In Vivo Neural Tract Tracing Procedures: Unravelling Neural Hodology Using Fluorescent and Non-fluorescent Neural Tract Tracers, Optogenetic Approach, and Diffusion Tensor Neurotractography Protocols.

Fluorescent and non-fluorescent neural tract tracers enable the investigation of neural pathways in both peripheral and central nervous systems in laboratory animals demonstrating images with high resolution and great anatomic precision. Anterograde and retrograde viral tracers are important cutting-edge tools for neuroanatomical mapping. The optogenetic consists of an advanced alternative for in vivo neural tract tracing procedures, fundamentally considering the possibility to dissect and modulate pathways either exciting or inhibiting neural circuits in laboratory animals. The neurotractography by diffusion tensor imaging in vivo procedures enables the study of neural pathways in humans with reasonable accuracy. Here we describe the procedure of classical anatomic neural tract tracing and modern optogenetic technique performed in anima vili in addition to different diffusion tensor neurotractography performed in anima nobili.

Optogenetic induction of mechanical muscle stress identifies myosin regulatory ubiquitin ligase NHL-1 in C. elegans.

Mechanical stress during muscle contraction is a constant threat to proteome integrity. However, there is a lack of experimental systems to identify critical proteostasis regulators under mechanical stress conditions. Here, we present the transgenic Caenorhabditis elegans model OptIMMuS (Optogenetic Induction of Mechanical Muscle Stress) to study changes in the proteostasis network associated with mechanical forces. Repeated blue light exposure of a muscle-expressed Chlamydomonas rheinhardii channelrhodopsin-2 variant results in sustained muscle contraction and mechanical stress. Using OptIMMuS, combined with proximity labeling and mass spectrometry, we identify regulators that cooperate with the myosin-directed chaperone UNC-45 in muscle proteostasis. One of these is the TRIM E3 ligase NHL-1, which interacts with UNC-45 and muscle myosin in genetic epistasis and co-immunoprecipitation experiments. We provide evidence that the ubiquitylation activity of NHL-1 regulates myosin levels and functionality under mechanical stress. In the future, OptIMMuS will help to identify muscle-specific proteostasis regulators of therapeutic relevance.

Periaqueductal gray activates antipredatory neural responses in the amygdala of foraging rats.

Pavlovian fear conditioning research suggests that the interaction between the dorsal periaqueductal gray (dPAG) and basolateral amygdala (BLA) acts as a prediction error mechanism in the formation of associative fear memories. However, their roles in responding to naturalistic predatory threats, characterized by less explicit cues and the absence of reiterative trial-and-error learning events, remain unexplored. In this study, we conducted single-unit recordings in rats during an 'approach food-avoid predator' task, focusing on the responsiveness of dPAG and BLA neurons to a rapidly approaching robot predator. Optogenetic stimulation of the dPAG triggered fleeing behaviors and increased BLA activity in naive rats. Notably, BLA neurons activated by dPAG stimulation displayed immediate responses to the robot, demonstrating heightened synchronous activity compared to BLA neurons that did not respond to dPAG stimulation. Additionally, the use of anterograde and retrograde tracer injections into the dPAG and BLA, respectively, coupled with c-Fos activation in response to predatory threats, indicates that the midline thalamus may play an intermediary role in innate antipredatory-defensive functioning.

Phosphatidic acid is an endogenous negative regulator of PIEZO2 channels and mechanical sensitivity.

