Optogenetic Manipulation Research Articles

GFAP-NpHR mediated optogenetic inhibition of trigeminal nucleus caudalis attenuates hypersensitive behaviors and thalamic discharge attributed to infraorbital nerve constriction injury

The significance of hyperactive astrocytes in neuropathic pain is crucial. However, the association between medullary astrocytes and trigeminal neuralgia (TN)-related pain processing is unclear. Here, we examined how optogenetic inhibition of medullary astrocytes in the trigeminal nucleus caudalis (TNC) regulates pain hypersensitivity in an infraorbital nerve (ION) constricted TN model. We used adult Sprague Dawley rats subjected to infraorbital nerve (ION) constriction to mimic TN symptoms, with naive and sham rats serving as controls. For in vivo optogenetic manipulations, rats stereotaxically received AAV8-GFAP-eNpHR3.0-mCherry or AAV8-GFAP-mCherry at the trigeminal nucleus caudalis (TNC). Open field, von Frey, air puff, and acetone tests measured pain behavioral flexibility. In vivo thalamic recordings were obtained simultaneously with optogenetic manipulation in the TNC. Orofacial hyperalgesia and thalamic hyperexcitability were both accompanied by medullary astrocyte hyperactivity, marked by upregulated GFAP. The yellow laser-driven inhibition of TNC astrocytes markedly improved behavioral responses and regulated thalamic neuronal responses. Halorhodopsin-mediated inhibition in medullary astrocytes may modify the nociceptive input transmitted through the trigeminothalamic tract and pain perception. Taken together, these findings imply that this subpopulation in the TNC and its thalamic connections play a significant role in regulating the trigeminal pain circuitry, which might aid in the identification of new therapeutic measures in TN management.Graphical

Application of optogenetic glial cells to neuron-glial communication.

Optogenetic techniques combine optics and genetics to enable cell-specific targeting and precise spatiotemporal control of excitable cells, and they are increasingly being employed. One of the most significant advantages of the optogenetic approach is that it allows for the modulation of nearby cells or circuits with millisecond precision, enabling researchers to gain a better understanding of the complex nervous system. Furthermore, optogenetic neuron activation permits the regulation of information processing in the brain, including synaptic activity and transmission, and also promotes nerve structure development. However, the optimal conditions remain unclear, and further research is required to identify the types of cells that can most effectively and precisely control nerve function. Recent studies have described optogenetic glial manipulation for coordinating the reciprocal communication between neurons and glia. Optogenetically stimulated glial cells can modulate information processing in the central nervous system and provide structural support for nerve fibers in the peripheral nervous system. These advances promote the effective use of optogenetics, although further experiments are needed. This review describes the critical role of glial cells in the nervous system and reviews the optogenetic applications of several types of glial cells, as well as their significance in neuron-glia interactions. Together, it briefly discusses the therapeutic potential and feasibility of optogenetics.

Paraventricular Hypothalamic Nucleus Upregulates Intraocular Pressure Via Glutamatergic Neurons.

The neuroregulatory center of intraocular pressure (IOP) is located in the hypothalamus. An efferent neural pathway exists between the hypothalamic nuclei and the autonomic nerve endings in the anterior chamber of the eye. This study was designed to investigate whether the paraventricular hypothalamic nucleus (PVH) regulates IOP as the other nuclei do. Optogenetic manipulation of PVH neurons was used in this study. Light stimulation was applied via an optical fiber embedded over the PVH to activate projection neurons after AAV2/9-CaMKIIα-hChR2-mCherry was injected into the right PVH of C57BL/6J mice. The same methods were used to inhibit projection neurons after AAV2/9-CaMKIIα-eNpHR3.0-mCherry was injected into the bilateral PVH of C57BL/6J mice. AAV2/9-EF1α-DIO-hChR2-mCherry was injected into the right PVH of Vglut2-Cre mice to elucidate the effect of glutamatergic neuron-specific activation. IOP was measured before and after light manipulation. Associated nuclei activation was clarified by c-Fos immunohistochemical staining. Only mice with accurate viral expression and fiber embedding were included in the statistical analysis. Activation of projection neurons in the right PVH induced significant bilateral IOP elevation (n = 11, P < 0.001); the ipsilateral IOP increased more noticeably (n = 11, P < 0.05); Bilateral inhibition of PVH projection neurons did not significantly influence IOP (n = 5, P > 0.05). Specific activation of glutamatergic neurons among PVH projection neurons also induced IOP elevation in both eyes (n = 5, P < 0.001). The dorsomedial hypothalamic nucleus, ventromedial hypothalamic nucleus, locus coeruleus and basolateral amygdaloid nucleus responded to light stimulation of PVH in AAV-ChR2 mice. The PVH may play a role in IOP upregulation via glutamatergic neurons.

