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The development of methods for the activity-dependent tagging of neurons enabled a new way to tackle the problem of engram identification at the cellular level, giving rise to groundbreaking findings in the field of memory studies. However, the resolution of activity-dependent tagging remains limited to the whole-cell level. Notably, events taking place at the synapse level play a critical role in the establishment of new memories, and strong experimental evidence shows that learning and synaptic plasticity are tightly linked. Here, we provide a comprehensive review of the currently available techniques that enable to identify and track the neuronal activity with synaptic spatial resolution. We also present recent technologies that allow to selectively interfere with specific subsets of synapses. Lastly, we discuss how these technologies can be applied to the study of learning and memory.

Fluorescent protein-based biosensors are indispensable molecular tools for life science research. The invention and development of high-fidelity biosensors for a particular molecule or molecular event often catalyze important scientific breakthroughs. Understanding the structural and functional organization of brain activities remain a subject for which optical sensors are in desperate need and of growing interest. Here, we review genetically encoded fluorescent sensors for imaging neuronal activities with a focus on the design principles and optimizations of various sensors. New bioluminescent sensors useful for deep-tissue imaging are also discussed. By highlighting the protein engineering efforts and experimental applications of these sensors, we can consequently analyze factors influencing their performance. Finally, we remark on how future developments can fill technological gaps and lead to new discoveries.

Activity recognition in live-cell imaging is labor-intensive and requires significant human effort. Existing automated analysis tools are largely limited in versatility. We present the Intelligent Vesicle Exocytosis Analysis (IVEA) platform, an ImageJ plugin for automated, reliable analysis of fluorescence-labeled vesicle fusion events and other burst-like activity. IVEA includes three specialized modules for detecting: (1) synaptic transmission in neurons, (2) single-vesicle exocytosis in any cell type, and (3) nano-sensor-detected exocytosis. Each module uses distinct techniques, including deep learning, allowing the detection of rare events often missed by humans at a speed estimated to be approximately 60 times faster than manual analysis. IVEA’s versatility can be expanded by refining or training new models via an integrated interface. With its impressive speed and remarkable accuracy, IVEA represents a seminal advancement in exocytosis image analysis and other burst-like fluorescence fluctuations applicable to a wide range of microscope types and fluorescent dyes. Activity recognition in live-cell imaging is laborious. Here, authors present, IVEA, a fully automated AI ImageJ plugin, that efficiently detects and classifies exocytosis events, from synaptic transmission to single-vesicle fusion, across cell types and imaging setups.

Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes. The authors present a fluorescent probe that tracks glutamine in live cells. They demonstrate the capabilities of the probe by providing direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions.

Microplastics (MP) are contaminants able to cause adverse effects on organisms. MPs are capable to interact with other environmental contaminants, including pesticides, altering their toxicity. The objective of the study was to research the sublethal effects (enzymatic activity) of pesticides alone and in combination with MPs. Cholinesterase enzymes are used as biomarkers to determine and evaluate the effects produced in organisms after exposure to pollutants. This study showed the acetylcholinesterase (AChE) enzymatic activity in the tissue of Solea senegalensis exposed to two environmental pollutants, the insecticide chlorpyrifos (CPF) and antibacterial triclosan (TCS) with and without microplastics (MPs). Solea senegalensis was chosen because it is a species in high demand because of its high economic value in southern Europe, as well as the use of this species in ecotoxicology and its increasing use as sentinel species, which justify using it to assess biological effects of pollutants. Toxicity tests were performed in organisms exposed to concentrations of between 5 and 80 μg/L CPF and 0.1 and 0.4 mg/L TCS for 96 h. In addition, each test incorporated MPs that were added at different concentrations in order to evaluate their role as a possible enhancer of the effects caused by the previous pollutants. In the case of CPF, the head and muscle tissue cholinesterase activity was inhibited from a concentration of 5 μg/L both without and with MPs, and the AChE enzymatic activity for the treatment with MPs was approximately half the activity for the treatment without MPs in the tissues studied. Besides, TCS inhibited the cholinesterase activity at a concentration of 0.3 mg/L in the muscle of S. senegalensis. In contrast, no significant differences were observed in the TCS + MP treatment compared to the controls. These results showed the importance of studies in assessing the anticholinesterase effects of pesticides combined with microplastics due to the abundance of these contaminants in the marine environment and the role of cholinesterase activity (biomarker) in the neurotransmission of key physiological processes.

