Differential encoding in prefrontal cortex projection neuron classes across cognitive tasks.

Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca2+ activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics.

Decoding and perturbing decision states in real time.

In dynamic environments, subjects often integrate multiple samples of a signal and combine them to reach a categorical judgment1. The process of deliberation can be described by a time-varying decision variable (DV), decoded from neural population activity, that predicts a subject's upcoming decision2. Within single trials, however, there are large moment-to-moment fluctuations in the DV, the behavioural significance of which is unclear. Here, using real-time, neural feedback control of stimulus duration, we show that within-trial DV fluctuations, decoded from motor cortex, are tightly linked to decision state in macaques, predicting behavioural choices substantially better than the condition-averaged DV or the visual stimulus alone. Furthermore, robust changes in DV sign have the statistical regularities expected from behavioural studies of changes of mind3. Probing the decision process on single trials with weak stimulus pulses, we find evidence for time-varying absorbing decision bounds, enabling us to distinguish between specific models of decision making.

Gut physiology of rainbow trout (Oncorhynchus mykiss) is influenced more by short-term fasting followed by refeeding than by feeding fishmeal-free diets.

Supplementing a fishmeal-free diet with yeast extract improves rainbow trout (Oncorhynchus mykiss) growth performance and modulates the hepatic and intestinal transcriptomic response. These effects are often observed in the long term but are not well documented after short periods of fasting. Fasting for a few days is a common practice in fish farming, especially before handling the fish, such as for short sorting, tank transfers, and vaccinations. In the present study, rainbow trout were subjected to a 4-day fast and then refed, for 8 days, a conventional diet containing fishmeal (control diet) or alternative diets composed of terrestrial animal by-products supplemented or not with a yeast extract. During the refeeding period alone, most of the parameters considered did not differ significantly in response to the different feeds. Only the expression of claudin-15 was upregulated in fish fed the yeast-supplemented diet compared to the control diet. Conversely, fasting followed by refeeding significantly influenced most of the parameters analyzed. In the proximal intestine, the surface area of villi significantly increased, and the density of goblet cell tended to decrease during refeeding. Although no distinct plasma immune response or major signs of gut inflammation were observed, some genes involved in the structure, complement pathway, antiviral functions, coagulation, and endoplasmic reticulum stress response of the liver and intestine were significantly regulated by refeeding after fasting. These results indicate that short-term fasting, as commonly practiced in fish farming, significantly alters the physiology of the liver and intestine regardless of the composition of the diet.

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida.

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Tryptophan Modulatory Role in European Seabass (Dicentrarchus labrax) Immune Response to Acute Inflammation under Stressful Conditions.

The present work aimed to study the role of dietary tryptophan supplementation in modulating the European seabass (Dicentrarchus labrax) immune condition during stressful rearing conditions (i.e., 15 days exposure to high density), as well as the immune response to acute inflammation after intraperitoneal injection of a bacterial pathogen. Stress alone did not compromise seabass health indicators. In contrast, a clear peripheral and local inflammatory response was observed in response to the inoculated bacteria. Moreover, exposure to a high stocking density seemed to exacerbate the inflammatory response at early sampling points, compared to fish stocked at a lower density. In contrast, stressed fish presented some immune-suppressing effects on the T-cell surface glycoprotein receptor expressions at a late sampling point following inflammation. Regarding the effects of dietary tryptophan, no changes were observed on seabass immune indicators prior to inflammation, while a small number of immunosuppressive effects were observed in response to inflammation, supporting tryptophan's role in the promotion of immune-tolerance signals during inflammation. Nonetheless, tryptophan dietary supplementation improved the inflammatory response against a bacterial pathogen during stressful conditions, supported by a reduction of plasma cortisol levels, an up-regulation of several immune-related genes at 48 h, and an inversion of the previously observed, stress-induced T-cell suppression. Finally, the involvement of tryptophan catabolism in macrophages was confirmed by the up-regulation of genes involved in the kynurenine pathway. The present study brings new insights regarding the immune modulatory role of tryptophan during stressful conditions in fish, thus allowing for the development of novel prophylactic protocols during vaccination by intraperitoneal injection in the European seabass.

