How the discovery of microbial opsins led to the development of optogenetics.

This year's Lasker Award recognizes Dieter Oesterhelt, Peter Hegemann, and Karl Deisseroth for their discovery of microbial opsins as light-activated ion conductors and the development of optogenetics using these proteins to regulate neural activity in awake, behaving animals. Optogenetics has revolutionized neuroscience and transformed our understanding of brain function.

Regulation of Energy Expenditure by Brainstem GABA Neurons.

Homeostatic control of core body temperature is essential for survival. Temperature is sensed by specific neurons, in turn eliciting both behavioral (i.e., locomotion) and physiologic (i.e., thermogenesis, vasodilatation) responses. Here, we report that a population of GABAergic (Vgat-expressing) neurons in the dorsolateral portion of the dorsal raphe nucleus (DRN), hereafter DRNVgat neurons, are activated by ambient heat and bidirectionally regulate energy expenditure through changes in both thermogenesis and locomotion. We find that DRNVgat neurons innervate brown fat via a descending projection to the raphe pallidus (RPa). These neurons also densely innervate ascending targets implicated in the central regulation of energy expenditure, including the hypothalamus and extended amygdala. Optogenetic stimulation of different projection targets reveals that DRNVgat neurons are capable of regulating thermogenesis through both a "direct" descending pathway through the RPa and multiple "indirect" ascending pathways. This work establishes a key regulatory role for DRNVgat neurons in controlling energy expenditure.

Identification of a Brainstem Circuit Controlling Feeding.

Hunger, driven by negative energy balance, elicits the search for and consumption of food. While this response is in part mediated by neurons in the hypothalamus, the role of specific cell types in other brain regions is less well defined. Here, we show that neurons in the dorsal raphe nucleus, expressing vesicular transporters for GABA or glutamate (hereafter, DRNVgat and DRNVGLUT3 neurons), are reciprocally activated by changes in energy balance and that modulating their activity has opposite effects on feeding-DRNVgat neurons increase, whereas DRNVGLUT3 neurons suppress, food intake. Furthermore, modulation of these neurons in obese (ob/ob) mice suppresses food intake and body weight and normalizes locomotor activity. Finally, using molecular profiling, we identify druggable targets in these neurons and show that local infusion of agonists for specific receptors on these neurons has potent effects on feeding. These data establish the DRN as an important node controlling energy balance. PAPERCLIP.

Sympathetic neuro-adipose connections mediate leptin-driven lipolysis.

Leptin is a hormone produced by the adipose tissue that acts in the brain, stimulating white fat breakdown. We find that the lipolytic effect of leptin is mediated through the action of sympathetic nerve fibers that innervate the adipose tissue. Using intravital two-photon microscopy, we observe that sympathetic nerve fibers establish neuro-adipose junctions, directly "enveloping" adipocytes. Local optogenetic stimulation of sympathetic inputs induces a local lipolytic response and depletion of white adipose mass. Conversely, genetic ablation of sympathetic inputs onto fat pads blocks leptin-stimulated phosphorylation of hormone-sensitive lipase and consequent lipolysis, as do knockouts of dopamine ß-hydroxylase, an enzyme required for catecholamine synthesis. Thus, neuro-adipose junctions are necessary and sufficient for the induction of lipolysis in white adipose tissue and are an efferent effector of leptin action. Direct activation of sympathetic inputs to adipose tissues may represent an alternative approach to induce fat loss, circumventing central leptin resistance. PAPERCLIP.

Molecular profiling of neurons based on connectivity.

The complexity and cellular heterogeneity of neural circuitry presents a major challenge to understanding the role of discrete neural populations in controlling behavior. While neuroanatomical methods enable high-resolution mapping of neural circuitry, these approaches do not allow systematic molecular profiling of neurons based on their connectivity. Here, we report the development of an approach for molecularly profiling projective neurons. We show that ribosomes can be tagged with a camelid nanobody raised against GFP and that this system can be engineered to selectively capture translating mRNAs from neurons retrogradely labeled with GFP. Using this system, we profiled neurons projecting to the nucleus accumbens. We then used an AAV to selectively profile midbrain dopamine neurons projecting to the nucleus accumbens. By comparing the captured mRNAs from each experiment, we identified a number of markers specific to VTA dopaminergic projection neurons. The current method provides a means for profiling neurons based on their projections.

Critical roles of transcriptional coactivator MED1 in the formation and function of mouse adipose tissues.

