Biomedical subjects

Stephen J Smith

Publications and source records attributed to Stephen J Smith.

9 recordsLinked to original sources

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↗