Optogenetic Manipulation Research Articles

Optogenetic modulation of long-range cortical circuits in awake nonhuman primates.

Causal control of short- and long-range projections between networks is necessary to study complex cognitive processes and cortical computations. Neural circuits can be studied via optogenetic approaches, which provide excellent genetic and temporal control and electrophysiological recordings. However, in nonhuman primates (NHPs), these approaches are commonly performed at a single location, missing out on the potential to test connections between separate networks. We have recently developed an approach for optogenetic manipulation in NHPs which targets intra- and interareal cortical projections. Here we describe the combination of optogenetic stimulation with standard chamber-based electrophysiological recordings in awake NHPs to monitor and manipulate both short- and long-range feedforward and feedback circuits. We describe the injection of viral constructs, the simultaneous electrophysiological recordings with the optical stimulation of neurons at various cortical distances and the evaluation of gene expression using a focal biopsy technique. We focus on details that are specific to NHP preparations, such as the precise targeting of injection sites, choosing appropriate viral constructs and considerations for behavioral measures. When combined with laminar electrode configurations (to functionally identify cortical layers) and complex cognitive behavioral tasks, our approach can be used to investigate an array of systems neuroscience questions, such as the role of feedback circuits in attention and the role of lateral connections in contrast normalization. The procedure requires 2-3 active days and 45 waiting days to transduce selected neural circuits and several weeks to complete experiments. The procedure is appropriate for users with expertise in in vivo, awake electrophysiology with NHPs.

Devaluing memories of reward: a case for dopamine

Midbrain dopamine cells encode differences in predictive and expected value to support learning through reward prediction error. Recent findings have questioned whether reward prediction error can fully account for dopamine function and suggest a more complex role for dopamine in encoding detailed features of the reward environment. In this series of studies, we describe a novel role for dopamine in devaluing sensory features of reward. Mesencephalic dopamine cells activated during a mediated devaluation phase were later chemogenetically reactivated. This retrieval of the devalued reward memory elicited a reduction in the hedonic evaluation of sucrose reward. Through optogenetic and chemogenetic manipulations, we confirm dopamine cells are both sufficient and necessary for mediated devaluation, and retrieval of these memories reflected dopamine release in the nucleus accumbens. Consistent with our computational modeling data, our findings indicate a critical role for dopamine in encoding predictive representations of the sensory features of reinforcement. Overall, we elucidate a novel role for dopamine function in mediated devaluation and illuminate a more elaborate framework through which dopamine encodes reinforcement signals.

Temporal Association Cortex Gates Sound-Evoked Arousal from NREM Sleep.

Sound-evoked wakefulness from sleep is crucial in daily life, yet its neural mechanisms remain poorly understood. It is found that CaMKIIα+ neurons in the temporal association cortex (TeA) of mice are not essential for natural awakening from sleep. However, optogenetic activation of these neurons reliably induces wakefulness from non-rapid eye movement (NREM) sleep but not from rapid eye movement (REM) sleep. In vivo electrophysiological and calcium recordings further demonstrated that TeA neurons are monotonically tuned to sound intensity but not frequency. More importantly, it is found that the activity of CaMKIIα+ neurons in TeA can gate sound-evoked arousal from NREM sleep, which is further confirmed by optogenetic manipulations. Further investigation reveals that the baseline excitability of TeA CaMKIIα+ neurons and the delta oscillations in the electroencephalogramare particularly important in regulating the evoked activity of TeA neurons. Anatomical and functional screening of downstream targets of TeA reveals that excitatory projections from TeA glutamatergic neurons to glutamatergic neurons in the basolateral/lateral amygdala are critical for modulating sound-evoked arousal from NREM sleep. These findings uncover a top-down regulatory circuit that selectively governs sound-evoked arousal from NREM sleep, with the TeA functioning as a key connecting cortex to subcortical regions.

Scaling of ventral hippocampal activity during anxiety.

