Brain-derived neurotrophic factor scales presynaptic calcium transients to modulate excitatory neurotransmission.

Brain-derived neurotrophic factor (BDNF) plays a critical role in synaptic physiology, as well as mechanisms underlying various neuropsychiatric diseases and their treatment. Despite its clear physiological role and disease relevance, BDNF's function at the presynaptic terminal, a fundamental unit of neurotransmission, remains poorly understood. In this study, we evaluated single synapse dynamics using optical imaging techniques in hippocampal cell cultures. We find that exogenous BDNF selectively increases evoked excitatory neurotransmission without affecting spontaneous neurotransmission. However, acutely blocking endogenous BDNF has no effect on evoked or spontaneous release, demonstrating that different approaches to studying BDNF may yield different results. When we suppressed BDNF-Tropomyosin receptor kinase B (TrkB) activity chronically over a period of days to weeks using a mouse line enabling conditional knockout of TrkB, we found that evoked glutamate release was significantly decreased while spontaneous release remained unchanged. Moreover, chronic blockade of BDNF-TrkB activity selectively downscales evoked calcium transients without affecting spontaneous calcium events. Via pharmacological blockade by voltage-gated calcium channel (VGCC) selective blockers, we found that the changes in evoked calcium transients are mediated by the P/Q subtype of VGCCs. These results suggest that BDNF-TrkB activity increases presynaptic VGCC activity to selectively increase evoked glutamate release.

Postsynaptic receptors regulate presynaptic transmitter stability through transsynaptic bridges.

Stable matching of neurotransmitters with their receptors is fundamental to synapse function and reliable communication in neural circuits. Presynaptic neurotransmitters regulate the stabilization of postsynaptic transmitter receptors. Whether postsynaptic receptors regulate stabilization of presynaptic transmitters has received less attention. Here, we show that blockade of endogenous postsynaptic acetylcholine receptors (AChR) at the neuromuscular junction destabilizes the cholinergic phenotype in motor neurons and stabilizes an earlier, developmentally transient glutamatergic phenotype. Further, expression of exogenous postsynaptic gamma-aminobutyric acid type A receptors (GABAA receptors) in muscle cells stabilizes an earlier, developmentally transient GABAergic motor neuron phenotype. Both AChR and GABAA receptors are linked to presynaptic neurons through transsynaptic bridges. Knockdown of specific components of these transsynaptic bridges prevents stabilization of the cholinergic or GABAergic phenotypes. Bidirectional communication can enforce a match between transmitter and receptor and ensure the fidelity of synaptic transmission. Our findings suggest a potential role of dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter.

Complexin has a dual synaptic function as checkpoint protein in vesicle priming and as a promoter of vesicle fusion.

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.

Differential nanoscale organization of excitatory synapses onto excitatory vs. inhibitory neurons.

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters (NCs) whose precise alignment across the cleft in a transsynaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses-those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses). As in Ex→Ex synapses, we find that in Ex→PV synapses, presynaptic Munc13-1 and postsynaptic PSD-95 both form NCs that demonstrate alignment, underscoring synaptic nanostructure and the transsynaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre- and postsynaptic Ex→PV nanostructure. Ex→PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex→Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell was also represented in Munc13-1 organization, as Ex→PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of transsynaptic alignment in these synapse types, revealing protein alignment in Ex→PV synapses over a distinct range of distances compared to Ex→Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses.

Identification of secretory autophagy as a mechanism modulating activity-induced synaptic remodeling.

The ability of neurons to rapidly remodel their synaptic structure and strength in response to neuronal activity is highly conserved across species and crucial for complex brain functions. However, mechanisms required to elicit and coordinate the acute, activity-dependent structural changes across synapses are not well understood, as neurodevelopment and structural plasticity are tightly linked. Here, using an RNAi screen in Drosophila against genes affecting nervous system functions in humans, we uncouple cellular processes important for synaptic plasticity and synapse development. We find mutations associated with neurodegenerative and mental health disorders are 2-times more likely to affect activity-induced synaptic remodeling than synapse development. We report that while both synapse development and activity-induced synaptic remodeling at the fly NMJ require macroautophagy (hereafter referred to as autophagy), bifurcation in the autophagy pathway differentially impacts development and synaptic plasticity. We demonstrate that neuronal activity enhances autophagy activation but diminishes degradative autophagy, thereby driving the pathway towards autophagy-based secretion. Presynaptic knockdown of Snap29, Sec22, or Rab8, proteins implicated in the secretory autophagy pathway, is sufficient to abolish activity-induced synaptic remodeling. This study uncovers secretory autophagy as a transsynaptic signaling mechanism modulating synaptic plasticity.