Dietary methionine as a strategy to improve innate immunity in rainbow trout (Oncorhynchus mykiss) juveniles.

Methionine ability to enhance fish immune status and inflammatory response by the modulation of methionine-related pathways has been verified in several fish species. However, no attention has been given to the role of methionine as an immune-modulatory additive in rainbow trout (Oncorhynchus mykiss). Hence, this study was designed to evaluate the effect of short-term feeding a diet supplemented with dl-methionine on the rainbow trout immune status. For this purpose, two diets were formulated: a control (CTRL) diet including the AA profile required to meet the ideal pattern estimated for rainbow trout; and an experimental diet (MET) identical to the CTRL but supplemented with dl-methionine two fold above its requirement level. After 2 and 4 weeks, fish haematological profile, peripheral cell populations, plasma and skin mucus humoral immune parameters as well as head-kidney gene expression were analysed. Results showed that methionine was able to improve peripheral neutrophil numbers after 4 weeks of feeding while reducing the expression of pro-inflammatory genes (i.e. IL1ß and IL8). Also, indications of fish physiological ability to regulate polyamine biosynthesis were found by the reduced expression of the spermine synthase enzyme (SMS). Together, these results point to some level of enhancement of the cellular-related innate immune status by dietary dl-methionine supplementation after a short-term feeding period, which could be used as a prophylactic strategy for rainbow trout health management.

Uptake and effects of different concentrations of spherical polymer microparticles on Artemia franciscana.

Artemia cysts have a huge economic importance for the aquaculture sector due to the fact that they are used as live feed for larviculture. Microplastics (MPs) are common and emergent pollutants in the aquatic environments, with unknown and potential long-term effects on planktonic species such as Artemia spp. When used as live feed, Artemia could transfer contaminants to fish along the food chain, with possible adverse effects on human health through their consumption. This study aims to assess the uptake of different concentrations of spherical polymer microparticles (FRM) (1-5 µm diameter) and their associated chronic effects on feeding, growth, mortality, and reproductive success from juvenile to adult stage of brine shrimp Artemia franciscana. Individuals were exposed for 44 days to 0.4, 0.8 and 1.6 mg.L-1 of FRM. No significant detrimental effects on growth, ingestion and mortality rates of A. franciscana were observed in all tested conditions. However, reproductive success was strongly affected by the increase of MP concentrations. The results of the present study showed that A. franciscana juveniles and adults were able to survive different experimental MP concentrations, but their reproductive success and progeny were significantly impacted by exposure to FRM particles.

Dietary tryptophan intervention counteracts stress-induced transcriptional changes in a teleost fish HPI axis during inflammation.

Immune nutrition is currently used to enhance fish health by incorporating functional ingredients into aquafeeds. This study aimed to investigate the connections between tryptophan nutrition and the network that regulates the communication pathways between neuroendocrine and immune systems in European seabass (Dicentrarchus labrax). When tryptophan was supplemented in the diet of unstressed fish, it induced changes in the hypothalamic-pituitary-interrenal axis response to stress. Tryptophan-mediated effects were observed in the expression of anti-inflammatory cytokines and glucocorticoid receptors. Tryptophan supplementation decreased pro-opiomelanocortin b-like levels, that are related with adrenocorticotropic hormone and cortisol secretion. When stressed fish fed a tryptophan-supplemented diet were subjected to an inflammatory stimulus, plasma cortisol levels decreased and the expression of genes involved in the neuroendocrine response was altered. Modulatory effects of tryptophan dietary intervention on molecular patterns seem to be mediated by altered patterns in serotonergic activity.

Dietary Tryptophan Plays a Role as an Anti-Inflammatory Agent in European Seabass (Dicentrarchus labrax) Juveniles during Chronic Inflammation.

