Frontiers in Synaptic Neuroscience

Super-resolution microscopy approaches have revealed that synaptic proteins are not randomly distributed within individual synapses. Rather, they form sub-synaptic clusters that may be coordinated between the synaptic compartments, thereby forming transsynaptic nanocolumns. However, the functional significance of this nano-architecture remains under debate. In particular, it is unclear whether the positions of synaptic proteins optimize a specific cost function. Here, we present a normative modeling framework that integrates synaptic nano-architecture imaged with STED and dSTORM microscopy with synaptic transmission recorded with electrophysiology at the Drosophila neuromuscular junction (NMJ). Model-based quantification showed that clusters of Unc13A, which likely demark presynaptic release sites, and postsynaptic glutamate receptors (GluRs) adopt aligned ring-like arrangements in individual synapses. We developed an analytical description of GluR activation relative to presynaptic release position, enabling to link super-resolution imaging data with recorded postsynaptic current amplitudes without relying on costly Markov chain Monte Carlo (MCMC) simulations. Our model predicts that glutamate released from a single vesicle spreads across the entire receptor population. Given this spread, the postsynaptic current amplitude is maximized when glutamate is released close to the active zone (AZ) center, in contradiction to the observed offset of Unc13A clusters. However, the observed relative positions of Unc13A and GluR clusters maximize the postsynaptic current amplitude and minimize the delay between vesicle release and receptor activation during multivesicular release. Hence, transsynaptically-aligned Unc13A-GluR rings optimize for multivesicular but not univesicular release. Given that multivesicular release is mainly observed during enhanced presynaptic release at the Drosophila NMJ, transsynaptic nano-architecture may be adapted to elevated presynaptic release states.

The small GTPase Rem2 is a key signaling molecule involved in synaptic formation, dendritic complexity, spine formation, and regulation of CaMKII-dependent long-term potentiation (LTP). However, it remains unclear how Rem2 explicitly impacts learning and memory. To address this issue, we deleted Rem2 specifically in dorsal CA1 neurons of male and female mice and assessed spatial learning using a spatial object recognition (SOR) task. We found that males outperform females in this behavioral assay and that deleting Rem2 did not impact spatial learning in males. In contrast, Rem2 deletion significantly improved spatial learning in females enabling them to perform as well as males in the SOR task. Using an automated Western blot system to assess all known AMPA- and NMDA-mediated glutamate receptor (AMPAR and NMDAR) subunits in each mouse, we found that the sex-dependent change in SOR we observed was likely due to Rem2 increasing GluN2D expression in the interneurons of female mice only. Furthermore, using S-statistics to compare the overall AMPAR and NMDAR composition among groups, we found that males and females normally have divergent covariance structures but this divergence is eliminated when Rem2 is deleted from CA1 neurons. These results reveal an unexpected role of Rem2 in maintaining sex differences in glutamate receptor composition and spatial learning abilities. To our knowledge, this is the first demonstration of a signaling molecule that confers sexual dimorphism to excitatory synapses. As such, Rem2 may play a critical role in understanding how sex-dependent symptoms of neurodevelopmental and neurodegenerative disorders arise.

For more than a century we have pictured the dendritic spine as the postsynaptic point where a single presynaptic bouton delivers its message. That picture is correct but incomplete, since it overlooks other neuronal elements surrounding each spine. Here we characterize that neighborhood for the first time by counting and measuring the proximity of the elements encircling each postsynaptic spine. We call this neighborhood the vicinome: the full set of neuronal and glial elements surrounding a dendritic spine. We used ground-truth three-dimensional meshes from the H01 petavoxel dataset, a reconstruction of the human cerebral cortex. We analyzed 4550 neighboring elements across 322 spines from 17 pyramidal neurons in cortical layers 2 through 5, with each spine classified as apical or basal. Neighbor number followed a depth gradient, falling from 17.2 {+/-} 4.2 elements per spine in layer 2 apical spines to 12.5 {+/-} 3.1 in layer 5 basal spines. A linear mixed model with a random intercept per neuron confirmed a strong effect of layer (p = 4.3 x 10-5) with large effect sizes (Cohen's d up to 1.45) and no effect of polarity. The minimum spine-to-neighbor distance stayed uniform, near 30 nm in every group, and although some depth-related contrasts were significant, all effect sizes were negligible (Cohen's d below 0.21). We conclude that cortical depth, and not pyramidal neuron polarity, was related to the number of neuronal elements neighboring the spine. We suggest that vicinome crowding could offer a measurable observable for comparison across regions, ages, species, and disease.