Dopamine control of social novelty preference is constrained by an interpeduncular-tegmentum circuit.

Animals are inherently motivated to explore social novelty cues over familiar ones, resulting in a novelty preference (NP), although the behavioral and circuit bases underlying NP are unclear. Combining calcium and neurotransmitter sensors with fiber photometry and optogenetics in mice, we find that mesolimbic dopamine (DA) neurotransmission is strongly and predominantly activated by social novelty controlling bout length of interaction during NP, a response significantly reduced by familiarity. In contrast, interpeduncular nucleus (IPN) GABAergic neurons that project to the lateral dorsal tegmentum (LDTg) were inhibited by social novelty but activated during terminations with familiar social stimuli. Inhibition of this pathway during NP increased interaction and bout length with familiar social stimuli, while activation reduced interaction and bout length with novel social stimuli via decreasing DA neurotransmission. These data indicate interest towards novel social stimuli is encoded by mesolimbic DA which is dynamically regulated by an IPN→LDTg circuit to control NP.

Synapsin 2a tetramerisation selectively controls the presynaptic nanoscale organisation of reserve synaptic vesicles.

Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are tightly packed within the presynaptic bouton of neurons. The mechanism by which SVs are clustered at the presynapse, while preserving their ability to dynamically recycle to support neuronal communication, remains unknown. Synapsin 2a (Syn2a) tetramerization has been suggested as a potential clustering mechanism. Here, we used Dual-pulse sub-diffractional Tracking of Internalised Molecules (DsdTIM) to simultaneously track single SVs from the recycling and the reserve pools, in live hippocampal neurons. The reserve pool displays a lower presynaptic mobility compared to the recycling pool and is also present in the axons. Triple knockout of Synapsin 1-3 genes (SynTKO) increased the mobility of reserve pool SVs. Re-expression of wild-type Syn2a (Syn2aWT), but not the tetramerization-deficient mutant K337Q (Syn2aK337Q), fully rescued these effects. Single-particle tracking revealed that Syn2aK337QmEos3.1 exhibited altered activity-dependent presynaptic translocation and nanoclustering. Therefore, Syn2a tetramerization controls its own presynaptic nanoclustering and thereby contributes to the dynamic immobilisation of the SV reserve pool.

New Light on Prions: Putative Role of PrPc in Pathophysiology of Mood Disorders.

Mood disorders are highly prevalent and heterogenous mental illnesses with devastating rates of mortality and treatment resistance. The molecular basis of those conditions involves complex interplay between genetic and environmental factors. Currently, there are no objective procedures for diagnosis, prognosis and personalization of patients' treatment. There is an urgent need to search for novel molecular targets for biomarkers in mood disorders. Cellular prion protein (PrPc) is infamous for its potential to convert its insoluble form, leading to neurodegeneration in Creutzfeldt-Jacob disease. Meanwhile, in its physiological state, PrPc presents neuroprotective features and regulates neurotransmission and synaptic plasticity. The aim of this study is to integrate the available knowledge about molecular mechanisms underlying the impact of PrPc on the pathophysiology of mood disorders. Our review indicates an important role of this protein in regulation of cognitive functions, emotions, sleep and biological rhythms, and its deficiency results in depressive-like behavior and cognitive impairment. PrPc plays a neuroprotective role against excitotoxicity, oxidative stress and inflammation, the main pathophysiological events in the course of mood disorders. Research indicates that PrPc may be a promising biomarker of cognitive decline. There is an urgent need of human studies to elucidate its potential utility in clinical practice.

High-speed measurements of SNARE-complexin interactions using magnetic tweezers.

In neuroscience, understanding the mechanics of synapses, especially the function of force-sensitive proteins at the molecular level, is essential. This need emphasizes the importance of precise measurement of synaptic protein interactions. Addressing this, we introduce high-resolution magnetic tweezers (MT) as a novel method to probe the mechanics of synapse-related proteins with high precision. We demonstrate this technique through studying SNARE-complexin interactions, crucial for synaptic transmission, showcasing its capability to apply specific forces to individual molecules. Our results reveal that high-resolution MT provides in-depth insights into the stability and dynamic transitions of synaptic protein complexes. This method is a significant advancement in synapse biology, offering a new tool for researchers to investigate the impact of mechanical forces on synaptic functions and their implications for neurological disorders.

