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Cultural involvement takes a significant role on quality of life of patients (QoL) as namely culture is an important domain for health and QoL simultaneously. Paper analyzes the possible connection between culture and health, and, attendance of patients the cultural events as one of the important dimensions of QoL. Research is based on social project “Cultural prescription”, organized by public cultural institution “Mokytojų namai” (Eng. Teacher’s House) in Vilnius and dedicated to Outpatient Clinic “Centro poliklinika” (Vilnius) patients, who were suffering from chronic disease to improve their QoL. The aim of the paper is to explore the possibilities of innovative social measures to increase social cultural engagement of patients, to contribute to their well-being, and to present the approach of health care practitioners towards the expediency and usefulness of inter-sectorial cooperation for the patients’ social welfare.

Introduction: In tauopathies, altered tau processing correlates with impairments in synaptic density and function. Changes in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to disease-associated abnormalities in multiple neurodegenerative diseases., Methods: To investigate the link between tau and HCN channels, we performed histological, biochemical, ultrastructural, and functional analyses of hippocampal tissues from Alzheimer's disease (AD), age-matched controls, Tau35 mice, and/or Tau35 primary hippocampal neurons., Results: Expression of specific HCN channels is elevated in post mortem AD hippocampus. Tau35 mice develop progressive abnormalities including increased phosphorylated tau, enhanced HCN channel expression, decreased dendritic branching, reduced synapse density, and vesicle clustering defects. Tau35 primary neurons show increased HCN channel expression enhanced hyperpolarization-induced membrane voltage "sag" and changes in the frequency and kinetics of spontaneous excitatory postsynaptic currents., Discussion: Our findings are consistent with a model in which pathological changes in tauopathies impact HCN channels to drive network-wide structural and functional synaptic deficits., Highlights: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are functionally linked to the development of tauopathy. Expression of specific HCN channels is elevated in the hippocampus in Alzheimer's disease and the Tau35 mouse model of tauopathy. Increased expression of HCN channels in Tau35 mice is accompanied by hyperpolarization-induced membrane voltage "sag" demonstrating a detrimental effect of tau abnormalities on HCN channel function. Tau35 expression alters synaptic organization, causing a loosened vesicle clustering phenotype in Tau35 mice., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Long-term memory formation is energetically costly. Neural mechanisms that guide an animal to identify fruitful associations therefore have important survival benefits. Here, we elucidate a circuit mechanism in Lymnaea , which enables past memory to shape new memory formation through changes in perception. Specifically, strong classical conditioning drives a positive shift in perception that facilitates the robust learning of a subsequent and otherwise ineffective weak association. Circuit dissection approaches reveal the neural control network responsible, characterized by a mutual inhibition motif. This both sets perceptual state and acts as the master controller for gating new learning. Pharmacological circuit manipulation in vivo fully substitutes for strong paradigm learning, shifting the network into a more receptive state to enable subsequent weak paradigm learning. Thus, perceptual change provides a conduit to link past and future memory storage. We propose that this mechanism alerts animals to learning-rich periods, lowering the threshold for new memory acquisition.

Alzheimer's disease is linked to increased levels of amyloid beta (Aβ) in the brain, but the mechanisms underlying neuronal dysfunction and neurodegeneration remain enigmatic. Here, we investigate whether organizational characteristics of functional presynaptic vesicle pools, key determinants of information transmission in the central nervous system, are targets for elevated Aβ. Using an optical readout method in cultured hippocampal neurons, we show that acute Aβ42 treatment significantly enlarges the fraction of functional vesicles at individual terminals. We observe the same effect in a chronically elevated Aβ transgenic model (APPSw,Ind) using an ultrastructure-function approach that provides detailed information on nanoscale vesicle pool positioning. Strikingly, elevated Aβ is correlated with excessive accumulation of recycled vesicles near putative endocytic sites, which is consistent with deficits in vesicle retrieval pathways. Using the glutamate reporter, iGluSnFR, we show that there are parallel functional consequences, where ongoing information signaling capacity is constrained. Treatment with levetiracetam, an antiepileptic that dampens synaptic hyperactivity, partially rescues these transmission defects. Our findings implicate organizational and dynamic features of functional vesicle pools as targets in Aβ-driven synaptic impairment, suggesting that interventions to relieve the overloading of vesicle retrieval pathways might have promising therapeutic value., (© The Author(s) 2022. Published by Oxford University Press.)

