Holographic optogenetic stimulation with calcium imaging as an all optical tool for cardiac electrophysiology.

All optical approaches to control and read out the electrical activity in a cardiac syncytium can improve our understanding of cardiac electrophysiology. Here, we demonstrate optogenetic stimulation of cardiomyocytes with high spatial precision using light foci generated with a ferroelectric spatial light modulator. Computer generated holograms binarized by bidirectional error diffusion create multiple foci with more even intensity distribution compared with thresholding approach. We evoke the electrical activity of cardiac HL1 cells expressing the channelrhodopsin-2 variant, ChR2(H134R) using single and multiple light foci and at the same time visualize the action potential using a calcium sensitive indicator called Cal-630. We show that localized regions in the cardiac monolayer can be stimulated enabling us to initiate signal propagation from a precise location. Furthermore, we demonstrate that probing the cardiac cells with multiple light foci enhances the excitability of the cardiac network. This approach opens new applications in manipulating and visualizing the electrical activity in a cardiac syncytium.

Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology.

Optogenetic approaches have evolved as potent means to investigate cardiac electrophysiology, with research ranging from the study of arrhythmia mechanisms to effects of cardiac innervation and heterocellular structural and functional interactions, both in healthy and diseased myocardium. Most commonly, these studies use channelrhodopsin-2 (ChR2)-expressing murine models that enable light-activated depolarization of the target cell population. However, each newly generated mouse line requires thorough characterization, as cell-type specific ChR2 expression cannot be taken for granted, and the electrophysiological response of its activation in the target cell should be evaluated. In this chapter, we describe detailed protocols for assessing ChR2 specificity using immunohistochemistry, isolation of specific cell populations to analyze electrophysiological effects of ChR2 activation with the patch-clamp technique, and whole-heart experiments to assess in situ effects of optical stimulation.

Cortical layer 6 control of sensory responses in higher-order thalamus.

KEY POINTS: Thalamic activity is regulated by corticothalamic feedback from layers 5B and 6. To selectively study the importance of the layer 6 corticothalamic (L6 CT) projection, a transgenic mouse line was used in which layer 6 cells projecting to posterior medial thalamus (POm) were targeted for expression of channelrhodopsin-2. Repetitive optogenetic stimulation of this sub-type of L6 cells caused a rapid adaptation in POm spiking output, but had little effect on the spiking activity in the other cortical layers. L6 photoactivation increased POm spiking to the first, but not to subsequent whisker deflections in a 4 Hz train. A sub-population of L6 CT cells that can cause an initial increase in POm activity, that is not sustained with repetitive stimulation, could indicate that this L6 projection does not modulate ongoing sensory processing, but rather serves to briefly increase POm activity in specific behavioural contexts. ABSTRACT: Thalamic activity is regulated by corticothalamic feedback from layers 5B and 6. The nature of these feedback systems differs, one difference being that whereas layer 5 provides 'driver' input, the layer 6 input is thought to be 'modulatory'. To selectively study the importance of the layer 6 corticothalamic (L6 CT) projection, a transgenic mouse line was used in which layer 6 cells projecting to posterior medial thalamus (POm) were targeted for expression of channelrhodopsin-2 and in vivo electrophysiology recordings were done in urethane-anaesthetized mice. Pre- and postsynaptic targets were identified using tracing techniques and light-sheet microscopy in cleared intact brains. We find that optogenetic activation of this subtype of L6 CT cells (L6-Drd1) has little effect on cortical activity, but activates POm. Repetitive photoactivation of L6-Drd1 cells evoked a reliable response following every photoactivation, whereas in the connected POm area spiking was only initially increased. The response to repetitive whisker stimulation showed a similar pattern with only an initial increase in whisker-evoked spiking. Furthermore, the increase in whisker-evoked spiking with optogenetic activation of L6-Drd1 cells is additive, rather than multiplicative, causing even cells that in the absence of L6 activation produce relatively few spikes to increase their spiking substantially. We show that layer 6 corticothalamic cells can provide a strong, albeit rapidly depressing, input to POm. This type of cortical L6 activity could be important for rapid gain control in POm, rather than providing a modulation in phase with the whisking cycle.