The endocytic pathway plays key roles in facilitating and regulating synaptic plasticity, but how its components are organized at spines undergoing plasticity is not fully understood. Here we use live-cell imaging and single-particle tracking to examine the localization of endosomes and lysosomes in response to plasticity-inducing stimuli. We find that endosomes and lysosomes are positioned and retained near the bases of stimulated spines after the induction of structural plasticity in a manner dependent on actin polymerization in the dendritic shaft. Spines with endocytic compartments near their bases exhibit greater glutamate receptor turnover and more sustained enlargement after stimulation than spines lacking them. These results suggest that actin polymerization in the dendritic shaft acts as a hub to organize critical cellular machinery at the bases of spines undergoing synaptic plasticity.

Volumetric imaging is increasingly important in fields such as neuroscience, where quantifying structure and mapping features are inherently three-dimensional problems. Block-face serial two-photon tomography (STPT) has emerged as a powerful approach for high-resolution, whole-organ volumetric imaging. STPT avoids complex sample preparation and balances image quality, speed, and scalability. However, STPT remains niche, in part because of the absence of flexible, general-purpose control software. Here we present BakingTray, an open-source software platform for STPT that extends ScanImage. BakingTrays modular framework accommodates diverse hardware configurations and allows users to build systems customised to their needs. Twelve such systems, which we call BrainSaws, have been constructed worldwide since 2016, with more under construction, and have so far contributed to 35 research papers. The three systems at the Sainsbury Wellcome Centre alone have acquired over 7,000 samples in a busy multi-user facility. We demonstrate the platform across its core neuroscience applications: electrode track localisation, brain-wide cell counting, and bulk projection mapping. Beyond these, we map the cell-type-specific synaptic output of inhibitory neurons in the ventral lateral geniculate nucleus and, with whole-mount staining, amyloid plaques brain-wide in a mouse model of Alzheimers disease. BakingTray is a mature platform for groups or facilities needing a customised volumetric imaging solution, whether built new or converted cheaply from an existing two-photon microscope.

Hippocampal sharp-wave ripple (SWR) events occur during post-experience rest and sleep and play a significant role in memory consolidation. Recent ex vivo studies have found orexin-driven neuromodulatory effects on SWR parameters and proposed that they can influence memory consolidation. Additionally, behavioral studies demonstrate contradictory results regarding orexinergic effects on memory acquisition depending on task structure and intervention time. This inconsistency, along with the diffuse spread of orexin receptors (OXR) in the CA1 and CA3 hippocampus, challenges theoretical interpretation of orexinergic effects on memory. To reconcile this problem, we propose a rate-based model linking orexinergic tone-dependent SWR statistics to memory performance. The model combines an encoding module whose gain scales with the global orexinergic tone with a consolidation module driven by OX2R-specific replay dynamics. The model is based on a plasticity rule that considers SWR events as the unit of induction. Therefore, weight change requires sufficient replay content and depolarizing drive. We observed that, depending on the task structure, encoding-dominant learning benefits discrimination as the orexin drive increases. Consolidation-dominant learning shows a non-monotonic relationship with an internal optimum level of orexinergic tone. This interaction highlights that stronger truncation at higher receptor engagement shortens memory replay and reduces the efficacy of the consolidation process. The model predicts that the optimal regime depends on task complexity reflected in sequence length. Together, these results demonstrate how task demands determine whether increased orexinergic drive enhances or impairs effective consolidation and SWR-dependent replay in the hippocampus.