Defects in DNA single-strand break repair (SSBR) are linked with neurological dysfunction but the underlying mechanisms remain poorly understood. Here, we show that hyperactivity of the DNA strand break sensor protein Parp1 in mice in which the central SSBR protein Xrcc1 is conditionally deleted (Xrcc1 Nes-Cre ) results in lethal seizures and shortened lifespan. Using electrophysiological recording and synaptic imaging approaches, we demonstrate that aberrant Parp1 activation triggers seizure-like activity in Xrcc1-defective hippocampus ex vivo and deregulated presynaptic calcium signalling in isolated hippocampal neurons in vitro. Moreover, we show that these defects are prevented by Parp1 inhibition or deletion and, in the case of Parp1 deletion, that the lifespan of Xrcc1 Nes-Cre mice is greatly extended. This is the first demonstration that lethal seizures can be triggered by aberrant Parp1 activity at unrepaired SSBs, highlighting PARP inhibition as a possible therapeutic approach in hereditary neurological disease., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Misfolding and aggregation of proteins is strongly linked to several neurodegenerative diseases, but how such species bring about their cytotoxic actions remains poorly understood. Here we used specifically-designed optical reporter probes and live fluorescence imaging in primary hippocampal neurons to characterise the mechanism by which prefibrillar, oligomeric forms of the Alzheimer's-associated peptide, Aβ42, exert their detrimental effects. We used a pH-sensitive reporter, Aβ42-CypHer, to track Aβ internalisation in real-time, demonstrating that oligomers are rapidly taken up into cells in a dynamin-dependent manner, and trafficked via the endo-lysosomal pathway resulting in accumulation in lysosomes. In contrast, a non-assembling variant of Aβ42 (vAβ42) assayed in the same way is not internalised. Tracking ovalbumin uptake into cells using CypHer or Alexa Fluor tags shows that preincubation with Aβ42 disrupts protein uptake. Our results identify a potential mechanism by which amyloidogenic aggregates impair cellular function through disruption of the endosomal-lysosomal pathway.

Background: The growing public health concern caused by non-communicable diseases in urban surroundings cannot be solved by health care alone; therefore a multidisciplinary approach is mandatory. This study aimed to evaluate the airborne aerosol pollution level in primary schools as possible factor influencing origin and course of the diseases in children., Methods: Seasonal aerosol particle number concentration (PNC) and mass concentration (PMC) were studied in the randomly selected eleven primary schools in the Lithuanian capital, Vilnius, as model of a middle-size Eastern European city. Total PNC in the size range from 0.01 to >1.0 μm in diameter was measured using a condensation particle counter. Using an optical particle sizer, PNC was measured and PMC estimated for particles from 0.3 to 10.0 μm. A descriptive statistics was used to estimate the aerosol pollution levels., Results: During all seasons, local cafeterias in the absence of ventilation were the main sources of the elevated levels of indoor PMC and PNC (up to 97,500 particles/cm). The other sources of airborne particulates were the children's activity during the lesson breaks with PMC up to 586 μg/m. Soft furniture, carpets in the classrooms and corridors were responsible for PMC up to 200 μg/m. Outdoor aerosol pollution (up to 18,170 particles/cm) was higher for schools in city center. Elevated air pollution in classrooms also resulted from intermittent sources, such as construction work during classes (200-1000 μg/m) and petrol-powered lawn trimmers (up to 66,400 particles/cm)., Conclusion: The results of our survey show that even in a relatively low polluted region of Eastern Europe there are big differences in aerosol pollution within middle-sized city. Additional efforts are needed to improve air quality in schools: more frequent wet cleaning, monitoring the operation of ventilation systems, a ban on construction works during school year, on a use of sandblasting mechanisms in the neighborhood of schools.

Aerosol particle number (PNC) and mass concentrations (PMC) were studied in 11 primary schools during the 2017-2018 school years (from September to May) in Vilnius, Lithuania, with the aim to evaluate the main aerosol pollution sources and its levels. Expeditious information on the total aerosol particle concentration over the full range of sizes (from 0.01 to > 1 μm) was estimated using a condensation particle counter (CPC). Particle number and mass concentrations in the size range of 0.3-10 μm were measured and estimated using an optical particle sizer (OPS). The use of aerosol particle size spectra (OPS) in school lodgements facilitated the identification of the main sources of indoor air pollution. The main sources responsible for the elevated levels of indoor PN and PM aerosol concentrations were determined: local canteens in the absence of ventilation (particle concentrations up to 97,500 part/cm 3 (CPC)), the school-grader activity during the lesson breaks (275-586 μg/m 3 ), soft furniture and carpets in the classrooms and corridors (~ 200 μg/m 3 ), and in some cases (city center) elevated outdoor aerosol pollution levels (16800-18,170 part/cm 3 ). Elevated aerosol pollution levels were also due to the occasional sources: construction works during lessons (200-1000 μg/m 3 ), scraping the exterior walls of buildings near schools (up to 1600 μg/m 3 ), and the use of petrol-powered trimmers during cutting of green plantings (22500-66,400 part/cm 3 (CPC)).

