Biomedical subjects

Stephen J Smith

Publications and source records attributed to Stephen J Smith.

18 recordsLinked to original sources

PubMed2006

Pregabalin reduces the release of synaptic vesicles from cultured hippocampal neurons.

Pregabalin [S-[+]-3-isobutylGABA or (S)-3-(aminomethyl)-5-methylhexanoic acid, Lyrica] is an anticonvulsant and analgesic medication that is both structurally and pharmacologically related to gabapentin (Neurontin; Pfizer Inc., New York, NY). Previous studies have shown that pregabalin reduces the release of neurotransmitters in several in vitro preparations, although the molecular details of these effects are less clear. The present study was performed using living cultured rat hippocampal neurons with the synaptic vesicle fluorescent dye probe FM4-64 to determine details of the action of pregabalin to reduce neurotransmitter release. Our results indicate that pregabalin treatment, at concentrations that are therapeutically relevant, slightly but significantly reduces the emptying of neurotransmitter vesicles from presynaptic sites in living neurons. Dye release is reduced in both glutamic acid decarboxylase (GAD)-immunoreactive and GAD-negative (presumed glutamatergic) synaptic terminals. Furthermore, both calcium-dependent release and hyperosmotic (calcium-independent) dye release are reduced by pregabalin. The effects of pregabalin on dye release are masked in the presence of l-isoleucine, consistent with the fact that both of these compounds have a high binding affinity to the calcium channel alpha(2)-delta protein. The effect of pregabalin is not apparent in the presence of an N-methyl-d-aspartate (NMDA) antagonist [D(-)-2-amino-5-phosphonopentanoic acid], suggesting that pregabalin action depends on NMDA receptor activation. Finally, the action of pregabalin on dye release is most apparent before and early during a train of electrical stimuli when vesicle release preferentially involves the readily releasable pool.

Animals↗

PubMed2006

Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms.

To explore the relationship between axon arbor growth and synaptogenesis, developing retinal ganglion cell (RGC) axon arbors in zebrafish optic tectum were imaged in vivo at high temporal and spatial resolution using two-photon microscopy. Individual RGC axons were dually labeled by expression of a cytosolic red fluorescent protein (DsRed Express) to mark arbor structure and a fusion of the synaptic vesicle protein synaptophysin with green fluorescent protein (Syp:GFP) to mark presynaptic vesicles. Analysis of time-lapse sequences acquired at 10 min intervals revealed unexpectedly rapid kinetics of both axon branch and vesicle cluster turnover. Nascent axonal branches exhibited short average lifetimes of 19 min, and only 17% of newly extended axonal processes persisted for periods exceeding 3 h. The majority (70%) of Syp:GFP puncta formed on newly extended axonal processes. Syp:GFP puncta also exhibited short average lifetimes of 30 min, and only 34% of puncta were stabilized for periods exceeding 3 h. Moreover, strongly correlated dynamics of Syp:GFP puncta and branch structure suggest that synaptogenesis exerts strong influences on both the extension and the selective stabilization of nascent branches. First, new branches form almost exclusively at newly formed Syp:GFP puncta. Second, stabilized nascent branches invariably bear Syp:GFP puncta, and the detailed dynamics of branch retraction suggest strongly that nascent synapses can act at branch tips to arrest retraction. These observations thus provide evidence that synaptogenesis guides axon arbor growth by first promoting initial branch extension and second by selective branch stabilization.

Aging↗

PubMed2006

Promoting vitality in health and physical education.

Vitality draws together the interests of health and physical education. Already these fields of education have come together, with health, fitness, wellness, and active and healthy living as shared curricular concepts. Vitality furthers these conjunctions by having us rethink prevailing views of the body of knowledge in health and physical education. More than a concept, vitality is promoted phenomenologically in terms of the essential movements of the body. It is explicated as vitality affects, specifically identifiable motions and developmental patterns of movement that provide curricular structure for teaching health and physical education. The promotional implications of this analysis relate to enlivening the baseline criteria currently used in health and physical education assessments; revitalizing the curricular concepts of body awareness, space, time, and relationships on which provincial programs are based; and expanding the reach of these programs to mental, emotional, spiritual, and, particularly, environmental health.