Where teleost fish are concerned, studies in tryptophan immunomodulation generally point to immunosuppressive properties, thus presenting a potential anti-inflammatory dietary strategy. The goal of the present work was to evaluate the effects of tryptophan dietary supplementation on immune and neuroendocrine responses of the European seabass, Dicentrarchus labrax, undergoing chronic inflammation. Juvenile European seabass were intraperitoneally injected with either Freund's Incomplete Adjuvant (FIA, inflamed group) or a saline solution (control group). Within each group, fish were fed a control (CTRL) and a CTRL-based diet supplemented with tryptophan (0.3% DM basis; TRP) for 4 weeks. Different tissues were sampled every week for the assessment of immune-related parameters. When TRP was provided to FIA-injected fish, mcsfr gene expression increased from 1 to 2 weeks and remained high until the end of the experiment. The same fish showed a concurrent increase in peripheral monocyte counts. Moreover, il34 expression at 1 week post-FIA injection was higher in TRP-fed than in CTRL-fed fish. After one week, molecular patterns of anti-inflammatory processes seemed to be favoured by TRP (mcsfr, gr1, il34 and tgfß). Altogether, the results show that the feeding period seems to be critical where tryptophan supplementation is concerned since at later inflammatory stages-and longer feeding periods-fish fed TRP displayed a molecular profile similar to that of the CTRL group. In contrast, shorter administration periods might accelerate immune regulatory pathways.

Early innate immune responses in European sea bass (Dicentrarchus labrax L.) following Tenacibaculum maritimum infection.

Introduction: The marine aquaculture industry has been witnessing a worldwide emergence of tenacibaculosis, a poorly understood bacterial disease caused by Tenacibaculum maritimum that affects commercially important fish. So far, knowledge on the T. maritimum virulence mechanisms is scarce and the pathogen-host interaction operating in tenacibaculosis remain to be disclosed. This study aimed at contributing to a better understanding of this disease, by evaluating the early innate immune response triggered in European sea bass (Dicentrarchus labrax) by a bath-challenge with T. maritimum. Methods: Groups of sea bass were bath-challenged with T. maritimum (challenged fish) or mock-challenged. Undisturbed fish were used as controls (time 0). Samples of blood, liver and mucosal organs (skin, gills and posterior-intestine) were collected at 0 h (control) and at 6, 24, 48 and 72 h post-challenge (n=12). Mucosal organs were used for analyzing the expression of immune-related genes by RT-qPCR, as well as blood samples for assessing haematological and innate humoral parameters and liver for oxidative stress assessment. Results: An increased expression of il-1ß, il8, mmp9 and hamp1 was detected in all mucosal organs of infected fish when compared with control and mock-challenged fish, suggesting a pro-inflammatory response against T. maritimum transversal to all organs. The faster induction of these pro-inflammatory genes was observed in the gills. Regarding the systemic response, challenged fish presented neutrophilia, monocytosis, signs of anemia, and a decrease of bactericidal and lysozyme activities in plasma. Almost no variations were observed regarding hepatic oxidative stress. Discussion/Conclusions: The present study suggests that T. maritimum induces a local innate immune response upon bath infection not only in the skin of European sea bass, but also in the gills and posterior-intestine, likely triggered by the T. maritimum's capacity to adhere, colonize and damage these organs that can function as entry ways to bacteria, leading ultimately to the seen host's systemic response.

Health-Promoting Additives Supplemented in Inert Microdiets for Whiteleg Shrimp (Penaeus vannamei) Post-Larvae: Effects on Growth, Survival, and Health Status.