The hippocampus supports a multiplicity of functions, with the dorsal region contributing to spatial representations and memory, and the ventral hippocampus (vH) being primarily involved in emotional processing. While spatial encoding has been extensively investigated, how the vH activity is tuned to emotional states, e.g. to different anxiety levels, is not well understood. We developed an adjustable linear track maze (aLTM) for male mice with which we could induce a scaling of behavioral anxiety levels within the same spatial environment. Using in vivo single-unit recordings, optogenetic manipulations and the application of a convolutional non-linear classifier, we examined the changes and causal effects of vH activity at different anxiety levels. We found that anxiogenic experiences activated the vH and that this activity scaled with increasing anxiety levels. We identified two processes that contributed to this scaling of anxiety-related activity: increased tuning and successive remapping of neurons to the anxiogenic compartment. Moreover, optogenetic inhibition of the vH reduced anxiety across different levels, while anxiety-related activity scaling could be decoded using a convolutional non-linear classifier. Collectively, our findings position the vH as a critical limbic region that functions as an 'anxiometer' by scaling its activity based on perceived anxiety levels. Our discoveries go beyond the traditional theory of cognitive maps in the hippocampus underlying spatial navigation and memory, by identifying hippocampal mechanisms selectively regulating anxiety.Significant statement This study reveals how the ventral hippocampus (vH) functions as an "anxiometer", tuning its activity to different anxiety levels. Using an adjustable linear track maze (aLTM) for mice, we demonstrated that vH activity scales with increased anxiety. By recording single-neuron activity and performing optogenetic manipulation of vH during the aLTM task, we identified key neuronal mechanisms for neuronal scaling during anxiety. Additionally, a convolutional non-linear classifier was used to decode anxiety-related activity scaling. Our findings advance the understanding of hippocampal function beyond spatial navigation and memory, offering new insights into how the brain regulates anxiety at the neuronal level.

Seeing in the Future - a Perspective on Combining Light with Chemical Biology Approaches to Treat Retinal Pathologies.

New concepts to treat eye diseases have emerged that elegantly combine unnatural light exposure with chemical biology approaches to achieve superior cellular specificity and, as a result, improvement of visual function. Historically, light exposure without further molecular eye treatment has offered limited success including photocoagulation to halt pathological blood vessel growth or low light exposure to stimulate retinal cell viability. To add cellular specificity to such treatments, researchers have introduced various biological or chemical light-sensing molecules and combined those with light exposure. (Pre-)clinical trials describe the use of optogenetics and channelrhodpsins, i. e. light-sensitive ion channels, in patient vision restoration. In the chemical arena, pharmacological agents, rendered light-sensitive by reversible modification with photosensitive protecting compounds ("caging"), have been applied to eyes of living mice to photo-release specific cellular activities. Among these were successful proof-of-principle experiments that were conducted to establish photo-sensitive gene therapies in the eye. For light-mediated treatment in combination with chemical biology, we wish to describe here the current frontiers of research in vision restoration with an eye on differences between biological and chemical light-sensing molecules, patient requirements, and future outlooks.

Dynamic representation of appetitive and aversive stimuli in nucleus accumbens shell D1- and D2-medium spiny neurons

The nucleus accumbens (NAc) is a key brain region for motivated behaviors, yet how distinct neuronal populations encode appetitive or aversive stimuli remains undetermined. Using microendoscopic calcium imaging in mice, we tracked NAc shell D1- or D2-medium spiny neurons’ (MSNs) activity during exposure to stimuli of opposing valence and associative learning. Despite drift in individual neurons’ coding, both D1- and D2-population activity was sufficient to discriminate opposing valence unconditioned stimuli, but not predictive cues. Notably, D1- and D2-MSNs were similarly co-recruited during appetitive and aversive conditioning, supporting a concurrent role in associative learning. Conversely, when contingencies changed, there was an asymmetric response in the NAc, with more pronounced changes in the activity of D2-MSNs. Optogenetic manipulation of D2-MSNs provided causal evidence of the necessity of this population in the extinction of aversive associations. Our results reveal how NAc shell neurons encode valence, Pavlovian associations and their extinction, and unveil mechanisms underlying motivated behaviors.

