Membrane contact sites (MCSs) are hubs allowing various cell organelles to coordinate their activities. The dynamic nature of these sites and their small size hinder analysis by current imaging techniques. To overcome these limitations, we here design a series of reversible chemogenetic reporters incorporating improved, low-affinity variants of splitFAST, and study the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrate that these versatile reporters suit different experimental setups well, allowing one to address challenging biological questions. Using these probes, we identify a pathway in which calcium (Ca²⁺) signalling dynamically regulates endoplasmic reticulum-mitochondria juxtaposition, characterizing the underlying mechanism. Finally, by integrating Ca²⁺-sensing capabilities into the splitFAST technology, we introduce PRINCESS (PRobe for INterorganelle Ca²⁺-Exchange Sites based on SplitFAST), a class of reporters to simultaneously detect MCSs and measure the associated Ca²⁺ dynamics using a single biosensor.

Recognition memory is the capacity to recognize previously encountered objects, events or places. This ability is crucial for many fitness-related activities, and it appears very early in the development of several species. In the laboratory, recognition memory is most often investigated using the novel object recognition test (NORt), which exploits the tendency of most vertebrates to explore novel objects over familiar ones. Despite that the use of larval zebrafish is rapidly increasing in research on brain, cognition and neuropathologies, it is unknown whether larvae possess recognition memory and whether the NORt can be used to assess it. Here, we tested a NOR procedure in zebrafish larvae of 7-, 14- and 21-days post-fertilization (dpf) to investigate when recognition memory first appears during ontogeny. Overall, we found that larvae explored a novel stimulus longer than a familiar one. This response was fully significant only for 14-dpf larvae. A control experiment evidenced that larvae become neophobic at 21-dpf, which may explain the poor performance at this age. The preference for the novel stimulus was also affected by the type of stimulus, being significant with tri-dimensional objects varying in shape and bi-dimensional geometrical figures but not with objects differing in colour. Further analyses suggest that lack of effect for objects with different colours was due to spontaneous preference for one colour. This study highlights the presence of recognition memory in zebrafish larvae but also revealed non-cognitive factors that may hinder the application of NORt paradigms in the early developmental stages of zebrafish.

The development of anxiety disorders is often linked to individuals’ negative experience. In many animals, development of anxiety‐like behavior is modeled by manipulating individuals’ exposure to environmental enrichment. We investigated whether environmental enrichment during early ontogenesis affects anxiety‐like behavior in larval zebrafish. Larvae were exposed from hatching to either an environment enriched with 3D‐objects of different color and shape or to a barren environment. Behavioral testing was conducted at different intervals during development (7, 14, and 21 days post‐fertilization, dpf). In a novel object exploration test, 7 dpf larvae of the two treatments displayed similar avoidance of the visual stimulus. However, at 14 and 21 dpf, larvae of the enriched environment showed less avoidance, indicating lower anxiety response. Likewise, larvae of the two treatments demonstrated comparable avoidance of a novel odor stimulus at 7 dpf, with a progressive reduction of anxiety behavior in the enriched treatment with development. In a control experiment, larvae treated before 7 dpf but tested at 14 dpf showed the effect of enrichment on anxiety, suggesting an early determination of the anxiety phenotype. This study confirms a general alteration of zebrafish anxiety‐like behavior due to a short enrichment period in first days of life.

Biological and living systems process information across spatiotemporal scales, exhibiting the hallmark ability to constantly modulate their behavior to ever-changing and complex environments. In the presence of repeated stimuli, a distinctive response is the progressive reduction of the activity at both sensory and molecular levels, known as habituation. In this work, we solve a minimal microscopic model devoid of biological details, where habituation to an external signal is driven by negative feedback provided by a slow storage mechanism. We show that our model recapitulates the main features of habituation, such as spontaneous recovery, potentiation, subliminal accumulation, and input sensitivity. Crucially, our approach enables a complete characterization of the stochastic dynamics, allowing us to compute how much information the system encodes on the input signal. We find that an intermediate level of habituation is associated with a steep increase in information. In particular, we are able to characterize this region of maximal information gain in terms of an optimal trade-off between information and energy consumption. We test our dynamical predictions against experimentally recorded neural responses in a zebrafish larva subjected to repeated looming stimulations, showing that our model captures the main components of the observed neural habituation. Our work makes a fundamental step towards uncovering the functional mechanisms that shape habituation in biological systems from an information-theoretic and thermodynamic perspective.

The effects of an early impoverished social or physical environment on vertebrate neural development and cognition has been known for decades. While existing studies have focused on the long-term effects, measuring adult cognitive phenotypes, studies on the effects of environmental complexity on the early stages of development are lacking. Zebrafish (Danio rerio) hatchlings are assumed to have minimal interaction with their environment and are routinely reared in small, bare containers. To investigate the effects of being raised under such conditions on development of behaviour and cognition, hatchlings housed for 10 days in either an enriched or a standard environment underwent two cognitive tasks. The results were mixed. Subjects of the two treatments did not differ in performance when required to discriminate two areas. Conversely, we found a significant effect in a number discrimination task, with subjects from impoverished condition performing significantly worse. In both experiments, larvae reared in impoverished environment showed a reduced locomotor activity. Given the effects that enrichment appears to exert on larvae, a third experiment explored whether hatchlings exhibit a spontaneous preference for more complex environments. When offered a choice between a bare setting and one with objects of different shapes and colors, larvae spent over 70% of time in the enriched sector. Deepening these effects of an early impoverished environment on cognitive development is crucial for the welfare of captive zebrafish populations and for enhancing the quality and reliability of studies on larval zebrafish.