Structural mechanisms of selectivity and gating in anion channelrhodopsins.

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anion channels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

Gamma oscillations organize top-down signalling to hypothalamus and enable food seeking.

Both humans and animals seek primary rewards in the environment, even when such rewards do not correspond to current physiological needs. An example of this is a dissociation between food-seeking behaviour and metabolic needs, a notoriously difficult-to-treat symptom of eating disorders. Feeding relies on distinct cell groups in the hypothalamus, the activity of which also changes in anticipation of feeding onset. The hypothalamus receives strong descending inputs from the lateral septum, which is connected, in turn, with cortical networks, but cognitive regulation of feeding-related behaviours is not yet understood. Cortical cognitive processing involves gamma oscillations, which support memory, attention, cognitive flexibility and sensory responses. These functions contribute crucially to feeding behaviour by unknown neural mechanisms. Here we show that coordinated gamma (30-90 Hz) oscillations in the lateral hypothalamus and upstream brain regions organize food-seeking behaviour in mice. Gamma-rhythmic input to the lateral hypothalamus from somatostatin-positive lateral septum cells evokes food approach without affecting food intake. Inhibitory inputs from the lateral septum enable separate signalling by lateral hypothalamus neurons according to their feeding-related activity, making them fire at distinct phases of the gamma oscillation. Upstream, medial prefrontal cortical projections provide gamma-rhythmic inputs to the lateral septum; these inputs are causally associated with improved performance in a food-rewarded learning task. Overall, our work identifies a top-down pathway that uses gamma synchronization to guide the activity of subcortical networks and to regulate feeding behaviour by dynamic reorganization of functional cell groups in the hypothalamus.

Variations in consumer acceptance, sensory engagement and method practicality across three remote consumer-testing modalities.

In the context of the COVID-19 pandemic, it has become challenging for sensory scientists to conduct in-person sensory tests, particularly large central location tests. Sensory literature comparing central location and home use tests shows no clear consensus about how each methodology affects sample ratings and panelist engagement. Research on instructional delivery suggests that the most effective method of increasing engagement involves interactive video conferencing. The objective of this study was to compare three methods of remote consumer testing regarding sample acceptance, sensory engagement, and method practicality. Eighty-four participants rated five chocolate-chip cookie products on a 9-pt hedonic scale in each of three methods: 1) a live (synchronous) Zoom session, 2) an asynchronous video-guided session, and 3) a fully written protocol session. Results showed no significant differences in sample liking pattern across the methods used. Engagement scores approached the limit of significance for the Active Involvement dimension, indicating panelists were least likely to feel distracted, zoned out or lose interest in the written protocol method. There were no significant differences in the time spent on the test by the panelists across the three methods. Asynchronous methods showed to be most suitable in terms of the convenience of the time of day at which the tests were completed, but showed no significant differences in other aspects of method practicality. Overall, a written protocol method of remote consumer testing is recommended, as it is less time-consuming for researchers while providing similar acceptance and engagement as other methods.

Compendium of sodium reduction strategies in foods: A scoping review.

In response to health concerns generated by increased sodium intake, many new approaches have been studied to reduce the sodium content in processed food. It has been suggested that reducing sodium in the food supply may be the most appropriate solution. The aim of this scoping review was to establish what sodium reduction strategies are effective in maintaining acceptable sensory qualities for various food industry applications. Studies that evaluate and report on the effectiveness of a sodium reduction strategy relevant to food and included outcomes detailing how the strategies were received by human subjects using sensory data are included, as well as book chapters, literature reviews, and patents focusing on sodium reduction strategies. Only those published in English and since 1970 were included. Literature was obtained through Scopus, PubMed, EBSCOhost, and ScienceDirect databases, whereas patents were obtained through US Patent Trademark Office, Google Patents, and PATENTSCOPE databases. Two-hundred and seventy-seven primary studies, 27 literature reviews, 10 book chapters, and 143 patents were selected for inclusion. Data extracted included details such as analytical methods, broad and specific treatment categories, significant outcomes, and limitations among other material. Sodium reduction methods were categorized as either salt removal, salt replacement, flavor modification, functional modification, or physical modification. Although salt removal and salt replacement were the majority of included studies, future research would benefit from combining methods from other categories while investigating the impact on sensory characteristics, technological aspects, and consumer perception of the strategy.

