Recent studies have shown evidence of behavioral recovery after transplantation of human pluripotent stem cell (PSC)-derived neural cells in animal models of neurological disease. However, little is ...known about the mechanisms underlying graft function. Here we use optogenetics to modulate in real time electrophysiological and neurochemical properties of mesencephalic dopaminergic (mesDA) neurons derived from human embryonic stem cells (hESCs). In mice that had recovered from lesion-induced Parkinsonian motor deficits, light-induced selective silencing of graft activity rapidly and reversibly re-introduced the motor deficits. The re-introduction of motor deficits was prevented by the dopamine agonist apomorphine. These results suggest that functionality depends on graft neuronal activity and dopamine release. Combining optogenetics, slice electrophysiology and pharmacological approaches, we further show that mesDA-rich grafts modulate host glutamatergic synaptic transmission onto striatal medium spiny neurons in a manner reminiscent of endogenous mesDA neurons. Thus, application of optogenetics in cell therapy can link transplantation, animal behavior and postmortem analysis to enable the identification of mechanisms that drive recovery.
In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization over large volumes has been largely restricted to labeling of proteins. But volumetric ...nucleic acid labeling may represent a far greater scientific and clinical opportunity, enabling detection of not only diverse coding RNA variants but also non-coding RNAs. Moreover, scaling immunohistochemical detection to large tissue volumes has limitations due to high cost, limited renewability/availability, and restricted multiplexing capability of antibody labels. With the goal of versatile, high-content, and scalable molecular phenotyping of intact tissues, we developed a method using carbodiimide-based chemistry to stably retain RNAs in clarified tissue, coupled with amplification tools for multiplexed detection. The resulting technology enables robust measurement of activity-dependent transcriptional signatures, cell-identity markers, and diverse non-coding RNAs in rodent and human tissue volumes. The growing set of validated probes is deposited in an online resource for nucleating related developments from across the scientific community.
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•Carbodiimide-based chemistry in CLARITY hydrogels for RNA preservation and detection•Rapid diffusion of DNA oligonucleotides for volumetric in situ hybridization•Detection of microRNAs and mRNAs in clarified mouse and human tissues•DNA-based amplification for multiplexed in situ hybridization in CLARITY
CLARITY with enhanced RNA coupling chemistry is developed for multiplexed, volumetric visualization of both long and short RNAs in a variety of intact tissues.
Whole-brain recordings give us a global perspective of the brain in action. In this study, we describe a method using light field microscopy to record near-whole brain calcium and voltage activity at ...high speed in behaving adult flies. We first obtained global activity maps for various stimuli and behaviors. Notably, we found that brain activity increased on a global scale when the fly walked but not when it groomed. This global increase with walking was particularly strong in dopamine neurons. Second, we extracted maps of spatially distinct sources of activity as well as their time series using principal component analysis and independent component analysis. The characteristic shapes in the maps matched the anatomy of subneuropil regions and, in some cases, a specific neuron type. Brain structures that responded to light and odor were consistent with previous reports, confirming the new technique's validity. We also observed previously uncharacterized behavior-related activity as well as patterns of spontaneous voltage activity.
We demonstrate a two-photon optogenetic method that generates action potentials in neurons with single-cell precision, using the red-shifted opsin C1V1(T). We applied the method to optically map ...synaptic circuits in mouse neocortical brain slices and to activate small dendritic regions and individual spines. Using a spatial light modulator, we split the laser beam onto several neurons and performed simultaneous optogenetic activation of selected neurons in three dimensions.
