Central mechanisms of pathological pain

Chronic pain is a major challenge to clinical practice and basic science. The peripheral and central neural networks that mediate nociception show extensive plasticity in pathological disease states. Disease-induced plasticity can occur at both structural and functional levels and is manifest as changes in individual molecules, synapses, cellular function and network activity. Recent work has yielded a better understanding of communication within the neural matrix of physiological pain and has also brought important advances in concepts of injury-induced hyperalgesia and tactile allodynia and how these might contribute to the complex, multidimensional state of chronic pain. This review focuses on the molecular determinants of network plasticity in the central nervous system (CNS) and discusses their relevance to the development of new therapeutic approaches.     

The diverse roles of ribbon synapses in sensory neurotransmission

Sensory synapses of the visual and auditory systems must faithfully encode a wide dynamic range of graded signals, and must be capable of sustained transmitter release over long periods of time. Functionally and morphologically, these sensory synapses are unique: their active zones are specialized in several ways for sustained, rapid vesicle exocytosis, but their most striking feature is an organelle called the synaptic ribbon, which is a proteinaceous structure that extends into the cytoplasm at the active zone and tethers a large pool of releasable vesicles. But precisely how does the ribbon function to support tonic release at these synapses? Recent genetic and biophysical advances have begun to open the 'black box' of the synaptic ribbon with some surprising findings and promise to resolve its function in vision and hearing.     

Optimal Learning Rules for Discrete Synapses

There is evidence that biological synapses have a limited number of discrete weight states. Memory storage with such synapses behaves quite differently from synapses with unbounded, continuous weights, as old memories are automatically overwritten by new memories. Consequently, there has been substantial discussion about how this affects learning and storage capacity. In this paper, we calculate the storage capacity of discrete, bounded synapses in terms of Shannon information. We use this to optimize the learning rules and investigate how the maximum information capacity depends on the number of synapses, the number of synaptic states, and the coding sparseness. Below a certain critical number of synapses per neuron (comparable to numbers found in biology), we find that storage is similar to unbounded, continuous synapses. Hence, discrete synapses do not necessarily have lower storage capacity.     

Neurotransmitter release at central synapses

Our understanding of synaptic transmission has grown dramatically during the 15 years since the first issue of Neuron was published, a growth rate expected from the rapid progress in modern biology. As in all of biology, new techniques have led to major advances in the cell and molecular biology of synapses, and the subject has evolved in ways (like the production of genetically engineered mice) that could not even be imagined 15 years ago. My plan for this review is to summarize what we knew about neurotransmitter release when Neuron first appeared and what we recognized we did not know, and then to describe how our views have changed in the intervening decade and a half. Some things we knew about synapses--"knew" in the sense that the field had reached a consensus--are no longer accepted, but for the most part, impressive advances have led to a new consensus on many issues. What I find fascinating is that in certain ways nothing has changed--many of the old arguments persist or recur in a different guise--but in other ways the field would be unrecognizable to a neurobiologist time-transported from 1988 to 2003.     

Ionic currents, transmitters and models of motor pattern generators

Recent work, combining direct study of ion channels and synapses with pharmacological manipulations and realistic computer simulations, has deepened our understanding of how motor circuits produce rhythmic outputs. In several preparations, both the roles of some key ionic currents in circuit operation and the mechanisms by which circuit operation may be modulated have been identified.     

Synaptic development: insights from Drosophila

In Drosophila, the larval neuromuscular junction is particularly tractable for studying how synapses develop and function. In contrast to vertebrate central synapses, each presynaptic motor neuron and postsynaptic muscle cell is unique and identifiable, and the wiring circuit is invariant. Thus, the full power of Drosophila genetics can be brought to bear on a single, reproducibly identifiable, synaptic terminal. Each individual neuromuscular junction encompasses hundreds of synaptic neurotransmitter release sites housed in a chain of synaptic boutons. Recent advances have increased our understanding of the mechanisms that shape the development of both individual synapses--that is, the transmitter release sites including active zones and their apposed glutamate receptor clusters--and the whole synaptic terminal that connects a pre- and post-synaptic cell.     