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.

Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain.

Real-time activity measurements from multiple specific cell populations and projections are likely to be important for understanding the brain as a dynamical system. Here we developed frame-projected independent-fiber photometry (FIP), which we used to record fluorescence activity signals from many brain regions simultaneously in freely behaving mice. We explored the versatility of the FIP microscope by quantifying real-time activity relationships among many brain regions during social behavior, simultaneously recording activity along multiple axonal pathways during sensory experience, performing simultaneous two-color activity recording, and applying optical perturbation tuned to elicit dynamics that match naturally occurring patterns observed during behavior.

Diverging neural pathways assemble a behavioural state from separable features in anxiety.

Behavioural states in mammals, such as the anxious state, are characterized by several features that are coordinately regulated by diverse nervous system outputs, ranging from behavioural choice patterns to changes in physiology (in anxiety, exemplified respectively by risk-avoidance and respiratory rate alterations). Here we investigate if and how defined neural projections arising from a single coordinating brain region in mice could mediate diverse features of anxiety. Integrating behavioural assays, in vivo and in vitro electrophysiology, respiratory physiology and optogenetics, we identify a surprising new role for the bed nucleus of the stria terminalis (BNST) in the coordinated modulation of diverse anxiety features. First, two BNST subregions were unexpectedly found to exert opposite effects on the anxious state: oval BNST activity promoted several independent anxious state features, whereas anterodorsal BNST-associated activity exerted anxiolytic influence for the same features. Notably, we found that three distinct anterodorsal BNST efferent projections-to the lateral hypothalamus, parabrachial nucleus and ventral tegmental area-each implemented an independent feature of anxiolysis: reduced risk-avoidance, reduced respiratory rate, and increased positive valence, respectively. Furthermore, selective inhibition of corresponding circuit elements in freely moving mice showed opposing behavioural effects compared with excitation, and in vivo recordings during free behaviour showed native spiking patterns in anterodorsal BNST neurons that differentiated safe and anxiogenic environments. These results demonstrate that distinct BNST subregions exert opposite effects in modulating anxiety, establish separable anxiolytic roles for different anterodorsal BNST projections, and illustrate circuit mechanisms underlying selection of features for the assembly of the anxious state.

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.

Questionnaire on Irritable Bowel Syndrome and Symptom Management Among Endurance Athletes Is Valid and Reliable.

BACKGROUND: Gastrointestinal symptoms are reported in a large proportion of endurance athletes, with similarities in symptom type and distribution to irritable bowel syndrome (IBS). AIMS: The objective of this study was to develop and validate a questionnaire to assess IBS diagnoses or fit to IBS diagnostic criteria in this population along with nutritional habits, gastrointestinal symptoms, and symptom management strategies. METHODS: A 93-item Endurance Athlete Questionnaire was developed to address the objective, targeted at American endurance athletes completing a marathon, ultra-marathon, half-distance triathlon, and/or full-distance triathlon that year. Content validity was established by expert reviewers (n = 6), and face validity was evaluated by endurance athletes (n = 9). Test-retest reliability was assessed by target athletes (n = 51). Participants completed two rounds of the questionnaire, separated by 1-2 weeks. Results were analyzed using Pearson and Spearman correlations and paired comparisons. RESULTS: Slight modifications in wording and three demographic questions were added based on the input of expert and athlete reviews. Pearson correlation coefficient of test-retest total questionnaire scores was significant at 0.839 (P < 0.001). Paired comparison of individual questions found significant differences in 10 of 236 analyzed responses; however, these did not affect fit to IBS diagnostic criteria for those without other GI diseases/disorders. CONCLUSIONS: The Endurance Athlete Questionnaire proved to be a valid and reliable measure of IBS diagnostic criteria, gastrointestinal symptoms, nutritional habits, and symptom management strategies among endurance athletes. Future implementation will help inform gastroenterologists with endurance athlete patients and can elucidate whether certain behaviors could be contributing to athlete gastrointestinal symptoms.

Targeting cells with single vectors using multiple-feature Boolean logic.

Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology.

Observed differences in child picky eating behavior between home and childcare locations.