Abstract The neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) controls behaviours that are essential for the parasitic lifestyle of plant parasitic nematodes (PPNs). Here, we used serotonin immunochemistry and in situ hybridization for the serotonin synthesis genes, Gpa-tph-1 and Gpa-bas-1, to map its signalling pathways in Globodera pallida J2 infective stages. We identified fourteen serotonin-immunoreactive cell bodies around the pharyngeal median bulb and revealed that eleven of them co-express Gpa-tph-1 and Gpa-bas-1, confirming these are serotonin-synthesising neurons. Serotonin immunoreactivity was also localised in structures consistent with labelling of amphids, and in sixteen non-neuronal cells located in the middle and posterior region of the nematode. Depletion of serotonin with reserpine reduced the serotonin immunoreactivity in all fourteen cell bodies confirming their neuronal identity, but not in non-neuronal tissue. We identified additional, but infrequent expression of Gpa-tph-1 in two cells and expression of Gpa-bas-1 in one pair of symmetrically bilateral cells anterior to the median bulb, that lacked serotonin immunoreactivity. The results reveal distinct pools of serotonin distribution in G. pallida and indicate serotonin signalling is more complex than in other parasitic nematodes and C. elegans, highlighting evolutionary divergence in neurotransmitter identity.

Sleep disorders and wakefulness constitute a pronounced and pervasive health risk that contributes to numerous cerebral pathologies such as stroke, depression, and neurodegenerative disorders. The hippocampus (HC) plays vital roles in learning and memory and is especially susceptible to the pathological insults imposed by sleep deprivation (SD). While SD disrupts neuronal network signaling and triggers inflammatory processes, the impact of SD on metabolism within the HC presently remains poorly understood. In this study, we assessed the progressive and cumulative effects of partial SD in the live mouse HC CA1 region using two-photon microscopy. Using a custom-designed implantable cannula enabling direct HC visualization and administration of dyes and pharmacological agents directly to HC tissue, we longitudinally monitored spontaneous neuronal activity and partial pressure of oxygen (pO2) within HC CA1 tissue before and during a 7-day period of partial SD and after a 7- day period of recovery. Our results revealed a progressive decline in spontaneous neural activity, alongside sustained elevation in tissue pO2; during the 7-day partial SD period. Notably, after 7 days of recovery, neuronal activity did not return to baseline, and tissue oxygenation continued to increase. Our findings emphasize chronic SD's cumulative and enduring impairments to neuronal function and metabolism in the HC and reveal uncoupling between metabolic and functional recovery that outlasts the sleep restriction period.

Nanopore sensors have transformed single-molecule analysis, enabling real-time detection of biomolecules with unprecedented resolution. Understanding and modelling noise in nanopore sensing is essential to unlocking their full analytical potential. Although mechanistic models of nanopore noise exist, a rigorous statistical framework that characterises noise structure across diverse experimental conditions is still lacking. Here, we present a comprehensive empirical analysis of nanopore noise using twelve diverse experimental conditions acquired with different nanopores, analytes, and electronic recording systems. By employing multiple, mutually reinforcing statistical methods, we establish that nanopore noise is well described by a composite fractional model comprising multiple fractional Gaussian noise and fractional Brownian motion components. This finding is supported by confirmation of Gaussianity, rigorous interpretation of second-order exponents, multifractal analysis, and evidence that transient deviations are attributable to deterministic or experimental artefacts rather than alternative stochastic mechanisms. We further resolve quasi-deterministic structures, such as baseline trends and structural breaks, and demonstrate their separability from the underlying stochastic profile. Collectively, these results provide the first formal statistical validation of a composite fractional model for nanopore noise and establish a principled basis for noise reconstruction, realistic simulation, and statistically grounded algorithm design for nanopore sensing applications. This study provides the foundation for the creation of high-fidelity performance evaluation datasets for signal processing, and the controlled generation and augmentation of data for machine learning while maintaining statistical fidelity to experimental conditions.

Extracellular vesicles (EVs) play a critical role in the propagation of Tau pathology in Alzheimer's disease (AD). The selective identification and characterization of Tau seed-carrying EVs would help developing new diagnostic tools and therapeutic strategies. While pathogenic forms of Tau can be detected biochemically in EV-enriched fractions, only super-resolution fluorescence imaging provides both morphological and compositional information at the level of single EVs on account of their small size and the low number of target molecules. This was the object of our study. Post-mortem human brain-derived EVs from AD patients and non-demented control individuals were characterized in depth using biochemistry, transmission electron microscopy (TEM) and single molecule localization microscopy (SMLM). Dual-colour SMLM of immobilized EVs, displaying size distributions that were consistent with TEM, enabled statistically meaningful measurements of thousands of EVs in parallel. Owing to the high spatial resolution and capability of SMLM to detect sparse epitopes, we identified pathological Tau proteins in individual EVs from AD samples. Reconstructed super-resolution images of permeabilized EVs demonstrate that pathological Tau forms are frequent cargo proteins of EVs from AD brains. Our data illustrate the power of SMLM to determine the ultrastructure, molecular composition and topology of single EVs and EV populations.