Presynaptic inhibition of excitatory synaptic transmission from the calcitonin gene-related peptide-containing parabrachial neurons to the central amygdala in mice - unexpected influence of systemic inflammation thereon.

The monosynaptic connection from the lateral parabrachial nucleus (LPB) to the central amygdala (CeA) serves as a fundamental pathway for transmitting nociceptive signals to the brain. The LPB receives nociceptive information from the dorsal horn and spinal trigeminal nucleus and sends it to the "nociceptive" CeA, which modulates pain-associated emotions and nociceptive sensitivity. To elucidate the role of densely expressed mu-opioid receptors (MORs) within this pathway, we investigated the effects of exogenously applied opioids on LPB-CeA synaptic transmission, employing optogenetics in mice expressing channelrhodopsin-2 in LPB neurons with calcitonin gene-related peptide (CGRP). A MOR agonist ([D-Ala2,N-Me-Phe4,Glycinol5]-enkephalin, DAMGO) significantly reduced the amplitude of light-evoked excitatory postsynaptic currents (leEPSCs), in a manner negatively correlated with an increase in the paired-pulse ratio. An antagonist of MORs significantly attenuated these effects. Notably, this antagonist significantly increased leEPSC amplitude when applied alone, an effect further amplified in mice subjected to lipopolysaccharide injection 2 h before brain isolation, yet not observed at the 24-h mark. We conclude that opioids could shut off the ascending nociceptive signal at the LPB-CeA synapse through presynaptic mechanisms. Moreover, this gating process might be modulated by endogenous opioids, and the innate immune system influences this modulation.

Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models.

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. Single fiber electromyography (SFEMG) is a clinically relevant and sensitive technique that measures single muscle fiber action potential responses during voluntary contractions or nerve stimulations to assess NMJ transmission. The assessment and quantification of NMJ transmission involves two parameters: jitter and blocking. Jitter refers to the variability in timing (latency) between consecutive single-fiber action potentials (SFAPs). Blocking signifies the failure of NMJ transmission to initiate an SFAP response. Although SFEMG is a well-established and sensitive test in clinical settings, its application in preclinical research has been relatively infrequent. This report outlines the steps and criteria employed in performing stimulated SFEMG to quantify jitter and blocking in rodent models. This technique can be used in preclinical and clinical studies to gain insights into NMJ function in the context of health, aging, and disease.

Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin.

Neurotransmission at synapses is mediated by the fusion and subsequent endocytosis of synaptic vesicle membranes. Actin has been suggested to be required for presynaptic endocytosis but the mechanisms that control actin polymerization and its mode of action within presynaptic nerve terminals remain poorly understood. We combine optical recordings of presynaptic membrane dynamics and ultrastructural analysis with genetic and pharmacological manipulations to demonstrate that presynaptic endocytosis is controlled by actin regulatory diaphanous-related formins mDia1/3 and Rho family GTPase signaling in mouse hippocampal neurons. We show that impaired presynaptic actin assembly in the near absence of mDia1/3 and reduced RhoA activity is partly compensated by hyperactivation of Rac1. Inhibition of Rac1 signaling further aggravates impaired presynaptic endocytosis elicited by loss of mDia1/3. Our data suggest that interdependent mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate synaptic vesicle endocytosis by controlling presynaptic F-actin.

Neonatal GABAergic transmission primes vestibular gating of output for adult spatial navigation.