Health Education↗

PubMed2005

Strong effects of subphysiological temperature on the function and plasticity of mammalian presynaptic terminals.

Most cellular processes are known to be strongly temperature dependent. Nevertheless, a large fraction of studies of mammalian synaptic function have been and are performed near room temperature (i.e., at least 10 degrees C below physiological temperature). Here, we examined the effects of temperature on presynaptic function in primary cultures of rat hippocampal neurons. FM dyes, VAMP (vesicle-associated membrane protein)-GFP (green fluorescent protein) transfection, and HRP uptake were used to quantify various aspects of synaptic vesicle recycling. Our results show that there are very substantial differences in synaptic vesicle recycling at physiological temperature as opposed to the common, lower experimental temperatures. At 37 degrees C, compared with 23 degrees C, the speed of both exocytosis and endocytosis was higher. The size of the recycling vesicle pool (in both number of vesicles and spatial extent) was twofold larger at 37 degrees C. In addition, although repeated 10 Hz electrical stimulation caused an NMDA receptor-dependent enlargement (averaging 170%) of the measurable recycling vesicle pool at 23 degrees C, the same stimulus repetition had no effect at 37 degrees C. These results show that it is potentially misleading to extend conclusions drawn about vesicle function or presynaptic plasticity at lowered experimental temperature to physiological conditions and that much new experimental work at the higher physiological temperature range will be needed to understand the true parameters of presynaptic functions.

Animals↗

PubMed2005

Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors.

Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery.

Animals↗

PubMed2005

Regulation of axon growth in vivo by activity-based competition.

The formation of functional neural networks requires precise regulation of the growth and branching of the terminal arbors of axons, processes known to be influenced by early network electrical activity. Here we show that a rule of activity-based competition between neighbouring axons appears to govern the growth and branching of retinal ganglion cell (RGC) axon arbors in the developing optic tectum of zebrafish. Mosaic expression of an exogenous potassium channel or a dominant-negative SNARE protein was used to suppress electrical or neurosecretory activity in subsets of RGC axons. Imaging in vivo showed that these forms of activity suppression strongly inhibit both net growth and the formation of new branches by individually transfected RGC axon arbors. The inhibition is relieved when the activity of nearby 'competing' RGC axons is also suppressed. These results therefore identify a new form of activity-based competition rule that might be a key regulator of axon growth and branch initiation.

Animals↗

PubMed2005

Functional imaging reveals rapid development of visual response properties in the zebrafish tectum.

The visual pathway from the retina to the optic tectum in fish and frogs has long been studied as a model for neural circuit formation. Although morphological aspects, such as axonal and dendritic arborization, have been well characterized, less is known about how this translates into functional properties of tectal neurons during development. We developed a system to provide controlled visual stimuli to larval zebrafish, while performing two-photon imaging of tectal neurons loaded with a fluorescent calcium indicator, allowing us to determine visual response properties in intact fish. In relatively mature larvae, we describe receptive field sizes, visual topography, and direction and size selectivity. We also characterize the onset and development of visual responses, beginning when retinal axons first arborize in the tectum. Surprisingly, most of these properties are established soon after dendrite growth and synaptogenesis begin and do not require patterned visual experience or a protracted period of refinement.

Animals↗

PubMed2005

Characterization of zebrafish PSD-95 gene family members.

The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism.

Animals↗

PubMed2004

In vivo trafficking and targeting of N-cadherin to nascent presynaptic terminals.

N-cadherin is a prominent component of developing and mature synapses, yet very little is known about its trafficking within neurons. To investigate N-cadherin dynamics in developing axons, we used in vivo two-photon time-lapse microscopy of N-cadherin--green fluorescent protein (Ncad-GFP), which was expressed in Rohon-Beard neurons of the embryonic zebrafish spinal cord. Ncad-GFP was present as either stable accumulations or highly mobile transport packets. The mobile transport packets were of two types: tubulovesicular structures that moved preferentially in the anterograde direction and discrete-punctate structures that exhibited bidirectional movement. Stable puncta of Ncad-GFP accumulated in the wake of the growth cone with a time course. Colocalization of Ncad-GFP puncta with synaptic markers suggests that N-cadherin is a very early component of nascent synapses. Expression of deletion mutants revealed a potential role of the extracellular domain in appropriate N-cadherin trafficking and targeting. These results are the first to characterize the trafficking of a synaptic cell-adhesion molecule in developing axons in vivo. In addition, we have begun to investigate the cell biology of N-cadherin trafficking and targeting in the context of an intact vertebrate embryo.