The MED1 subunit has been shown to mediate ligand-dependent binding of the Mediator coactivator complex to multiple nuclear receptors, including the adipogenic PPARγ, and to play an essential role in ectopic PPARγ-induced adipogenesis of mouse embryonic fibroblasts. However, the precise roles of MED1, and its various domains, at various stages of adipogenesis and in adipose tissue have been unclear. Here, after establishing requirements for MED1, including specific domains, for differentiation of 3T3L1 cells and both primary white and brown preadipocytes, we used multiple genetic approaches to assess requirements for MED1 in adipocyte formation, maintenance, and function in mice. We show that MED1 is indeed essential for the differentiation and/or function of both brown and white adipocytes, as its absence in these cells leads to, respectively, defective brown fat function and lipodystrophy. This work establishes MED1 as an essential transcriptional coactivator that ensures homeostatic functions of adipocytes.

Brainstem ADCYAP1+ neurons control multiple aspects of sickness behaviour.

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2-4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.

Molecular profiling of activated neurons by phosphorylated ribosome capture.

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue.

A leptin-BDNF pathway regulating sympathetic innervation of adipose tissue.

Mutations in the leptin gene (ob) result in a metabolic disorder that includes severe obesity1, and defects in thermogenesis2 and lipolysis3, both of which are adipose tissue functions regulated by the sympathetic nervous system. However, the basis of these sympathetic-associated abnormalities remains unclear. Furthermore, chronic leptin administration reverses these abnormalities in adipose tissue, but the underlying mechanism remains to be discovered. Here we report that ob/ob mice, as well as leptin-resistant diet-induced obese mice, show significant reductions of sympathetic innervation of subcutaneous white and brown adipose tissue. Chronic leptin treatment of ob/ob mice restores adipose tissue sympathetic innervation, which in turn is necessary to correct the associated functional defects. The effects of leptin on innervation are mediated via agouti-related peptide and pro-opiomelanocortin neurons in the hypothalamic arcuate nucleus. Deletion of the gene encoding the leptin receptor in either population leads to reduced innervation in fat. These agouti-related peptide and pro-opiomelanocortin neurons act via brain-derived neurotropic factor-expressing neurons in the paraventricular nucleus of the hypothalamus (BDNFPVH). Deletion of BDNFPVH blunts the effects of leptin on innervation. These data show that leptin signalling regulates the plasticity of sympathetic architecture of adipose tissue via a top-down neural pathway that is crucial for energy homeostasis.

Dynamic processing of hunger and thirst by common mesolimbic neural ensembles.

The nucleus accumbens (NAc) is a canonical reward center that regulates feeding and drinking but it is not known whether these behaviors are mediated by same or different neurons. We employed two-photon calcium imaging in awake, behaving mice and found that during the appetitive phase, both hunger and thirst are sensed by a nearly identical population of individual D1 and D2 neurons in the NAc that respond monophasically to food cues in fasted animals and water cues in dehydrated animals. During the consummatory phase, we identified three distinct neuronal clusters that are temporally correlated with action initiation, consumption, and cessation shared by feeding and drinking. These dynamic clusters also show a nearly complete overlap of individual D1 neurons and extensive overlap among D2 neurons. Modulating D1 and D2 neural activities revealed analogous effects on feeding versus drinking behaviors. In aggregate, these data show that a highly overlapping set of D1 and D2 neurons in NAc detect food and water reward and elicit concordant responses to hunger and thirst. These studies establish a general role of this mesolimbic pathway in mediating instinctive behaviors by controlling motivation-associated variables rather than conferring behavioral specificity.

Restriction of food intake by PPP1R17-expressing neurons in the DMH.

Leptin-deficient ob/ob mice eat voraciously, and their food intake is markedly reduced by leptin treatment. In order to identify potentially novel sites of leptin action, we used PhosphoTRAP to molecularly profile leptin-responsive neurons in the hypothalamus and brainstem. In addition to identifying several known leptin responsive populations, we found that neurons in the dorsomedial hypothalamus (DMH) of ob/ob mice expressing protein phosphatase 1 regulatory subunit 17 (PPP1R17) constitutively express cFos and that this is suppressed by leptin treatment. Because ob mice are hyperphagic, we hypothesized that activating PPP1R17 neurons would increase food intake. However, chemogenetic activation of PPP1R17 neurons decreased food intake and body weight of ob/ob mice while inhibition of PPP1R17 neurons increased them. Similarly, in a scheduled feeding protocol that elicits increased consumption, mice also ate more when PPP1R17 neurons were inhibited and ate less when they were activated. Finally, we found that pair-feeding of ob mice reduced cFos expression to a similar extent as leptin and that reducing the amount of food available during scheduled feeding in DMHPpp1r17 neurons also decreased cFos in DMHPpp1r17 neurons. Finally, these neurons do not express the leptin receptor, suggesting that the effect of leptin on these neurons is indirect and secondary to reduced food intake. In aggregate, these results show that PPP1R17 neurons in the DMH are activated by increased food intake and in turn restrict intake to limit overconsumption, suggesting that they function to constrain binges of eating.

