Altered glucocorticoid reactivity and behavioral phenotype in rx3-/- larval zebrafish.

Introduction: The transcription factor rx3 is important for the formation of the pituitary and parts of the hypothalamus. Mutant animals lacking rx3 function have been well characterized in developmental studies, but relatively little is known about their behavioral phenotypes. Methods: We used cell type staining to reveal differences in stress axis architecture, and performed cortisol measurements and behavior analysis to study both hormonal and behavioral stress responses in rx3 mutants. Results and Discussion: Consistent with the role of rx3 in hypothalamus and pituitary development, we show a distinct loss of corticotrope cells involved in stress regulation, severe reduction of pituitary innervation by hypothalamic cells, and lack of stress-induced cortisol release in rx3 mutants. Interestingly, despite these deficits, we report that rx3-/- larval zebrafish can still display nominal behavioral responses to both stressful and non-stressful stimuli. However, unlike wildtypes, mutants lacking proper pituitary-interrenal function do not show enhanced behavioral performance under moderate stress level, supporting the view that corticotroph cells are not required for behavioral responses to some types of stressful stimuli but modulate subtle behavioral adjustments under moderate stress.

Active behaviour during early development shapes glucocorticoid reactivity.

Glucocorticoids are the final effectors of the stress axis, with numerous targets in the central nervous system and the periphery. They are essential for adaptation, yet currently it is unclear how early life events program the glucocorticoid response to stress. Here we provide evidence that involuntary swimming at early developmental stages can reconfigure the cortisol response to homotypic and heterotypic stress in larval zebrafish (Danio rerio), also reducing startle reactivity and increasing spontaneous activity as well as energy efficiency during active behaviour. Collectively, these data identify a role of the genetically malleable zebrafish for linking early life stress with glucocorticoid function in later life.

Impaired path integration in mice with disrupted grid cell firing.

Path integration (PI) is a highly conserved, self-motion-based navigation strategy. Since the discovery of grid cells in the medial entorhinal cortex, neurophysiological data and computational models have suggested that these neurons serve PI. However, more direct empirical evidence supporting this hypothesis has been missing due to a lack of selective manipulations of grid cell activity and suitable behavioral assessments. Here we report that selective disruption of grid cell activity in mice can be achieved by removing NMDA glutamate receptors from the retro-hippocampal region and that disrupted grid cell firing accounts for impaired PI performance. Notably, the genetic manipulation did not affect the activity of other spatially selective cells in the medial entorhinal cortex and the hippocampus. By directly linking grid cell activity to PI, these results contribute to a better understanding of how grid cells support navigation and spatial memory.

Performance on innate behaviour during early development as a function of stress level.

What is the relationship between the level of acute stress and performance on innate behaviour? The diversity of innate behaviours and lack of sufficient data gathered under the same experimental conditions leave this question unresolved. While evidence points to an inverted-U shaped relationship between the level of acute stress and various measures of learning and memory function, it is unknown the extent to which such a non-linear function applies to performance on innate behaviour, which develops without example or practice under natural circumstances. The fundamental prediction of this view is that moderate stress levels will improve performance, while higher levels will not. Testing this proposition has been difficult because it entails an overall effect that must be invariant to the nature of the stressor, the behaviour under scrutiny and the stimulus that drives it. Here, we report new experimental results showing that developing zebrafish (Danio rerio) under moderate but not higher levels of stress improved their performance on instinctive activities driven by visual, hydrodynamic and thermal inputs. Our findings reveal, for the first time, the existence of an inverted-U shaped performance function according to stress level during early development in a series of innate behaviours.

Optogenetically enhanced pituitary corticotroph cell activity post-stress onset causes rapid organizing effects on behaviour.

The anterior pituitary is the major link between nervous and hormonal systems, which allow the brain to generate adequate and flexible behaviour. Here, we address its role in mediating behavioural adjustments that aid in coping with acutely threatening environments. For this we combine optogenetic manipulation of pituitary corticotroph cells in larval zebrafish with newly developed assays for measuring goal-directed actions in very short timescales. Our results reveal modulatory actions of corticotroph cell activity on locomotion, avoidance behaviours and stimulus responsiveness directly after the onset of stress. Altogether, the findings uncover the significance of endocrine pituitary cells for rapidly optimizing behaviour in local antagonistic environments.