Mechanosensitive PIEZO2 ion channels play roles in touch, proprioception, and inflammatory pain. Currently, there are no small molecule inhibitors that selectively inhibit PIEZO2 over PIEZO1. The TMEM120A protein was shown to inhibit PIEZO2 while leaving PIEZO1 unaffected. Here we find that TMEM120A expression elevates cellular levels of phosphatidic acid and lysophosphatidic acid (LPA), aligning with its structural resemblance to lipid-modifying enzymes. Intracellular application of phosphatidic acid or LPA inhibits PIEZO2 but not PIEZO1 activity. Extended extracellular exposure to the non-hydrolyzable phosphatidic acid and LPA analog carbocyclic phosphatidic acid (ccPA) also inhibits PIEZO2. Optogenetic activation of phospholipase D (PLD), a signaling enzyme that generates phosphatidic acid, inhibits PIEZO2 but not PIEZO1. Conversely, inhibiting PLD leads to increased PIEZO2 activity and increased mechanical sensitivity in mice in behavioral experiments. These findings unveil lipid regulators that selectively target PIEZO2 over PIEZO1, and identify the PLD pathway as a regulator of PIEZO2 activity.

Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag.

Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function - dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles. Here we show that light-gated recruitment of a solubilizing domain, maltose-binding protein (MBP), results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP by disrupting condensation of the oncogenic fusion protein FUS-CHOP and reverting FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

The critical dynamics of hippocampal seizures.

Epilepsy is defined by the abrupt emergence of harmful seizures, but the nature of these regime shifts remains enigmatic. From the perspective of dynamical systems theory, such critical transitions occur upon inconspicuous perturbations in highly interconnected systems and can be modeled as mathematical bifurcations between alternative regimes. The predictability of critical transitions represents a major challenge, but the theory predicts the appearance of subtle dynamical signatures on the verge of instability. Whether such dynamical signatures can be measured before impending seizures remains uncertain. Here, we verified that predictions on bifurcations applied to the onset of hippocampal seizures, providing concordant results from in silico modeling, optogenetics experiments in male mice and intracranial EEG recordings in human patients with epilepsy. Leveraging pharmacological control over neural excitability, we showed that the boundary between physiological excitability and seizures can be inferred from dynamical signatures passively recorded or actively probed in hippocampal circuits. Of importance for the design of future neurotechnologies, active probing surpassed passive recording to decode underlying levels of neural excitability, notably when assessed from a network of propagating neural responses. Our findings provide a promising approach for predicting and preventing seizures, based on a sound understanding of their dynamics.

Prediction-error signals in anterior cingulate cortex drive task-switching.

Task-switching is a fundamental cognitive ability that allows animals to update their knowledge of current rules or contexts. Detecting discrepancies between predicted and observed events is essential for this process. However, little is known about how the brain computes cognitive prediction-errors and whether neural prediction-error signals are causally related to task-switching behaviours. Here we trained mice to use a prediction-error to switch, in a single trial, between responding to the same stimuli using two distinct rules. Optogenetic silencing and un-silencing, together with widefield and two-photon calcium imaging revealed that the anterior cingulate cortex (ACC) was specifically required for this rapid task-switching, but only when it exhibited neural prediction-error signals. These prediction-error signals were projection-target dependent and were larger preceding successful behavioural transitions. An all-optical approach revealed a disinhibitory interneuron circuit required for successful prediction-error computation. These results reveal a circuit mechanism for computing prediction-errors and transitioning between distinct cognitive states.

Bidirectional fear modulation by discrete anterior insular circuits in male mice.

The brain's ability to appraise threats and execute appropriate defensive responses is essential for survival in a dynamic environment. Humans studies have implicated the anterior insular cortex (aIC) in subjective fear regulation and its abnormal activity in fear/anxiety disorders. However, the complex aIC connectivity patterns involved in regulating fear remain under investigated. To address this, we recorded single units in the aIC of freely moving male mice that had previously undergone auditory fear conditioning, assessed the effect of optogenetically activating specific aIC output structures in fear, and examined the organization of aIC neurons projecting to the specific structures with retrograde tracing. Single-unit recordings revealed that a balanced number of aIC pyramidal neurons' activity either positively or negatively correlated with a conditioned tone-induced freezing (fear) response. Optogenetic manipulations of aIC pyramidal neuronal activity during conditioned tone presentation altered the expression of conditioned freezing. Neural tracing showed that non-overlapping populations of aIC neurons project to the amygdala or the medial thalamus, and the pathway bidirectionally modulated conditioned fear. Specifically, optogenetic stimulation of the aIC-amygdala pathway increased conditioned freezing, while optogenetic stimulation of the aIC-medial thalamus pathway decreased it. Our findings suggest that the balance of freezing-excited and freezing-inhibited neuronal activity in the aIC and the distinct efferent circuits interact collectively to modulate fear behavior.