Site-specific recombinases, such as Cre recombinase (Cre) and FLP recombinase (FLPase), are popular tools that enable precise spatiotemporal control over fluorescent reporter genes. However, studies show that increased Cre expression can lead to cytotoxic phenotypes including DNA damage, cell cycle arrest, cell death, and aneuploidy. In contrast, there is little-to-no known cytotoxicity associated with FLPase. FLPase is considered to be the safer, if less efficient, recombinase. To compensate for decreased efficiency, researchers will often increase expression of the FLPase to ensure a complete gene edit. Despite previous evidence that FLPase is nontoxic at high doses, we report that high expression of FLPase from multi-copy extrachromosomal arrays can disrupt synaptic protein trafficking in C. elegans. We discovered several off-target effects of high FLPase expression including mislocalization of the pre-synaptic marker FLPon::mScarlet::SYD-2 and decreased synaptic abundance, transport, and expression of the ionotropic glutamate receptor subunit GLR-1. Neither overexpression of an inactive FLPase, low activity FLPase, nor mCherry under the same promoter elicited these effects. However, single-copy expression of the FLPase construct reliably tags FLPon::mScarlet::SYD-2 and does not lead to these off-target effects. This work supports growing awareness that tyrosine recombinases can become cytotoxic at high doses.

Microlasers based on whispering-gallery modes (WGMs) provide bright, spectrally narrow emission, making them ideal optical probes for high-resolution cellular tagging, single-cell tracking, and biosensing. To reproducibly apply WGM microlasers in biomedical or tissue-engineering contexts, control over the number of lasers in a cell population is highly desirable. Here we use a commercial instrument for flow cytometry-based cell sorting to obtain HEK293T cell populations with a controlled number of polystyrene laser particles (LPs). By modifying the particle surface with poly(ethyleneimine) (PEI) or lipofectamine, we enhance LP-association to cells. We demonstrate that our sorting procedure yields viable cells showing unchanged proliferation rates. Manufacturer-dependent differences in particle-cell interactions were observed and investigated by scanning and transmission electron microscopy (SEM/TEM) and flow cytometry. Finally, we show that the same sorting approach enables the generation of multi-cellular spheroids with a defined ratio of LP-containing and LP-free cells. This work lays the foundation for larger tissue engineering studies with defined LP populations and control over LP-placement with respect to the cell.

Adolescence is characterized by increased novelty seeking, impulsivity, locomotion and risk-taking, behaviours that facilitate the transition to adulthood but may also increase vulnerability to psychiatric illness. Maturation of dopamine (DA) transmission within the striatum is thought to contribute to these behavioural changes. However, the developmental trajectory of striatal DA release remains unclear, with conflicting evidence regarding age- and sex-dependent changes, particularly across dorsal and ventral striatal regions. Complicating interpretation, many studies classify mice younger than 2 months as 'adults,' potentially obscuring important developmental differences. Here, we examined how age, sex and striatal subregion influence DA release during adolescence and early adulthood. DA release was measured in dorsal and ventral striatal brain slices from male and female C57BL/6J mice at 2 and 4 months of age, corresponding to adolescence and young adulthood. Ultrafast imaging of the genetically encoded DA sensor, dLight, was used to quantify extracellular DA transients evoked by low- and high-frequency electrical stimulation. Glutamatergic and cholinergic transmission was pharmacologically blocked to isolate DA release intrinsic to dopaminergic axons. DA release measures were related to exploratory and locomotor behaviour. We identified a male-specific increase in dorsal striatal DA release between 2 and 4 months of age that coincided with increased locomotion and risk-taking. In contrast, DA release in the ventral striatum did not show comparable age- or sex-dependent changes. These differences were not attributable to altered glutamatergic or cholinergic contributions to DA release. Together, these findings demonstrate that maturation of striatal DA transmission is region- and sex-dependent, with prominent developmental changes occurring in the dorsal striatum during the transition from adolescence to adulthood. These findings help reconcile conflicting reports of developmental DA release and emphasize the importance of considering age, sex and striatal subregion when defining mature dopaminergic function in rodents.