Vesicle pool properties are known determinants of synaptic efficacy, but their potential role as modifiable substrates in forms of Hebbian plasticity is still unclear. Here, we investigate this using a nanoscale readout of functionally recycled vesicles in natively wired hippocampal CA3→CA1 circuits undergoing long-term potentiation (LTP). We show that the total recycled vesicle pool is larger after plasticity induction, with the smallest terminals exhibiting the greatest relative expansion. Changes in the spatial organization of vesicles accompany potentiation including a specific increase in the number of recycled vesicles at the active zone, consistent with an ultrastructural remodeling component of synaptic strengthening. The cAMP-PKA pathway activator, forskolin, selectively mimics some features of LTP-driven changes, suggesting that distinct and independent modules of regulation accompany plasticity expression. Our findings provide evidence for a presynaptic locus of LTP encoded in the number and arrangement of functionally recycled vesicles, with relevance for models of long-term plasticity storage., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Discriminating, extracting and encoding temporal regularities is a critical requirement in the brain, relevant to sensory-motor processing and learning. However, the cellular mechanisms responsible remain enigmatic; for example, whether such abilities require specific, elaborately organized neural networks or arise from more fundamental, inherent properties of neurons. Here, using multi-electrode array technology, and focusing on interval learning, we demonstrate that sparse reconstituted rat hippocampal neural circuits are intrinsically capable of encoding and storing sub-second-order time intervals for over an hour timescale, represented in changes in the spatial-temporal architecture of firing relationships among populations of neurons. This learning is accompanied by increases in mutual information and transfer entropy, formal measures related to information storage and flow. Moreover, temporal relationships derived from previously trained circuits can act as templates for copying intervals into untrained networks, suggesting the possibility of circuit-to-circuit information transfer. Our findings illustrate that dynamic encoding and stable copying of temporal relationships are fundamental properties of simple in vitro networks, with general significance for understanding elemental principles of information processing, storage and replication.

Hunger state can substantially alter the perceived value of a stimulus, even to the extent that the same sensory cue can trigger antagonistic behaviors. How the nervous system uses these graded perceptual shifts to select between opposed motor patterns remains enigmatic. Here, we challenged food-deprived and satiated Lymnaea to choose between two mutually exclusive behaviors, ingestion or egestion, produced by the same feeding central pattern generator. Decoding the underlying neural circuit reveals that the activity of central dopaminergic interneurons defines hunger state and drives network reconfiguration, biasing satiated animals toward the rejection of stimuli deemed palatable by food-deprived ones. By blocking the action of these neurons, satiated animals can be reconfigured to exhibit a hungry animal phenotype. This centralized mechanism occurs in the complete absence of sensory retuning and generalizes across different sensory modalities, allowing food-deprived animals to increase their perception of food value in a stimulus-independent manner to maximize potential calorific intake.

Action potential shape is a major determinant of synaptic transmission, and mechanisms of spike tuning are therefore of key functional significance. We demonstrate that synaptic activity itself modulates future spikes in the same neuron via a rapid feedback pathway. Using Ca 2+ imaging and targeted uncaging approaches in layer 5 neocortical pyramidal neurons, we show that the single spike-evoked Ca 2+ rise occurring in one proximal bouton or first node of Ranvier drives a significant sharpening of subsequent action potentials recorded at the soma. This form of intrinsic modulation, mediated by the activation of large-conductance Ca 2+ /voltage-dependent K + channels (BK channels), acts to maintain high-frequency firing and limit runaway spike broadening during repetitive firing, preventing an otherwise significant escalation of synaptic transmission. Our findings identify a novel short-term presynaptic plasticity mechanism that uses the activity history of a bouton or adjacent axonal site to dynamically tune ongoing signaling properties.

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease., Competing Interests: The authors declare that there is no conflict of interest.