Cross-modal cortical circuit for sound sensitivity in neuropathic pain.

Hyperacusis, exaggerated sensitivity to sound, frequently accompanies chronic pain in humans, suggesting interactions between different sensory systems in the brain. However, the neural mechanisms underlying this comorbidity remain largely unexplored. In this study, behavioral tests measuring sound-evoked pupil dilation and reaction times to lick water following auditory stimuli showed hyperacusis-like behaviors in neuropathic pain model mice. Through viral tracing, fiber photometry, and multi-electrode recordings, we identified glutamatergic projections from primary somatosensory cortex (S1HLGlu) to the auditory cortex (ACx) that participate in amplifying sound-evoked neuronal activity following spared nerve injury in the hindlimb. Chemo- or optogenetic manipulation and electrophysiology recordings confirmed that the S1HLGlu → ACx pathway is essential for this heightened response to sound. Specifically, activating this pathway intensified glutamatergic neuronal activity in the ACx and induced hyperacusis-like behaviors, while chemogenetic suppression reversed these effects in neuropathic pain model mice. These findings illustrate the mechanism by which central gain increases in the ACx of neuropathic pain mice, improving our understanding of cross-modal sensory system interactions and suggesting circuit pathway targets for developing interventions to treat pain-associated hyperacusis in clinic.

Role of Median Raphe Serotonergic Neurons in Positive and Negative Information Processing

Serotonergic neurons play a critical role in processing reward and aversive information. Rewarding stimuli activate serotonergic neurons in the dorsal raphe nucleus (DRN), whereas optogenetic activation of DRN serotonergic neurons induces reward-like effects. However, the pharmacological enhancement of serotonin neurotransmission does not induce rewarding or aversive effects. These findings suggest the presence of another serotonergic neuron that plays a role opposite to that of the DRN in processing reward and aversion information. Previous reports suggested that the median raphe nucleus (MRN) processes negative emotional stimuli. To elucidate the function of MRN serotonergic neurons in these processes, we recorded the changes in serotonergic activity in mice in response to rewarding and aversive stimuli. We also used optogenetic manipulation to determine whether these changes could induce rewarding and aversive behaviors. The activity of MRN serotonergic neurons decreased in response to rewarding stimuli and increased after aversive stimuli. Optogenetic inhibition of MRN serotonergic neurons induced reward-related behavior, while optogenetic stimulation induced aversion-related behavior. Furthermore, we found that the projection pathway from MRN serotonergic neurons to the interpeduncular nucleus is crucial for these processes. These results indicate that MRN serotonergic neurons play a pivotal role in processing reward and aversive information, functioning oppositely to DRN neurons.

The current landscape of optogenetics for the enhancement of adoptive T-cell therapy

Abstract Immunotherapy, medicinal modulation of a host’s immune response to better combat a pathogen or disease, has transformed cancer treatments in recent decades. T-cells, an important component of the adaptive immune system, are further paramount for therapy success. Recent immunotherapeutic modalities have therefore more frequently targeted T-cells for cancer treatments and other pathologies and are termed adoptive T-cell (ATC) therapies. ATC therapies characterise various types of immunotherapies but predominantly fall into three established techniques: Tumour infiltrating lymphocyte (TIL), Chimeric Antigen Receptor T-cell (CAR-T) and engineered T-cell receptor (eTCR) therapies. Despite promising clinical results, all ATC therapy types fall short in providing long-term sustained tumour clearance whilst being particularly ineffective against solid tumours, with substantial developments aiming to understand and prevent the typical drawbacks of ATC therapy. Optogenetics is a relatively recent development, incorporating light-sensitive protein domains into cells or tissues of interest to optically tune specific biological processes. Optogenetic manipulation of immunological functions is rapidly becoming an investigative tool in immunology, with light-sensitive systems now being used to optimise many cellular therapeutic modalities and ATC therapies. This review focuses on how optogenetic approaches are currently utilised to improve ATC therapy in clinical settings by deepening our understanding of the molecular rationale behind therapy success. Moreover, this review further critiques current immuno-optogenetic systems and speculates on the expansion of recent developments, enhancing current ATC-based therapeutic modalities to pave the way for clinical progress.