Dietary additives have the potential to stimulate the whiteleg shrimp immune system, but information is scarce on their use in diets for larval/post-larval stages. The potential beneficial effects of vitamins C and E, ß-glucans, taurine, and methionine were evaluated. Four experimental microdiets were tested: a positive control diet (PC); the PC with decreased levels of vitamin C and E as negative control (NC); the PC with increased taurine and methionine levels (T + M); and the PC supplemented with ß-glucans (BG). No changes in growth performance and survival were observed. However, post-larvae shrimp fed the NC had lower relative expressions of pen-3 than those fed the PC, suggesting that lower levels of vitamins C and E may impact the shrimp immune status. Lipid peroxidation levels dropped significantly in the BG compared to the PC, indicating that ß-glucans improved the post-larvae antioxidant mechanisms. Furthermore, when compared with the NC diet, PL fed with BG showed significant increases in tGSH levels and in the relative expression of crus and pen-3, suggesting a synergistic effect between vitamins C and E and ß-glucans. Amongst the additives tested, ß-glucans seems to be the most promising even when compared to a high-quality control diet.

Chronic Inflammation Modulates Opioid Receptor Gene Expression and Triggers Respiratory Burst in a Teleost Model.

Stress-inducing husbandry and rearing conditions, bacterial infections or parasitic diseases may all lead to chronic inflammation. The immune response will then channel energy away from growth, reproduction and other important physiological processes, to fuel immune-related metabolic responses. The present study aims to unravel the mechanisms and contribute with new information on the molecular, cellular and humoral parameters of European seabass (Dicentrarchus labrax) undergoing chronic inflammation that can be used as health indicators for application in fish health management. European seabass individuals were intra-peritoneally injected with either Freund's Incomplete Adjuvant (FIA) to induce inflammation or Hanks Balanced Salt Solution (HBSS) to serve as sham. Fish were sampled at 24 h, 7, 14 and 21 days post-injection and blood, plasma and head-kidney were collected. The results found were clear indicators of an inflamed peritoneal cavity and an ongoing systemic immune response that persisted for at least 21 days. Locally, inflammation was characterized by an intense recruitment of immune cells that was still evident 21 days after injection, thus illustrating the chronic character of the immune response. Cellular response was also noticed peripherally with leukocyte numbers rising in the blood of FIA-injected fish. Furthermore, the cellular-mediated respiratory burst peaked at 21 days post-FIA injection, suggesting that phagocytes were still actively fighting the phlogistic agent. Regarding the head-kidney molecular analysis, cxcr4 and il34 appear to be good markers of a chronic inflammation response due to their importance for pathways with high relevance in chronic inflammation settings. In addition, opioid receptor nopr seems to be a good marker of a chronic inflammation response due to its role in detecting noxious stimuli. The present study can serve as a baseline to assess long-term immune-related responses in future studies. For that, more research is nonetheless required to select more responsive and specific molecular markers.

Effect of short-term exposure to fluorescent red polymer microspheres on Artemia franciscana nauplii and juveniles.

Microplastics (MPs) are ubiquitously present in the world's seas with unknown potential toxic effects on aquatic ecosystems. The aim of this study was to evaluate biochemical responses caused by 1-5 µm diameter plastic fluorescent red polymer microspheres (FRM), under short-term exposure of nauplii and juveniles of Artemia franciscana, using a set of biomarkers involved in important physiological processes such as biotransformation, neuronal transmission and oxidative stress. Two FRM concentrations (0.4 and 1.6 mg mL-1) present in the water at ecologically relevant concentrations were used to study their toxicity. No significant differences were found in growth, survival and feeding behaviour of nauplii, after 2 days of exposure to both FRM concentrations. However, in juveniles, survival decreased after 5 days of exposure to FRM1.6; but no significant differences were found in either growth or feeding behaviour. It was observed that nauplii and juveniles, under short-term exposure, had the ability to ingest and egest FRM particles, although their accumulation was higher in nauplii than in juveniles, maybe related with the capacity of the latter to empty their gut content faster, in the presence of food. Regarding biomarkers responses in nauplii, all enzymatic activities increased significantly, after short-term exposure to the higher FRM concentration tested (FRM1.6), which could be related with detoxifying MPs-triggered oxidative stress. In juveniles, the inhibition of ChE and the decrease in the activity of antioxidant enzymes, after 5 days of exposure to FRM1.6, might indicate a neurotoxic effect and oxidative damage induced by FRM. This study provides further evidences that accumulation of MPs in the gut by nauplii and juveniles of A. franciscana can induce negative effects on important physiological processes with influence on their health, highlighting the general concern about the negative effects of MPs pollution on aquatic species, as well as the need to understand the mechanism of MPs toxicity and its possible impacts on environmental safety. Graphical abstract.