The genetic structure of the Turkish population reveals high levels of variation and admixture.

The construction of population-based variomes has contributed substantially to our understanding of the genetic basis of human inherited disease. Here, we investigated the genetic structure of Turkey from 3,362 unrelated subjects whose whole exomes (n = 2,589) or whole genomes (n = 773) were sequenced to generate a Turkish (TR) Variome that should serve to facilitate disease gene discovery in Turkey. Consistent with the history of present-day Turkey as a crossroads between Europe and Asia, we found extensive admixture between Balkan, Caucasus, Middle Eastern, and European populations with a closer genetic relationship of the TR population to Europeans than hitherto appreciated. We determined that 50% of TR individuals had high inbreeding coefficients (≥0.0156) with runs of homozygosity longer than 4 Mb being found exclusively in the TR population when compared to 1000 Genomes Project populations. We also found that 28% of exome and 49% of genome variants in the very rare range (allele frequency < 0.005) are unique to the modern TR population. We annotated these variants based on their functional consequences to establish a TR Variome containing alleles of potential medical relevance, a repository of homozygous loss-of-function variants and a TR reference panel for genotype imputation using high-quality haplotypes, to facilitate genome-wide association studies. In addition to providing information on the genetic structure of the modern TR population, these data provide an invaluable resource for future studies to identify variants that are associated with specific phenotypes as well as establishing the phenotypic consequences of mutations in specific genes.

Change in medial frontal cerebral metabolite concentrations following bariatric surgery.

Obesity is associated with adverse effects on brain health, including an increased risk of neurodegenerative diseases. Changes in cerebral metabolism may underlie or precede structural and functional brain changes. While bariatric surgery is known to be effective in inducing weight loss and improving obesity-related medical comorbidities, few studies have examined whether it may be able to improve brain metabolism. In the present study, we examined changes in cerebral metabolite concentrations in participants with obesity who underwent bariatric surgery. Thirty-five patients with obesity (body mass index ≥ 35 kg/m2 ) were recruited from a bariatric surgery candidate nutrition class. They completed single voxel proton magnetic resonance spectroscopy at baseline (presurgery) and within 1 year postsurgery. Spectra were obtained from a large medial frontal brain region using a PRESS sequence on a 3-T Siemens Verio scanner. The acquisition parameters were TR = 3000 ms and TE = 37 ms. Tissue-corrected metabolite concentrations were determined using Osprey. Paired t-tests were used to examine within-subject change in metabolite concentrations, and correlations were used to relate these changes to other health-related outcomes, including weight loss and glycated hemoglobin (HbA1c ), a measure of blood sugar levels. Bariatric surgery was associated with a reduction in cerebral choline-containing compounds (Cho; t [34] = - 3.79, p < 0.001, d = -0.64) and myo-inositol (mI; t [34] = - 2.81, p < 0.01, d = -0.47) concentrations. There were no significant changes in N-acetyl-aspartate, creatine, or glutamate and glutamine concentrations. Reductions in Cho were associated with greater weight loss (r = 0.40, p < 0.05), and reductions in mI were associated with greater reductions in HbA1c (r = 0.44, p < 0.05). In conclusion, participants who underwent bariatric surgery exhibited reductions in cerebral Cho and mI concentrations, which were associated with improvements in weight loss and glycemic control. Given that elevated levels of Cho and mI have been implicated in neuroinflammation, reduction in these metabolites after bariatric surgery may reflect amelioration of obesity-related neuroinflammatory processes. As such, our results provide evidence that bariatric surgery may improve brain health and metabolism in individuals with obesity.

Identification of white adipocyte progenitor cells in vivo.