The Severity of Acute Stress Is Represented by Increased Synchronous Activity and Recruitment of Hypothalamic CRH Neurons.

The hypothalamo-pituitary-adrenocortical (HPA) axis regulates stress physiology and behavior. To achieve an optimally tuned adaptive response, it is critical that the magnitude of the stress response matches the severity of the threat. Corticotropin-releasing hormone (CRH) released from the paraventricular nucleus of the hypothalamus is a major regulator of the HPA axis. However, how CRH-producing neurons in an intact animal respond to different stressor intensities is currently not known. Using two-photon calcium imaging on intact larval zebrafish, we recorded the activity of CRH cells, while the larvae were exposed to stressors of varying intensity. By combining behavioral and physiological measures, we first determined how sudden alterations in environmental conditions lead to different levels of stress axis activation. Then, we measured changes in the frequency and amplitude of Ca(2+) transients in individual CRH neurons in response to such stressors. The response magnitude of individual CRH cells covaried with stressor intensity. Furthermore, stressors caused the recruitment of previously inactive CRH neurons in an intensity-dependent manner, thus increasing the pool of responsive CRH cells. Strikingly, stressor-induced activity appeared highly synchronized among CRH neurons, and also across hemispheres. Thus, the stressor strength-dependent output of CRH neurons emerges by a dual mechanism that involves both the increased activity of individual cells and the recruitment of a larger pool of responsive cells. The synchronicity of CRH neurons within and across hemispheres ensures that the overall output of the HPA axis matches the severity of the threat. SIGNIFICANCE STATEMENT: Stressors trigger adaptive responses in the body that are essential for survival. How the brain responds to acute stressors of varying intensity in an intact animal, however, is not well understood. We address this question using two-photon Ca(2+) imaging in larval zebrafish with transgenically labeled corticotropin-releasing hormone (CRH) cells, which represent a major regulator of the stress axis. We show that stressor strength-dependent responses of CRH neurons emerge via an intensity-dependent increase in the activity of individual CRH cells, and by an increase in the pool of responsive CRH cells at the population level. Furthermore, we report striking synchronicity among CRH neurons even across hemispheres, which suggests tight intrahypothalamic and interhypothalamic coordination. Thus, our work reveals how CRH neurons respond to different levels of acute stress in vivo.

Manipulation of Interrenal Cell Function in Developing Zebrafish Using Genetically Targeted Ablation and an Optogenetic Tool.

Zebrafish offer an opportunity to study conserved mechanisms underlying the ontogeny and physiology of the hypothalamic-pituitary-adrenal/interrenal axis. As the final effector of the hypothalamic-pituitary-adrenal/interrenal axis, glucocorticoids exert both rapid and long-term regulatory functions. To elucidate their specific effects in zebrafish, transgenic approaches are necessary to complement pharmacological studies. Here, we report a robust approach to specifically manipulate endogenous concentrations of cortisol by targeting heterologous proteins to interrenal cells using a promoter element of the steroidogenic acute regulatory protein. To test this approach, we first used this regulatory region to generate a transgenic line expressing the bacterial nitroreductase protein, which allows conditional targeted ablation of interrenal cells. We demonstrate that this line can be used to specifically ablate interrenal cells, drastically reducing both basal and stress-induced cortisol concentrations. Next, we coupled this regulatory region to an optogenetic actuator, Beggiatoa photoactivated adenylyl cyclase, to increase endogenous cortisol concentrations in a blue light-dependent manner. Thus, our approach allows specific manipulations of steroidogenic interrenal cell activity for studying the effects of both hypo- and hypercortisolemia in zebrafish.

Positive taxis and sustained responsiveness to water motions in larval zebrafish.

Larval zebrafish (Danio rerio) have become favored subjects for studying the neural bases of behavior. Here, we report a highly stereotyped response of zebrafish larvae to hydrodynamic stimuli. It involves positive taxis, motion damping and sustained responsiveness to flows derived from local, non-stressful water motions. The response depends on the lateral line and has a high sensitivity to stimulus frequency and strength, sensory background and rearing conditions--also encompassing increased threshold levels of response to parallel input. The results show that zebrafish larvae can use near-field detection to locate sources of minute water motions, and offer a unique handle for analyses of hydrodynamic sensing, sensory responsiveness and arousal with accurate control of stimulus properties.

