The editorial offers principles of everyday leadership, with hope for the new year.

Valentina Emiliani, CNRS research director at the Vision Institute in Paris, discusses her pioneering work in neurophotonics, in an interview with fellow Neurophotonics Editorial Board Member Amanda Foust, Senior Lecturer in Biomedical Engineering at University College London.

Rats are used in neuroscience research because of their physiological similarities with humans and accessibility as model organisms, trainability, and behavioral repertoire. In particular, rats perform a wide range of sophisticated social, cognitive, motor, and learning behaviors within the contexts of both naturalistic and laboratory environments. Further progress in neuroscience can be facilitated by using advanced imaging methods to measure the complex neural and physiological processes during behavior in rats. However, compared with the mouse, the rat nervous system offers a set of challenges, such as larger brain size, decreased neuron density, and difficulty with head restraint. Here, we review recent advances in in vivo imaging techniques in rats with a special focus on open-source solutions for calcium imaging. Finally, we provide suggestions for both users and developers of in vivo imaging systems for rats.

The ability to accurately map and manipulate the dynamic subcellular distribution of proteins is key for a mechanistic understanding of neuronal functioning. Current fluorescence microscopy techniques provide access to subcellular protein organization at increasing resolution but are often restricted by the availability of methods that reliably label endogenous proteins. Excitingly, recent development in CRISPR/Cas9 genome editing now allows researchers to specifically tag and visualize endogenous proteins, overcoming limitations associated with current labeling strategies. This article will discuss the progress that has been made in the last years that has led to the development of CRISPR/Cas9 genome editing tools for the reliable mapping of endogenous proteins in neurons. Furthermore, recently developed tools enable the duplex labeling of two proteins simultaneously and acute manipulation of protein distribution. Future implementations of this generation of genome editing technologies will undoubtedly drive progress in molecular and cellular neurobiology.

Membrane trafficking of post-synaptic cargo is a key determinant of synaptic transmission and synaptic plasticity. We describe here the latest developments in visualizing individual exocytosis and endocytosis events in neurons using pH-sensitive tags. We show how these tools help decipher the spatial and temporal regulation of membrane trafficking steps during synaptic plasticity.

Machine learning has revolutionized the way data are processed, allowing information to be extracted in a fraction of the time it would take an expert. In the field of neurophotonics, machine learning approaches are used to automatically detect and classify features of interest in complex images. One of the key challenges in applying machine learning methods to the field of neurophotonics is the scarcity of available data and the complexity associated with labeling them, which can limit the performance of data-driven algorithms. We present an overview of various strategies, such as weakly supervised learning, active learning, and domain adaptation that can be used to address the problem of labeled data scarcity in neurophotonics. We provide a comprehensive overview of the strengths and limitations of each approach and discuss their potential applications to bioimaging datasets. In addition, we highlight how different strategies can be combined to increase model performance on those datasets. The approaches we describe can help to improve the accessibility of machine learning-based analysis with limited number of annotated images for training and can enable researchers to extract more meaningful insights from small datasets.

Despite decades of research on the noradrenergic system, our understanding of its impact on brain function and behavior remains incomplete. Traditional recording techniques are challenging to implement for investigating in vivo noradrenergic activity, due to the relatively small size and the position in the brain of the locus coeruleus (LC), the primary location for noradrenergic neurons. However, recent advances in optical and fluorescent methods have enabled researchers to study the LC more effectively. Use of genetically encoded calcium indicators to image the activity of noradrenergic neurons and biosensors that monitor noradrenaline release with fluorescence can be an indispensable tool for studying noradrenergic activity. In this review, we examine how these methods are being applied to record the noradrenergic system in the rodent brain during behavior.