Dietary Strategies to Modulate the Health Condition and Immune Responses in Gilthead Seabream (Sparus aurata) Juveniles Following Intestinal Inflammation.

Several feed additives have proved to be beneficial in eliciting fish health. Β-glucans and curcumin are compounds with immunomodulatory capacities known to increase growth performance, stimulate immunity, improve general health, and enhance disease resistance in fish. The present study aimed to evaluate the effects of dietary Phaeodactylum tricornutum extracts rich in ß-glucans and curcumin on gilthead seabream health status prior to and following an intestinal inflammatory stimulus. Three experimental diets were formulated: a practical commercial-type diet (CTRL), a CTRL diet supplemented with 1% microalgae-derived ß-glucans extract (BG), and a CTRL diet supplemented with 0.2% of curcumin (CUR). After 30 days of the feeding trial, fish were sampled and subjected to an oral administration of 1% dextran sodium sulphate (DSS) to induce intestinal inflammation. Four groups were considered: a group of fish continued to be fed on the CTRL diet while the remaining groups were exposed to DSS, including CTRL-D (CTRL + DSS), BG-D (BG + DSS), and CUR-D (CUR + DSS), for 6 days. Growth, plasma and gut humoral immunity, liver and gut oxidative stress biomarkers, and intestinal gene expression were evaluated. No significant differences were found in growth after 30 days of feeding; however, seabream fed BG had decreased anti-protease activity and nitric oxide concentration in plasma while those fed CUR had increased mRNA levels of the tnfα, csf1r, and hep genes compared to those fed CTRL. After the inflammatory stimulus, hematocrit was enhanced in fish fed BG-D and CUR-D while red blood cell counts increased in those fed CTRL-D. Superoxide dismutase activity decreased in the intestine of all DSS groups while lipid peroxidation increased in the gut of fish fed CTRL-D and BG-D compared to CTRL. Moreover, the mRNA expression levels of csfr1 and sod decreased in fish fed CTRL-D and BG-D compared to CTRL, respectively. Despite the mild intestinal inflammatory condition induced by DSS, CUR was able to partially ameliorate its effects, improving the hematological profile and assisting against the oxidative stress.

Macaque dorsal premotor cortex exhibits decision-related activity only when specific stimulus-response associations are known.

How deliberation on sensory cues and action selection interact in decision-related brain areas is still not well understood. Here, monkeys reached to one of two targets, whose colors alternated randomly between trials, by discriminating the dominant color of a checkerboard cue composed of different numbers of squares of the two target colors in different trials. In a Targets First task the colored targets appeared first, followed by the checkerboard; in a Checkerboard First task, this order was reversed. After both cues appeared in both tasks, responses of dorsal premotor cortex (PMd) units covaried with action choices, strength of evidence for action choices, and RTs- hallmarks of decision-related activity. However, very few units were modulated by checkerboard color composition or the color of the chosen target, even during the checkerboard deliberation epoch of the Checkerboard First task. These findings implicate PMd in the action-selection but not the perceptual components of the decision-making process in these tasks.

Lymnaea stagnalis as a freshwater model invertebrate for ecotoxicological studies.