Prefrontal cortical regulation of REM sleep.

Rapid eye movement (REM) sleep is accompanied by intense cortical activity, underlying its wake-like electroencephalogram. The neural activity inducing REM sleep is thought to originate from subcortical circuits in brainstem and hypothalamus. However, whether cortical neurons can also trigger REM sleep has remained unknown. Here we show in mice that the medial prefrontal cortex (mPFC) strongly promotes REM sleep. Bidirectional optogenetic manipulations demonstrate that excitatory mPFC neurons promote REM sleep through their projections to the lateral hypothalamus and regulate phasic events, reflected in accelerated electroencephalogram theta oscillations and increased eye movement density during REM sleep. Calcium imaging reveals that the majority of lateral hypothalamus-projecting mPFC neurons are maximally activated during REM sleep and a subpopulation is recruited during phasic theta accelerations. Our results delineate a cortico-hypothalamic circuit for the top-down control of REM sleep and identify a critical role of the mPFC in regulating phasic events during REM sleep.

Single cell in vivo optogenetic stimulation by two-photon excitation fluorescence transfer

Optogenetic manipulation with single-cell resolution can be achieved by two-photon excitation. However, this frequently requires relatively high laser powers. Here, we developed a novel strategy that can improve the efficiency of current two-photon stimulation technologies by positioning fluorescent proteins or small fluorescent molecules with high two-photon cross-sections in the vicinity of opsins. This generates a highly localized source of endogenous single-photon illumination that can be tailored to match the optimal opsin absorbance. Through neuronal and vascular stimulation in the live mouse brain, we demonstrate the utility of this technique to achieve efficient opsin stimulation, without loss of cellular resolution. We also provide a theoretical framework for understanding the potential advantages and constrains of this methodology, with directions for future improvements. Altogether, this fluorescence transfer illumination method opens new possibilities for experiments difficult to implement in the live brain such as all-opticalneural interrogation and control of regional cerebral blood flow.

Distinct Circuits From the Central Lateral Amygdala to the Ventral Part of the Bed Nucleus of Stria Terminalis Regulate Different Fear Memory

BackgroundThe ability to differentiate stimuli that predict fear is critical for survival; however, the underlying molecular and circuit mechanisms remain poorly understood. MethodsWe combined transgenic mice, in vivo transsynaptic circuit-dissecting anatomical approaches, optogenetics, pharmacological methods, and electrophysiological recording to investigate the involvement of specific extended amygdala circuits in different fear memory. ResultsWe identified the projections from central lateral amygdala (CeL) protein kinase C δ (PKCδ)–positive neurons and somatostatin (SST)-positive neurons to GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons in the ventral part of the bed nucleus of stria terminalis (vBNST). Prolonged optogenetic activation or inhibition of the PKCδCeL-vBNST pathway specifically reduced context fear memory, whereas the SSTCeL-vBNST pathway mainly reduced tone fear memory. Intriguingly, optogenetic manipulation of vBNST neurons that received the projection from PKCδCeL neurons exerted bidirectional regulation of context fear, whereas manipulation of vBNST neurons that received the projection from SSTCeL neurons could bidirectionally regulate both context and tone fear memory. We subsequently demonstrated the presence of δ and κ opioid receptor protein expression within the CeL-vBNST circuits, potentially accounting for the discrepancy between prolonged activation of GABAergic circuits and inhibition of downstream vBNST neurons. Finally, administration of an opioid receptor antagonist cocktail on the PKCδCeL-vBNST or SSTCeL-vBNST pathway successfully restored context or tone fear memory reduction induced by prolonged activation of the circuits. ConclusionsTogether, these findings establish a functional role for distinct CeL-vBNST circuits in the differential regulation and appropriate maintenance of fear.