Cell type designations label uniquely-identifiable groups of related cells that typically function in similar ways. Cell types divide the Drosophila melanogaster central nervous system into functional units, but remain challenging to target and identify. Previously we generated a driver line and image resource for targeting many neuron types (Meissner et al., 2025). Here we improve on identifying the targeted neurons by automated searching against electron microscopy datasets and manual scoring of search results to add 6,368 annotations across 1,772 driver lines in the collection. These annotations provide a potential training resource for future search algorithms. We then estimate the number of cell types and thus the fraction of the central nervous system labeled by the collection as a whole.

Background: Nervous system regeneration requires creation of new neurons and their integration into new and existing networks. How new neurons find their targets and reconnect in the environment of regenerating tissue remains a major gap in knowledge. The planarian Schmidtea mediterranea is well suited to the study of neural regeneration due to the remarkable ability of planarians to regenerate their entire body, including the nervous system. However, we have not yet been able to explore synaptogenesis in regeneration using planarians due to a lack of tools to study synapses. Here we investigate potential players in planarian synapse, focusing on the conserved syntaxin protein family, which functions in neurotransmitter vesicle fusion at the synapse. Results: We found that reduction of either syntaxin-1 (stx1) or syntaxin-binding protein 1-1 (stxbp1-1) decreased locomotion and impeded brain regeneration, specifically in synapse-rich regions. Conclusions: Our observations support STX-1 and STXBP1-1 as bona fide synapse components, providing a starting point for studies neuronal connectivity during homeostatic remodeling and regeneration.

Abstract The importance of hypothalamic kisspeptin (Kiss1) neurons in controlling the reproductive axis is well established, while a population of extra-hypothalamic Kiss1 neurons is found in the medial amygdala (MeA) of both males and females in rodents and is less well understood. The MeA is connected with the olfactory bulbs, the hypothalamus and cortical areas. MeA Kiss1 neurons have been implicated in sexual behaviour and modulation of the reproductive axis; however, little is known about their action potential firing properties, which would influence kisspeptin release in target brain regions, nor whether their physiological properties are sex- or estrous cycle-dependent. In this study, whole-cell patch-clamp recordings were made from Kiss1 neurons in brain slices containing the MeA of adult female (estrous and diestrous) and male mice. MeA Kiss1 (Kiss1MeA) neurons were sexually dimorphic, with greater numbers in male compared to female mice and a slower change in excitability in estrous females compared with males. However, combining a range of electrophysiological parameters using principal component analysis did not reveal a distinct sex- or estrous cycle-linked phenotype, suggesting that the properties of Kiss1MeA neurons- and therefore kisspeptin release- are broadly similar. Interestingly, Kiss1MeA neurons had unique electrophysiological properties compared with anteroventral periventricular (AVPV) hypothalamic Kiss1 (Kiss1AVPV) neurons in female mice; with their reduced excitability, achieving action potential firing frequencies greater than 10Hz required a larger current input, indicating that Kiss1MeA neurons require stronger synaptic input to achieve firing frequencies that drive kisspeptin release. These findings provide new information about the physiological properties of Kiss1MeA neurons, showing subtle sex differences and profound regional differences between the MeA and AVPV.

