Neuronal Dynamics Regulating Brain and Behavioral State Transitions.

Prolonged behavioral challenges can cause animals to switch from active to passive coping strategies to manage effort-expenditure during stress; such normally adaptive behavioral state transitions can become maladaptive in psychiatric disorders such as depression. The underlying neuronal dynamics and brainwide interactions important for passive coping have remained unclear. Here, we develop a paradigm to study these behavioral state transitions at cellular-resolution across the entire vertebrate brain. Using brainwide imaging in zebrafish, we observed that the transition to passive coping is manifested by progressive activation of neurons in the ventral (lateral) habenula. Activation of these ventral-habenula neurons suppressed downstream neurons in the serotonergic raphe nucleus and caused behavioral passivity, whereas inhibition of these neurons prevented passivity. Data-driven recurrent neural network modeling pointed to altered intra-habenula interactions as a contributory mechanism. These results demonstrate ongoing encoding of experience features in the habenula, which guides recruitment of downstream networks and imposes a passive coping behavioral strategy.

Ancestral Circuits for the Coordinated Modulation of Brain State.

Internal states of the brain profoundly influence behavior. Fluctuating states such as alertness can be governed by neuromodulation, but the underlying mechanisms and cell types involved are not fully understood. We developed a method to globally screen for cell types involved in behavior by integrating brain-wide activity imaging with high-content molecular phenotyping and volume registration at cellular resolution. We used this method (MultiMAP) to record from 22 neuromodulatory cell types in behaving zebrafish during a reaction-time task that reports alertness. We identified multiple monoaminergic, cholinergic, and peptidergic cell types linked to alertness and found that activity in these cell types was mutually correlated during heightened alertness. We next recorded from and controlled homologous neuromodulatory cells in mice; alertness-related cell-type dynamics exhibited striking evolutionary conservation and modulated behavior similarly. These experiments establish a method for unbiased discovery of cellular elements underlying behavior and reveal an evolutionarily conserved set of diverse neuromodulatory systems that collectively govern internal state.

SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function.

The goal of understanding living nervous systems has driven interest in high-speed and large field-of-view volumetric imaging at cellular resolution. Light sheet microscopy approaches have emerged for cellular-resolution functional brain imaging in small organisms such as larval zebrafish, but remain fundamentally limited in speed. Here, we have developed SPED light sheet microscopy, which combines large volumetric field-of-view via an extended depth of field with the optical sectioning of light sheet microscopy, thereby eliminating the need to physically scan detection objectives for volumetric imaging. SPED enables scanning of thousands of volumes-per-second, limited only by camera acquisition rate, through the harnessing of optical mechanisms that normally result in unwanted spherical aberrations. We demonstrate capabilities of SPED microscopy by performing fast sub-cellular resolution imaging of CLARITY mouse brains and cellular-resolution volumetric Ca(2+) imaging of entire zebrafish nervous systems. Together, SPED light sheet methods enable high-speed cellular-resolution volumetric mapping of biological system structure and function.

Neural Circuit Transitions Supporting Developmentally Specific Social Behavior.

Environmentally appropriate social behavior is critical for survival across the lifespan. To support this flexible behavior, the brain must rapidly perform numerous computations taking into account sensation, memory, motor-control, and many other systems. Further complicating this process, individuals must perform distinct social behaviors adapted to the unique demands of each developmental stage; indeed, the social behaviors of the newborn would not be appropriate in adulthood and vice versa. However, our understanding of the neural circuit transitions supporting these behavioral transitions has been limited. Recent advances in neural circuit dissection tools, as well as adaptation of these tools for use at early time points, has helped uncover several novel mechanisms supporting developmentally appropriate social behavior. This review, and associated Minisymposium, bring together social neuroscience research across numerous model organisms and ages. Together, this work highlights developmentally regulated neural mechanisms and functional transitions in the roles of the sensory cortex, prefrontal cortex, amygdala, habenula, and the thalamus to support social interaction from infancy to adulthood. These studies underscore the need for synthesis across varied model organisms and across ages to advance our understanding of flexible social behavior.

Sleeve Gastrectomy Protects Against Hypertension in Rats due to Changes in the Gut Microbiome.