Training-induced circuit-specific excitatory synaptogenesis in mice is required for effort control

Synaptogenesis is essential for circuit development; however, it is unknown whether it is critical for the establishment and performance of goal-directed voluntary behaviors. Here, we show that operant conditioning via lever-press for food reward training in mice induces excitatory synapse formation onto a subset of anterior cingulate cortex neurons projecting to the dorsomedial striatum (ACC→DMS). Training-induced synaptogenesis is controlled by the Gabapentin/Thrombospondin receptor α2δ−1, which is an essential neuronal protein for proper intracortical excitatory synaptogenesis. Using germline and conditional knockout mice, we found that deletion of α2δ−1 in the adult ACC→DMS circuit diminishes training-induced excitatory synaptogenesis. Surprisingly, this manipulation does not impact learning but results in a significant increase in effort exertion without affecting sensitivity to reward value or changing contingencies. Bidirectional optogenetic manipulation of ACC→DMS neurons rescues or phenocopies the behaviors of the α2δ−1 cKO mice, highlighting the importance of synaptogenesis within this cortico-striatal circuit in regulating effort exertion.

Central regulation of stress-evoked peripheral immune responses.

Stress-linked psychiatric disorders, including anxiety and major depressive disorder, are associated with systemic inflammation. Recent studies have reported stress-induced alterations in haematopoiesis that result in monocytosis, neutrophilia, lymphocytopenia and, consequently, in the upregulation of pro-inflammatory processes in immunologically relevant peripheral tissues. There is now evidence that this peripheral inflammation contributes to the development of psychiatric symptoms as well as to common co-morbidities of psychiatric disorders such as metabolic syndrome and immunosuppression. Here, we review the specific brain and spinal regions, and the neuronal populations within them, that respond to stress and transmit signals to peripheral tissues via the autonomic nervous system or neuroendocrine pathways to influence immunological function. We comprehensively summarize studies that have employed retrograde tracing to define neurocircuits linking the brain to the bone marrow, spleen, gut, adipose tissue and liver. Moreover, we highlight studies that have used chemogenetic or optogenetic manipulation or intracerebroventricular administration of peptide hormones to control somatic immune responses. Collectively, this growing body of literature illustrates potential mechanisms through which stress signals are conveyed from the CNS to immune cells to regulate stress-relevant behaviours and comorbid pathophysiology.

GIRK2 potassium channels expressed by the AgRP neurons decrease adiposity and body weight in mice.

It is well known that the neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons increase appetite and decrease thermogenesis. Previous studies demonstrated that optogenetic and/or chemogenetic manipulations of NPY/AgRP neuronal activity alter food intake and/or energy expenditure (EE). However, little is known about intrinsic molecules regulating NPY/AgRP neuronal excitability to affect long-term metabolic function. Here, we found that the G protein-gated inwardly rectifying K+ (GIRK) channels are key to stabilize NPY/AgRP neurons and that NPY/AgRP neuron-selective deletion of the GIRK2 subunit results in a persistently increased excitability of the NPY/AgRP neurons. Interestingly, increased body weight and adiposity observed in the NPY/AgRP neuron-selective GIRK2 knockout mice were due to decreased sympathetic activity and EE, while food intake remained unchanged. The conditional knockout mice also showed compromised adaptation to coldness. In summary, our study identified GIRK2 as a key determinant of NPY/AgRP neuronal excitability and driver of EE in physiological and stress conditions.