Perceptual choice and motor signals in mouse somatosensory cortex.

Somatosensory cortex activity relates both to sensation and movement, reflecting their intimate relationship, but the extent and nature of sensory-motor interactions in the somatosensory cortex remain unclear. Here, we investigated perception-related sensory and motor signals in the whisker areas of mouse primary (wS1) and secondary (wS2) somatosensory cortices. We recorded neuronal activity while mice performed a whisker detection task using two alternative lickports, one each to indicate the presence or absence of a whisker deflection on a given trial. One group of mice reported the presence of the whisker stimulus by licking at the port on the same ("congruent") side of the animal as the stimulated whisker, whereas a second group of mice did so by licking at the opposite ("incongruent") side. Activity of single neurons in wS1 and wS2 correlated with perceptual choice. This choice-related activity was enhanced when responding to the congruent side. wS2 neurons projecting along two output pathways---to wS1 or to whisker secondary motor cortex, wM2---also showed choice-related activity, but differed in their dependence on congruence and in the effects of optogenetic manipulation. Thus, somatosensory cortex contains pathway- and action-specific choice-related activity.

GABAergic neurons from the ventral tegmental area represent and regulate force vectors.

The ventral tegmental area (VTA), a midbrain region associated with motivated behaviors, consists predominantly of dopaminergic (DA) neurons and GABAergic (GABA) neurons. Previous work has suggested that VTA GABA neurons provide a reward prediction, which is used in computing a reward prediction error. In this study, using in vivo electrophysiology and continuous quantification of force exertion in head-fixed mice, we discovered distinct populations of VTA GABA neurons that exhibited precise force tuning independently of learning, reward prediction, and outcome valence. Their activity usually preceded force exertion, and selective optogenetic manipulations of these neurons systematically modulated force exertion without influencing reward prediction. Together, these findings show that VTA GABA neurons continuously regulate force vectors during motivated behavior.

Self-experience of a negative event alters responses to others in similar states through prefrontal cortex CRF mechanisms.

Our own experience of emotional events influences how we approach and react to others' emotions. Here we observe that mice exhibit divergent interindividual responses to others in stress (that is, preference or avoidance) only if they have previously experienced the same aversive event. These responses are estrus dependent in females and dominance dependent in males. Notably, silencing the expression of the corticotropin-releasing factor (CRF) within the medial prefrontal cortex (mPFC) attenuates the impact of stress self-experience on the reaction to others' stress. In vivo microendoscopic calcium imaging revealed that mPFC CRF neurons are activated more toward others' stress only following the same negative self-experience. Optogenetic manipulations confirmed that higher activation of mPFC CRF neurons is responsible for the switch from preference to avoidance of others in stress, but only following stress self-experience. These results provide a neurobiological substrate underlying how an individual's emotional experience influences their approach toward others in a negative emotional state.

Ketamine induces plasticity in a norepinephrine-astroglial circuit to promote behavioral perseverance.

Transient exposure to ketamine can trigger lasting changes in behavior and mood. We found that brief ketamine exposure causes long-term suppression of futility-induced passivity in larval zebrafish, reversing the "giving-up" response that normally occurs when swimming fails to cause forward movement. Whole-brain imaging revealed that ketamine hyperactivates the norepinephrine-astroglia circuit responsible for passivity. After ketamine washout, this circuit exhibits hyposensitivity to futility, leading to long-term increased perseverance. Pharmacological, chemogenetic, and optogenetic manipulations show that norepinephrine and astrocytes are necessary and sufficient for ketamine's long-term perseverance-enhancing aftereffects. Invivo calcium imaging revealed that astrocytes in adult mouse cortex are similarly activated during futility in the tail suspension test and that acute ketamine exposure also induces astrocyte hyperactivation. The cross-species conservation of ketamine's modulation of noradrenergic-astroglial circuits and evidence that plasticity in this pathway can alter the behavioral response to futility hold promise for identifying new strategies to treat affective disorders.