INTRODUCTION: Sleeve gastrectomy (SG), results in improvement in hypertension. We have previously published that rodent SG improves hypertension independent of weight loss associated with unique shifts in the gut microbiome. We tested if the gut microbiome directly improves blood pressure by performing fecal material transfer (FMT) from post-SG rats to surgery-naïve animals. METHODS: We performed SG or Sham surgery in male, Zucker rats (n = 6-7) with obesity. Stool was collected postop from surgical donors for treatment of recipient rats. Three nonsurgical groups received daily, oral consumption of SG stool, sham stool, or vehicle alone (Nutella) for 10 wk (n = 7-8). FMT treatment was assessed for effects on body weight, food intake, oral glucose tolerance, and blood pressure. Genomic deoxyribonucleic acid of stool from donor and recipient groups were sequenced by 16S ribosomal ribonucleic acid and analyzed for diversity, abundance, and importance. RESULTS: Ten weeks of SG-FMT treatment significantly lowered systolic blood pressures in surgery-naïve, recipient rats compared to vehicle treatment alone (126.8 ± 13.3 mmHg versus 151.8 ± 12.2 mmHg, P = 0.001). SG-FMT treatment also significantly altered beta diversity metrics compared to Sham-FMT and vehicle treatment. In random forest analysis, amplicon sequence variant level significantly predicted FMT group, P = 0.01. CONCLUSIONS: We have found a direct link between gut microbial changes after SG and regulation of blood pressure. Future mechanistic studies are required to learn what specific gut microbial changes are required to induce improvements in obesity-associated hypertension and translation to clinical, metabolic surgery.

Brain-wide perception of the emotional valence of light is regulated by distinct hypothalamic neurons.

Salient sensory stimuli are perceived by the brain, which guides both the timing and outcome of behaviors in a context-dependent manner. Light is such a stimulus, which is used in treating mood disorders often associated with a dysregulated hypothalamic-pituitary-adrenal stress axis. Relationships between the emotional valence of light and the hypothalamus, and how they interact to exert brain-wide impacts remain unclear. Employing larval zebrafish with analogous hypothalamic systems to mammals, we show in free-swimming animals that hypothalamic corticotropin releasing factor (CRFHy) neurons promote dark avoidance, and such role is not shared by other hypothalamic peptidergic neurons. Single-neuron projection analyses uncover processes extended by individual CRFHy neurons to multiple targets including sensorimotor and decision-making areas. In vivo calcium imaging uncovers a complex and heterogeneous response of individual CRFHy neurons to the light or dark stimulus, with a reduced overall sum of CRF neuronal activity in the presence of light. Brain-wide calcium imaging under alternating light/dark stimuli further identifies distinct and distributed photic response neuronal types. CRFHy neuronal ablation increases an overall representation of light in the brain and broadly enhances the functional connectivity associated with an exploratory brain state. These findings delineate brain-wide photic perception, uncover a previously unknown role of CRFHy neurons in regulating the perception and emotional valence of light, and suggest that light therapy may alleviate mood disorders through reducing an overall sum of CRF neuronal activity.

A single, peri-operative antibiotic can persistently alter the post-operative gut microbiome after Roux-en-Y gastric bypass.

INTRODUCTION: Roux-en-Y gastric bypass (RYGB) significantly alters the gut microbiome and may be a mechanism for post-operative cardiovascular disease improvement. We have previously found an association between the class of peri-operative, intravenous antibiotic administered at the time of RYGB and the resolution rate of hypertension suggesting the gut microbiome as a mechanism. In this study, we performed a prospective study of RYGB to determine if a single intravenous antibiotic could alter the gastrointestinal microbial composition. METHODS: Patients undergoing RYGB were randomized to a single, peri-operative antibiotic of intravenous cefazolin (n = 8) or clindamycin (n = 8). Stool samples were collected from four-time points: 2 weeks pre-op (- 2w), 2 days pre-op (- 2d), 2 weeks post-op (+ 2w) and 3 months post-op (+ 3m). Stool samples were processed for genomic DNA followed by Illumina 16S rRNA gene sequencing and shotgun metagenomic sequencing (MGS). RESULTS: A total of 60 stool samples (- 2w, n = 16; - 2d, n = 15; + 2w, n = 16; + 3m, n = 13) from 16 patients were analyzed. 87.5% of patients were female with an average age of 48.6 ± 12.2 years and pre-operative BMI of 50.9 ± 23.3 kg/m2. RYGB induced statistically significant differences in alpha and beta diversity. There were statistically significant differences in alpha diversity at + 2w and beta diversity at + 3m due to antibiotic treatment. MGS revealed significantly distinct gut microbiota with 11 discriminatory metagenomic assembled genomes driven by antibiotic treatment at 3 months post-op, including increased Bifidobacterium spp. with clindamycin. CONCLUSION: RYGB induces significant changes in the gut microbiome at 2 weeks that are maintained 3 months after surgery. However, the single peri-operative dose of antibiotic administered at the time of RYGB induces unique and persisting changes to the gut microbiome that are antibiotic-specific. Increased Bifidobacterium spp. with clindamycin administration may improve the metabolic efficacy of RYGB when considering gut-microbiome driven mechanisms for blood pressure resolution.