Neural circuits that determine the perception and modulation of pain remain poorly understood. The prefrontal cortex (PFC) provides top-down control of sensory and affective processes. While animal ...and human imaging studies have shown that the PFC is involved in pain regulation, its exact role in pain states remains incompletely understood. A key output target for the PFC is the nucleus accumbens (NAc), an important component of the reward circuitry. Interestingly, recent human imaging studies suggest that the projection from the PFC to the NAc is altered in chronic pain. The function of this corticostriatal projection in pain states, however, is not known. Here we show that optogenetic activation of the PFC produces strong antinociceptive effects in a rat model (spared nerve injury model) of persistent neuropathic pain. PFC activation also reduces the affective symptoms of pain. Furthermore, we show that this pain-relieving function of the PFC is likely mediated by projections to the NAc. Thus, our results support a novel role for corticostriatal circuitry in pain regulation.
Optimally orchestrating complex behavioral states, such as the pursuit and consumption of food, is critical for an organism’s survival. The lateral hypothalamus (LH) is a neuroanatomical region ...essential for appetitive and consummatory behaviors, but whether individual neurons within the LH differentially contribute to these interconnected processes is unknown. Here, we show that selective optogenetic stimulation of a molecularly defined subset of LH GABAergic (Vgat-expressing) neurons enhances both appetitive and consummatory behaviors, whereas genetic ablation of these neurons reduced these phenotypes. Furthermore, this targeted LH subpopulation is distinct from cells containing the feeding-related neuropeptides, melanin-concentrating hormone (MCH), and orexin (Orx). Employing in vivo calcium imaging in freely behaving mice to record activity dynamics from hundreds of cells, we identified individual LH GABAergic neurons that preferentially encode aspects of either appetitive or consummatory behaviors, but rarely both. These tightly regulated, yet highly intertwined, behavioral processes are thus dissociable at the cellular level.
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•Activation of LH GABAergic neurons produces appetitive and consummatory behaviors•Ablation of LH GABAergic cells attenuates weight gain, consumption, and motivation•LH GABAergic neuronal subsets are molecularly distinct from MCH and Orx cells•In vivo Ca2+ imaging reveals separate appetitive and consummatory encoding networks
Deep brain calcium imaging in freely behaving mice reveals that appetitive and consummatory behaviors are encoded in distinct neurons in the hypothalamus, suggesting the existence of separate networks regulating motivation to eat and food consumption.
Pain thresholds are, in part, set as a function of emotional and internal states by descending modulation of nociceptive transmission in the spinal cord. Neurons of the rostral ventromedial medulla ...(RVM) are thought to critically contribute to this process; however, the neural circuits and synaptic mechanisms by which distinct populations of RVM neurons facilitate or diminish pain remain elusive. Here we used in vivo opto/chemogenetic manipulations and trans-synaptic tracing of genetically identified dorsal horn and RVM neurons to uncover an RVM-spinal cord-primary afferent circuit controlling pain thresholds. Unexpectedly, we found that RVM GABAergic neurons facilitate mechanical pain by inhibiting dorsal horn enkephalinergic/GABAergic interneurons. We further demonstrate that these interneurons gate sensory inputs and control pain through temporally coordinated enkephalin- and GABA-mediated presynaptic inhibition of somatosensory neurons. Our results uncover a descending disynaptic inhibitory circuit that facilitates mechanical pain, is engaged during stress, and could be targeted to establish higher pain thresholds.
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•Primary afferents and descending pain pathways project onto spinal Penk+ neurons•A population of GABA+ RVM neurons control spinal Penk+ neurons and mechanical pain•Together, spinal enkephalins and GABA presynaptically modulate mechanonociception•Brain regions processing stress recruit this RVM/spinal/primary afferent circuit
François et al. identified a neural circuit that controls mechanical pain thresholds. They demonstrated that GABAergic brainstem neurons regulate the release of the endogenous opioid enkephalin in the spinal cord to modulate inputs from sensory pain fibers.