Single vesicle tracking for studying synaptic vesicle dynamics in small central synapses

Sustained neurotransmission is driven by a continuous supply of synaptic vesicles to the release sites and modulated by synaptic vesicle dynamics. However, synaptic vesicle dynamics in synapses remain elusive because of technical limitations. Recent advances in fluorescence imaging techniques have enabled the tracking of single synaptic vesicles in small central synapses in living neurons. Single vesicle tracking has uncovered a wealth of new information about synaptic vesicle dynamics both within and outside presynaptic terminals, showing that single vesicle tracking is an effective tool for studying synaptic vesicle dynamics. Particularly, single vesicle tracking with high spatiotemporal resolution has revealed the dependence of synaptic vesicle dynamics on the location, stages of recycling, and neuronal activity. This review summarizes the recent findings from single synaptic vesicle tracking in small central synapses and their implications in synaptic transmission and pathogenic mechanisms of neurodegenerative diseases.     

Mechanisms of synapse assembly and disassembly

The mechanisms that govern synapse formation and elimination are fundamental to our understanding of neural development and plasticity. The wiring of neural circuitry requires that vast numbers of synapses be formed in a relatively short time. The subsequent refinement of neural circuitry involves the formation of additional synapses coincident with the disassembly of previously functional synapses. There is increasing evidence that activity-dependent plasticity also involves the formation and disassembly of synapses. While we are gaining insight into the mechanisms of both synapse assembly and disassembly, we understand very little about how these phenomena are related to each other and how they might be coordinately controlled to achieve the precise patterns of synaptic connectivity in the nervous system. Here, we review our current understanding of both synapse assembly and disassembly in an effort to unravel the relationship between these fundamental developmental processes.     

Late-phase long-term potentiation: getting to the nucleus

New mRNA must be transcribed in order to consolidate changes in synaptic strength. But how are events at the synapse communicated to the nucleus? Some research has shown that proteins can move from activated synapses to the nucleus. However, other work has shown that action potentials can directly inform the nucleus about cellular activation. Here we contend that action potential-induced signalling to the nucleus best meets the requirements of the consolidation of synapse-specific plasticity, which include both timing and stoichiometric constraints.     

A developmental switch in neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation

Formation of glutamatergic synapses entails development of "silent" immature contacts into mature functional synapses. To determine how this transformation occurs, we investigated the development of neurotransmission at single synapses in vitro. Maturation of presynaptic function, assayed with endocytotic markers, followed accumulation of synapsin I. During this period, synaptic transmission was primarily mediated by activation of NMDA receptors, suggesting that most synapses were functionally silent. However, local glutamate application to silent synapses indicated that these synapses contained functional AMPA receptors, suggesting a possible presynaptic locus for silent transmission. Interference with presynaptic vesicle fusion by exposure to tetanus toxin reverted functional to silent transmission, implicating SNARE-mediated fusion as a determinant of the ratio of NMDA:AMPA receptor activation. This work reveals that functional maturation of synaptic transmission involves transformation of presynaptic silent secretion into mature synaptic transmitter release.     

Optical detection of synaptic vesicle exocytosis and endocytosis.

Recent advances in optical methods have catalyzed a detailed study of individual visualized synapses in several model systems. Quantal events at small central synapses, as well as single granule exocytosis in secretory cells, have been detected using quantitative fluorescence imaging. Sensitive detection of exocytosis and endocytosis at individual synapses has advanced our knowledge of synaptic vesicle trafficking.     

Efficacy of thalamocortical and intracortical synaptic connections: quanta, innervation, and reliability.

Thalamocortical (TC) synapses carry information into the neocortex, but they are far outnumbered by excitatory intracortical (IC) synapses. We measured the synaptic properties that determine the efficacy of TC and IC axons converging onto spiny neurons of layer 4 in the mouse somatosensory cortex. Quantal events from TC and IC synapses were indistinguishable. However, TC axons had, on average, about 3 times more release sites than IC axons, and the mean release probability at TC synapses was about 1.5 times higher than that at IC synapses. Differences of innervation ratio and release probability make the average TC connection several times more effective than the average IC connection, and may allow small numbers of TC axons to dominate the activity of cortical layer 4 cells during sensory inflow.     

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer''s attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.     

Receptor trafficking and the plasticity of excitatory synapses

Newly discovered features of the trafficking of AMPA receptors to and from the postsynaptic membrane of excitatory synapses are now bringing the mechanisms of synaptic plasticity into focus. Recent advances, including the existence of slots, anchors, transport factors and pathways for activity-dependent control, have elucidated the role of the individual AMPA receptor subunits and their binding partners. The latest views describe how subunit type dictates the assembly of heteromeric receptors, and how these heteromers interact with the receptor trafficking machinery and synaptic anchorage factors. Moreover, phosphorylation may play an important role in receptor transport and synaptic turnover.     