Sleeve gastrectomy prevents hypertension associated with unique shifts in the gut microbiome.

INTRODUCTION: Bariatric surgery results in resolution of hypertension in over 50% of patients. While weight loss is a critical component to hypertension resolution after bariatric surgery, there may also be weight loss-independent mechanisms. OBJECTIVES: We hypothesized that sleeve gastrectomy (SG) initiates changes in the gut microbiome which reduce postoperative blood pressure. METHODS: Male, obese Zucker rats underwent SG, pair-fed sham, or ad-lib-fed sham surgery. Blood pressure measurements were performed 1 week pre-operatively, and at 2 and 6 weeks post-operatively. The stool microbiome composition was determined by 16S rDNA gene at 6 weeks post-operatively. Regression Random Forest modeling was performed to determine an association of the microbial composition with blood pressure. RESULTS: SG and pair-fed rats weighed significantly less than ad-lib-fed sham rats throughout the post-surgical period. At 6 weeks after surgery, SG rats had a significantly lower systolic blood pressure (149.2 ± 1.99 mmHg) than pair-fed (164.7 ± 7.87, p < 0.001) or ad-lib-fed sham rats (167.1 ± 2.41 mmHg, p < 0.001). There was a significant difference in multiple measures of beta diversity between SG rats and pair-fed and ad-lib-fed sham rats. 45.11% of the difference in blood pressure variability between samples was explained with the regression Random Forest model. CONCLUSION: SG in a rat model prevented hypertension progression independent of weight loss with changes in beta diversity and gut bacterial composition associated with the blood pressure outcome. These findings further support the metabolic efficacy of SG in treating hyperglycemia, cardiac dysfunction, and now hypertension, independent of obesity class.

Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: the matrix sandwich method.

RATIONALE: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation. OBJECTIVE: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis. METHODS AND RESULTS: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. CONCLUSIONS: Dynamic extracellular matrix application promoted epithelial-mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Understanding collective behavior through neurobiology.

A variety of organisms exhibit collective movement, including schooling fish and flocking birds, where coordinated behavior emerges from the interactions between group members. Despite the prevalence of collective movement in nature, little is known about the neural mechanisms producing each individual's behavior within the group. Here we discuss how a neurobiological approach can enrich our understanding of collective behavior by determining the mechanisms by which individuals interact. We provide examples of sensory systems for social communication during collective movement, highlight recent discoveries about neural systems for detecting the position and actions of social partners, and discuss opportunities for future research. Understanding the neurobiology of collective behavior can provide insight into how nervous systems function in a dynamic social world.

Immediate postoperative radiographs after shoulder arthroplasty are often poor quality and do not alter care.

BACKGROUND: It is technically difficult to obtain high-quality, postoperative shoulder radiographs immediately after surgery. Further, poor-quality radiographs may be unlikely to change clinical practice or improve patient outcomes. We therefore questioned the value of routine postoperative radiographs after shoulder arthroplasty. QUESTIONS/PURPOSES: We asked whether (1) postanesthesia care unit (PACU) radiographs can reasonably serve as a baseline for future studies; and (2) routine PACU radiographs change clinical care. (3) We also determined the charges associated with routine PACU radiographs and formal radiographic interpretation of these images. METHODS: We retrospectively compared the radiographs of 283 patients who had shoulder arthroplasties (Group 1) who underwent PACU radiographs with those of 241 patients (Group 2) who had their first postoperative radiographs at a later date. Radiographs were compared for quality, ability to serve as a baseline, and their influence on clinical course. Orthopaedic evaluation of each radiograph and the radiographic report were compared and charges were analyzed. RESULTS: All images in Group 1 were single-view radiographs (88% internal rotation), most were underpenetrated (71%); no images changed postoperative management or were considered adequate to serve as a baseline. Group 2 radiographs were multiview radiographs, and 83% were deemed adequate to serve as baseline radiographs. Radiographic interpretation of immediate postoperative radiographs did not change the clinical course or treatment. The charges billed from radiographic evaluation in this study were $64,524 for Group 1. CONCLUSIONS: Routine PACU radiographs, in the absence of a specific indication, may result in poor-quality images. Elimination of these radiographs and radiographic interpretation after shoulder arthroplasty may reduce charges without changing clinical care. LEVEL OF EVIDENCE: Level III, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.