Optogenetic manipulation of nuclear Dorsal reveals temporal requirements and consequences for transcription.

Morphogen gradients convey essential spatial information during tissue patterning. While both concentration and timing of morphogen exposure are crucial, how cells interpret these graded inputs remains challenging to address. We employed an optogenetic system to acutely and reversibly modulate the nuclear concentration of the morphogen Dorsal (DL), homologue of NF-κB, which orchestrates dorso-ventral patterning in the Drosophila embryo. By controlling DL nuclear concentration while simultaneously recording target gene outputs in real time, we identified a critical window for DL action that is required to instruct patterning, and characterized the resulting effect on spatio-temporal transcription of target genes in terms of timing, coordination, and bursting. We found that a transient decrease in nuclear DL levels at nuclear cycle 13 leads to reduced expression of the mesoderm-associated gene snail (sna) and partial derepression of the neurogenic ectoderm-associated target short gastrulation ( sog) in ventral regions. Surprisingly, the mispatterning elicited by this transient change in DL is detectable at the level of single cell transcriptional bursting kinetics, specifically affecting long inter-burst durations. Our approach of using temporally-resolved and reversible modulation of a morphogen in vivo , combined with mathematical modeling, establishes a framework for understanding the stimulus-response relationships that govern embryonic patterning.

An Integrated Method for Crafting Flexible and Convenient Electrophysiological Optrodes for Multi-Region In Vivo Recording.

Epilepsy is a neurological disorder characterized by synchronized abnormal discharges involving multiple brain regions. Focal lesions facilitate the propagation of epileptic signals through associated neural circuits. Therefore, in vivo recording of local field potential (LFP) from the critical brain regions isessential for deciphering the circuits involved in seizure propagation. However, current methods for electrode fabrication and implantation lack flexibility. Here, we present a handy device designed for electrophysiological recordings (LFPs and electroencephalography [EEG]) across multiple regions. Additionally, we seamlessly integrated optogenetic manipulation and calcium signaling recording with LFP recording. Robust after-discharges were observed in several separate regions during epileptic seizures, accompanied by increasingcalcium signaling. The approach used in this study offers a convenient and flexible strategy for synchronous neural recordings across diverse regions of the brain. It holds the potential for advancing research on neurological disorders by providing insights into the neural profiles of multiple regions involved in these disorders.

Sensory History Drives Adaptive Neural Geometry in LP/Pulvinar-Prefrontal Cortex Circuits.

Prior expectations guide attention and support perceptual filtering for efficient processing during decision-making. Here we show that during a visual discrimination task, mice adaptively use prior stimulus history to guide ongoing choices by estimating differences in evidence between consecutive trials (| Δ Dir |). The thalamic lateral posterior (LP)/pulvinar nucleus provides robust inputs to the Anterior Cingulate Cortex (ACC), which has been implicated in selective attention and predictive processing, but the function of the LP-ACC projection is unknown. We found that optogenetic manipulations of LP-ACC axons disrupted animals' ability to effectively estimate and use information across stimulus history, leading to | Δ Dir |-dependent ipsilateral biases. Two-photon calcium imaging of LP-ACC axons revealed an engagement-dependent low-dimensional organization of stimuli along a curved manifold. This representation was scaled by | Δ Dir | in a manner that emphasized greater deviations from prior evidence. Thus, our work identifies the LP-ACC pathway as essential for selecting and evaluating stimuli relative to prior evidence to guide decisions.

Charge-neutralized polyethylenimine-lipid nanoparticles for gene transfer to human embryonic stem cells

Gene delivery is fundamentally crucial for the genetic manipulation of stem cells. Here, we present a straightforward approach to create a library of charge-neutralized polyethylenimine (PEI)-lipid nanoparticles designed for stem cell transfection. These lipid nanoparticles were formulated using small, branched PEI and lipidic anhydrides. Remarkably, over 15% of the lipid nanoparticles demonstrated high transfection efficiency across various cell types, surpassing the efficiency of both Lipofectamine 2000 and FuGENE HD. A structure–activity analysis indicated that the length and ratio of hydrophobic alkyl substitutions were critical parameters for efficient gene delivery. Notably, the transfection efficiency was higher than that of the original cation PEI. Our optimized PEI-lipid system enabled highly effective plasmid DNA delivery and successfully co-transferred two plasmid DNAs into difficult-to-transfect human embryonic stem cells (hESCs), facilitating optogenetic manipulation within these cells.

