Targeting Neural Circuits.

Optogenetic methodology enables direct targeting of specific neural circuit elements for inhibition or excitation while spanning timescales from the acute (milliseconds) to the chronic (many days or more). Although the impact of this temporal versatility and cellular specificity has been greater for basic science than clinical research, it is natural to ask whether the dynamic patterns of neural circuit activity discovered to be causal in adaptive or maladaptive behaviors could become targets for treatment of neuropsychiatric diseases. Here, we consider the landscape of ideas related to therapeutic targeting of circuit dynamics. Specifically, we highlight optical, ultrasonic, and magnetic concepts for the targeted control of neural activity, preclinical/clinical discovery opportunities, and recently reported optogenetically guided clinical outcomes.

Basomedial amygdala mediates top-down control of anxiety and fear.

Anxiety-related conditions are among the most difficult neuropsychiatric diseases to treat pharmacologically, but respond to cognitive therapies. There has therefore been interest in identifying relevant top-down pathways from cognitive control regions in medial prefrontal cortex (mPFC). Identification of such pathways could contribute to our understanding of the cognitive regulation of affect, and provide pathways for intervention. Previous studies have suggested that dorsal and ventral mPFC subregions exert opposing effects on fear, as do subregions of other structures. However, precise causal targets for top-down connections among these diverse possibilities have not been established. Here we show that the basomedial amygdala (BMA) represents the major target of ventral mPFC in amygdala in mice. Moreover, BMA neurons differentiate safe and aversive environments, and BMA activation decreases fear-related freezing and high-anxiety states. Lastly, we show that the ventral mPFC-BMA projection implements top-down control of anxiety state and learned freezing, both at baseline and in stress-induced anxiety, defining a broadly relevant new top-down behavioural regulation pathway.

Projections from neocortex mediate top-down control of memory retrieval.

Top-down prefrontal cortex inputs to the hippocampus have been hypothesized to be important in memory consolidation, retrieval, and the pathophysiology of major psychiatric diseases; however, no such direct projections have been identified and functionally described. Here we report the discovery of a monosynaptic prefrontal cortex (predominantly anterior cingulate) to hippocampus (CA3 to CA1 region) projection in mice, and find that optogenetic manipulation of this projection (here termed AC-CA) is capable of eliciting contextual memory retrieval. To explore the network mechanisms of this process, we developed and applied tools to observe cellular-resolution neural activity in the hippocampus while stimulating AC-CA projections during memory retrieval in mice behaving in virtual-reality environments. Using this approach, we found that learning drives the emergence of a sparse class of neurons in CA2/CA3 that are highly correlated with the local network and that lead synchronous population activity events; these neurons are then preferentially recruited by the AC-CA projection during memory retrieval. These findings reveal a sparsely implemented memory retrieval mechanism in the hippocampus that operates via direct top-down prefrontal input, with implications for the patterning and storage of salient memory representations.

Dopamine neurons modulate neural encoding and expression of depression-related behaviour.

Major depression is characterized by diverse debilitating symptoms that include hopelessness and anhedonia. Dopamine neurons involved in reward and motivation are among many neural populations that have been hypothesized to be relevant, and certain antidepressant treatments, including medications and brain stimulation therapies, can influence the complex dopamine system. Until now it has not been possible to test this hypothesis directly, even in animal models, as existing therapeutic interventions are unable to specifically target dopamine neurons. Here we investigated directly the causal contributions of defined dopamine neurons to multidimensional depression-like phenotypes induced by chronic mild stress, by integrating behavioural, pharmacological, optogenetic and electrophysiological methods in freely moving rodents. We found that bidirectional control (inhibition or excitation) of specified midbrain dopamine neurons immediately and bidirectionally modulates (induces or relieves) multiple independent depression symptoms caused by chronic stress. By probing the circuit implementation of these effects, we observed that optogenetic recruitment of these dopamine neurons potently alters the neural encoding of depression-related behaviours in the downstream nucleus accumbens of freely moving rodents, suggesting that processes affecting depression symptoms may involve alterations in the neural encoding of action in limbic circuitry.