In-silico predicted mouse melanopsins with blue spectral shifts deliver efficient subcellular signaling.

Melanopsin is a photopigment belonging to the G Protein-Coupled Receptor (GPCR) family expressed in a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) and responsible for a variety of processes. The bistability and, thus, the possibility to function under low retinal availability would make melanopsin a powerful optogenetic tool. Here, we aim to utilize mouse melanopsin to trigger macrophage migration by its subcellular optical activation with localized blue light, while simultaneously imaging the migration with red light. To reduce melanopsin's red light sensitivity, we employ a combination of in silico structure prediction and automated quantum mechanics/molecular mechanics modeling to predict minimally invasive mutations to shift its absorption spectrum towards the shorter wavelength region of the visible spectrum without compromising the signaling efficiency. The results demonstrate that it is possible to achieve melanopsin mutants that resist red light-induced activation but are activated by blue light and display properties indicating preserved bistability. Using the A333T mutant, we show that the blue light-induced subcellular melanopsin activation triggers localized PIP3 generation and macrophage migration, which we imaged using red light, demonstrating the optogenetic utility of minimally engineered melanopsins.

Stress pathway outputs are encoded by pH-dependent clustering of kinase components.

Signal processing by intracellular kinases controls near all biological processes but how signal pathway functions evolve with changed cellular context is poorly understood. Functional specificity of c-Jun N-terminal Kinases (JNK) are partly encoded by signal strength. Here we reveal that intracellular pH (pHi) is a significant component of the JNK network and defines signal response to specific stimuli. We show pHi regulates JNK activity in response to cell stress, with the relationship between pHi and JNK activity dependent on specific stimuli and upstream kinases activated. Using the optogenetic clustering tag CRY2, we show that an increase in pHi promotes the light-induced phase transition of ASK1 to augment JNK activation. While increased pHi similarly promoted CRY2-tagged JNK2 to form light-induced condensates, this attenuated JNK activity. Mathematical modelling of feedback signalling incorporating pHi and differential contributions by ASK1 and JNK2 condensates was sufficient to delineate signal responses to specific stimuli. Taking pHi and ASK1/JNK2 signal contributions into consideration may delineate oncogenic versus tumour suppressive JNK functions and cancer cell drug responses.

The effect of P2X7 antagonism on subcortical spread of optogenetically-triggered cortical spreading depression and neuroinflammation.

Migraine is a neurological disorder characterized by episodes of severe headache. Cortical spreading depression (CSD), the electrophysiological equivalent of migraine aura, results in opening of pannexin 1 megachannels that release ATP and triggers parenchymal neuroinflammatory signaling cascade in the cortex. Migraine symptoms suggesting subcortical dysfunction bring subcortical spread of CSD under the light. Here, we investigated the role of purinergic P2X7 receptors on the subcortical spread of CSD and its consequent neuroinflammation using a potent and selective P2X7R antagonist, JNJ-47965567. P2X7R antagonism had no effect on the CSD threshold and characteristics but increased the latency to hypothalamic voltage deflection following CSD suggesting that ATP acts as a mediator in the subcortical spread. P2X7R antagonism also prevented cortical and subcortical neuronal activation following CSD, revealed by bilateral decrease in c-fos positive neuron count, and halted CSD-induced neuroinflammation revealed by decreased neuronal HMGB1 release and decreased nuclear translocation of NF-kappa B-p65 in astrocytes. In conclusion, our data suggest that P2X7R plays a role in CSD-induced neuroinflammation, subcortical spread of CSD and CSD-induced neuronal activation hence can be a potential target.

Dorsal raphe nucleus-hippocampus serotonergic circuit underlies the depressive and cognitive impairments in 5×FAD male mice.