Astrocytes Amplify Neuronal Dendritic Volume Transmission Stimulated by Norepinephrine.

In addition to their support role in neurotransmitter and ion buffering, astrocytes directly regulate neurotransmission at synapses via local bidirectional signaling with neurons. Here, we reveal a form of neuronal-astrocytic signaling that transmits retrograde dendritic signals to distal upstream neurons in order to activate recurrent synaptic circuits. Norepinephrine activates α1 adrenoreceptors in hypothalamic corticotropin-releasing hormone (CRH) neurons to stimulate dendritic release, which triggers an astrocytic calcium response and release of ATP; ATP stimulates action potentials in upstream glutamate and GABA neurons to activate recurrent excitatory and inhibitory synaptic circuits to the CRH neurons. Thus, norepinephrine activates a retrograde signaling mechanism in CRH neurons that engages astrocytes in order to extend dendritic volume transmission to reach distal presynaptic glutamate and GABA neurons, thereby amplifying volume transmission mediated by dendritic release.

µ-opioid receptor-mediated downregulation of midline thalamic pathways to basal and central amygdala.

Brain µ-opioid receptors (MOR) mediate reward and help coping with pain, social rejection, anxiety and depression. The dorsal midline thalamus (dMT) integrates visceral/emotional signals and biases behavior towards aversive or defensive states through projections to the amygdala. While a dense MOR expression in the dMT has been described, the exact cellular and synaptic mechanisms of µ-opioidergic modulation in the dMT-amygdala circuitry remain unresolved. Here, we hypothesized that MORs are important negative modulators of dMT-amygdala excitatory networks. Using retrograde tracers and targeted channelrhodopsin expression in combination with patch-clamp electrophysiology, we found that projections of dMT neurons onto both basal amygdala principal neurons (BA PN) and central amygdala (CeL) neurons are attenuated by stimulation of somatic or synaptic MORs. Importantly, dMT efferents to the amygdala drive feedforward excitation of centromedial amygdala neurons (CeM), which is dampened by MOR activation. This downregulation of excitatory activity in dMT-amygdala networks puts the µ-opioid system in a position to ameliorate aversive or defensive behavioral states associated with stress, withdrawal, physical pain or social rejection.

Ipsilateral-Dominant Control of Limb Movements in Rodent Posterior Parietal Cortex.

It is well known that the posterior parietal cortex (PPC) and frontal motor cortices in primates preferentially control voluntary movements of contralateral limbs. The PPC of rats has been defined based on patterns of thalamic and cortical connectivity. The anatomical characteristics of this area suggest that it may be homologous to the PPC of primates. However, its functional roles in voluntary forelimb movements have not been well understood, particularly in the lateralization of motor limb representation; that is, the limb-specific activity representations for right and left forelimb movements. We examined functional spike activity of the PPC and two motor cortices, the primary motor cortex (M1) and the secondary motor cortex (M2), when head-fixed male rats performed right or left unilateral movements. Unlike primates, PPC neurons in rodents were found to preferentially represent ipsilateral forelimb movements, in contrast to the contralateral preference of M1 and M2 neurons. Consistent with these observations, optogenetic activation of PPC and motor cortices, respectively, evoked ipsilaterally and contralaterally biased forelimb movements. Finally, we examined the effects of optogenetic manipulation on task performance. PPC or M1 inhibition by optogenetic GABA release shifted the behavioral limb preference contralaterally or ipsilaterally, respectively. In addition, weak optogenetic PPC activation, which was insufficient to evoke motor responses by itself, shifted the preference ipsilaterally; although similar M1 activation showed no effects on task performance. These paradoxical observations suggest that the PPC plays evolutionarily different roles in forelimb control between primates and rodents.SIGNIFICANCE STATEMENT In rodents, the primary and secondary motor cortices (M1 and M2, respectively) are involved in voluntary movements with contralateral preference. However, it remains unclear whether and how the posterior parietal cortex (PPC) participates in controlling multiple limb movements. We recorded functional activity from these areas using a behavioral task to monitor movements of the right and left forelimbs separately. PPC neurons preferentially represented ipsilateral forelimb movements and optogenetic PPC activation evoked ipsilaterally biased forelimb movements. Optogenetic PPC inhibition via GABA release shifted the behavioral limb preference contralaterally during task performance, whereas weak optogenetic PPC activation, which was insufficient to evoke motor responses by itself, shifted the preference ipsilaterally. Our findings suggest rodent PPC contributes to ipsilaterally biased motor response and/or planning.