We propose a biologically plausible architecture for unsupervised ensemble learning in a population of spiking neural network classifiers. A mixture of experts type organisation is shown to be effective, with the individual classifier outputs combined via a gating network whose operation is driven by input timing dependent plasticity (ITDP). The ITDP gating mechanism is based on recent experimental findings. An abstract, analytically tractable model of the ITDP driven ensemble architecture is derived from a logical model based on the probabilities of neural firing events. A detailed analysis of this model provides insights that allow it to be extended into a full, biologically plausible, computational implementation of the architecture which is demonstrated on a visual classification task. The extended model makes use of a style of spiking network, first introduced as a model of cortical microcircuits, that is capable of Bayesian inference, effectively performing expectation maximization. The unsupervised ensemble learning mechanism, based around such spiking expectation maximization (SEM) networks whose combined outputs are mediated by ITDP, is shown to perform the visual classification task well and to generalize to unseen data. The combined ensemble performance is significantly better than that of the individual classifiers, validating the ensemble architecture and learning mechanisms. The properties of the full model are analysed in the light of extensive experiments with the classification task, including an investigation into the influence of different input feature selection schemes and a comparison with a hierarchical STDP based ensemble architecture., Competing Interests: The authors have declared that no competing interests exist.

Amyloid β1-42 (Aβ1-42) plays a central role in Alzheimer's disease. The link between structure, assembly and neuronal toxicity of this peptide is of major current interest but still poorly defined. Here, we explored this relationship by rationally designing a variant form of Aβ1-42 (vAβ1-42) differing in only two amino acids. Unlike Aβ1-42, we found that the variant does not self-assemble, nor is it toxic to neuronal cells. Moreover, while Aβ1-42 oligomers impact on synaptic function, vAβ1-42 does not. In a living animal model system we demonstrate that only Aβ1-42 leads to memory deficits. Our findings underline a key role for peptide sequence in the ability to assemble and form toxic structures. Furthermore, our non-toxic variant satisfies an unmet demand for a closely related control peptide for Aβ1-42 cellular studies of disease pathology, offering a new opportunity to decipher the mechanisms that accompany Aβ1-42-induced toxicity leading to neurodegeneration.

During goal-directed decision-making, animals must integrate information from the external environment and their internal state to maximize resource localization while minimizing energy expenditure. How this complex problem is solved by the nervous system remains poorly understood. Here, using a combined behavioural and neurophysiological approach, we demonstrate that the mollusc Lymnaea performs a sophisticated form of decision-making during food-searching behaviour, using a core system consisting of just two neuron types. The first reports the presence of food and the second encodes motivational state acting as a gain controller for adaptive behaviour in the absence of food. Using an in vitro analogue of the decision-making process, we show that the system employs an energy management strategy, switching between a low- and high-use mode depending on the outcome of the decision. Our study reveals a parsimonious mechanism that drives a complex decision-making process via regulation of levels of tonic inhibition and phasic excitation.

Efficient recycling of synaptic vesicles is thought to be critical for sustained information transfer at central terminals. However, the specific contribution that retrieved vesicles make to future transmission events remains unclear. Here we exploit fluorescence and time-stamped electron microscopy to track the functional and positional fate of vesicles endocytosed after readily releasable pool (RRP) stimulation in rat hippocampal synapses. We show that most vesicles are recovered near the active zone but subsequently take up random positions in the cluster, without preferential bias for future use. These vesicles non-selectively queue, advancing towards the release site with further stimulation in an actin-dependent manner. Nonetheless, the small subset of vesicles retrieved recently in the stimulus train persist nearer the active zone and exhibit more privileged use in the next RRP. Our findings reveal heterogeneity in vesicle fate based on nanoscale position and timing rules, providing new insights into the origins of future pool constitution.

Fast activity-driven turnover of neurotransmitter-filled vesicles at presynaptic terminals is a crucial step in information transfer in the CNS. Characterization of the relationship between the nanoscale organization of synaptic vesicles and their functional properties during transmission is currently of interest. Here we outline a procedure for ultrastructural investigation of functional vesicles in synapses from native mammalian brain tissue. FM dye is injected into the target region of a brain slice and upstream axons are electrically activated to stimulate vesicle turnover and dye uptake. In the presence of diaminobenzidine (DAB), photoactivation of dye-filled vesicles yields an osmiophilic precipitate that is visible in electron micrographs. When combined with serial-section electron microscopy, fundamental ultrastructure-function relationships of presynaptic terminals in native circuits are revealed. We outline the utility of this protocol for the 3D reconstruction of a recycling vesicle pool in CA3-CA1 synapses from an acute hippocampal slice and for the characterization of its anatomically defined docked pool. This protocol requires 6-7 d.