The increased white adipose tissue (WAT) mass associated with obesity is the result of both hyperplasia and hypertrophy of adipocytes. However, the mechanisms controlling adipocyte number are unknown in part because the identity of the physiological adipocyte progenitor cells has not been defined in vivo. In this report, we employ a variety of approaches, including a noninvasive assay for following fat mass reconstitution in vivo, to identify a subpopulation of early adipocyte progenitor cells (Lin(-):CD29(+):CD34(+):Sca-1(+):CD24(+)) resident in adult WAT. When injected into the residual fat pads of A-Zip lipodystrophic mice, these cells reconstitute a normal WAT depot and rescue the diabetic phenotype that develops in these animals. This report provides the identification of an undifferentiated adipocyte precursor subpopulation resident within the adipose tissue stroma that is capable of proliferating and differentiating into an adipose depot in vivo.

Functional analysis of distinct populations of subthalamic nucleus neurons on Parkinson's disease and OCD-like behaviors in mice.

The subthalamic nucleus (STN) is a component of the basal ganglia and plays a key role to control movement and limbic-associative functions. STN modulation with deep brain stimulation (DBS) improves the symptoms of Parkinson's disease (PD) and obsessive-compulsive disorder (OCD) patients. However, DBS does not allow for cell-type-specific modulation of the STN. While extensive work has focused on elucidating STN functionality, the understanding of the role of specific cell types is limited. Here, we first performed an anatomical characterization of molecular markers for specific STN neurons. These studies revealed that most STN neurons express Pitx2, and that different overlapping subsets express Gabrr3, Ndnf, or Nos1. Next, we used optogenetics to define their roles in regulating locomotor and limbic functions in mice. Specifically, we showed that optogenetic photoactivation of STN neurons in Pitx2-Cre mice or of the Gabrr3-expressing subpopulation induces locomotor changes, and improves locomotion in a PD mouse model. In addition, photoactivation of Pitx2 and Gabrr3 cells induced repetitive grooming, a phenotype associated with OCD. Repeated stimulation prompted a persistent increase in grooming that could be reversed by fluoxetine treatment, a first-line drug therapy for OCD. Conversely, repeated inhibition of STNGabrr3 neurons suppressed grooming in Sapap3 KO mice, a model for OCD. Finally, circuit and functional mapping of STNGabrr3 neurons showed that these effects are mediated via projections to the globus pallidus/entopeduncular nucleus and substantia nigra reticulata. Altogether, these data identify Gabrr3 neurons as a key population in mediating the beneficial effects of STN modulation thus providing potential cellular targets for PD and OCD drug discovery.

Changes in overactive bladder medication following bariatric surgery: segmented regression analysis.

PURPOSE: Bariatric surgery has shown reductions in overactive bladder (OAB) symptoms; however, the impacts on OAB treatment is unknown. The goal of our study is to evaluate the impact of bariatric surgery on OAB medication utilization. METHODS: We used IBM® MarketScan® commercial databases from 2005 to 2018. We included patients aged ≥ 18 years with 360 days of continuous enrollment before and after bariatric surgery (Roux-en-Y Gastric Bypass and Sleeve Gastrectomy) with at least one fill of an OAB medication in the 360 days prior to bariatric surgery. We evaluated all included patients and stratified by surgery type and patient sex. Segmented regression analyses were used to assess the proportion of patients on OAB medications before and after bariatric surgery. We replicated our findings using hip or knee replacement surgery as a negative control. RESULTS: Among the included patients (n = 3069), 92.2% were females, 58.6% underwent Roux-en-Y Gastric Bypass. Immediately following bariatric surgery, the proportion of patients treated with an OAB medication reduced from 34.8 to 14.1% (p < 0.001) resulting in a 59.5% relative reduction. Patients who underwent Roux-en-Y Gastric Bypass vs. Sleeve Gastrectomy (63.8% vs. 55.1%) relative reduction (p = 0.009)) and females versus males [62.3% vs. 52.9% relative reduction (p < 0.001)] had a more pronounced reduction in OAB medication use. There was slight decrease in OAB medication use in the negative control analysis. CONCLUSIONS: A reduction in OAB medication use following bariatric surgery may be associated with a reduction in OAB symptoms suggesting an additional benefit of bariatric surgery.

Impact of Laparoscopic Sleeve Gastrectomy on Cardiovascular Pharmacotherapy in Left Ventricular Assist Device Patients.