GABAergic interneurons are poised with the capacity to shape circuit output via inhibitory gating. How early in the development of medial vestibular nucleus (MVN) are GABAergic neurons recruited for feedforward shaping of outputs to higher centers for spatial navigation? The role of early GABAergic transmission in assembling vestibular circuits for spatial navigation was explored by neonatal perturbation. Immunohistochemistry and confocal imaging were utilized to reveal the expression of parvalbumin (PV)-expressing MVN neurons and their perineuronal nets. Whole-cell patch-clamp recording, coupled with optogenetics, was conducted in vitro to examine the synaptic function of MVN circuitry. Chemogenetic targeting strategy was also employed in vivo to manipulate neuronal activity during navigational tests. We found in rats a neonatal critical period before postnatal day (P) 8 in which competitive antagonization of GABAergic transmission in the MVN retarded maturation of inhibitory neurotransmission, as evidenced by deranged developmental trajectory for excitation/inhibition ratio and an extended period of critical period-like plasticity in GABAergic transmission. Despite increased number of PV-expressing GABAergic interneurons in the MVN, optogenetic-coupled patch-clamp recording indicated null-recruitment of these neurons in tuning outputs along the ascending vestibular pathway. Such perturbation not only offset output dynamics of ascending MVN output neurons, but was further accompanied by impaired vestibular-dependent navigation in adulthood. The same perturbations were however non-consequential when applied after P8. Results highlight neonatal GABAergic transmission as key to establishing feedforward output dynamics to higher brain centers for spatial cognition and navigation.

ß2* nAChR sensitivity modulates acquisition of cocaine self-administration in male rats.

Signaling through nicotinic acetylcholine receptors (nAChRs) plays a role in cocaine reward and reinforcement, suggesting that the cholinergic system could be manipulated with therapeutics to modulate aspects of cocaine use disorder (CUD). We examined the interaction between nAChRs and cocaine reinforcement by expressing a hypersensitive ß2 nAChR subunit (ß2Leu9'Ser) in the ventral tegmental area of male Sprague Dawley rats. Compared to control rats, ß2Leu9'Ser rats acquired (fixed ratio) intravenous cocaine self-administration faster and with greater likelihood. By contrast, ß2Leu9'Ser rats were approximately equivalent to controls in their intake of cocaine on a progressive ratio schedule of reinforcement, suggesting differential effects of cholinergic signaling depending on experimental parameters. Like progressive ratio cocaine SA, ß2Leu9'Ser rats and controls did not differ significantly in food SA assays, including acquisition on a fixed ratio schedule or in progressive ratio sessions. These results highlight the specific role of high-affinity, heteropentameric ß2* (ß2-containing) nAChRs in acquisition of cocaine SA, suggesting that mesolimbic acetylcholine signaling is active during this process.

Liprin-α proteins are master regulators of human presynapse assembly.

The formation of mammalian synapses entails the precise alignment of presynaptic release sites with postsynaptic receptors but how nascent cell-cell contacts translate into assembly of presynaptic specializations remains unclear. Guided by pioneering work in invertebrates, we hypothesized that in mammalian synapses, liprin-α proteins directly link trans-synaptic initial contacts to downstream steps. Here we show that, in human neurons lacking all four liprin-α isoforms, nascent synaptic contacts are formed but recruitment of active zone components and accumulation of synaptic vesicles is blocked, resulting in 'empty' boutons and loss of synaptic transmission. Interactions with presynaptic cell adhesion molecules of either the LAR-RPTP family or neurexins via CASK are required to localize liprin-α to nascent synaptic sites. Liprin-α subsequently recruits presynaptic components via a direct interaction with ELKS proteins. Thus, assembly of human presynaptic terminals is governed by a hierarchical sequence of events in which the recruitment of liprin-α proteins by presynaptic cell adhesion molecules is a critical initial step.

Synaptotagmin 7 docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release.

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

Anti-GluA3 autoantibodies define a new sub-population of frontotemporal lobar degeneration patients with distinct neuropathological features.

Autoantibodies directed against the GluA3 subunit (anti-GluA3 hIgGs) of AMPA receptors have been identified in 20%-25% of patients with frontotemporal lobar degeneration (FTLD). Data from patients and in vitro/ex vivo pre-clinical studies indicate that anti-GluA3 hIgGs negatively affect glutamatergic neurotransmission. However, whether and how the chronic presence of anti-GluA3 hIgGs triggers synaptic dysfunctions and the appearance of FTLD-related neuropathological and behavioural signature has not been clarified yet. To address this question, we developed and characterized a pre-clinical mouse model of passive immunization with anti-GluA3 hIgGs purified from patients. In parallel, we clinically compared FTLD patients who were positive for anti-GluA3 hIgGs to negative ones. Clinical data showed that the presence of anti-GluA3 hIgGs defined a subgroup of patients with distinct clinical features. In the preclinical model, anti-GluA3 hIgGs administration led to accumulation of phospho-tau in the postsynaptic fraction and dendritic spine loss in the prefrontal cortex. Remarkably, the preclinical model exhibited behavioural disturbances that mostly reflected the deficits proper of patients positive for anti-GluA3 hIgGs. Of note, anti-GluA3 hIgGs-mediated alterations were rescued in the animal model by enhancing glutamatergic neurotransmission with a positive allosteric modulator of AMPA receptors. Overall, our study clarified the contribution of anti-GluA3 autoantibodies to central nervous system symptoms and pathology and identified a specific subgroup of FTLD patients. Our findings will be instrumental in the development of a therapeutic personalised medicine strategy for patients positive for anti-GluA3 hIgGs.