Thalamocortical and Intracortical Inputs Differentiate Layer-Specific Mouse Auditory Corticocollicular Neurons.

Long-range descending projections from the auditory cortex play key roles in shaping response properties in the inferior colliculus. The auditory corticocollicular projection is massive and heterogeneous, with axons emanating from cortical layers 5 and 6, and plays a key role in directing plastic changes in the inferior colliculus. However, little is known about the cortical and thalamic networks within which corticocollicular neurons are embedded. Here, laser scanning photostimulation glutamate uncaging and photoactivation of channelrhodopsin-2 were used to probe the local and long-range network differences between preidentified layer 5 and layer 6 auditory corticocollicular neurons from male and female mice in vitro Layer 5 corticocollicular neurons were found to vertically integrate supragranular excitatory and inhibitory input to a substantially greater degree than their layer 6 counterparts. In addition, all layer 5 corticocollicular neurons received direct and large thalamic inputs from channelrhodopsin-2-labeled thalamocortical fibers, whereas such inputs were less common in layer 6 corticocollicular neurons. Finally, a new low-calcium/synaptic blockade approach to separate direct from indirect inputs using laser photostimulation was validated. These data demonstrate that layer 5 and 6 corticocollicular neurons receive distinct sets of cortical and thalamic inputs, supporting the hypothesis that they have divergent roles in modulating the inferior colliculus. Furthermore, the direct connection between the auditory thalamus and layer 5 corticocollicular neurons reveals a novel and rapid link connecting ascending and descending pathways.SIGNIFICANCE STATEMENT Descending projections from the cortex play a critical role in shaping the response properties of sensory neurons. The projection from the auditory cortex to the inferior colliculus is a massive, yet poorly understood, pathway emanating from two distinct cortical layers. Here we show, using a range of optical techniques, that mouse auditory corticocollicular neurons from different layers are embedded into different cortical and thalamic networks. Specifically, we observed that layer 5 corticocollicular neurons integrate information across cortical lamina and receive direct thalamic input. The latter connection provides a hyperdirect link between acoustic sensation and descending control, thus demonstrating a novel mechanism for rapid "online" modulation of sensory perception.

Dopamine receptor D2, but not D1, mediates descending dopaminergic pathway-produced analgesic effect in a trigeminal neuropathic pain mouse model.

Neuropathic pain represents a challenge to clinicians because it is resistant to commonly prescribed analgesics due to its largely unknown mechanisms. Here, we investigated a descending dopaminergic pathway-mediated modulation of trigeminal neuropathic pain. We performed chronic constriction injury of the infraorbital nerve from the maxillary branch of trigeminal nerve to induce trigeminal neuropathic pain in mice. Our retrograde tracing showed that the descending dopaminergic projection from hypothalamic A11 nucleus to spinal trigeminal nucleus caudalis is bilateral. Optogenetic/chemogenetic manipulation of dopamine receptors D1 and D2 in the spinal trigeminal nucleus caudalis produced opposite effects on the nerve injury-induced trigeminal neuropathic pain. Specific excitation of dopaminergic neurons in the A11 nucleus attenuated the trigeminal neuropathic pain through the activation of D2 receptors in the spinal trigeminal nucleus caudalis. Conversely, specific ablation of the A11 dopaminergic neurons exacerbated such pain. Our results suggest that the descending A11-spinal trigeminal nucleus caudalis dopaminergic projection is critical for the modulation of trigeminal neuropathic pain and could be manipulated to treat such pain.

Frontal cortical control of posterior sensory and association cortices through the claustrum.