The direct visualization of subcellular dynamic processes is often hampered by limitations in the resolving power achievable with conventional microscopy techniques. Fluorescence recovery after photobleaching has emerged as a highly informative approach to address this challenge, permitting the quantitative measurement of the movement of small organelles and proteins in living functioning cells, and offering detailed insights into fundamental cellular phenomena of physiological importance. In recent years, its implementation has benefited from the increasing availability of confocal microscopy systems and of powerful labeling techniques based on genetically encoded fluorescent proteins or other chemical markers. In this review, we present fluorescence recovery after photobleaching and related techniques in the context of contemporary neurobiological research and discuss quantitative and semi-quantitative approaches to their interpretation., (© 2013 International Society for Neurochemistry.)

Natural antisense transcripts (NATs) are endogenous RNA molecules that are complementary to known RNA transcripts. The functional significance of NATs is poorly understood, but their prevalence in the CNS suggests a role in brain function. Here we investigated a long NAT (antiNOS-2 RNA) associated with the regulation of nitric oxide (NO) production in the CNS of Lymnaea, an established model for molecular analysis of learning and memory. We show the antiNOS-2 RNA is axonally trafficked and demonstrate that this is regulated by classical conditioning. Critically, a single conditioning trial changes the amount of antiNOS-2 RNA transported along the axon. This occurs within the critical time window when neurotransmitter NO is required for memory formation. Our data suggest a role for the antiNOS-2 RNA in establishing memories through the regulation of NO signaling at the synapse.

At small central synapses, efficient turnover of vesicles is crucial for stimulus-driven transmission, but how the structure of this recycling pool relates to its functional role remains unclear. Here we characterize the organizational principles of functional vesicles at native hippocampal synapses with nanoscale resolution using fluorescent dye labeling and electron microscopy. We show that the recycling pool broadly scales with the magnitude of the total vesicle pool, but its average size is small (∼45 vesicles), highly variable, and regulated by CDK5/calcineurin activity. Spatial analysis demonstrates that recycling vesicles are preferentially arranged near the active zone and this segregation is abolished by actin stabilization, slowing the rate of activity-driven exocytosis. Our approach reveals a similarly biased recycling pool distribution at synapses in visual cortex activated by sensory stimulation in vivo. We suggest that in small native central synapses, efficient release of a limited pool of vesicles relies on their favored spatial positioning within the terminal., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Dynamic clamp is a powerful approach for electrophysiological investigations allowing researchers to introduce artificial electrical components into target neurons to simulate ionic conductances, chemical or electrotonic inputs or connections to other cells. Due to the rapidly changing and potentially large current injections during dynamic clamp, problematic voltage artifacts appear on the electrode used to inject dynamic clamp currents into a target neuron. Dynamic clamp experiments, therefore, typically use two separate electrodes in the same cell, one for recording membrane potential and one for injecting currents. The requirement for two independent electrodes has been a limiting factor for the use of dynamic clamp in applications where dual recordings of this kind are difficult or impossible to achieve. The recent development of an active electrode compensation (AEC) method has overcome some of these prior limitations, permitting artifact-free dynamic clamp experimentation with a single electrode. Here we describe an AEC method for the free dynamic clamp software StdpC. The AEC component of StdpC is the first such system implemented for the use of non-expert users and comes with a set of semi-automated configuration and calibration procedures that facilitate its use. We briefly introduce the AEC method and its implementation in StdpC and then validate it with an electronic model cell and in two different biological preparations., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Most presynaptic terminals in the central nervous system are characterized by two functionally distinct vesicle populations: a recycling pool, which supports action potential-driven neurotransmitter release via vesicle exocytosis, and a resting pool. The relative proportions of these two pools are highly variable between individual synapses, prompting speculation on their specific relationship, and on the possible functions of the resting pool.Using fluorescence imaging of FM-styryl dyes and synaptophysinI-pHluorin(sypHy) as well as correlative electronmicroscopy approaches, we show here that Hebbian plasticity-dependent changes in synaptic strength in rat hippocampal neurons can increase the recycling pool fraction at the expense of the resting pool in individual synaptic terminals. This recruitment process depends on NMDA-receptor activation, nitric oxide signalling and calcineurin and is accompanied by an increase in the probability of neurotransmitter release at individual terminals. Blockade of actin-mediated intersynaptic vesicle exchange does not prevent recycling pool expansion demonstrating that vesicle recruitment is intrasynaptic.We propose that the conversion of resting pool vesicles to the functionally recycling pool provides a rapid mechanism to implement long-lasting changes in presynaptic efficacy.