Optogenetic manipulation of Gq- and Gi/o-coupled receptor signaling in neurons and heart muscle cells.

G-protein-coupled receptors (GPCRs) transmit signals into cells depending on the G protein type. To analyze the functions of GPCR signaling, we assessed the effectiveness of animal G-protein-coupled bistable rhodopsins that can be controlled into active and inactive states by light application using zebrafish. We expressed Gq- and Gi/o-coupled bistable rhodopsins in hindbrain reticulospinal V2a neurons, which are involved in locomotion, or in cardiomyocytes. Light stimulation of the reticulospinal V2a neurons expressing Gq-coupled spider Rh1 resulted in an increase in the intracellular Ca2+ level and evoked swimming behavior. Light stimulation of cardiomyocytes expressing the Gi/o-coupled mosquito Opn3, pufferfish TMT opsin, or lamprey parapinopsin induced cardiac arrest, and the effect was suppressed by treatment with pertussis toxin or barium, suggesting that Gi/o-dependent regulation of inward-rectifier K+ channels controls cardiac function. These data indicate that these rhodopsins are useful for optogenetic control of GPCR-mediated signaling in zebrafish neurons and cardiomyocytes.

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of BeGC1 or PACs achieved it at long latencies. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the function and regulation of zebrafish neurons and cardiomyocytes.

Near-infrared voltage-sensitive dyes based on chromene donor

Voltage-sensitive dyes (VSDs) are used to image electrical activity in cells and tissues with submillisecond time resolution. Most of these fast sensors are constructed from push-pull chromophores whose fluorescence spectra are modulated by the electric field across the cell membrane. It was found that the substitution of naphthalene with chromene produces a 60 to 80 nm red-shift in absorption and emission spectra while maintaining fluorescence quantum efficiency and voltage sensitivity. One dye was applied to ex vivo murine heart with excitation at 730 nm, by far the longest wavelength reported in voltage imaging. This VSD resolves cardiac action potentials in single trials with 12% ΔF/F per action potential. The well-separated excitation spectra between these long-wavelength VSDs and channelrhodopsin (ChR2) enabled monitoring of action potential propagation in ChR2 hearts without any perturbation of electrical dynamics. Importantly, by employing spatially localized optogenetic manipulation, action potential dynamics can be assessed in an all-optical fashion with no artifact related to optical cross-talk between the reporter and actuator. These new environmentally sensitive chromene-based chromophores are also likely to have applications outside voltage imaging.

Pathway selective optogenetic manipulations on the oculomotor circuits of non-human primates

Pathway selective optogenetic manipulations on the oculomotor circuits of non-human primates

Anti-Hebbian plasticity in the motor cortex promotes defensive freezing

Regional brain activity often decreases from baseline levels in response to external events, but how neurons develop such negative responses is unclear. To study this, we leveraged the negative response that develops in the primary motor cortex (M1) after classical fear learning. We trained mice with a fear conditioning paradigm while imaging their brains with standard two-photon microscopy. This enabled monitoring changes in neuronal responses to the tone with synaptic resolution through learning. We found that M1 layer 5 pyramidal neurons (L5 PNs) developed negative tone responses within an hour after conditioning, which depended on the weakening of their dendritic spines that were active during training. Blocking this form of anti-Hebbian plasticity using an optogenetic manipulation of CaMKII activity disrupted negative tone responses and freezing. Therefore, reducing the strength of spines active at the time of memory encoding leads to negative responses of L5 PNs. In turn, these negative responses curb M1's capacity for promoting movement, thereby aiding freezing. Collectively, this work provides a mechanistic understanding of how area-specific negative responses to behaviorally relevant cues can be achieved.