The behavior of larval zebrafish reveals stressor-mediated anorexia during early vertebrate development.

The relationship between stress and food consumption has been well documented in adults but less so in developing vertebrates. Here we demonstrate that an encounter with a stressor can suppress food consumption in larval zebrafish. Furthermore, we provide indication that food intake suppression cannot be accounted for by changes in locomotion, oxygen consumption and visual responses, as they remain unaffected after exposure to a potent stressor. We also show that feeding reoccurs when basal levels of cortisol (stress hormone in humans and teleosts) are re-established. The results present evidence that the onset of stress can switch off the drive for feeding very early in vertebrate development, and add a novel endpoint for analyses of metabolic and behavioral disorders in an organism suitable for high-throughput genetics and non-invasive brain imaging.

Optogenetic elevation of endogenous glucocorticoid level in larval zebrafish.

The stress response is a suite of physiological and behavioral processes that help to maintain or reestablish homeostasis. Central to the stress response is the hypothalamic-pituitary-adrenal (HPA) axis, as it releases crucial hormones in response to stress. Glucocorticoids (GCs) are the final effector hormones of the HPA axis, and exert a variety of actions under both basal and stress conditions. Despite their far-reaching importance for health, specific GC effects have been difficult to pin-down due to a lack of methods for selectively manipulating endogenous GC levels. Hence, in order to study stress-induced GC effects, we developed a novel optogenetic approach to selectively manipulate the rise of GCs triggered by stress. Using this approach, we could induce both transient hypercortisolic states and persistent forms of hypercortisolaemia in freely behaving larval zebrafish. Our results also established that transient hypercortisolism leads to enhanced locomotion shortly after stressor exposure. Altogether, we present a highly specific method for manipulating the gain of the stress axis with high temporal accuracy, altering endocrine and behavioral responses to stress as well as basal GC levels. Our study offers a powerful tool for the analysis of rapid (non-genomic) and delayed (genomic) GC effects on brain function and behavior, feedbacks within the stress axis and developmental programming by GCs.

Controlled variations in stimulus similarity during learning determine visual discrimination capacity in freely moving mice.

The mouse is receiving growing interest as a model organism for studying visual perception. However, little is known about how discrimination and learning interact to produce visual conditioned responses. Here, we adapted a two-alternative forced-choice visual discrimination task for mice and examined how training with equiprobable stimuli of varying similarity influenced conditioned response and discrimination performance as a function of learning. Our results indicate that the slope of the gradients in similarity during training determined the learning rate, the maximum performance and the threshold for successful discrimination. Moreover, the learning process obeyed an inverse relationship between discrimination performance and discriminative resolution, implying that sensitivity within a similarity range cannot be improved without sacrificing performance in another. Our study demonstrates how the interplay between discrimination and learning controls visual discrimination capacity and introduces a new training protocol with quantitative measures to study perceptual learning and visually-guided behavior in freely moving mice.

Discrimination learning with variable stimulus 'salience'.

BACKGROUND: In nature, sensory stimuli are organized in heterogeneous combinations. Salient items from these combinations 'stand-out' from their surroundings and determine what and how we learn. Yet, the relationship between varying stimulus salience and discrimination learning remains unclear. PRESENTATION OF THE HYPOTHESIS: A rigorous formulation of the problem of discrimination learning should account for varying salience effects. We hypothesize that structural variations in the environment where the conditioned stimulus (CS) is embedded will be a significant determinant of learning rate and retention level. TESTING THE HYPOTHESIS: Using numerical simulations, we show how a modified version of the Rescorla-Wagner model, an influential theory of associative learning, predicts relevant interactions between varying salience and discrimination learning. IMPLICATIONS OF THE HYPOTHESIS: If supported by empirical data, our model will help to interpret critical experiments addressing the relations between attention, discrimination and learning.

Honeybees learn the sign and magnitude of reward variations.