BACKGROUND: Depressive symptoms often occur in patients with Alzheimer's disease (AD) and exacerbate the pathogenesis of AD. However, the neural circuit mechanisms underlying the AD-associated depression remain unclear. The serotonergic system plays crucial roles in both AD and depression. METHODS: We used a combination of in vivo trans-synaptic circuit-dissecting anatomical approaches, chemogenetic manipulations, optogenetic manipulations, pharmacological methods, behavioral testing, and electrophysiological recording to investigate dorsal raphe nucleus serotonergic circuit in AD-associated depression in AD mouse model. RESULTS: We found that the activity of dorsal raphe nucleus serotonin neurons (DRN5-HT) and their projections to the dorsal hippocampal CA1 (dCA1) terminals (DRN5-HT-dCA1CaMKII) both decreased in brains of early 5×FAD mice. Chemogenetic or optogenetic activation of the DRN5-HT-dCA1CaMKII neural circuit attenuated the depressive symptoms and cognitive impairments in 5×FAD mice through serotonin receptor 1B (5-HT1BR) and 4 (5-HT4R). Pharmacological activation of 5-HT1BR or 5-HT4R attenuated the depressive symptoms and cognitive impairments in 5×FAD mice by regulating the DRN5-HT-dCA1CaMKII neural circuit to improve synaptic plasticity. CONCLUSIONS: These findings provide a new mechanistic connection between depression and AD and provide potential pharmaceutical prevention targets for AD.

STX-bpc: "Brightening" the path to neuronal inhibition.

In this issue of Cell Chemical Biology, Elleman et al.1 introduce a transformative chemical approach to control neuronal activity with high spatial and temporal resolution. The authors present STX-bpc, a potent neurotoxin that naturally inhibits voltage-gated sodium channels (NaVs), complementing available optogenetic methods for manipulating neuronal activity, cellular communication, and behavior.

A new light on PLCß!

In this issue of Cell Chemical Biology, Kim et al.1 present a novel optogenetic tool, opto-PLCß, to control PLCß signaling optically. In addition to eliciting PIP2 hydrolysis and downstream signaling in cells, opto-PLCß also enabled probing the impact of PLCß signaling on amygdala synaptic plasticity and fear learning in mice.

Using synchronized brain rhythms to bias memory-guided decisions.

Functional interactions between the prefrontal cortex and hippocampus, as revealed by strong oscillatory synchronization in the theta (6-11 Hz) frequency range, correlate with memory-guided decision-making. However, the degree to which this form of long-range synchronization influences memory-guided choice remains unclear. We developed a brain-machine interface that initiated task trials based on the magnitude of prefrontal-hippocampal theta synchronization, then measured choice outcomes. Trials initiated based on strong prefrontal-hippocampal theta synchrony were more likely to be correct compared to control trials on both working memory-dependent and -independent tasks. Prefrontal-thalamic neural interactions increased with prefrontal-hippocampal synchrony and optogenetic activation of the ventral midline thalamus primarily entrained prefrontal theta rhythms, but dynamically modulated synchrony. Together, our results show that prefrontal-hippocampal theta synchronization leads to a higher probability of a correct choice and strengthens prefrontal-thalamic dialogue. Our findings reveal new insights into the neural circuit dynamics underlying memory-guided choices and highlight a promising technique to potentiate cognitive processes or behavior via brain-machine interfacing.

Efferent pathways from the suprachiasmatic nucleus to the horizontal limbs of diagonal band promote NREM sleep during the dark phase in mice.