Wirelessly powered, fully internal optogenetics for brain, spinal and peripheral circuits in mice.

To enable sophisticated optogenetic manipulation of neural circuits throughout the nervous system with limited disruption of animal behavior, light-delivery systems beyond fiber optic tethering and large, head-mounted wireless receivers are desirable. We report the development of an easy-to-construct, implantable wireless optogenetic device. Our smallest version (20 mg, 10 mm(3)) is two orders of magnitude smaller than previously reported wireless optogenetic systems, allowing the entire device to be implanted subcutaneously. With a radio-frequency (RF) power source and controller, this implant produces sufficient light power for optogenetic stimulation with minimal tissue heating (<1 °C). We show how three adaptations of the implant allow for untethered optogenetic control throughout the nervous system (brain, spinal cord and peripheral nerve endings) of behaving mice. This technology opens the door for optogenetic experiments in which animals are able to behave naturally with optogenetic manipulation of both central and peripheral targets.

The separate effects of lipids and proteins on brain MRI contrast revealed through tissue clearing.

Despite the widespread use of magnetic resonance imaging (MRI) of the brain, the relative contribution of different biological components (e.g. lipids and proteins) to structural MRI contrasts (e.g., T1, T2, T2*, proton density, diffusion) remains incompletely understood. This limitation can undermine the interpretation of clinical MRI and hinder the development of new contrast mechanisms. Here, we determine the respective contribution of lipids and proteins to MRI contrast by removing lipids and preserving proteins in mouse brains using CLARITY. We monitor the temporal dynamics of tissue clearance via NMR spectroscopy, protein assays and optical emission spectroscopy. MRI of cleared brain tissue showed: 1) minimal contrast on standard MRI sequences; 2) increased relaxation times; and 3) diffusion rates close to free water. We conclude that lipids, present in myelin and membranes, are a dominant source of MRI contrast in brain tissue.

Satb2 Regulates the Differentiation of Both Callosal and Subcerebral Projection Neurons in the Developing Cerebral Cortex.

The chromatin-remodeling protein Satb2 plays a role in the generation of distinct subtypes of neocortical pyramidal neurons. Previous studies have shown that Satb2 is required for normal development of callosal projection neurons (CPNs), which fail to extend axons callosally in the absence of Satb2 and instead project subcortically. Here we conditionally delete Satb2 from the developing neocortex and find that neurons in the upper layers adopt some electrophysiological properties characteristic of deep layer neurons, but projections from the superficial layers do not contribute to the aberrant subcortical projections seen in Satb2 mutants. Instead, axons from deep layer CPNs descend subcortically in the absence of Satb2. These data demonstrate distinct developmental roles of Satb2 in regulating the fates of upper and deep layer neurons. Unexpectedly, Satb2 mutant brains also display changes in gene expression by subcerebral projection neurons (SCPNs), accompanied by a failure of corticospinal tract (CST) formation. Altering the timing of Satb2 ablation reveals that SCPNs require an early expression of Satb2 for differentiation and extension of the CST, suggesting that early transient expression of Satb2 in these cells plays an essential role in development. Collectively these data show that Satb2 is required by both CPNs and SCPNs for proper differentiation and axon pathfinding.

Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins.

Diverse optogenetic tools have allowed versatile control over neural activity. Many depolarizing and hyperpolarizing tools have now been developed in multiple laboratories and tested across different preparations, presenting opportunities but also making it difficult to draw direct comparisons. This challenge has been compounded by the dependence of performance on parameters such as vector, promoter, expression time, illumination, cell type and many other variables. As a result, it has become increasingly complicated for end users to select the optimal reagents for their experimental needs. For a rapidly growing field, critical figures of merit should be formalized both to establish a framework for further development and so that end users can readily understand how these standardized parameters translate into performance. Here we systematically compared microbial opsins under matched experimental conditions to extract essential principles and identify key parameters for the conduct, design and interpretation of experiments involving optogenetic techniques.

Clinical Spectrum of West Nile Virus Neuroinvasive Disease.