Earthworms are key organisms of the soil ecosystem and bioindicators for soil quality. While pesticides are used for the improvement of crop yields, they also present a burden for soil organisms. To understand the complex effects of pesticides on soil organisms, it is important to test these effects in soil exposures to include influences of the soil matrix on the toxicity. Therefore, the aim of this study was the assessment of the effects pesticides on earthworm Eisenia andrei. In an initial screening, active ingredients and commercial preparations were tested for comparison. Since the commercial preparations showed a higher toxicity, all further investigations (biomarkers, multixenobiotic resistance (MXR) activity, and avoidance behavior) were performed using the commercial pesticide formulations only: Sumialfa (esfenvalerate), Calypso (thiacloprid), Frontier (dimethenamid-p), and Filon (prosulfocarb). Significant differences in avoidance behavior were observed for Filon and Frontier. All pesticides inhibited the MXR activity and affected oxidative stress-related markers. Frontier was the only pesticide that did not affect enzymatic biomarkers related to neurotransmission. The results show the potential hazards associated with the usage of the tested pesticides and the importance of evaluating the effects of commercial pesticide preparations for a more realistic insight into the adverse effects on the environment.

Styryl dyes and genetically encoded pH-sensitive fluorescent proteins like pHluorin are well-established tools for the optical analysis of synaptic vesicle (SV) recycling at presynaptic boutons. Here, we describe the development of a new class of fluorescent probes based on pH-sensitive organic dyes covalently bound to lipids, providing a promising complementary assay to genetically encoded fluorescent probes. These new optical tracers allow a pure read out of membrane turnover during synaptic activity and visualization of multiple rounds of stimulation-dependent SV recycling without genetic perturbation. Measuring the incorporation efficacy of different dye-labeled lipids into budding SVs, we did not observe an enrichment of lipids with affinity for liquid ordered membrane domains. But most importantly, we found no evidence for a static segregation of SVs into recycling and resting pools. A small but significant fraction of SVs that is reluctant to release during a first round of evoked activity can be exocytosed during a second bout of stimulation, showing fast intermixing of SV pools within seconds. Furthermore, we found that SVs recycling spontaneously have a higher chance to re-occupy release sites than SVs recycling during high-frequency evoked activity. In summary, our data provide strong evidence for a highly dynamic and use-dependent control of the fractions of releasable or resting SVs.

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) represent a promising young-state stem cell source for cell-based therapy. hUCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of hUCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that hUCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with hUCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of hUCMSCs in peripheral nerve repair.

The entry of calcium into dendritic spines can trigger a sequence of biochemical reactions that begins with the activation of calmodulin (CaM) and ends with long-term changes to synaptic strengths. The degree of activation of CaM can depend on highly local elevations in the concentration of calcium and the duration of transient increases in calcium concentration. Accurate measurement of these local changes in calcium is difficult because the spaces are so small and the numbers of molecules are so low. We have therefore developed a Monte Carlo model of intracellular calcium dynamics within the spine that included calcium binding proteins, calcium transporters and ion channels activated by voltage and glutamate binding. The model reproduced optical recordings using calcium indicator dyes and showed that without the dye the free intracellular calcium concentration transient was much higher than predicted from the fluorescent signal. Excitatory postsynaptic potentials induced large, long-lasting calcium gradients across the postsynaptic density, which activated CaM. When glutamate was released at the synapse 10 ms before an action potential occurred, simulating activity patterns that strengthen hippocampal synapses, the calcium gradient and activation of CaM in the postsynaptic density were much greater than when the order was reversed, a condition that decreases synaptic strengths, suggesting a possible mechanism underlying the induction of long-term changes in synaptic strength. The spatial and temporal mechanisms for selectivity in CaM activation demonstrated here could be used in other signaling pathways.

Spino-cerebral (projection) neurons localized in lamina I of the spinal gray substance play an important role in the transmission of pain-related information to the brain. We examined spontaneous excitatory postsynaptic currents (sEPSC) recorded from lamina I spino-pontine neurons in isolated preparations of the rat lumbar spinal cord; the respective neurons were retrogradely labeled by a fluorescent dye. We tried to find out how experimentally induced peripheral inflammation affects the amplitude/time characteristics of these currents. It was found that, in preparations obtained from animals with inflammation of hind limb tissues, the frequency and (to a lesser extent) amplitude of sEPSC in projection neurons are, on average, higher than those measured in neurons of the control animals. It is belived that such changes result mostly from plastic modifications of neuron-to-neuron interactions in neuronal networks of lamina II, which form main synaptic inputs to neurons of lamina I. Increased frequency and amplitude of sEPSC in lamina I neurons should lead to some facilitation of transmission of nociceptive information to the cerebral structures. Such hyperexcitability of lamina 1 projection neurons can provide a notable contribution to the development of hyperalgesia in chronic inflammatory states and to facilitation of generation of pain-related emotions.