Animals↗

PubMed2004

In vivo imaging of synapse formation on a growing dendritic arbor.

The form of a neuron's dendritic arbor determines the set of axons with which it may form synaptic contacts, thus establishing connectivity within neural circuits. However, the dynamic relationship between dendrite growth and synaptogenesis is not well understood. To observe both processes simultaneously, we performed long-term imaging of non-spiny dendritic arbors expressing a fluorescent postsynaptic marker protein as they arborized within the optic tectum of live zebrafish larvae. Our results indicate that almost all synapses form initially on newly extended dendritic filopodia. A fraction of these nascent synapses are maintained, which in turn stabilizes the subset of filopodia on which they form. Stabilized filopodia mature into dendritic branches, and successive iterations of this process result in growth and branching of the arbor. These findings support a 'synaptotropic model' in which synapse formation can direct dendrite arborization.

Animals↗

PubMed2004

Neural activity and the dynamics of central nervous system development.

Recent imaging studies show that the formation of neural connections in the central nervous system is a highly dynamic process. The iterative formation and elimination of synapses and neuronal branches result in the formation of a much larger number of trial connections than is maintained in the mature brain. Neural activity modulates development through biasing this process of formation and elimination, promoting the formation and stabilization of appropriate synaptic connections on the basis of functional activity patterns.

Animals↗

PubMed2004

Live optical imaging of nervous system development.

Although development of the nervous system is inherently a process of dynamic change, until recently it has generally been investigated by inference from static images. However, advances in live optical imaging are now allowing direct observation of growth, synapse formation, and even incipient function in the developing nervous system, at length scales from molecules to cortical regions, and over timescales from milliseconds to months. In this review, we provide technical background and present examples of how these new methods, including confocal and two-photon microscopy, GFP-based markers, and functional indicators, are being applied to provide fresh insight into long-standing questions of neural development.

Animals↗

PubMed2003

Mitochondria and release at hippocampal synapses.

Mitochondria are present in some, but not all presynaptic terminals in the hippocampus. Mitochondria are capable of sequestering and storing large amounts of calcium, but it is unclear whether they influence release probability at these synapses. Using FM dye imaging techniques and confocal microscopy, we have examined the relationship between mitochondrial presence/absence and presynaptic vesicle release from rat hippocampal neurones in primary dissociated culture at room temperature. Following staining with the mitochondrial dye mitotracker green, we were able to resolve putative individual mitochondria associated with neuronal processes. The majority of mitochondria were positionally stable, although some exhibited periods of rapid motility (up to 0.4 microm/s) interspersed with periods of immobility. Co-staining with mitotracker green and the synaptic vesicle dye FM 4-64 indicated that 180 of 506 (36%) synapses were devoid of mitochondria. A comparison of vesicular release in response to stimulation at 1 Hz and at 10 Hz revealed no differences in release properties between synapses with and without mitochondria.

Animals↗

PubMed2003

Integrated bio-fluorescence sensor.

Due to the recent explosion in optoelectronics for telecommunication applications, novel optoelectronic sensing structures can now be realized. In this work, we explore the integration of optoelectronic components towards miniature and portable fluorescence sensors. The integration of these micro-fabricated sensors with microfluidics and capillary networks may reduce the cost and complexity of current research instruments and open up a world of new applications in portable biological analysis systems. A novel optoelectronic design that capitalizes on current vertical-cavity surface-emitting laser (VCSEL) technology is explored. Specifically, VCSELs, optical emission filters and PIN photodetectors are fabricated as part of a monolithically integrated near-infrared fluorescence detection system. High-performance lasers and photodetectors have been characterized and integrated to form a complete sensor. Experimental results show that sensor sensitivity is limited by laser background. The laser background is caused by spontaneous emission emitted from the side of the VCSEL excitation source. Laser background will limit sensitivity in most integrated sensing designs due to locating excitation sources and photodetectors in such close proximity, and methods are proposed to reduce the laser background in such designs so that practical fluorescent detection limits can be achieved.