The claustrum is a telencephalic gray matter nucleus that is richly interconnected with the neocortex. This structure subserves top-down executive functions that require frontal cortical control of posterior cortical regions. However, functional anatomical support for the claustrum allowing for long-range intercortical communication is lacking. To test this, we performed a channelrhodopsin-assisted long-circuit mapping strategy in mouse brain slices. We find that anterior cingulate cortex input to the claustrum is transiently amplified by claustrum neurons that, in turn, project to parietal association cortex or to primary and secondary visual cortices. Additionally, we observe that claustrum drive of cortical neurons in parietal association cortex is layer-specific, eliciting action potential generation briefly in layers II/III, IV, and VI but not V. These data are the first to provide a functional anatomical substrate through claustrum that may underlie top-down functions, such as executive attention or working memory, providing critical insight to this most interconnected and enigmatic nucleus.

Circuit-selective properties of glutamatergic inputs to the rat prelimbic cortex and their alterations in neuropathic pain.

Functional deactivation of the prefrontal cortex (PFC) is a critical step in the neuropathic pain phenotype. We performed optogenetic circuit dissection to study the properties of ventral hippocampal (vHipp) and thalamic (MDTh) inputs to L5 pyramidal cells in acute mPFC slices and to test whether alterations in these inputs contribute to mPFC deactivation in neuropathic pain. We found that: (1) both the vHipp and MDTh inputs elicit monosynaptic excitatory and polysynaptic inhibitory currents. (2) The strength of the excitatory MDTh input is uniform, while the vHipp input becomes progressively stronger along the dorsal-ventral axis. (3) Synaptic current kinetics suggests that the MDTh inputs contact distal, while the vHipp inputs contact proximal dendritic sections. (4) The longer delay of inhibitory currents in response to vHipp compared to MDTh inputs suggests that they are activated by feedback and feed-forward circuitries, respectively. (5) One week after a peripheral neuropathic injury, both glutamatergic inputs are modified: MDTh responses are smaller, without evidence of presynaptic changes, while the probability of release at vHipp-mPFC synapses becomes lower, without significant change in current amplitude. Thus, dysregulation of both these inputs likely contributes to the mPFC deactivation in neuropathic pain and may impair PFC-dependent cognitive tasks.

Spectrally distinct channelrhodopsins for two-colour optogenetic peripheral nerve stimulation.

Technologies for peripheral nerve stimulation have conventionally relied on the anatomic placement of electrodes adjacent to subsets of sensory fibres or motor fibres that selectively target an end effector. Here, we demonstrate the use of optogenetics to directly target the innervating fibres of an end effector by relying on retrograde transfection of adeno-associated virus serotype 6 to restrict axonal opsin expression to the desired fibre targets. By using an in vivo screen in rats, we identify the first channelrhodopsins as well as a halorhodopsin that respond to red light in the peripheral nerve. Combining two channelrhodopsins with spectrally distinct activation profiles allowed us to drive opposing muscle activity via two-colour illumination of the same mixed nerve. We also show halorhodopsin-mediated reductions in electrically evoked muscle tremor spectrally optimized for deep peripheral nerves. Our non-invasive peripheral neurostimulator with targeted multi-fascicle resolution enables scientific and clinical exploration, such as motor control in paralysis, biomimetic sensation feedback for amputees and targeted inhibition of muscle tremor.

Accumbal D2 cells orchestrate innate risk-avoidance according to orexin signals.

Excitation of accumbal D2 cells governs vital actions, including avoidance of learned risks, but the origins of this excitation and roles of D2 cells in innate risk-avoidance are unclear. Hypothalamic neurons producing orexins (also called hypocretins) enhance innate risk-avoidance via poorly understood neurocircuits. We describe a direct orexin→D2 excitatory circuit and show that D2 cell activity is necessary for orexin-dependent innate risk-avoidance in mice, thus revealing an unsuspected hypothalamus-accumbens interplay in action selection.

Termination of re-entrant atrial tachycardia via optogenetic stimulation with optimized spatial targeting: insights from computational models.