Neurophotonic approaches have fostered substantial progress in our understanding of the brain by providing an assortment of means to either monitor or manipulate neural processes. Among these approaches, the development of two-photon uncaging provides a useful and flexible approach to manipulate the activity of individual synapses. In this short piece, we explore how this technique has emerged at the intersection of chemistry, optics, and electrophysiology to enable spatially and temporally precise photoactivation for studying functional aspects of synaptic transmission and dendritic integration. We discuss advantages and limitations of this approach, focusing on our efforts to study several functional aspects of glutamate receptors using uncaging of glutamate. Among other advancements, this approach has contributed to further our understanding of the subcellular regulation, trafficking, and biophysical features of glutamate receptors (e.g., desensitization and silent synapse regulation), the dynamics of spine calcium, and the integrative features of dendrites, and how these functions are altered by several forms of plasticity.

The increasingly widespread use of calcium imaging to explore the nature of neuronal activity and circuits has unexpectedly revealed the ubiquitous presence and significance of astrocytic activity. Here, we present a brief review of visual system development, placing it in the context of recently identified roles of astrocytes in the modulation of neuronal responses and circuit plasticity, through their responses to sensory stimuli and the release of gliotransmitters.

In the past two decades, digital brain atlases have emerged as essential tools for sharing and integrating complex neuroscience datasets. Concurrently, the larval zebrafish has become a prominent vertebrate model offering a strategic compromise for brain size, complexity, transparency, optogenetic access, and behavior. We provide a brief overview of digital atlases recently developed for the larval zebrafish brain, intersecting neuroanatomical information, gene expression patterns, and connectivity. These atlases are becoming pivotal by centralizing large datasets while supporting the generation of circuit hypotheses as functional measurements can be registered into an atlas’ standard coordinate system to interrogate its structural database. As challenges persist in mapping neural circuits and incorporating functional measurements into zebrafish atlases, we emphasize the importance of collaborative efforts and standardized protocols to expand these resources to crack the complex codes of neuronal activity guiding behavior in this tiny vertebrate brain.

Brain and gut barriers have been receiving increasing attention in health and diseases including in psychiatry. Recent studies have highlighted changes in the blood–brain barrier and gut barrier structural properties, notably a loss of tight junctions, leading to hyperpermeability, passage of inflammatory mediators, stress vulnerability, and the development of depressive behaviors. To decipher the cellular processes actively contributing to brain and gut barrier function in health and disease, scientists can take advantage of neurophotonic tools and recent advances in super-resolution microscopy techniques to complement traditional imaging approaches like confocal and electron microscopy. Here, we summarize the challenges, pros, and cons of these innovative approaches, hoping that a growing number of scientists will integrate them in their study design exploring barrier-related properties and mechanisms.

Imaging in the freely moving animal gives unparalleled access to circuit activity as the animal interacts with its environment in a self-guided way. Over the past few years, new imaging technologies have enabled the interrogation of neuronal populations located at any depth of the cortex in freely moving mice while preserving the animal’s behavioral repertoire. This commentary gives an updated overview of the recent advances that have enabled the link between behavior and the underlying neuronal activity to be explored.

Accurate capture of animal behavior and posture requires the use of multiple cameras to reconstruct three-dimensional (3D) representations. Typically, a paper ChArUco (or checker) board works well for correcting distortion and calibrating for 3D reconstruction in stereo vision. However, measuring the error in two-dimensional (2D) is also prone to bias related to the placement of the 2D board in 3D. We proposed a procedure as a visual way of validating camera placement, and it also can provide some guidance about the positioning of cameras and potential advantages of using multiple cameras. We propose the use of a 3D printable test object for validating multi-camera surround-view calibration in small animal video capture arenas. The proposed 3D printed object has no bias to a particular dimension and is designed to minimize occlusions. The use of the calibrated test object provided an estimate of 3D reconstruction accuracy. The approach reveals that for complex specimens such as mice, some view angles will be more important for accurate capture of keypoints. Our method ensures accurate 3D camera calibration for surround image capture of laboratory mice and other specimens.

The erratum corrects errors in Fig. 3 and Table 2 of the original published article.