Picky eating (PE) is a common mealtime difficulty that is reported by up to 50% of caregivers. Most of the research to date on PE has focused on parents, even though millions of children also eat meals in home- or center-based childcare settings. Currently, little is known about PE behaviors manifested by the child across the home and childcare settings, or how these behaviors differ between home-based childcare (HBCC) and center-based childcare (CBCC) locations. The objectives of this study were to compare PE behaviors between the child's home and HBCC or CBCC environments, and compare PE behaviors between HBCC and CBCC environments. Children, ages 3-5 years, were recruited from CBCC (n = 26) or HBCC (n = 24) locations. Caregivers and children were videotaped consuming two different lunchtime meals in their home and childcare. Picky eating behaviors were coded from the videos using a codebook created for the study. Observational results showed that children in CBCC displayed more PE behaviors when at home than at childcare, while HBCC children displayed PE behaviors more similarly between the two locations. Thus, interventions to reduce PE behaviors should be personalized for location-specific intervention programs focused on raising healthy eaters across multiple locations.

Influence of seasoning on vegetable selection, liking and intent to purchase.

Low vegetable intake continues to be a health concern, and strategies to increase vegetable intake have resulted in only small increases. One strategy that has received less attention is the use of seasonings. This study's objective was to determine the impact of seasoning on vegetable selection, liking, and intent to purchase. We conducted a 3-week study in a public café on a university campus. Customers buying a main dish could select a vegetable side (seasoned [SS] or steamed [ST]) at no cost. Based on café data and power analysis (alpha 0.05, 80% power), 2 days per vegetable pair were conducted with carrot, broccoli, and green bean pairs randomized 3 days/week 1 and 3, with normal service week 2. Selection was greater for SS vs ST, n = 335 vs. 143 for all 3 vegetables combined; n = 97 vs 47 for carrots; n = 114 vs. 55 for broccoli; n = 124 vs. 41 for green beans (p < 0.001 Chi-Square). Liking responses were similar for SS vs ST and were high for all vegetables. Response distribution was not significantly different for SS vs ST vegetables when people were asked if they would purchase the vegetable that they selected. More customers chose the 'somewhat likely' and 'very likely' (n = 353) than the 'not likely' and 'definitely would not' (n = 121) purchase responses. Regression showed that people who did not often consume a vegetable with lunch while dining out were 1.59 times more likely to select the SS vegetables over the ST (p = 0.007). Given a choice, consumers were more likely to select a seasoned vegetable. With low vegetable consumption as a predictor of seasoned vegetable choice, offering seasoned vegetables may increase intake in those with poor vegetable intake in a café setting.

Functionality of Sugars in Foods and Health.

Overweight and obesity are global health problems that affect more than 1.9 billion adults who are overweight, and of these 600 million are obese. In the United States, these problems affect 60% of the population. Critical to these statistics is the association with increased risk of cardiovascular disease, type 2 diabetes, and metabolic syndrome among other noncommunicable diseases. Many factors, including sugars, have been charged as potential causes. However, obesity and overweight and their attendant health problems continue to increase despite the fact that there is a decline in the consumption of sugars. Sugars vary in their types and structure. From a food science perspective, sugars present an array of attributes that extend beyond taste, flavor, color, and texture to aspects such as structure and shelf-life of foods. From a public health perspective, there is considerable controversy about the effect of sugar relative to satiety, digestion, and noncommunicable diseases. This comprehensive overview from experts in food science, nutrition and health, sensory science, and biochemistry describes the technical and functional roles of sugar in food production, provides a balanced evidence-based assessment of the literature and addresses many prevalent health issues commonly ascribed to sugar by the media, consumer groups, international scientific organizations, and policy makers. The preponderance of the evidence indicates that sugar as such does not contribute to adverse health outcomes when consumed under isocaloric conditions. The evidence generally indicates, as noted by the 2010 Dietary Guidelines Advisory Committee, that sugar, like any other caloric macronutrient, such as protein and fat, when consumed in excess leads to conditions such as obesity and related comorbidities. More recently, the 2015-2020 Dietary Guidelines for Americans recommended limiting dietary sugar to 10% of total energy in an effort to reduced the risk of these noncommunicable diseases.