Hippocampal ensemble dynamics and memory performance are modulated by respiration during encoding

During offline brain states, such as sleep and memory consolidation, respiration coordinates hippocampal activity. However, the role of breathing during online memory traces remains unclear. Here, we show that respiration can be recruited during online memory encoding. Optogenetic manipulation was used to control activation of the primary inspiratory rhythm generator PreBötzinger complex (PreBötC) in transgenic mice. When intermittent PreBötC-induced apnea covered the object exploration time during encoding, novel object detection was impaired. Moreover, the mice did not exhibit freezing behavior during presentation of fear-conditioned stimuli (CS+) when PreBötC-induced apnea occurred at the exact time of encoding. This apnea did not evoke changes in CA3 cell ensembles between presentations of CS+ and conditioned inhibition (CS−), whereas in normal breathing, CS+ presentations produced dynamic changes. Our findings demonstrate that components of central respiratory activity (e.g., frequency) during online encoding strongly contribute to shaping hippocampal ensemble dynamics and memory performance.

The ortholog of human ssDNA-binding protein SSBP3 influences neurodevelopment and autism-like behaviors in Drosophila melanogaster.

1p32.3 microdeletion/duplication is implicated in many neurodevelopmental disorders-like phenotypes such as developmental delay, intellectual disability, autism, macro/microcephaly, and dysmorphic features. The 1p32.3 chromosomal region harbors several genes critical for development; however, their validation and characterization remain inadequate. One such gene is the single-stranded DNA-binding protein 3 (SSBP3) and its Drosophila melanogaster ortholog is called sequence-specific single-stranded DNA-binding protein (Ssdp). Here, we investigated consequences of Ssdp manipulations on neurodevelopment, gene expression, physiological function, and autism-associated behaviors using Drosophila models. We found that SSBP3 and Ssdp are expressed in excitatory neurons in the brain. Ssdp overexpression caused morphological alterations in Drosophila wing, mechanosensory bristles, and head. Ssdp manipulations also affected the neuropil brain volume and glial cell number in larvae and adult flies. Moreover, Ssdp overexpression led to differential changes in synaptic density in specific brain regions. We observed decreased levels of armadillo in the heads of Ssdp overexpressing flies, as well as a decrease in armadillo and wingless expression in the larval wing discs, implicating the involvement of the canonical Wnt signaling pathway in Ssdp functionality. RNA sequencing revealed perturbation of oxidative stress-related pathways in heads of Ssdp overexpressing flies. Furthermore, Ssdp overexpressing brains showed enhanced reactive oxygen species (ROS), altered neuronal mitochondrial morphology, and up-regulated fission and fusion genes. Flies with elevated levels of Ssdp exhibited heightened anxiety-like behavior, altered decisiveness, defective sensory perception and habituation, abnormal social interaction, and feeding defects, which were phenocopied in the pan-neuronal Ssdp knockdown flies, suggesting that Ssdp is dosage sensitive. Partial rescue of behavioral defects was observed upon normalization of Ssdp levels. Notably, Ssdp knockdown exclusively in adult flies did not produce behavioral and functional defects. Finally, we show that optogenetic manipulation of Ssdp-expressing neurons altered autism-associated behaviors. Collectively, our findings provide evidence that Ssdp, a dosage-sensitive gene in the 1p32.3 chromosomal region, is associated with various anatomical, physiological, and behavioral defects, which may be relevant to neurodevelopmental disorders like autism. Our study proposes SSBP3 as a critical gene in the 1p32.3 microdeletion/duplication genomic region and sheds light on the functional role of Ssdp in neurodevelopmental processes in Drosophila.