Social interaction is a complex behavior essential for many species and is impaired in major neuropsychiatric disorders. Pharmacological studies have implicated certain neurotransmitter systems in ...social behavior, but circuit-level understanding of endogenous neural activity during social interaction is lacking. We therefore developed and applied a new methodology, termed fiber photometry, to optically record natural neural activity in genetically and connectivity-defined projections to elucidate the real-time role of specified pathways in mammalian behavior. Fiber photometry revealed that activity dynamics of a ventral tegmental area (VTA)-to-nucleus accumbens (NAc) projection could encode and predict key features of social, but not novel object, interaction. Consistent with this observation, optogenetic control of cells specifically contributing to this projection was sufficient to modulate social behavior, which was mediated by type 1 dopamine receptor signaling downstream in the NAc. Direct observation of deep projection-specific activity in this way captures a fundamental and previously inaccessible dimension of mammalian circuit dynamics.
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•Fiber photometry enables recording of neural projection activity in behaving mice•VTA-NAc projection activity encodes and predicts social interaction•Optogenetic control of VTA DA neurons bidirectionally modulates social behavior•Elevating VTA-NAc activity and NAc D1R signaling increases social behavior
A new method called fiber photometry allows recording of the natural neuronal activity of neuronal projections in behaving mice. Such recordings in mice partaking in social interactions reveal that the dynamics of a neural projection between the ventral tegmental area (VTA) and nucleus accumbens (NAc) encode and predict key features of social behavior.
Cholinergic transmission in the striatal complex is critical for the modulation of the activity of local microcircuits and dopamine release. Release of acetylcholine has been considered to originate ...exclusively from a subtype of striatal interneuron that provides widespread innervation of the striatum. Cholinergic neurons of the pedunculopontine (PPN) and laterodorsal tegmental (LDT) nuclei indirectly influence the activity of the dorsal striatum and nucleus accumbens through their innervation of dopamine and thalamic neurons, which in turn converge at the same striatal levels. Here we show that cholinergic neurons in the brainstem also provide a direct innervation of the striatal complex. By the expression of fluorescent proteins in choline acetyltransferase (ChAT)::Cre(+) transgenic rats, we selectively labeled cholinergic neurons in the rostral PPN, caudal PPN, and LDT. We show that cholinergic neurons topographically innervate wide areas of the striatal complex: rostral PPN preferentially innervates the dorsolateral striatum, and LDT preferentially innervates the medial striatum and nucleus accumbens core in which they principally form asymmetric synapses. Retrograde labeling combined with immunohistochemistry in wild-type rats confirmed the topography and cholinergic nature of the projection. Furthermore, transynaptic gene activation and conventional double retrograde labeling suggest that LDT neurons that innervate the nucleus accumbens also send collaterals to the thalamus and the dopaminergic midbrain, thus providing both direct and indirect projections, to the striatal complex. The differential activity of cholinergic interneurons and cholinergic neurons of the brainstem during reward-related paradigms suggest that the two systems play different but complementary roles in the processing of information in the striatum.
Light field microscopy has been proposed as a new high-speed volumetric computational imaging method that enables reconstruction of 3-D volumes from captured projections of the 4-D light field. ...Recently, a detailed physical optics model of the light field microscope has been derived, which led to the development of a deconvolution algorithm that reconstructs 3-D volumes with high spatial resolution. However, the spatial resolution of the reconstructions has been shown to be non-uniform across depth, with some z planes showing high resolution and others, particularly at the center of the imaged volume, showing very low resolution. In this paper, we enhance the performance of the light field microscope using wavefront coding techniques. By including phase masks in the optical path of the microscope we are able to address this non-uniform resolution limitation. We have also found that superior control over the performance of the light field microscope can be achieved by using two phase masks rather than one, placed at the objective's back focal plane and at the microscope's native image plane. We present an extended optical model for our wavefront coded light field microscope and develop a performance metric based on Fisher information, which we use to choose adequate phase masks parameters. We validate our approach using both simulated data and experimental resolution measurements of a USAF 1951 resolution target; and demonstrate the utility for biological applications with in vivo volumetric calcium imaging of larval zebrafish brain.
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