Lymnaea stagnalis, also referred to as great or common pond snail, is an abundant and widespread invertebrate species colonizing temperate limnic systems. Given the species importance, studies involving L. stagnalis have the potential to produce scientifically relevant information, leading to a better understanding of the damage caused by aquatic contamination, as well as the modes of action of toxicants. Lymnaea stagnalis individuals are easily maintained in laboratory conditions, with a lifespan of about two years. The snails are hermaphrodites and sexual maturity occurs about three months after egg laying. Importantly, they can produce a high number of offspring all year round and are considered well suited for use in investigations targeting the identification of developmental and reproductive impairments. The primary aims of this review were two-fold: i) to provide an updated and insightful compilation of established toxicological measures determined in both chronic and acute toxicity assays, as useful tool to the design and development of future research; and ii) to provide a state of the art related to direct toxicant exposure and its potentially negative effects on this species. Relevant and informative studies were analysed and discussed. Knowledge gaps in need to be addressed in the near future were further identified.

Laminar differences in decision-related neural activity in dorsal premotor cortex.

Dorsal premotor cortex is implicated in somatomotor decisions. However, we do not understand the temporal patterns and laminar organization of decision-related firing rates in dorsal premotor cortex. We recorded neurons from dorsal premotor cortex of monkeys performing a visual discrimination task with reaches as the behavioral report. We show that these neurons can be organized along a bidirectional visuomotor continuum based on task-related firing rates. "Increased" neurons at one end of the continuum increased their firing rates ~150 ms after stimulus onset and these firing rates covaried systematically with choice, stimulus difficulty, and reaction time-characteristics of a candidate decision variable. "Decreased" neurons at the other end of the continuum reduced their firing rate after stimulus onset, while "perimovement" neurons at the center of the continuum responded only ~150 ms before movement initiation. These neurons did not show decision variable-like characteristics. "Increased" neurons were more prevalent in superficial layers of dorsal premotor cortex; deeper layers contained more "decreased" and "perimovement" neurons. These results suggest a laminar organization for decision-related responses in dorsal premotor cortex.Dorsal premotor cortex (PMd) is thought to be involved in making somatomotor decisions. Chandrasekaran et al. investigated the temporal response dynamics of PMd neurons across cortical layers and show stronger and earlier decision-related responses in the superficial layers and more action execution-related signals in the deeper layers.

Sensory integration dynamics in a hierarchical network explains choice probabilities in cortical area MT.

Neuronal variability in sensory cortex predicts perceptual decisions. This relationship, termed choice probability (CP), can arise from sensory variability biasing behaviour and from top-down signals reflecting behaviour. To investigate the interaction of these mechanisms during the decision-making process, we use a hierarchical network model composed of reciprocally connected sensory and integration circuits. Consistent with monkey behaviour in a fixed-duration motion discrimination task, the model integrates sensory evidence transiently, giving rise to a decaying bottom-up CP component. However, the dynamics of the hierarchical loop recruits a concurrently rising top-down component, resulting in sustained CP. We compute the CP time-course of neurons in the medial temporal area (MT) and find an early transient component and a separate late contribution reflecting decision build-up. The stability of individual CPs and the dynamics of noise correlations further support this decomposition. Our model provides a unified understanding of the circuit dynamics linking neural and behavioural variability.

Natural grouping of neural responses reveals spatially segregated clusters in prearcuate cortex.

A fundamental challenge in studying the frontal lobe is to parcellate this cortex into "natural" functional modules despite the absence of topographic maps, which are so helpful in primary sensory areas. Here we show that unsupervised clustering algorithms, applied to 96-channel array recordings from prearcuate gyrus, reveal spatially segregated subnetworks that remain stable across behavioral contexts. Looking for natural groupings of neurons based on response similarities, we discovered that the recorded area includes at least two spatially segregated subnetworks that differentially represent behavioral choice and reaction time. Importantly, these subnetworks are detectable during different behavioral states and, surprisingly, are defined better by "common noise" than task-evoked responses. Our parcellation process works well on "spontaneous" neural activity, and thus bears strong resemblance to the identification of "resting-state" networks in fMRI data sets. Our results demonstrate a powerful new tool for identifying cortical subnetworks by objective classification of simultaneously recorded electrophysiological activity.