, respectively. Using injectable Ce:GAGG microparticles, we successfully activated and inhibited midbrain dopamine neurons in freely moving mice by X-ray irradiation, producing bidirectional modulation of place preference behavior. Ce:GAGG microparticles are non-cytotoxic and biocompatible, allowing for chronic implantation. Pulsed X-ray irradiation at a clinical dose level is sufficient to elicit behavioral changes without reducing the number of radiosensitive cells in the brain and bone marrow. Thus, scintillator-mediated optogenetics enables minimally invasive, wireless control of cellular functions at any tissue depth in living animals, expanding X-ray applications to functional studies of biology and medicine.

Accumbal D1 and D2 medium spiny neurons control distinct learning parameters in complex behavior

Value-based decision-making is at the core of nearly all motivated behaviors and requires the ability to associate outcomes with specific actions and make adaptive decisions about future behavioral action. At the core of value-based decision-making and reinforcement is the nucleus accumbens (NAc), which is integrally involved in learning, selecting, and executing goal-oriented behaviors. The NAc is a heterogeneous population primarily composed of D1 and D2 medium spiny projection (MSN) neurons that are thought to have opposing roles in behavior, in which D1 MSNs promote reward and D2 MSNs promote aversion. By expressing channelrhodopsin selectively in D1- and D2- populations (using D1-Cre and A2A-Cre mice) in the NAc core, we show that mice will nose poke for optical self-stimulation of both cell types, suggesting D2-MSN activity is not inherently aversive. While optogenetic approaches give some information about how cellular activation can modulate behavior, they eliminate the temporally specific neural activity patterns that encode information in behaving animals. To understand how real-time activity in these populations is linked to behavioral execution, we expressed the genetically encoded calcium indicator (GCaMP6f) within D1 and D2 MSNs coupled with in vivo fiber photometry to record from these cell populations in awake and behaving animals during pavlovian and operant conditioning tasks. Utilizing complex reinforcement schedules that allow dissociation of stimulus value, outcome, cue learning, and action, we show that D1 MSNs respond to the presence and intensity of unconditioned stimuli – regardless of value. Conversely, D2 MSNs respond to a mismatch between what is expected and what is received and thus encode errors in prediction in a value-independent fashion. To establish a causal link between the information encoded within these neuronal populations and behavioral output, we next expressed an inhibitory opsin (halorhodopsin) selectively in either D1- or D2-MSNs to inhibit these cells at specific time points during a fear conditioning task. We show that inhibition at select time points, either by inhibiting D2-MSN activity during the predictive cue or D1-MSN activity during the shock outcome, disrupts learning of the association. We thus provide foundational evidence for the discrete aspects of information that are encoded within these cellular populations.

Experience in the Use of Optogenetic Approaches to Studies of Brain Functions and Prosthetization of Degenerative Retinas

Optogenetics – a method allowing light to be used to control neuron activity via expression of light-activated proteins within cells – is a powerful tool in neurophysiological research. Optogenetics has made significant progress in studies of brain functions in the last decade. Progress in optogenetics depends decisively on the development of new molecular tools, i.e., light-activated proteins. The most widely used molecule for excitation of cells in optogenetics is the natural light-activated cation channelrhodopsin 2 (ChR2). Studies in 2015 identified the natural light-activated chloride channel GtACR2, which in optogenetic experiments can efficiently suppress neuron activity. We identified the unique properties of this channel and showed that not only do GtACR2-expressing neurons respond to the light signal with strong inhibition, but also that action potentials can be generated, evidently in the axon terminals of the neuron due to changes in the chloride reversion potential is this cell compartment. Our studies used optogenetic methods to investigate the cellular mechanisms of learning and memory. Using channelrhodopsin 2 expression in networks of presynaptic neurons, we employed light stimulation to study the properties of synaptic connections and their plasticity in whole populations of neurons in a single experiment. One potential direction in the clinical use of optogenetics is its use for prosthetization of degenerative retinas. One version of this approach consists of creating an ON/OFF receptor field of retinal ganglion cells by targeted expression of the excitatory light-activated protein in the central part of a ganglion cell and an inhibitory protein in the periphery. Within the framework of this approach, we created a two-layer construct carrying genes for excitatory and inhibitory opsins whose expression was able to lead to restoration of ON/OFF interactions typical of ganglion neurons.

Off-Peak 594-nm Light Surpasses On-Peak 532-nm Light in Silencing Distant ArchT-Expressing Neurons InVivo.

SummaryFor large brain volume manipulations using optogenetics, both effective opsin excitation and efficient light delivery with minimal light absorption are required to minimize the illuminating light intensity and concomitant off-target effects. ArchT, a widely used potent inhibitory opsin, is commonly activated by 532-nm light, which lies on its in vitro excitation peak. However, 532-nm light also lies on a peak range of the hemoglobin absorption spectrum. Therefore, we predicted that 594-nm light is superior in suppressing distant ArchT-expressing neurons, which is slightly off the ArchT-excitation-plateau and largely off the peak of the hemoglobin absorption spectrum. We quantitatively tested this prediction by the electrophysiological recording of the rat cortex in vivo. At illumination distances greater than 500 μm, 594-nm light was more effective than 532-nm light. Its superiority increased with distance. These results validate our prediction and highlight the significance of excitation-absorption trade-off in selecting illumination wavelength for optogenetics in vivo.