In this study, we asked whether honeybees learn the sign and magnitude of variations in the level of reward. We designed an experiment in which bees first had to forage on a three-flower patch offering variable reward levels, and then search for food at the site in the absence of reward and after a long foraging pause. At the time of training, we presented the bees with a decrease in reward level or, instead, with either a small or a large increase in reward level. Testing took place as soon as they visited the patch on the day following training, when we measured the bees' food-searching behaviours. We found that the bees that had experienced increasing reward levels searched for food more persistently than the bees that had experienced decreasing reward levels, and that the bees that had experienced a large increase in reward level searched for food more persistently than the bees that had experienced a small increase in reward level. Because these differences at the time of testing cannot be accounted for by the bees' previous crop loads and food-intake rates, our results unambiguously demonstrate that honeybees adjust their investment of time/energy during foraging in relation to both the sign and the magnitude of past variations in the level of reward. It is likely that such variations lead to the formation of reward expectations enhancing a forager's reliance on a feeding site. Ultimately, this would make it more likely for honeybees to find food when forage is scarce.

Side-specific reward memories in honeybees.

We report a hitherto unknown form of side-specific learning in honeybees. We trained bees individually by coupling gustatory and mechanical stimulation of each antenna with either increasing or decreasing volumes of sucrose solution offered to the animal's proboscis along successive learning trials. Next, we examined their proboscis extension response (PER) after stimulation of each antenna 1, 2, 3, and 24 h after training. The bees extended their proboscises earlier after stimulation of the antenna that had been coupled with increasing volumes than after stimulation of the antenna that had been coupled with decreasing volumes, thereby revealing short- and long-term side differences in the bees' PE reaction time. The bees' reaction time correlated well with the reaction time of the muscles M17. Long-term side differences in reaction time were prevented by repetitive antennal stimulation. Mechanosensory input was indispensable and sufficient for revealing side differences in reaction time. Such differences were specific to the gustatory input that the bees experienced during training. Our results show that side differences in the bees' PE reaction time depend upon the activation of side-specific reward memories. These memories are formed via the combined effect of a specific property of reward, i.e., that its magnitude increases or decreases over time, and side information seemingly relying on mechanosensory input. We present a learning procedure suitable to study reward learning in honeybees, which includes precise behavioral measures, physiological correlates of behavior, and within-animal controls. This procedure will prove fruitful in pharmacological and electrophysiological analyses of the neural substrates underlying reward memories in honeybees.

The response of the honeybee dance to uncertain rewards.

This work focuses on the responses of dancing bees to uncertain rewards. We varied the distribution of a fixed amount of sugar solution among the several flowers of a patch and recorded the foraging and subsequent dance behaviour of single honeybees collecting such a reward at that patch. Concurrently, we aimed to minimize the well-known modulatory effects of sugar reward on both the probability and the strength of a honeybee's dance. It was under these circumstances that we conceived the honeybee dance as an autonomous information-processing system and asked whether or not such a system is sensitive to uncertainty of reward. Our results suggest that bees can tune their dancing according to the distribution of sugar reward among the several flowers of a patch, and that they seemingly do this based on the number - or the frequency - of their non-rewarding inspections to these flowers: the higher the number of non-rewarding inspections the lower the probability of dancing. As a result, a honeybee's dance appears as 'risk-averse', meaning that dances for uncertain resources are less likely. Presumably, the ultimate result of having 'risk-averse' dances is a colony's ability to diminish delayed rewards and the effects of competition with other flower visitors for limited resources. We conclude that a systems approach to the honeybee dance will help to further analyse the regulation of a honeybee's threshold for dancing, and that theoretical accounts of ;risk-sensitive' dances would prove fruitful in broader studies of honeybee foraging, particularly if one were to examine how recruitment actually translates into fitness.

Does an insect's unconditioned response to sucrose reveal expectations of reward?

We asked whether and how a sequence of a honeybee's experience with different reward magnitudes changes its subsequent unconditioned proboscis extension response (PER) to sucrose stimulation of the antennae, 24 hours after training, in the absence of reward, and under otherwise similar circumstances. We found that the bees that had experienced an increasing reward schedule extended their probosces earlier and during longer periods in comparison to bees that had experienced either decreasing or constant reward schedules, and that these effects at a later time depend upon the activation of memories formed on the basis of a specific property of the experienced reward, namely, that its magnitude increased over time. An anticipatory response to reward is typically thought of as being rooted in a subject's expectations of reward. Therefore our results make us wonder to what extent a long-term 'anticipatory' adjustment of a honeybee's PER is based upon an expectation of reward. Further experiments will aim to elucidate the neural substrates underlying reward anticipation in harnessed honeybees.