KEY POINTS: Optogenetics has emerged as a potential alternative to electrotherapy for treating heart rhythm disorders, but its applicability for terminating atrial arrhythmias remains largely unexplored. We used computational models reconstructed from clinical MRI scans of fibrotic patient atria to explore the feasibility of optogenetic termination of atrial tachycardia (AT), comparing two different illumination strategies: distributed vs. targeted. We show that targeted optogenetic stimulation based on automated, non-invasive flow-network analysis of patient-specific re-entry morphology may be a reliable approach for identifying the optimal illumination target in each individual (i.e. the critical AT isthmus). The above-described approach yields very high success rates (up to 100%) and requires dramatically less input power than distributed illumination We conclude that simulations in patient-specific models show that targeted light pulses lasting longer than the AT cycle length can efficiently and reliably terminate AT if the human atria can be successfully light-sensitized via gene delivery of ChR2. ABSTRACT: Optogenetics has emerged as a potential alternative to electrotherapy for treating arrhythmia, but feasibility studies have been limited to ventricular defibrillation via epicardial light application. Here, we assess the efficacy of optogenetic atrial tachycardia (AT) termination in human hearts using a strategy that targets for illumination specific regions identified in an automated manner. In three patient-specific models reconstructed from late gadolinium-enhanced MRI scans, we simulated channelrhodopsin-2 (ChR2) expression via gene delivery. In all three models, we attempted to terminate re-entrant AT (induced via rapid pacing) via optogenetic stimulation. We compared two strategies: (1) distributed illumination of the endocardium by multi-optrode grids (number of optrodes, Nopt  = 64, 128, 256) and (2) targeted illumination of the critical isthmus, which was identified via analysis of simulated activation patterns using an algorithm based on flow networks. The illuminated area and input power were smaller for the targeted approach (19-57.8 mm2 ; 0.6-1.8 W) compared to the sparsest distributed arrays (Nopt  = 64; 124.9 ± 6.3 mm2 ; 3.9 ± 0.2 W). AT termination rates for distributed illumination were low, ranging from <5% for short pulses (1/10 ms long) to ∼20% for longer stimuli (100/1000 ms). When we attempted to terminate the same AT episodes with targeted illumination, outcomes were similar for short pulses (1/10 ms long: 0% success) but improved for longer stimuli (100 ms: 54% success; 1000 ms: 90% success). We conclude that simulations in patient-specific models show that light pulses lasting longer than the AT cycle length can efficiently and reliably terminate AT in atria light-sensitized via gene delivery. We show that targeted optogenetic stimulation based on analysis of AT morphology may be a reliable approach for defibrillation and requires less power than distributed illumination.

Widespread optogenetic expression in macaque cortex obtained with MR-guided, convection enhanced delivery (CED) of AAV vector to the thalamus.

BACKGROUND: In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Previous work has shown that parenchymal delivery of adeno-associated virus (AAV) in the thalamus by convection-enhanced delivery (CED) can lead to large-scale transduction via axonal transport in distal areas including cortex. We used this approach to obtain widespread cortical expression of light-sensitive ion channels. NEW METHOD: AAV vectors co-expressing channelrhodopsin-2 (ChR2) and yellow fluorescent protein (YFP) genes were infused into thalamus of three rhesus macaques under MR-guided CED. After six to twelve weeks recovery, in vivo optical stimulation and single cell recording in the cortex was carried out using an optrode in anesthetized animals. Post-mortem immunostaining against YFP was used to estimate the distribution and level of expression of ChR2 in thalamus and cortex. RESULTS: Histological analysis revealed high levels of transduction in cortical layers. The patterns of expression were consistent with known thalamo-cortico-thalamic circuits. Dense expression was seen in thalamocortiocal axonal fibers in layers III, IV and VI and in pyramidal neurons in layers V and VI, presumably corticothalamic neurons. In addition we obtained reliable in vivo light-evoked responses in cortical areas with high levels of expression. COMPARISON WITH EXISTING METHODS: Thalamic CED is very efficient in achieving large expressing areas in comparison to convectional techniques both in minimizing infusion time and in minimizing damage to the brain. CONCLUSION: MR-guided CED infusion into thalamus provides a simplified approach to transduce large cortical areas by thalamo-cortico-thalamic projections in primate brain.

Frequency-dependent drug screening using optogenetic stimulation of human iPSC-derived cardiomyocytes.