Aβ42 [amyloid-β peptide-(1-42)] plays a central role in Alzheimer's disease and is known to have a detrimental effect on neuronal cell function and survival when assembled into an oligomeric form. In the present study we show that administration of freshly prepared Aβ42 oligomers to a neuroblastoma (SH-SY5Y) cell line results in a reduction in survival, and that Aβ42 enters the cells prior to cell death. Immunoconfocal and immunogold electron microscopy reveal the path of the Aβ42 with time through the endosomal system and shows that it accumulates in lysosomes. A 24 h incubation with Aβ results in cells that have damaged lysosomes showing signs of enzyme leakage, accumulate autophagic vacuoles and exhibit severely disrupted nuclei. Endogenous Aβ is evident in the cells and the results of the present study suggest that the addition of Aβ oligomers disrupts a crucial balance in Aβ conformation and concentration inside neuronal cells, resulting in catastrophic effects on cellular function and, ultimately, in cell death.

Fast neuronal signalling relies on highly regulated vesicle fusion and recycling at specialized presynaptic terminals. Recently, examples of non-classical neurotransmission have also been reported, where fusion of vesicles can occur at sites remote from conventional synapses. This has potentially broad biological implications, but the underlying mechanisms are not well established. Here we show that a complete vesicle recycling pathway can occur at discrete axonal sites in mature hippocampal neurons and that extrasynaptic fusion is a robust feature of native tissue. We demonstrate that laterally mobile vesicle clusters trafficking between synaptic terminals become transiently stabilized by evoked action potentials and undergo complete but delayed Ca(2+)-dependent fusion along axons. This fusion is associated with dynamic actin accumulation and, subsequently, vesicles can be locally recycled, re-acidified and re-used. Immunofluorescence and ultrastructural work demonstrates that extrasynaptic fusion sites can have apposed postsynaptic specializations, suggesting that mobile vesicle recycling may underlie highly dynamic neuron-neuron communication.

Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, for spike timing-dependent plasticity clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real-time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments through an intuitive and powerful interface with a minimal initial lead time of a few hours. After initial configuration, experimental results can be generated within minutes of establishing cell recording.

Release probability (p(r)) is a fundamental presynaptic parameter which is critical in defining synaptic strength. Knowledge of how synapses set and regulate their p(r) is a fundamental step in understanding synaptic transmission and communication between neurons. Despite its importance, p(r) is difficult to measure directly at single synapses. One important strategy to achieve this has relied on the application of fluorescence-based imaging methods, but this is always limited by the lack of detailed information on the morphological and structural properties of the individual synapses under study, and thus precludes an investigation of the relationship between p(r) and synaptic anatomy. Here we outline a powerful methodology based on using FM-styryl dyes, photoconversion and correlative ultrastructural analysis in dissociated hippocampal cultured neurons, which provides both a direct readout of p(r) as well as nanoscale detail on synaptic organization and structure. We illustrate the value of this approach by investigating, at the level of individual reconstructed terminals, the relationship between release probability and defined vesicle pools. We show that in our population of synapses, p(r) is highly variable, and while it is positively correlated with the number of vesicles docked at the active zone it shows no relationship with the total number of synaptic vesicles. The lack of a direct correlation between total synaptic size and performance in these terminals suggests that factors other than the absolute magnitude of the synapse are the most important determinants of synaptic efficacy., (Copyright © 2009 Elsevier Inc. All rights reserved.)

Presynaptic terminals in hippocampal neurons house functionally distinct vesicle pools, the size, structure and biochemical features of which are major determinants of presynaptic strength and performance. In classical models of synaptic function these vesicle pools are synapse-specific, but accumulating evidence is now demonstrating that some vesicles are laterally mobile along axons and readily shared in a functional manner across adjacent terminals. In effect then, these mobile vesicles represent a further class of synapse-spanning vesicle pool, or "superpool". Here we outline the characteristics of this additional pool type, discussing its structural organization within axons and presynaptic terminals as well as its relationship with conventional vesicle pools. We draw comparisons between extrasynaptic vesicle dynamics and the growing literature on extrasynaptic mobility of non-vesicular synaptic elements which, taken together, raise important questions about the operational independence of adjacent release sites. We also examine the functional implications of lateral vesicle sharing, from the notion that extrasynaptic vesicles can contribute to the release capabilities of individual terminals, to its potential role as a substrate for facilitating changes in synaptic weight as a basis for plasticity.