Biosensing Techniques↗

PubMed2003

Retrograde regulation of synaptic vesicle endocytosis and recycling.

Sustained release of neurotransmitter depends upon the recycling of synaptic vesicles. Until now, it has been assumed that vesicle recycling is regulated by signals from the presynaptic bouton alone, but results from rat hippocampal neurons reported here indicate that this need not be the case. Fluorescence imaging and pharmacological analysis show that a nitric oxide (NO) signal generated postsynaptically can regulate endocytosis and at least one later step in synaptic vesicle recycling. The proposed retrograde pathway involves an NMDA receptor (NMDAR)-dependent postsynaptic production of NO, diffusion of NO to a presynaptic site, and a cGMP-dependent increase in presynaptic phosphatidylinositol 4,5-biphosphate (PIP2). These results indicate that the regulation of synaptic vesicle recycling may integrate a much broader range of neural activity signals than previously recognized, including postsynaptic depolarization and the activation of NMDARs at both immediate and nearby postsynaptic active zones.

Animals↗

PubMed2002

Vesicle pool partitioning influences presynaptic diversity and weighting in rat hippocampal synapses.

Hippocampal synapses display a range of release probabilities. This is partially the result of scaling of release probability with the total number of releasable vesicles at each synapse. We have compared synaptic release and vesicle pool sizes across a large number of hippocampal synapses using FM 1-43 and confocal fluorescence microscopy. We found that the relationship between the number of recycling vesicles at a synapse and its release probability is dependent on firing frequency. During firing at 10 Hz, the release probability of each synapse is closely related to the number of recycling vesicles that it contains. In contrast, during firing at 1 Hz, different synapses turn over their recycling vesicle pools at different rates leading to an indirect relationship between recycling vesicle pool size and release probability. Hence two synapses may release vesicles at markedly different rates during low frequency firing, even if they contain similar numbers of vesicles. Both further kinetic analyses and manipulation of the number of vesicles in the readily releasable pool using phorbol ester treatment suggested that this imprecise scaling observed during firing at 1 Hz resulted from synapse-to-synapse differences in the proportion of recycling vesicles partitioned into the readily releasable pool. Hence differential partitioning between vesicle pools affects presynaptic weighting in a frequency-dependent manner. Since hippocampal single unit firing rates shift between 1 Hz and 10 Hz regimes with behavioural state, differential partitioning may be a mechanism for encoding information in hippocampal circuits.

Action Potentials↗

PubMed2002

Knowing a nascent synapse when you see it.

To understand brain development, we must learn how synapse formation shapes functional neural circuits. At the heart of this process lies the nascent synapse--an enigmatic structure spanning the developmental gap between initial cell-cell contact and the mature synapse. New experimental techniques are beginning to illuminate the processes involved in synaptogenesis, but much remains to be learned, including simply how to recognize the synapse in its nascent form.

Animals↗

PubMed2002

Stability and plasticity of developing synapses in hippocampal neuronal cultures.

To explore mechanisms governing the formation, stability, and elimination of synapses during neuronal development, we used FM 1-43 fluorescence imaging to track vesicle turnover at >7000 individually identified developing synapses between embryonic rat hippocampal neurons in culture. The majority of presynaptic boutons were stable in efficacy and position over a period of 1.5 hr. Activity, evoked by burst-patterned field stimulation, decreased presynaptic function across the population of boutons, an effect that required NMDA receptor activation. Decreased FM 1-43 staining correlated with low synapsin-I and synaptophysin immunoreactivities, suggesting that decreased presynaptic function was commensurate with synaptic disassembly. These observations provide new information on the stability of developing presynaptic function and suggest that NMDA receptor activation may regulate the stability of developing synapses.

Animals↗