The regulation of circadian rhythms and the sleep-wake states involves in multiple neural circuits. The suprachiasmatic nucleus (SCN) is a circadian pacemaker that controls the rhythmic oscillation of mammalian behaviors. The basal forebrain (BF) is a critical brain region of sleep-wake regulation, which is the downstream of the SCN. Retrograde tracing of cholera toxin subunit B showed a direct projection from the SCN to the horizontal limbs of diagonal band (HDB), a subregion of the BF. However, the underlying function of the SCN-HDB pathway remains poorly understood. Herein, activation of this pathway significantly increased non-rapid eye movement (NREM) sleep during the dark phase by using optogenetic recordings. Moreover, activation of this pathway significantly induced NREM sleep during the dark phase for first 4 h by using chemogenetic methods. Taken together, these findings reveal that the SCN-HDB pathway participates in NREM sleep regulation and provides direct evidence of a novel SCN-related pathway involved in sleep-wake states regulation.

A discrete subpopulation of PFC-LHb neurons govern cocaine place preference.

Addiction is a complex behavioral disorder characterized by compulsive drug-seeking and drug use despite harmful consequences. The prefrontal cortex (PFC) plays a crucial role in cocaine addiction, involving decision-making, impulse control, memory, and emotional regulation. The PFC interacts with the brain's reward system, including the ventral tegmental area (VTA) and nucleus accumbens (NAc). The PFC also projects to the lateral habenula (LHb), a brain region critical for encoding negative reward and regulating the reward system. In the current study, we examined the role of PFC-LHb projections in regulating cocaine reward-related behaviors. We found that optogenetic stimulation of the PFC-LHb circuit during cocaine conditioning abolished cocaine preference without causing aversion. In addition, increased c-fos expression in LHb neurons was observed in animals that received optic stimulation during cocaine conditioning, supporting the circuit's involvement in cocaine preference regulation. Molecular analysis in animals that received optic stimulation revealed that cocaine-induced alterations in the expression of GluA1 subunit of AMPA receptor was normalized to saline levels in a region-specific manner. Moreover, GluA1 serine phosphorylation on S845 and S831 were differentially altered in LHb and VTA but not in the PFC. Together these findings highlight the critical role of the PFC-LHb circuit in controlling cocaine reward-related behaviors and shed light on the underlying mechanisms. Understanding this circuit's function may provide valuable insights into addiction and contribute to developing targeted treatments for substance use disorders.

A chemogenetic approach for dopamine imaging with tunable sensitivity.

Genetically-encoded dopamine (DA) sensors enable high-resolution imaging of DA release, but their ability to detect a wide range of extracellular DA levels, especially tonic versus phasic DA release, is limited by their intrinsic affinity. Here we show that a human-selective dopamine receptor positive allosteric modulator (PAM) can be used to boost sensor affinity on-demand. The PAM enhances DA detection sensitivity across experimental preparations (in vitro, ex vivo and in vivo) via one-photon or two-photon imaging. In vivo photometry-based detection of optogenetically-evoked DA release revealed that DETQ administration produces a stable 31 minutes window of potentiation without effects on animal behavior. The use of the PAM revealed region-specific and metabolic state-dependent differences in tonic DA levels and enhanced single-trial detection of behavior-evoked phasic DA release in cortex and striatum. Our chemogenetic strategy can potently and flexibly tune DA imaging sensitivity and reveal multi-modal (tonic/phasic) DA signaling across preparations and imaging approaches.

Attention: The blue spot reveals one of its secrets.

The locus coeruleus is the seat of a brain-wide neuromodulatory circuit. Using optogenetic and electrophysiological tools to selectively interrogate noradrenergic neurons in non-human primates, Ghosh and Maunsell show how locus coeruleus neurons contribute to a specific aspect of visual attention.

Controlling the Subcellular Localization of Signaling Proteins Using Chemically Induced Dimerization and Optogenetics.

A given protein can perform numerous roles in a cell with its participation in protein complexes and distinct localization within the cell playing a critical role in its diverse functions. Thus, the ability to artificially dimerize proteins and recruit proteins to specific locations in a cell has become a powerful tool for the investigation of protein function and the understanding of cell biology. Here, we discuss two systems that have been used to activate signal transduction pathways, a chemically inducible dimerization (CID) and a light-inducible (LI) system to control signaling and cytoskeletal regulation in a spatial and temporal manner.