West Nile virus (WNV) is the most common arbovirus infection in the United States. The diagnosis requires consideration of not only a broad spectrum of presenting symptoms, ranging from a mild febrile illness to severe encephalitis and acute flaccid paralysis, but also public health risk factors and seasonality. There is no approved targeted therapy for WNV, so treatment relies on supportive care, management of neurologic sequelae and airway, treatment of other systems including the eye, and aggressive rehabilitation. Here, we describe a series of 3 cases of WNV encountered in September 2018 at one institution. First, we describe a case of WNV encephalitis with worsened dyskinesias and a relatively good recovery. Second, we describe a severe WNV encephalitis with overlying motor neuron involvement with a poor outcome. Finally, we describe a case of a WNV meningitis with significant bilateral chorioretinitis, an underappreciated complication of WNV infections. Through these cases, we review the epidemiology of WNV, risk factors for infection, the neurologic sequalae and long-term outcomes, and the importance of recognizing ocular involvement to prevent ophthalmologic complications.

New-Onset Delusions Heralding an Underlying Neurodegenerative Condition: A Case Report and Review of the Literature.

OBJECTIVE: To present a striking case of new-onset psychosis in a middle-aged woman subsequently diagnosed with behavioral variant frontotemporal dementia (bvFTD). To review the data regarding key red-flag features that may suggest a diagnosis of a neurodegenerative process, and specifically bvFTD, rather than a primary psychotic disorder. To examine the role of genetics, especially mutations of the microtubule-associated protein tau (MAPT) gene, in familial cases of frontotemporal dementia (FTD). DATA SOURCES: The pertinent literature was searched online (PubMed, Google Scholar) using the following search terms: frontotemporal dementia (FTD), Pick's disease, behavioral variant FTD (bvFTD), psychosis, delusions, MAPT, and genetics. No date or language limit was applied. STUDY SELECTION: The case report was generated through detailed assessment of clinical notes, imaging studies, and laboratory results. The brain autopsy was carried out and summarized by our neuropathology team. Previously published literature was selected for inclusion in the review section based on relevance to the topic. RESULTS: A neurodegenerative etiology for psychosis (and specifically bvFTD) should be suspected in patients with progressive deficits in executive function, language, or memory. Other key warning features include the presence of a strong family history of a late-life psychotic disorder (or institutional placement or suicide), loss of empathy, impaired recognition of facial expression, or the development of emotional blunting and apathy, abnormal movements, or seizures. CONCLUSIONS: Neurodegenerative disease should be on the differential diagnosis for any patient presenting with new-onset psychosis and behavioral changes in mid to late adulthood. Should red-flag features be present, early referral to a clinic specializing in dementia is recommended for further evaluation. This case highlights that MAPT mutations can be associated with psychosis in FTD and should be considered in the genetic workup. Ongoing research into the cellular and neural circuit mechanisms of psychosis in neurodegenerative disease may shed light on pathologic processes underlying psychosis in primary psychiatric disorders.

Spinal Decompression Sickness in an Experienced Scuba Diver: A Case Report and Review of Literature.

Decompression sickness from diving is a rare but potentially reversible cause of spinal injury. Early treatment with hyperbaric oxygen is associated with a better neurologic outcome, making prompt recognition and management clinically important. We describe a case of a 65-year-old diver who presented with thoracic back pain and bilateral leg weakness after a 70 feet of sea water (fsw) (21 meters of sea water [msw]) dive, with no acute abnormality on spinal magnetic resonance imaging (MRI). He made a partial recovery after extended hyperbaric oxygen therapy. We discuss the epidemiology and pathophysiology of central nervous system injury in decompression sickness, as well as acute management and prognostic factors for recovery, including the role of adjunctive therapies and the implications of negative MRI. Ultimately, clinicians should make the diagnosis of spinal cord decompression sickness based primarily on clinical evaluation, not on MRI findings.

Principles of Optogenetic Methods and Their Application to Cardiac Experimental Systems.