Variability in the encoding of spatial information by dancing bees.

A honeybee's waggle dance is an intriguing example of multisensory convergence, central processing and symbolic information transfer. It conveys to bees and human observers the position of a relatively small area at the endpoint of an average vector in a two-dimensional system of coordinates. This vector is often computed from a collection of waggle phases from the same or different dancers. The question remains, however, of how informative a small sample of waggle phases can be to the bees, and how the spatial information encoded in the dance is actually mapped to the followers' searches in the field. Certainly, it is the variability of a dancer's performance that initially defines the level of uncertainty that followers must cope with if they were to successfully decode information in the dance. Understanding how a dancer's behaviour is mapped to that of its followers initially relies on the analysis of both the accuracy and precision with which the dancer encodes spatial information in the dance. Here we describe within-individual variations in the encoding of the distance to and direction of a goal. We show that variations in the number of a dancer's wagging movements, a measure that correlates well with the distance to the goal, do not depend upon the dancer's travelled distance, meaning that there is a constant variance of wagging movements around the mean. We also show that the duration of the waggle phases and the angular dispersion and divergence of successive waggle phases co-vary with a dancer's orientation in space. Finally, using data from dances recorded through high-speed video techniques, we present the first analysis of the accuracy and precision with which an increasing number of waggle phases conveys spatial information to a human observer.

Learning reward expectations in honeybees.

The aim of this study was to test whether honeybees develop reward expectations. In our experiment, bees first learned to associate colors with a sugar reward in a setting closely resembling a natural foraging situation. We then evaluated whether and how the sequence of the animals' experiences with different reward magnitudes changed their later behavior in the absence of reinforcement and within an otherwise similar context. We found that the bees that had experienced increasing reward magnitudes during training assigned more time to flower inspection 24 and 48 h after training. Our design and behavioral measurements allowed us to uncouple the signal learning and the nutritional aspects of foraging from the effects of subjective reward values. We thus found that the animals behaved differently neither because they had more strongly associated the related predicting signals nor because they were fed more or faster. Our results document for the first time that honeybees develop long-term expectations of reward; these expectations can guide their foraging behavior after a relatively long pause and in the absence of reinforcement, and further experiments will aim toward an elucidation of the neural mechanisms involved in this form of learning.

Apis mellifera bees acquire long-term olfactory memories within the colony.

Early studies indicate that Apis mellifera bees learn nectar odours within their colonies. This form of olfactory learning, however, has not been analysed by measuring well-quantifiable learning performances and the question remains whether it constitutes a 'robust' form of learning. Hence, we asked whether bees acquire long-term olfactory memories within the colony. To this end, we used the bee proboscis extension response. We found that within-the-nest bees do indeed associate the odour (as the conditioned stimulus) with the sugar (as the unconditioned stimulus) present in the incoming nectar, and that the distribution of scented nectar within the colony allows them to establish long-term olfactory memories. This finding is discussed in the context of efficient foraging.

Spatial memory, navigation and dance behaviour in Apis mellifera.

Navigation and dance communication in Apis mellifera have been extensively studied on the level of sensory processing, but the structure and content of the spatial memory underlying such phenomena have yet to be addressed. Here we survey new findings indicating that the memory used by bees to navigate within the range of their orientation flights is much more complex than hitherto thought. It appears to allow them to decide between at least two goals in the field, and to steer towards them over considerable distances. Two models concerning the structure of bees' spatial memory are developed from new empirical evidence. The first one relies on the integration of at least two flight vectors, while the second assumes the existence of a 'functional' map based on the information available on-site. These findings also raise questions about the process of encoding and decoding information in the context of the waggle dance. We review published data and recent evidence indicating that memories of topographical features might also be involved in dance communication, and point out what needs to be addressed to elucidate the corresponding memory demands. The flight paths of recruited bees can now be traced by means of radar techniques, and thus tools are available to tackle these questions.