Animals show periods of quiescence interspersed with periods of motor activity. In a number of invertebrate and vertebrate systems, quiescence is achieved by active suppression of motor behavior is due to tonic inhibition induced by sensory input or changes in internal state. Removal of this inhibition (disinhibition) has the converse effect tending to increase the level of motor activity. We show that tonic inhibition and disinhibition can have a variety of roles. It can simply switch off specific unwanted motor behaviors, or modulate the occurrence of a motor response, a type of 'threshold' controlling function, or be involved in the selection of a particular motor program by inhibiting 'competing' motor mechanisms that would otherwise interfere with the carrying out of a desired movement. A suggested general function for tonic inhibition is to prevent unnecessary non-goal directed motor activity that would be energetically expensive. The reason why basic motor programs might be a particular target for tonic inhibition is that many of them involve central pattern generator circuits that are often spontaneously active and need to be actively suppressed for energy saving. Based on this hypothesis, tonic inhibition represents the default state for energy saving and motor programs are switched-on when required by removal of this inhibition.

Synapse-specific vesicle pools have been widely characterized at central terminals. Here, we demonstrate a vesicle pool that is not confined to a synapse but spans multiple terminals. Using fluorescence imaging, correlative electron microscopy, and modeling of vesicle dynamics, we show that some recycling pool vesicles at synapses form part of a larger vesicle "superpool." The vesicles within this superpool are highly mobile and are rapidly exchanged between terminals (turnover: approximately 4% of total pool/min), significantly changing vesicular composition at synapses over time. In acute hippocampal slices we show that the mobile vesicle pool is also a feature of native brain tissue. We also demonstrate that superpool vesicles are available to synapses during stimulation, providing an extension of the classical recycling pool. Experiments using focal BDNF application suggest the involvement of a local TrkB-receptor-dependent mechanism for synapse-specific regulation of presynaptic vesicle pools through control of vesicle release and capture to or from the extrasynaptic pool., (Copyright 2010 Elsevier Inc. All rights reserved.)

The arrival of an action potential at a synapse triggers neurotransmitter release with a limited probability, p(r). Although p(r) is a fundamental parameter in defining synaptic efficacy, it is not uniform across all synapses, and the mechanisms by which a given synapse sets its basal release probability are unknown. By measuring p(r) at single presynaptic terminals in connected pairs of hippocampal neurons, we show that neighboring synapses on the same dendritic branch have very similar release probabilities, and p(r) is negatively correlated with the number of synapses on the branch. Increasing dendritic depolarization elicits a homeostatic decrease in p(r), and equalizing activity in the dendrite significantly reduces its variability. Our results indicate that local dendritic activity is the major determinant of basal release probability, and we suggest that this feedback regulation might be required to maintain synapses in their operational range.

Central presynaptic terminals harbour synaptic vesicles (SVs) and synapse-specific proteins necessary for neurotransmission. Classically, these elements were thought to reside more or less stably at individual mature synapses, giving rise to the idea that each terminal was essentially an independent functional unit. However, emerging evidence from fluorescence imaging studies in hippocampal cultured neurons is now challenging this view, suggesting that neighbouring synapses along axons share vesicles, and also other synaptic elements, at high levels. This raises the possibility that control of import and export might be an important regulatory target for the maintenance of release sites, modulation of synaptic efficacy and formation of new synaptic contacts. Here, temporal synaptic stability and the functional consequences for presynaptic operation will be considered.

Background: It is now well established that persistent nonsynaptic neuronal plasticity occurs after learning and, like synaptic plasticity, it can be the substrate for long-term memory. What still remains unclear, though, is how nonsynaptic plasticity contributes to the altered neural network properties on which memory depends. Understanding how nonsynaptic plasticity is translated into modified network and behavioral output therefore represents an important objective of current learning and memory research., Results: By using behavioral single-trial classical conditioning together with electrophysiological analysis and calcium imaging, we have explored the cellular mechanisms by which experience-induced nonsynaptic electrical changes in a neuronal soma remote from the synaptic region are translated into synaptic and circuit level effects. We show that after single-trial food-reward conditioning in the snail Lymnaea stagnalis, identified modulatory neurons that are extrinsic to the feeding network become persistently depolarized between 16 and 24 hr after training. This is delayed with respect to early memory formation but concomitant with the establishment and duration of long-term memory. The persistent nonsynaptic change is extrinsic to and maintained independently of synaptic effects occurring within the network directly responsible for the generation of feeding. Artificial membrane potential manipulation and calcium-imaging experiments suggest a novel mechanism whereby the somal depolarization of an extrinsic neuron recruits command-like intrinsic neurons of the circuit underlying the learned behavior., Conclusions: We show that nonsynaptic plasticity in an extrinsic modulatory neuron encodes information that enables the expression of long-term associative memory, and we describe how this information can be translated into modified network and behavioral output.