Exploring the Ca2+-dependent synaptic dynamics in vibro-dissociated cells

Dynamic alteration of the synaptic strength is one of the most important processes occurring in the nervous system. Combination of electrophysiology, confocal imaging and molecular biology led to significant advances in this research field. Yet, a progress in this area, in particular in studies of changes in the quantal behavior of central synapses and impact of glial cells on individual synapses, is hampered by technical difficulties of resolving small quantal synaptic currents. In this paper we will show how the technique of non-enzymatic vibro-dissociation, which enables to isolate living neurons avoiding artifacts of cell culture and preserving functional synapse, can be used to obtain a valuable information on fine details and mechanisms of synaptic plasticity. In particular, we will describe our recent results on Ca²⁺-dependent modulation of the postsynaptic AMPA and NMDA receptors in the individual synaptic boutons.     

Acetylcholinesterase dynamics at the neuromuscular junction of live animals

At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely slow. This fluorescent probe was used to study the removal and insertion of AChE at individual synapses of living adult mice. After a one-time blockade of AChEs with fluorescent fasciculin 2, AChEs are removed from synapses initially at a faster rate (t(1/2) of approximately 3 days) and later at a slower rate (t(1/2) of approximately 12 days). Most of the removed AChEs are replaced by newly inserted AChEs over time. However, when AChEs are continuously blocked with fasciculin 2, the removal rate increases substantially (t(1/2) of approximately 12 h), and most of the lost AChEs are not replaced by newly inserted AChE. Furthermore, complete one-time inactivation of AChE activity significantly increases the removal of postsynaptic nicotinic acetylcholine receptors (AChRs). Finally, time lapse imaging reveals that synaptic AChEs and AChRs that are removed from synapses are co-localized in the same pool after being internalized. These results demonstrate a remarkable AChE dynamism and argue for a potential link between AChE function and postsynaptic receptor lifetime.     

Drosophila memory: will Orb(2) predict the future?

Genetically tractable organisms with relatively simple nervous systems offer a realistic platform to understand how and where memories are formed and stored in defined neural circuits. Recent work in Drosophila provides promise that this analysis may soon reach the resolution of identifiable synapses.     

Collecting,processing, and storing avian blood: a review

ABSTRACT With recent advances in molecular techniques, collecting blood from birds has become a common practice among field ornithologists. There are a variety of techniques for collecting blood samples and numerous caveats for how samples should be processed, depending on the research question being asked. Currently, few resources are available for individuals learning how to collect blood from birds or needing more information about how to process blood samples. Here, I describe commonly used methods for collecting, processing, and storing blood for particular research objectives, and provide answers to frequently asked questions about blood collection. The information provided is intended primarily for investigators working with passerines, but many techniques and suggestions are applicable to other avian taxa.     

Energy Efficient Sparse Connectivity from Imbalanced Synaptic Plasticity Rules

It is believed that energy efficiency is an important constraint in brain evolution. As synaptic transmission dominates energy consumption, energy can be saved by ensuring that only a few synapses are active. It is therefore likely that the formation of sparse codes and sparse connectivity are fundamental objectives of synaptic plasticity. In this work we study how sparse connectivity can result from a synaptic learning rule of excitatory synapses. Information is maximised when potentiation and depression are balanced according to the mean presynaptic activity level and the resulting fraction of zero-weight synapses is around 50%. However, an imbalance towards depression increases the fraction of zero-weight synapses without significantly affecting performance. We show that imbalanced plasticity corresponds to imposing a regularising constraint on the L ₁-norm of the synaptic weight vector, a procedure that is well-known to induce sparseness. Imbalanced plasticity is biophysically plausible and leads to more efficient synaptic configurations than a previously suggested approach that prunes synapses after learning. Our framework gives a novel interpretation to the high fraction of silent synapses found in brain regions like the cerebellum.     

Neuroenergetics and the kinetic design of excitatory synapses

Why is the characteristic timescale of neural information processing in the millisecond range, corresponding to a 'clock speed' of about 1 kHz, whereas the clock speed of modern computers is about 3 GHz? Here we investigate how the brain's energy supply limits the maximum rate at which the brain can compute, and how the molecular components of excitatory synapses have evolved properties that are matched to the information processing they perform.