Optogenetic techniques permit studies of excitable tissue through genetically expressed light-gated microbial channels or pumps permitting transmembrane ion movement. Light activation of these proteins modulates cellular excitability with millisecond precision. This review summarizes optogenetic approaches, using examples from neurobiological applications, and then explores their application in cardiac electrophysiology. We review the available opsins, including depolarizing and hyperpolarizing variants, as well as modulators of G-protein coupled intracellular signaling. We discuss the biophysical properties that determine the ability of microbial opsins to evoke reliable, precise stimulation or silencing of electrophysiological activity. We also review spectrally shifted variants offering possibilities for enhanced depth of tissue penetration, combinatorial stimulation for targeting different cell subpopulations, or all-optical read-in and read-out studies. Expression of the chosen optogenetic tool in the cardiac cell of interest then requires, at the single-cell level, introduction of opsin-encoding genes by viral transduction, or coupling "spark cells" to primary cardiomyocytes or a stem-cell derived counterpart. At the system-level, this requires construction of transgenic mice expressing ChR2 in their cardiomyocytes, or in vivo injection (myocardial or systemic) of adenoviral expression systems. Light delivery, by laser or LED, with widespread or multipoint illumination, although relatively straightforward in vitro may be technically challenged by cardiac motion and light-scattering in biological tissue. Physiological read outs from cardiac optogenetic stimulation include single cell patch clamp recordings, multi-unit microarray recordings from cell monolayers or slices, and electrical recordings from isolated Langendorff perfused hearts. Optical readouts of specific cellular events, including ion transients, voltage changes or activity in biochemical signaling cascades, using small detecting molecules or genetically encoded sensors now offer powerful opportunities for all-optical control and monitoring of cellular activity. Use of optogenetics has expanded in cardiac physiology, mainly using optically controlled depolarizing ion channels to control heart rate and for optogenetic defibrillation. ChR2-expressing cardiomyocytes show normal baseline and active excitable membrane and Ca2+ signaling properties and are sensitive even to ~1 ms light pulses. They have been employed in studies of the intrinsic cardiac adrenergic system and of cardiac arrhythmic properties.

Publisher Correction: Development of an optogenetic toolkit for neural circuit dissection in squirrel monkeys.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intraobserver variability in grading severity of repeated identical cases of mitral regurgitation.

BACKGROUND: In clinical practice, mitral regurgitation (MR) is often assessed from the visual impression of the color Doppler image. It is recognized that repeated scanning can give different images and that different observers may grade the same images differently. In this study, we focus on a more intrinsic source of variability-intraobserver variability in grading of identical images, presented more than once at the same sitting. Furthermore, we look for evidence that observer grading is influenced by the severity of the immediately preceding case viewed. METHODS: Anonymous, 4-chamber color Doppler 2-dimensional video clips of MR were obtained from 60 unselected patients with MR. Six clips were identified by 2 observers as of contentious severity (either between mild and moderate or between moderate and severe). A 72-clip sequence was constructed from the 54 "uncontentious" selected cases of MR intermingled with 3 presentations of each of the 6 "contentious" images. Each contentious image was shown once without a designed order, once preceded by 3 clips of less severe MR, and once preceded by 3 clips of more severe MR. RESULTS: Only 1 (8%) of 12 observers gave consistent gradings for the triply presented images. More than 90% (11/12) of the observers reported a different grading of the same clip of MR in at least 1 of the 6 cases. The MR severity was changed in 51.4% of the triplets of identical images shown. Of 12 reporters, 5 (42%) showed classification variability between severe and nonsevere grades in at least 1 of the 6 cases. The direction of change showed no sign of consistency between observers (P = .375). CONCLUSION: Even a skilled observer cannot be relied upon to give an identical grading to an identical simple video clip of MR, when re-presented surreptitiously within a few minutes. Interobserver variability cannot therefore simply be blamed on differential levels of skill. Because, even under these ideal and dramatically simplified conditions, visual assessment is so variable, the future emphasis of echocardiographic MR grading may lie in integrating qualitative analysis with simple quantification methods.

Glioblastoma arising within sites of encephalomalacia from cerebrovascular insult: two cases and a review of the literature.