Two-photon calcium imaging enables measurements of neural activity from individual neurons and their subcellular processes in awake animals. Axial brain motion can change measured fluorescence independently of neural activity and move small structures out of the imaging plane, confounding two-photon calcium measurements in behaving animals. We developed two complementary post-acquisition methods, selected according to whether the imaged structure remains detectable across the encountered depth range. When it does, z-stack correction estimates imaging depth from a separately acquired reference z-stack and uses an axial fluorescence profile specific to each neural structure to rescale fluorescence to a common reference depth. When a structure can leave a single imaging plane, volume-based z-registration reconstructs a common-depth movie from repeated acquisitions of closely spaced planes. In awake mice, both methods tracked experimentally imposed axial displacements within the sampled range, with depth-estimation root mean squared errors (RMSEs) below 1 m for z-stack correction and 0.69-1.03 m for volume-based z-registration. Correction changed fluorescence substantially for a subset of somata, with larger effects in axonal boutons. Both methods are implemented in suite2p-in-depth, a Python package that integrates with Suite2p and provides diagnostics for depth estimation, correction quality and data reliability. Together, these methods enable axial-motion artifacts to be assessed and corrected within established Suite2p workflows.

Hypothalamic supramammillary nucleus (SuM) neurons project to the dentate gyrus and co-release glutamate and GABA, thereby regulating hippocampal information processing. However, how synapses mediating glutamate/GABA co-transmission are established during development remains unclear. Here, we investigated the functional and structural maturation of SuM inputs onto adult-born granule cells (GCs). We found that most newborn GCs received GABAergic responses without detectable glutamatergic responses, whereas a smaller population exhibited glutamate/GABA co-transmission. Newborn GCs with glutamate/GABA co-transmission showed more mature anatomical and physiological properties than GABA-only neurons, with greater dendritic complexity, longer dendritic length, lower input resistance, and stronger medial perforant path inputs. Postsynaptic depolarization failed to induce synapse unsilencing in GABA-only neurons but induced robust long-term potentiation in newborn GCs with glutamatergic transmission, indicating that plasticity at SuM inputs emerges after functional glutamatergic transmission is established. Morphological analysis showed that SuM terminals apposed to newborn GCs contained both VGluT2 and VIAAT, whereas postsynaptic receptor organization remained immature. Moreover, environmental enrichment and chemogenetic activation of SuM neurons increased the proportion of newborn GCs receiving glutamate/GABA co-transmission, suggesting that SuM activity promotes the emergence of glutamatergic transmission. Together, these results suggest that SuM inputs onto newborn GCs undergo sequential maturation from initial GABAergic transmission to glutamate/GABA co-transmission, a process likely supported by SuM activity-dependent postsynaptic maturation.

Axons of substantia nigra dopaminergic neurons "die back" in Parkinson's disease (PD), exhibiting early degeneration relative to cell bodies, a key feature also observed in PD models. A critical step towards understanding the process of axonal neurodegeneration is to determine the physiological properties of dopaminergic axons that are altered during the early stages of cellular dysfunction. Here, we compared the electrophysiology and calcium dynamics of dopaminergic axons in the MitoPark model of PD with age-matched controls. Our experiments show a pronounced reduction in action potential (AP)-evoked calcium signals in MitoPark axons that emerges well before the onset of dopaminergic fiber loss and motor deficits. In addition, calcium imaging and direct axon recording revealed deficits in AP propagation accompanied by alterations in the axonal AP waveform. Therefore, our experimental findings identify the progression of axon-specific physiological deficits that occur long before neurodegeneration, more precisely defining the period that precedes axonal loss.

Dopamine D1 receptors (D1R) and muscarinic M4 receptors (M4R) regulate overlapping functions in the cortex and the striatum, but the cellular relationship between D1R- and M4R-associated populations across brain regions remains incompletely defined. Using D1R-tdTomato/M4R-EGFP double-reporter mice, we quantified tdTomato-only, EGFP-only, and coexpressing cellular populations across the dorsal striatum, nucleus accumbens, and medial prefrontal cortex in 18 animals. Reporter composition differed strongly by anatomical region. Coexpression was prominent throughout the dorsal striatum and greatest in the dorsomedial striatum, whereas the nucleus accumbens shifted toward greater tdTomato-only and reduced EGFP-only representation, particularly in the core. In contrast, the medial prefrontal cortex was dominated by EGFP-only cells. These regional patterns were broadly retained across sex and age. HCR-FISH further supported spatial correspondence between endogenous Drd1 and Chrm4 transcript signal and the respective fluorescent reporters. Together, these findings demonstrate region-dependent organization of D1R- and M4R-associated cellular populations across the cortex and striatum and define distinct anatomical contexts in which M4R-directed signaling may influence D1R-associated neurons.