ABSTRACT: Left ventricular assist device (LVAD) implantation is increasingly utilized in patients with advanced heart failure and morbid obesity. Laparoscopic sleeve gastrectomy (LSG) can facilitate weight loss in this population and can ultimately change the pharmacokinetics of heart failure therapeutics. In this study, we aimed to explore the changes in cardiovascular pharmacotherapy post LSG intervention. We conducted a retrospective observational cohort study of morbidly obese LVAD patients between 2013 and 2019 at the University of Florida with available pharmacotherapeutic data at 1 and 6 months. Thirteen post-LSG patients and 13 control subjects were included in the final analysis. In the post-LSG group, the mean body mass index decreased significantly (44 ± 5 vs. 34 ± 4.9, P < 0.001), and 7 patients were successfully bridged to cardiac transplantation. Only 3 patients required adjustment of their LVAD speed. Mean return to flow decreased by 8 mm Hg, despite a 45% reduction in the mean number of vasodilators per patient (1.2 vs. 0.7, P = 0.03). Mean weekly warfarin dose decreased by 35% after 6 months (32.9 ± 20.9 vs. 50.7 ± 26.6, P = 0.01). The use of diuretics, vasodilators, and beta-blockers was significantly reduced by 50%, 45%, and 35%, respectively. None of these changes were observed in the control group at 6-month follow-up post LVAD. In this single-center experience, weight loss post LSG is associated with decreased vasodilator, diuretic, and anticoagulant medication requirements in LVAD patients.

Bidirectional electromagnetic control of the hypothalamus regulates feeding and metabolism.

Targeted, temporally regulated neural modulation is invaluable in determining the physiological roles of specific neural populations or circuits. Here we describe a system for non-invasive, temporal activation or inhibition of neuronal activity in vivo and its use to study central nervous system control of glucose homeostasis and feeding in mice. We are able to induce neuronal activation remotely using radio waves or magnetic fields via Cre-dependent expression of a GFP-tagged ferritin fusion protein tethered to the cation-conducting transient receptor potential vanilloid 1 (TRPV1) by a camelid anti-GFP antibody (anti-GFP-TRPV1). Neuronal inhibition via the same stimuli is achieved by mutating the TRPV1 pore, rendering the channel chloride-permeable. These constructs were targeted to glucose-sensing neurons in the ventromedial hypothalamus in glucokinase-Cre mice, which express Cre in glucose-sensing neurons. Acute activation of glucose-sensing neurons in this region increases plasma glucose and glucagon, lowers insulin levels and stimulates feeding, while inhibition reduces blood glucose, raises insulin levels and suppresses feeding. These results suggest that pancreatic hormones function as an effector mechanism of central nervous system circuits controlling blood glucose and behaviour. The method we employ obviates the need for permanent implants and could potentially be applied to study other neural processes or used to regulate other, even dispersed, cell types.

Comparative Safety of Sleeve Gastrectomy and Roux-en-Y: A Propensity Score Analysis.

BACKGROUND: Use of bariatric surgery has increased dramatically in the USA. However, there are growing concerns regarding the safety outcomes of different bariatric procedures. We aim to compare the safety of sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB), which includes hospital readmissions, emergency room (ER) visits, gastrointestinal bleeding, and revisional surgery. METHODS: A retrospective cohort analysis was conducted for adults (≥ 18 years) who received SG and RYGB in the USA. We used Truven MarketScan Commercial and Medicare supplemental claims databases from January 1, 2005, to October 1, 2015. To adjust for baseline demographic and clinical characteristics, we used stabilized inverse probability of treatment weighting using propensity score. Cox proportional hazard models was used to compare safety outcomes between SG and RYGB after bariatric surgery. RESULTS: A total of 194,248 patients met inclusion criteria; 79,813 patients (41%) received SG and 114,435 patients (59%) received RYGB. The use of SG was associated with a significantly lower 30-day hospital readmission rate [adjusted hazard ratios (aHRs) 0.77; 95% confidence interval (CI), 0.74-0.81] and ER visits [aHR, 0.82; 95% CI, 0.80-0.83], and decreased risk of gastrointestinal bleeding [aHR, 0.87; 95% CI, 0.78-0.98] compared to RYGB. However, SG was associated with an increased risk of revisional surgery, compared to RYGB [aHR,1.21; 95% CI, 1.08-1.35]. CONCLUSIONS: Among patients receiving bariatric surgery in a real-world setting, SG was associated with lower complication rate but a higher risk of revisional surgery compared to RYGB. Further longitudinal studies are needed to assess long-term findings.