The synaptic vesicle cycle is vital for sustained neurotransmitter release. It has been assumed that functional synaptic vesicles are replenished autonomously at individual presynaptic terminals. Here we tested this assumption by using FM dyes in combination with fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured rat hippocampal neurons. After photobleaching, synapses acquired recently recycled FM dye-labeled vesicles originating from nonphotobleached synapses by a process requiring dynamic actin turnover. The imported vesicles entered the functional pool at their host synapses, as revealed by the exocytic release of the dye upon stimulation. FM1-43 photoconversion and ultrastructural analysis confirmed the incorporation of imported vesicles into the presynaptic terminal, where they mixed with the native vesicle pools. Our results demonstrate that synaptic vesicle recycling is not confined to individual presynaptic terminals as is widely believed; rather, a substantial proportion of recycling vesicles are shared constitutively between boutons.

Fluorescence recovery after photobleaching (FRAP) provides an important quantitative readout of the mobility of fluorescently tagged structures in live tissue. Here we present a protocol for visualizing FRAP signal at the ultrastructural level, permitting the nature of recovered fluorescence signal to be studied at greater resolution than afforded by conventional light microscopy. Specifically we use FRAP, fixation, photoconversion and correlative light and electron microscopy (CLEM) to examine the ultrastructural organization of mobile FM1-43-labeled vesicles in synapses of cultured hippocampal neurons. At photobleached synapses, the FRAP signal can be visualized as photoconverted electron-dense vesicles. The combination of FRAP and CLEM provides a powerful tool for examining the specific localization of imported vesicles in relation to synaptic architecture. Moreover, with the increasing availability of photoconvertible fluorophores, this approach should be readily applicable to other systems where an ultrastructural characterization of FRAP signal is desirable. After cultures are prepared and ready to use, this protocol takes 2-3 days.

Background: Rhythmic motor behaviors can be generated continuously (e.g., breathing) or episodically (e.g., locomotion, swallowing), when short or long bouts of rhythmic activity are interspersed with periods of quiescence. Although the mechanisms of rhythm generation are known in detail in many systems, there is very little understanding of how the episodic nature of rhythmic behavior is produced at the neuronal level., Results: Using a well-established episodic rhythm-generating neural circuit controlling molluscan feeding, we demonstrate that quiescence between bouts of activity arises from active, maintained inhibition of an otherwise rhythmically active network. We show that the source of the suppressive drive is within the circuit itself; a single central pattern generator (CPG) interneuron type that fires tonically to inhibit feeding during quiescence. Suppression of the tonic activity of this neuron by food is sufficient to change the network from an inactive to a rhythmically active state, with the cell switching function to fire phasically as part of the food-evoked rhythmogenesis. Furthermore, the absolute level of intrinsic suppressive control is modulated extrinsically by the animal's behavioral state (e.g., hunger/satiety), increasing the probability of episodes of feeding when the animal is hungry., Conclusions: By utilizing the same intrinsic member of a CPG network in both rhythm-generation and suppression, this system has developed a simple and efficient mechanism for generating a variable level of response to suit the animal's changing behavioral demands.

Understanding central pattern generator (CPG) circuits requires a detailed knowledge of the intrinsic cellular properties of the constituent neurons. These properties are poorly understood in most CPGs because of the complexity resulting from interactions with other neurons of the circuit. This is also the case in the feeding network of the snail, Lymnaea, one of the best-characterized CPG networks. We addressed this problem by isolating the interneurons comprising the feeding CPG in cell culture, which enabled us to study their basic intrinsic electrical and pharmacological cellular properties without interference from other network components. These results were then related to the activity patterns of the neurons in the intact feeding network. The most striking finding was the intrinsic generation of plateau potentials by medial N1 (N1M) interneurons. This property is probably critical for rhythm generation in the whole feeding circuit because the N1M interneurons are known to play a pivotal role in the initiation of feeding cycles in response to food. Plateau potential generation in another cell type, the ventral N2 (N2v), appeared to be conditional on the presence of acetylcholine. Examination of the other isolated feeding CPG interneurons [lateral N1 (N1L), dorsal N2 (N2d), phasic N3 (N3p)] and the modulatory slow oscillator (SO) revealed no significant intrinsic properties in relation to pattern generation. Instead, their firing patterns in the circuit appear to be determined largely by cholinergic and glutamatergic synaptic inputs from other CPG interneurons, which were mimicked in culture by application of these transmitters. This is an example of a CPG system where the initiation of each cycle appears to be determined by the intrinsic properties of a key interneuron, N1M, but most other features of the rhythm are probably determined by network interactions.