Histaminergic responses by hypothalamic neurons that regulate lordosis and their modulation by estradiol.

How do fluctuations in the level of generalized arousal of the brain affect the performance of specific motivated behaviors, such as sexual behaviors that depend on sexual arousal? A great deal of previous work has provided us with two important starting points in answering this question: (i) that histamine (HA) serves generalized CNS arousal and (ii) that heightened electrical activity of neurons in the ventromedial nucleus of the hypothalamus (VMN) is necessary and sufficient for facilitating the primary female sex behavior in laboratory animals, lordosis behavior. Here we used patch clamp recording technology to analyze HA effects on VMN neuronal activity. The results show that HA acting through H1 receptors (H1R) depolarizes these neurons. Further, acute administration of estradiol, an estrogen necessary for lordosis behavior to occur, heightens this effect. Hyperpolarization, which tends to decrease excitability and enhance inhibition, was not affected by acute estradiol or mediated by H1R but was mediated by other HA receptor subtypes, H2 and H3. Sampling of mRNA from individual VMN neurons showed colocalization of expression of H1 receptor mRNA with estrogen receptor (ER)-alpha mRNA but also revealed ER colocalization with the other HA receptor subtypes and colocalization of different subtypes with each other. The latter finding provides the molecular basis for complex "push-pull" regulation of VMN neuronal excitability by HA. Thus, in the simplest causal route, HA, acting on VMN neurons through H1R provides a mechanism by which elevated states of generalized CNS arousal can foster a specific estrogen-dependent, aroused behavior, sexual behavior.

Sleeve Gastrectomy Provides Cardioprotection from Oxidative Stress In Vitro Due to Reduction of Circulating Myeloperoxidase.

Bariatric surgery, including sleeve gastrectomy (SG), improves systolic and diastolic function, which is independent of weight loss in rodent models. The cause of weight loss-independent improvements in cardiac function are unknown but may originate from the gastrointestinal tract. In this study, we investigated whether a circulating blood factor is a mechanism for acute cardioprotection after SG by testing the utility of rodent SG plasma to reduce metabolic stress in vitro. For the initial experiment, obese male Zucker rats underwent SG, ad lib sham, or pair-fed sham surgeries (n = six SG, n = eight SH, n = eight PF). For all other studies, a second group of Zucker rats underwent SG or ad lib sham surgeries (n = eight SH, n = six SG). Six weeks following surgery, plasma was collected from each group, both in the fasting and post-prandial (pp) state. This plasma was then pooled per surgical group and nutrient state and tested in multiple in vitro cell culture and extra-cellular assays to determine the effect of SG on myotubular metabolic stress compared to the sham surgeries. Post-prandial SG plasma (ppSG), but not fasting SG, pp, or fasting sham plasma, reduced the metabolic stress of the H9c2 cells as measured by lactate dehydrogenase (LDH) release (p < 0.01). Unlike SG, weight reduction through pair-feeding did not prevent H9c2 metabolic stress. The PpSG plasma had the slowest rate of extracellular hydrogen peroxide consumption and peroxidatic activity compared to the pp sham, fasting SG, and fasting sham groups. Redox testing of plasma with aminiobenzoic acid hydrazide and edaravone suggested a pattern supporting myeloperoxidase (MPO), or other peroxidases, as the primary component responsible for reduced metabolic stress with ppSG plasma. The PpSG plasma contained 35% less circulating MPO protein as compared to the pp sham and fasting SG plasma. The plasma from an MPO global knockout rat also prevented metabolic stress of the H9c2 cells, compared to the significant increase in LDH release from the plasma of the WT controls (p < 0.01). The MPO global knockout plasma also had a rate of extracellular hydrogen peroxide consumption and peroxidatic activity comparable to the ppSG plasma. These studies suggest that one of the weight loss-independent mechanisms by which SG improves myocellular function could be a reduced pro-oxidative environment due to lower circulating levels of MPO. It appears that the gastrointestinal tract is of critical importance to these findings, as the MPO levels were only lowered after enteral, nutrient stimulation in the SG rats. If this surgical effect is confirmed in humans, SG may be a unique surgical treatment for multiple diseases with a pathogenesis of inflammation and oxidative damage, including obesity-associated heart failure with preserved ejection fraction.

A medley of gene expression adds new depth to zebrafish brain maps.

The integration of large-scale gene expression mapping into a multifaceted larval zebrafish brain atlas accelerates the characterization of neurons in behaviorally relevant circuits.

Development of neural circuits for social motion perception in schooling fish.