Glioblastoma is the most common primary parenchymal brain malignancy, with median survival of less than one year. While there are likely multiple predisposing genetic and environmental factors in glioblastoma formation, chronic inflammation resulting from non-traumatic vascular brain injury is one proposed risk factor for oncogenesis. Here, we report two instances of glioblastoma arising within areas of encephalomalacia caused by remote vascular insults (one following aneurysmal subarachnoid hemorrhage and one following ischemic infarction), review the literature associating glioblastoma with prior brain injury, and discuss potential mechanisms for malignant transformation in injured brain tissue.

Development of an optogenetic toolkit for neural circuit dissection in squirrel monkeys.

Optogenetic tools have opened a rich experimental landscape for understanding neural function and disease. Here, we present the first validation of eight optogenetic constructs driven by recombinant adeno-associated virus (AAV) vectors and a WGA-Cre based dual injection strategy for projection targeting in a widely-used New World primate model, the common squirrel monkey Saimiri sciureus. We observed opsin expression around the local injection site and in axonal projections to downstream regions, as well as transduction to thalamic neurons, resembling expression patterns observed in macaques. Optical stimulation drove strong, reliable excitatory responses in local neural populations for two depolarizing opsins in anesthetized monkeys. Finally, we observed continued, healthy opsin expression for at least one year. These data suggest that optogenetic tools can be readily applied in squirrel monkeys, an important first step in enabling precise, targeted manipulation of neural circuits in these highly trainable, cognitively sophisticated animals. In conjunction with similar approaches in macaques and marmosets, optogenetic manipulation of neural circuits in squirrel monkeys will provide functional, comparative insights into neural circuits which subserve dextrous motor control as well as other adaptive behaviors across the primate lineage. Additionally, development of these tools in squirrel monkeys, a well-established model system for several human neurological diseases, can aid in identifying novel treatment strategies.

Prefrontal cortical regulation of brainwide circuit dynamics and reward-related behavior.

Motivation for reward drives adaptive behaviors, whereas impairment of reward perception and experience (anhedonia) can contribute to psychiatric diseases, including depression and schizophrenia. We sought to test the hypothesis that the medial prefrontal cortex (mPFC) controls interactions among specific subcortical regions that govern hedonic responses. By using optogenetic functional magnetic resonance imaging to locally manipulate but globally visualize neural activity in rats, we found that dopamine neuron stimulation drives striatal activity, whereas locally increased mPFC excitability reduces this striatal response and inhibits the behavioral drive for dopaminergic stimulation. This chronic mPFC overactivity also stably suppresses natural reward-motivated behaviors and induces specific new brainwide functional interactions, which predict the degree of anhedonia in individuals. These findings describe a mechanism by which mPFC modulates expression of reward-seeking behavior, by regulating the dynamical interactions between specific distant subcortical regions.

Optogenetic approaches addressing extracellular modulation of neural excitability.

The extracellular ionic environment in neural tissue has the capacity to influence, and be influenced by, natural bouts of neural activity. We employed optogenetic approaches to control and investigate these interactions within and between cells, and across spatial scales. We began by developing a temporally precise means to study microdomain-scale interactions between extracellular protons and acid-sensing ion channels (ASICs). By coupling single-component proton-transporting optogenetic tools to ASICs to create two-component optogenetic constructs (TCOs), we found that acidification of the local extracellular membrane surface by a light-activated proton pump recruited a slow inward ASIC current, which required molecular proximity of the two components on the membrane. To elicit more global effects of activity modulation on 'bystander' neurons not under direct control, we used densely-expressed depolarizing (ChR2) or hyperpolarizing (eArch3.0, eNpHR3.0) tools to create a slow non-synaptic membrane current in bystander neurons, which matched the current direction seen in the directly modulated neurons. Extracellular protons played contributory role but were insufficient to explain the entire bystander effect, suggesting the recruitment of other mechanisms. Together, these findings present a new approach to the engineering of multicomponent optogenetic tools to manipulate ionic microdomains, and probe the complex neuronal-extracellular space interactions that regulate neural excitability.