The CHD family chromatin remodeling enzyme, Kismet, promotes both clathrin-mediated and activity-dependent bulk endocytosis.

Chromodomain helicase DNA binding domain (CHD) proteins, including CHD7 and CHD8, remodel chromatin to enable transcriptional programs. Both proteins are important for proper neural development as heterozygous mutations in Chd7 and Chd8 are causative for CHARGE syndrome and correlated with autism spectrum disorders, respectively. Their roles in mature neurons are poorly understood despite influencing the expression of genes required for cell adhesion, neurotransmission, and synaptic plasticity. The Drosophila homolog of CHD7 and CHD8, Kismet (Kis), promotes neurotransmission, endocytosis, and larval locomotion. Endocytosis is essential in neurons for replenishing synaptic vesicles, maintaining protein localization, and preserving the size and composition of the presynaptic membrane. Several forms of endocytosis have been identified including clathrin-mediated endocytosis, which is coupled with neural activity and is the most prevalent form of synaptic endocytosis, and activity-dependent bulk endocytosis, which occurs during periods of intense stimulation. Kis modulates the expression of gene products involved in endocytosis including promoting shaggy/GSK3ß expression while restricting PI3K92E. kis mutants electrophysiologically phenocopy a liquid facets mutant in response to paradigms that induce clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Further, kis mutants do not show further reductions in endocytosis when activity-dependent bulk endocytosis or clathrin-mediated endocytosis are pharmacologically inhibited. We find that Kis is important in postsynaptic muscle for proper endocytosis but the ATPase domain of Kis is dispensable for endocytosis. Collectively, our data indicate that Kis promotes both clathrin-mediated endocytosis and activity-dependent bulk endocytosis possibly by promoting transcription of several endocytic genes and maintaining the size of the synaptic vesicle pool.

The kainate receptor GluK2 mediates cold sensing in mice.

Thermosensors expressed in peripheral somatosensory neurons sense a wide range of environmental temperatures. While thermosensors detecting cool, warm and hot temperatures have all been extensively characterized, little is known about those sensing cold temperatures. Though several candidate cold sensors have been proposed, none has been demonstrated to mediate cold sensing in somatosensory neurons in vivo, leaving a knowledge gap in thermosensation. Here we characterized mice lacking the kainate-type glutamate receptor GluK2, a mammalian homolog of the Caenorhabditis elegans cold sensor GLR-3. While GluK2 knockout mice respond normally to heat and mechanical stimuli, they exhibit a specific deficit in sensing cold but not cool temperatures. Further analysis supports a key role for GluK2 in sensing cold temperatures in somatosensory DRG neurons in the periphery. Our results reveal that GluK2-a glutamate-sensing chemoreceptor mediating synaptic transmission in the central nervous system-is co-opted as a cold-sensing thermoreceptor in the periphery.

The phase of tACS-entrained pre-SMA beta oscillations modulates motor inhibition.

Inhibitory control has been linked to beta oscillations in the fronto-basal ganglia network. Here we aim to investigate the functional role of the phase of this oscillatory beta rhythm for successful motor inhibition. We applied 20 Hz transcranial alternating current stimulation (tACS) to the pre-supplementary motor area (pre-SMA) while presenting stop signals at 4 (Experiment 1) and 8 (Experiment 2) equidistant phases of the tACS entrained beta oscillations. Participants showed better inhibitory performance when stop signals were presented at the trough of the beta oscillation whereas their inhibitory control performance decreased with stop signals being presented at the oscillatory beta peak. These results are consistent with the communication through coherence theory, in which postsynaptic effects are thought to be greater when an input arrives at an optimal phase within the oscillatory cycle of the target neuronal population. The current study provides mechanistic insights into the neural communication principles underlying successful motor inhibition and may have implications for phase-specific interventions aimed at treating inhibitory control disorders such as PD or OCD.