Cell class-specific electric field entrainment of neural activity.

Electric fields affect the activity of neurons and brain circuits, yet how this interaction happens at the cellular level remains enigmatic. Lack of understanding on how to stimulate the human brain to promote or suppress specific activity patterns significantly limits basic research and clinical applications. Here we study how electric fields impact the subthreshold and spiking properties of major cortical neuronal classes. We find that cortical neurons in rodent neocortex and hippocampus as well as human cortex exhibit strong and cell class-dependent entrainment that depends on the stimulation frequency. Excitatory pyramidal neurons with their typically slower spike rate entrain to slow and fast electric fields, while inhibitory classes like Pvalb and SST with their fast spiking predominantly phase lock to fast fields. We show this spike-field entrainment is the result of two effects: non-specific membrane polarization occurring across classes and class-specific excitability properties. Importantly, these properties of spike-field and class-specific entrainment are present in cells across cortical areas and species (mouse and human). These findings open the door to the design of selective and class-specific neuromodulation technologies.

Cell-class-specific electric field entrainment of neural activity.

Electric fields affect the activity of neurons and brain circuits, yet how this happens at the cellular level remains enigmatic. Lack of understanding of how to stimulate the brain to promote or suppress specific activity significantly limits basic research and clinical applications. Here, we study how electric fields impact subthreshold and spiking properties of major cortical neuronal classes. We find that neurons in the rodent and human cortex exhibit strong, cell-class-dependent entrainment that depends on stimulation frequency. Excitatory pyramidal neurons, with their slower spike rate, entrain to both slow and fast electric fields, while inhibitory classes like Pvalb and Sst (with their fast spiking) predominantly phase-lock to fast fields. We show that this spike-field entrainment is the result of two effects: non-specific membrane polarization occurring across classes and class-specific excitability properties. Importantly, these properties are present across cortical areas and species. These findings allow for the design of selective and class-specific neuromodulation.

Consumer acceptance of reduced sodium white and multigrain bread: Impact of flavor enhancement and ingredient information on sample liking.

Chronic consumption of sodium in quantities exceeding recommendations has led to sodium being designated as a nutrient of health concern for overconsumption. As a result of the prevalence of sodium overconsumption, the Food and Drug Administration (FDA) released voluntary sodium reduction goals for a wide variety of products on both short- and long-term timespans. As food palatability may decrease when sodium is reduced, flavor enhancers such as monosodium glutamate (MSG) may provide a promising solution to mitigate such palatability loss. The objective of this research was to investigate consumer acceptance of white and multigrain breads with either a 43% or 60% reduction in sodium content and with and without MSG as well as to investigate the influence of information on consumer acceptance of these breads under blind, informed, and informed with education conditions. Seventy-eight frequent bread consumers participated in the evaluations. A significant difference was evidenced across breads with different levels of sodium content and MSG status, although no difference was seen across the different evaluation conditions. Consumer segmentation found multiple consumer clusters showing different liking patterns of the bread treatments for both white and multigrain breads. Breads with sodium content set at the FDA's long-term goal with and without MSG were liked no differently in nearly all attributes evaluated than the full-sodium bread demonstrating the feasibility of producing acceptable reduced-sodium breads. Future research characterizing the predominant sensory attributes of full-sodium and reduced-sodium breads with and without MSG would be valuable for identifying the drivers of liking in such products. Practical Application: The findings of our study suggest that consumer liking of reduced sodium white and multigrain breads could be improved with the addition of monosodium glutamate. Increasing the acceptance of reduced sodium food products could help to reduce the risk of hypertension and subsequently heart attacks and stroke for the American population.

Characterizing the effect of sodium reduction and monosodium glutamate supplementation on white and multigrain breads.