Age-dependent cerebral vasodilation induced by volatile anesthetics is mediated by NG2+ vascular mural cells

Anesthesia can influence cerebral blood flow by altering vessel diameter. Using in vivo two-photon imaging, we examined the effects of volatile anesthetics, sevoflurane and isoflurane, on vessel diameter in young and adult mice. Our results show that these anesthetics induce robust dilation of cortical arterioles and arteriole-proximate capillaries in adult mice, with milder effects in juveniles and no dilation in infants. This anesthesia-induced vasodilation correlates with decreased cytosolic Ca2+ levels in NG2+ vascular mural cells. Optogenetic manipulation of these cells bidirectionally regulates vessel diameter, and their ablation abolishes the vasodilatory response to anesthetics. In immature brains, NG2+ mural cells are fewer in number and express lower levels of Kir6.1, a subunit of ATP-sensitive potassium channels. This likely contributes to the age-dependent differences in vasodilation, as Kir6.1 activation promotes, while its inhibition reduces, anesthesia-induced vasodilation. These findings highlight the essential role of NG2+ mural cells in mediating anesthesia-induced cerebral vasodilation.

Sodium-Selective Channelrhodopsins.

Channelrhodopsins (ChRs) are light-gated ion channels originally discovered in algae and are commonly used in neuroscience for controlling the electrical activity of neurons with high precision. Initially-discovered ChRs were non-selective cation channels, allowing the flow of multiple ions, such as Na+, K+, H+, and Ca2+, leading to membrane depolarization and triggering action potentials in neurons. As the field of optogenetics has evolved, ChRs with more specific ion selectivity were discovered or engineered, offering more precise optogenetic manipulation. This review highlights the natural occurrence and engineered variants of sodium-selective channelrhodopsins (NaChRs), emphasizing their importance in optogenetic applications. These tools offer enhanced specificity in Na+ ion conduction, reducing unwanted effects from other ions, and generating strong depolarizing currents. Some of the NaChRs showed nearly no desensitization upon light illumination. These characteristics make them particularly useful for experiments requiring robust depolarization or direct Na+ ion manipulation. The review further discusses the molecular structure of these channels, recent advances in their development, and potential applications, including a proposed drug delivery system using NaChR-expressing red blood cells that could be triggered to release therapeutic agents upon light activation. This review concludes with a forward-looking perspective on expanding the use of NaChRs in both basic research and clinical settings.

Direct Piriform-to-Auditory Cortical Projections Shape Auditory-Olfactory Integration.

In a real-world environment, the brain must integrate information from multiple sensory modalities, including the auditory and olfactory systems. However, little is known about the neuronal circuits governing how odors influence and modulate sound processing. Here, we investigated the mechanisms underlying auditory-olfactory integration using anatomical, electrophysiological, and optogenetic approaches, focusing on the auditory cortex as a key locus for cross-modal integration. First, retrograde and anterograde viral tracing strategies revealed a direct projection from the piriform cortex to the auditory cortex. Next, using in vivo electrophysiological recordings of neuronal activity in the auditory cortex of awake male or female mice, we found that odors modulate auditory cortical responses to sound. Finally, we used in vivo optogenetic manipulations during electrophysiology to demonstrate that olfactory modulation in the auditory cortex, specifically, odor-driven enhancement of sound responses, depends on direct input from the piriform cortex. Together, our results identify a novel role of piriform-to-auditory cortical circuitry in shaping olfactory modulation in the auditory cortex, shedding new light on the neuronal mechanisms underlying auditory-olfactory integration.