Parvalbumin neurons enhance temporal coding and reduce cortical noise in complex auditory scenes

Cortical representations supporting many cognitive abilities emerge from underlying circuits comprised of several different cell types. However, cell type-specific contributions to rate and timing-based cortical coding are not well-understood. Here, we investigated the role of parvalbumin neurons in cortical complex scene analysis. Many complex scenes contain sensory stimuli which are highly dynamic in time and compete with stimuli at other spatial locations. Parvalbumin neurons play a fundamental role in balancing excitation and inhibition in cortex and sculpting cortical temporal dynamics; yet their specific role in encoding complex scenes via timing-based coding, and the robustness of temporal representations to spatial competition, has not been investigated. Here, we address these questions in auditory cortex of mice using a cocktail party-like paradigm, integrating electrophysiology, optogenetic manipulations, and a family of spike-distance metrics, to dissect parvalbumin neurons’ contributions towards rate and timing-based coding. We find that suppressing parvalbumin neurons degrades cortical discrimination of dynamic sounds in a cocktail party-like setting via changes in rapid temporal modulations in rate and spike timing, and over a wide range of time-scales. Our findings suggest that parvalbumin neurons play a critical role in enhancing cortical temporal coding and reducing cortical noise, thereby improving representations of dynamic stimuli in complex scenes.

Optogenetic control of medaka behavior with channelrhodopsin.

Optogenetics enables the manipulation of neural activity with high spatiotemporal resolution in genetically defined neurons. The method is widely used in various model animals in the neuroscience and physiology fields. Channelrhodopsins are robust tools for optogenetic manipulation, but they have not yet been used for studies in medaka. In the present study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated knock-in approach to establish a transgenic medaka strain expressing the Chloromonas oogama channelrhodopsin (CoChR) in the ISL LIM homeobox 1 (isl1) locus. We demonstrated that light stimuli elicited specific behavioral responses, such as bending or turning locomotion in the embryos and pectoral fin movements in the larvae and adults. The response probabilities and intensities of these movements could be controlled by adjusting the intensity, duration, or wavelength of each light stimulus. Furthermore, we demonstrated that the pectoral fin movements in the adult stage could be elicited using a laser pointer to irradiate region including the caudal hind brain and the rostral spinal cord. Our results indicate that CoChR allows for manipulation of medaka behaviors by activating targeted neurons, which will further our understanding of the detailed neural mechanisms of motor control or social behaviors in medaka.

Disentangling the role of NAc D1 and D2 cells in hedonic eating

Overeating is driven by both the hedonic component (‘liking’) of food, and the motivation (‘wanting’) to eat it. The nucleus accumbens (NAc) is a key brain center implicated in these processes, but how distinct NAc cell populations encode ‘liking’ and ‘wanting’ to shape overconsumption remains unclear. Here, we probed the roles of NAc D1 and D2 cells in these processes using cell-specific recording and optogenetic manipulation in diverse behavioral paradigms that disentangle reward traits of ‘liking’ and ‘wanting’ related to food choice and overeating in healthy mice. Medial NAc shell D2 cells encoded experience-dependent development of ‘liking’, while D1 cells encoded innate ‘liking’ during the first food taste. Optogenetic control confirmed causal links of D1 and D2 cells to these aspects of ‘liking’. In relation to ‘wanting’, D1 and D2 cells encoded and promoted distinct aspects of food approach: D1 cells interpreted food cues while D2 cells also sustained food-visit-length that facilitates consumption. Finally, at the level of food choice, D1, but not D2, cell activity was sufficient to switch food preference, programming subsequent long-lasting overconsumption. By revealing complementary roles of D1 and D2 cells in consumption, these findings assign neural bases to ‘liking’ and ‘wanting’ in a unifying framework of D1 and D2 cell activity.

Theoretical Considerations for Optimizing the Use of Optogenetics with Complex Behavior.

Optogenetic approaches have allowed researchers to address complex questions about behavior that were previously unanswerable. However, as optogenetic procedures involve a large parameter space across multiple dimensions, it is crucial to consider such parameters in conjunction with the behaviors under study. Here, we discuss strategies to optimize optogenetic approaches with complex behavior by identifying critical experimental design considerations, including frequency specificity, temporal precision, activity-controlled optogenetics, stimulation pattern, and cell-type specificity. We highlight potential limitations or theoretical considerations to be made when manipulating each of these factors of optogenetic experiments. This overview emphasizes the importance of optimizing optogenetic study design to enhance the conclusions that can be drawn about the neuroscience of behavior. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.