Sodium overconsumption has become a serious health concern resulting in the Food and Drug Administration (FDA) publishing voluntary sodium reduction guidelines for a wide spectrum of packaged and processed foods. Reducing sodium through the removal of salt may decrease the palatability of foods, thus increasing the need for new approaches to prevent such palatability loss. The objective of this research was to characterize white and multigrain breads with either 43% or 60% reduction in sodium and with and without monosodium glutamate (MSG) using descriptive analysis methodology as well as to identify the drivers of liking for white and multigrain breads of varying sodium contents with and without MSG. Significant differences were identified in the attributes of salty taste and aftertaste, savory aftertaste, and chewy and firm textures in white bread and of salty taste and aftertaste, sweet taste, and density in multigrain bread. By regressing consumer test data of these breads onto their principal component analysis biplots, textural attributes and salty taste and aftertaste were identified as primary drivers of liking in white and multigrain breads. Flavor enhancers such as MSG show promise in mitigating palatability loss that occurs when the sodium content of bread is greatly reduced and thus provide a promising solution to produce breads with an improved nutritional profile. Future research on flavor enhancement in other food matrices would be valuable as well as in other bread and carbohydrate-based food categories. PRACTICAL APPLICATION: The findings of our study suggest that texture and a salty taste and aftertaste drive consumer liking of reduced-sodium white and multigrain breads and liking of breads could be improved with the addition of monosodium glutamate. Increasing the acceptance of reduced-sodium food products could help to improve the health of the American population by resulting in a reduced risk of hypertension and subsequently heart attacks and stroke.

A qualitative look at perception and experience of sodium reduction strategies in the food industry through focus groups and individual interviews.

The high incidence of sodium overconsumption in the general population has led sodium reduction in commercial food products to become a topic of importance in the food industry. In order to bridge the gap between sodium reduction understanding in the food industry and academia, focus groups and individual interviews of food industry professionals were conducted. Sodium reduction and influence from external entities such as federal regulations and consumer insight were prominent in the nutritional concerns of food industry professionals. A large variety of sodium reduction strategies were introduced with discussion on the many factors that contribute to their potential for success. Flavor modification methods were most prevalent in the discussion, with particular focus on potassium chloride and incorporating umami taste. Factors that frequently positively contributed to a strategy's success include maintaining functionality and/or important sensory attributes, inexpensive to implement, and being perceived as clean label. Conversely, factors that negatively affect success include adversely impacting flavor, being considered not clean label, and high costs of implementation. Foods important for future sodium reduction varied widely, although those were largely products with high sodium density. Future efforts toward reducing sodium overconsumption and sodium content in the food supply fell into three categories: consumer-focused, industry-focused, and research-focused. Of particular importance for future efforts included greater regulatory pressure and more consumer nutritional education. Findings suggest that future efforts to reduce the incidence of sodium overconsumption should be carried out through multiple avenues rather than focusing on the agency of consumers, the food industry, or research alone.

Associations between in vitro, in vivo and in silico cell classes in mouse primary visual cortex.

The brain consists of many cell classes yet in vivo electrophysiology recordings are typically unable to identify and monitor their activity in the behaving animal. Here, we employed a systematic approach to link cellular, multi-modal in vitro properties from experiments with in vivo recorded units via computational modeling and optotagging experiments. We found two one-channel and six multi-channel clusters in mouse visual cortex with distinct in vivo properties in terms of activity, cortical depth, and behavior. We used biophysical models to map the two one- and the six multi-channel clusters to specific in vitro classes with unique morphology, excitability and conductance properties that explain their distinct extracellular signatures and functional characteristics. These concepts were tested in ground-truth optotagging experiments with two inhibitory classes unveiling distinct in vivo properties. This multi-modal approach presents a powerful way to separate in vivo clusters and infer their cellular properties from first principles.

Associations between in vitro , in vivo and in silico cell classes in mouse primary visual cortex.

The brain consists of many cell classes yet in vivo electrophysiology recordings are typically unable to identify and monitor their activity in the behaving animal. Here, we employed a systematic approach to link cellular, multi-modal in vitro properties from experiments with in vivo recorded units via computational modeling and optotagging experiments. We found two one-channel and six multi-channel clusters in mouse visual cortex with distinct in vivo properties in terms of activity, cortical depth, and behavior. We used biophysical models to map the two one- and the six multi-channel clusters to specific in vitro classes with unique morphology, excitability and conductance properties that explain their distinct extracellular signatures and functional characteristics. These concepts were tested in ground-truth optotagging experiments with two inhibitory classes unveiling distinct in vivo properties. This multi-modal approach presents a powerful way to separate in vivo clusters and infer their cellular properties from first principles.