Side effects on cardiac ion channels are one major reason for new drugs to fail during preclinical evaluation. Herein we propose a simple optogenetic screening tool measuring extracellular field potentials (FP) from paced cardiomyocytes to identify drug effects over the whole physiological heart range, which is essential given the rate-dependency of ion channel function and drug action. Human induced pluripotent stem cell-derived cardiomyocytes were transduced with an adeno-associated virus to express Channelrhodopsin2 and plated on micro-electrode arrays. Global pulsed illumination (470 nm, 1 ms, 0.9 mW/mm2) was applied at frequencies from 1 to 2.5 Hz, which evoked FP simultaneously in all cardiomyocytes. This synchronized activation allowed averaging of FP from all electrodes resulting in one robust FP signal for analysis. Field potential duration (FPD) was ~25% shorter at 2.5 Hz compared to 1 Hz. Inhibition of hERG channels prolonged FPD only at low heart rates whereas Ca2+ channel block shortened FPD at all heart rates. Optogenetic pacing also allowed analysis of the maximum downstroke velocity of the FP to detect drug effects on Na+ channel availability. In principle, the presented method is well scalable for high content cardiac toxicity screening or personalized medicine for inherited cardiac channelopathies.

Optogenetic rewiring of thalamocortical circuits to restore function in the stroke injured brain.

To regain sensorimotor functions after stroke, surviving neural circuits must reorganize and form new connections. Although the thalamus is critical for processing and relaying sensory information to the cortex, little is known about how stroke affects the structure and function of these connections, or whether a therapeutic approach targeting these circuits can improve recovery. Here we reveal with in vivo calcium imaging that stroke in somatosensory cortex dampens the excitability of surviving thalamocortical circuits. Given this deficit, we hypothesized that chronic transcranial window optogenetic stimulation of thalamocortical axons could facilitate recovery. Using two-photon imaging, we show that optogenetic stimulation promotes the formation of new and stable thalamocortical synaptic boutons, without impacting axon branch dynamics. Stimulation also enhances the recovery of somatosensory cortical circuit function and forepaw sensorimotor abilities. These results demonstrate that an optogenetic approach can rewire thalamocortical circuits and restore function in the damaged brain.

Activation of hypothalamic astrocytes suppresses feeding without altering emotional states.

Emerging evidence shows that hypothalamic astrocytes react to and counteract energy surfeit produced by high-fat diet (HFD) feeding. However, the functional role of astrocytes in the control of energy states and the underlying molecular mechanism(s) during physiological conditions remain largely underexplored. In the present study, by taking advantage of spatiotemporally precise optogenetic approaches, real-time measurements of extracellular adenosine, and behavioral assays, we find that optogenetic stimulation of astrocytes localized in the medial basal hypothalamus (MBH) suppresses food intake in a frequency dependent manner with high frequency, but not low frequency, stimulation of astrocytes reducing food intake. Furthermore, stimulation of MBH astrocytes diminishes orexigenic ghrelin or fasting-induced hyperphagia without effecting anxiety-related behavior. Consistent with a frequency dependent role for MBH astrocytes in feeding behavior, optogenetic stimulation of MBH astrocytes increases extracellular levels of adenosine in a frequency dependent manner. Collectively, our results provide new insights into the role of astrocytes in physiological functions during naturally occurring behaviors, such as feeding. GLIA 2016;64:2263-2273.

Optogenetic and chemogenetic strategies for sustained inhibition of pain.

Spatially targeted, genetically-specific strategies for sustained inhibition of nociceptors may help transform pain science and clinical management. Previous optogenetic strategies to inhibit pain have required constant illumination, and chemogenetic approaches in the periphery have not been shown to inhibit pain. Here, we show that the step-function inhibitory channelrhodopsin, SwiChR, can be used to persistently inhibit pain for long periods of time through infrequent transdermally delivered light pulses, reducing required light exposure by >98% and resolving a long-standing limitation in optogenetic inhibition. We demonstrate that the viral expression of the hM4D receptor in small-diameter primary afferent nociceptor enables chemogenetic inhibition of mechanical and thermal nociception thresholds. Finally, we develop optoPAIN, an optogenetic platform to non-invasively assess changes in pain sensitivity, and use this technique to examine pharmacological and chemogenetic inhibition of pain.