Many animals move in groups, where collective behavior emerges from the interactions amongst individuals. These social interactions produce the coordinated movements of bird flocks and fish schools, but little is known about their developmental emergence and neurobiological foundations. By characterizing the visually-based schooling behavior of the micro glassfish Danionella cerebrum, here we found that social development progresses sequentially, with animals first acquiring the ability to aggregate, followed by postural alignment with social partners. This social maturation was accompanied by the development of neural populations in the midbrain and forebrain that were preferentially driven by visual stimuli that resemble the shape and movements of schooling fish. The development of these neural circuits enables the social coordination required for collective movement.

Sleeve Gastrectomy Improves High-Fat Diet-Associated Hepatic Steatosis Independent of the Glucagon-like-Petpide-1 Receptor in Rats.

BACKGROUND: The gastrointestinal hormone glucagon-like peptide-1 (GLP-1) is increased after sleeve gastrectomy (SG). Rat and clinical studies support, while mouse studies refute, a role for GLP-1R signaling after SG. Therefore, we developed a global GLP-1R knockout (KO) rat to test the hypothesis that a functional GLP-1R is critical to induce weight loss and metabolic disease improvement after SG. METHODOLOGY: A 4 bp deletion was created in exon 2 of the GLP-1R gene on a Lewis strain background to create a global GLP-1R KO rat. KO and Lewis rats were placed on a high-fat or low-fat diet and phenotyped followed by SG or Sham surgery and assessed for the effect of GLP-1R KO on surgical and metabolic efficacy. RESULTS: Loss of the GLP-1R created an obesity-prone rodent without changes in energy expenditure. Both male and female KO rats had significantly greater insulin concentrations after an oral glucose gavage, augmented by a high-fat diet, compared to Lewis rats despite similar glucose concentrations. GLP-1R KO caused hepatomegaly and increased triglyceride deposition compared to Lewis rats. We found no difference between SG GLP-1R KO and Lewis groups when considering efficacy on body weight, glucose tolerance, and a robustly preserved improvement in fatty liver disease. CONCLUSIONS: Loss of the GLP-1R in rats resulted in increased adiposity, insulin resistance, and severe steatosis. A functional GLP-1R is not critical to the metabolic efficacy of SG in Lewis rats, similar to mouse studies, but importantly including steatosis, supporting a GLP-1R-independent mechanism for the improvement in fatty liver disease after SG.

Serial evaluation of segmental esophageal reconstruction using a polyurethane scaffold in a pig model.

Background: Many esophageal pathologies are clinically treated by resection and reconstruction of the esophagus. Surgical esophagectomy remains a morbid procedure and despite minimally invasive advances, has changed little in decades. Novel approaches to esophageal segmental resection and reconstruction are an unmet need. Methods: Circumferential thoracic esophageal transection was performed in both male and female pigs and the defects reconstructed using 5 or 10 cm polyurethane (PU) tubular grafts and stented. A subset were treated with stent only. Animals were survived to 14, 30, 60, and 399 days. Tissues were evaluated histologically, and via non-invasive serial endoscopy and contrast swallowing studies in long-term animals. Results: Luminal patency was achieved in all animals with no clinical evidence of leak. In short-term animals, there was healing noted in all cases with a variably sized region of ulceration remaining at the most central part of the repaired tube (between the proximal and distal anastomosis). In four long-term animals following stent removal, two resumed normal diet and thrived, while two animals were euthanized prior to the proposed endpoint because of stricture formation and inability to tolerate a normal diet. Re-epithelialization was observed in all groups, and more complete over time. Conclusions: The PU scaffold provides a matrix across which formation of new tissue can occur. The mechanisms through which this happens remain unclear, but likely a combination of fibrosis and tissue contraction, in conjunction with new tissue formation.

The emergence and influence of internal states.

Animal behavior is shaped by a variety of "internal states"-partially hidden variables that profoundly shape perception, cognition, and action. The neural basis of internal states, such as fear, arousal, hunger, motivation, aggression, and many others, is a prominent focus of research efforts across animal phyla. Internal states can be inferred from changes in behavior, physiology, and neural dynamics and are characterized by properties such as pleiotropy, persistence, scalability, generalizability, and valence. To date, it remains unclear how internal states and their properties are generated by nervous systems. Here, we review recent progress, which has been driven by advances in behavioral quantification, cellular manipulations, and neural population recordings. We synthesize research implicating defined subsets of state-inducing cell types, widespread changes in neural activity, and neuromodulation in the formation and updating of internal states. In addition to highlighting the significance of these findings, our review advocates for new approaches to clarify the underpinnings of internal brain states across the animal kingdom.