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C F Stevens

Publications and source records attributed to C F Stevens.

At least 37 records · Page 2Linked to original sources
PubMed1998

Crystal structure of the tetramerization domain of the Shaker potassium channel.

Voltage-dependent, ion-selective channels such as Na+, Ca2+ and K+ channel proteins function as tetrameric assemblies of identical or similar subunits. The clustering of four subunits is thought to create an aqueous pore centred at the four-fold symmetry axis. The highly conserved, amino-terminal cytoplasmic domain (approximately 130 amino acids) immediately preceding the first putative transmembrane helix S1 is designated T1. It is known to confer specificity for tetramer formation, so the heteromeric assembly of K+-channel subunits is an important mechanism for the observed channel diversity. We have determined the crystal structure of the T1 domain of a Shaker potassium channel at 1.55 A resolution. The structure reveals that four identical subunits are arranged in a four-fold symmetry surrounding a centrally located pore about 20 A in length. Subfamily-specific assembly is provided primarily by polar interactions encoded in a conserved set of amino acids at its tetramerization interface. Most highly conserved amino acids in the T1 domain of all known potassium channels are found in the core of the protein, indicating a common structural framework for the tetramer assembly.

Amino Acid Sequence↗
PubMed1998

Synaptic vesicles retain their identity through the endocytic cycle.

After fusion of synaptic vesicles with presynaptic membrane and secretion of the contents of the vesicles into the synaptic cleft (a process known as exocytosis), the vesicular membrane is retrieved by endocytosis (internalization) for re-use. Several issues regarding endocytosis at central synapses are unresolved, including the location of membrane retrieval (relative to the active zone, where exocytosis occurs), the time course of various endocytic steps, and the recycling path taken by newly endocytosed membranes. The classical model of synaptic-vesicle recycling, proposed by analogy to other cellular endocytic pathways, involves retrieval of the membrane, fusion of the membrane with endosome-like compartments and, finally, budding of new synaptic vesicles from endosomes, although the endosomal station may not be obligatory. Here we test the classical model by using the fluorescent membrane dye FM1-43 with quantitative fluorescence microscopy. We find that the amount of dye per vesicle taken up by endocytosis equals the amount of dye a vesicle releases on exocytosis; therefore, we conclude that the internalized vesicles do not, as the classical picture suggests, communicate with intermediate endosome-like compartments during the recycling process.

Action Potentials↗
PubMed1998

Readily releasable pool size changes associated with long term depression.

We have estimated, for hippocampal neurons in culture, the size of the autaptic readily releasable pool before and after stimulation of the sort that produces culture long term depression (LTD). This stimulation protocol causes a decrease in the pool size that is proportional to the depression of synaptic currents. To determine if depression in this system is synapse specific rather than general, we have also monitored synaptic transmission between pairs of cultured hippocampal neurons that are autaptically and reciprocally interconnected. We find that the change in synaptic strength is restricted to the synapses on the target neuron that were active during LTD induction. When viewed from the perspective of the presynaptic neuron, however, synapse specificity is partial rather than complete: synapses active during induction that were not on the target neuron were partially depressed.

Animals↗
PubMed1998

Activity-dependent modulation of the rate at which synaptic vesicles become available to undergo exocytosis.

The number of vesicles contained in the readily releasable pool at excitatory hippocampal synapses has recently been identified as a major determinant of the strength of these synapses. Here, we show that the rate at which this pool refills following depletion is variable from neuron to neuron and can be increased by the accumulation of intracellular calcium during action potential-mediated activation of the synapses. The refilling rate nearly doubles during the first second of a high frequency train of action potentials and does not increase further with additional stimulation. During periods of rest, the rate relaxes back to its original value, with a time constant of about 10 s. Since this refilling rate helps set the strength of synapses during high frequency bursts of action potentials and is modulated by physiological signaling, it is an attractive candidate point of control in the storage and manipulation of information by the central nervous system.

Action Potentials↗
PubMed1998

Regulation of the readily releasable vesicle pool by protein kinase C.

Modulation of the size of the readily releasable vesicle pool has recently come under scrutiny as a candidate for the regulation of synaptic strength. Using electrophysiological and optical measurement techniques, we show that phorbol esters increase the size of the readily releasable pool at glutamatergic hippocampal synapses in culture through a protein kinase C (PKC)-dependent mechanism. Phorbol ester activation of PKC also increases the rate at which the pool refills. These results identify two powerful ways that activation of the PKC pathway may regulate synaptic strength by modulating the readily releasable pool of vesicles.

Electric Stimulation↗
PubMed1998

An essential role for retinoid receptors RARbeta and RXRgamma in long-term potentiation and depression.

Hippocampal long-term potentiation (LTP) and long-term depression (LTD) are the most widely studied forms of synaptic plasticity thought to underlie spatial learning and memory. We report here that RARbeta deficiency in mice virtually eliminates hippocampal CA1 LTP and LTD. It also results in substantial performance deficits in spatial learning and memory tasks. Surprisingly, RXRgamma null mice exhibit a distinct phenotype in which LTD is lost whereas LTP is normal. Thus, while retinoid receptors contribute to both LTP and LTD, they do so in different ways. These findings not only genetically uncouple LTP and LTD but also reveal a novel and unexpected role for vitamin A in higher cognitive functions.

Animals↗
PubMed1998

Input synchrony and the irregular firing of cortical neurons.

Cortical neurons in the waking brain fire highly irregular, seemingly random, spike trains in response to constant sensory stimulation, whereas in vitro they fire regularly in response to constant current injection. To test whether, as has been suggested, this high in vivo variability could be due to the postsynaptic currents generated by independent synaptic inputs, we injected synthetic synaptic current into neocortical neurons in brain slices. We report that independent inputs cannot account for this high variability, but this variability can be explained by a simple alternative model of the synaptic drive in which inputs arrive synchronously. Our results suggest that synchrony may be important in the neural code by providing a means for encoding signals with high temporal fidelity over a population of neurons.

Action Potentials↗
PubMed1998

The matter of mind: molecular control of memory.

A widely accepted hypothesis suggests that changes in synaptic strength underlie the formation of memories in the brain. LTP is a mechanism of synaptic strengthening. Induction of LTP depends on NMDA receptor activation, and its expression depends in part on protein kinase activity. Studies of knock-out mice suggest that LTP is critical for hippocampus-based memory. Genetic studies in Drosophila implicate cAMP metabolism in classical conditioning, a form of unconscious memory. Consolidating memories for long-term retention depends on the cAMP-inducible transcription factor CREB.

Animals↗
PubMed1997

Very short-term plasticity in hippocampal synapses.

Hippocampal pyramidal neurons often fire in bursts of action potentials with short interspike intervals (2-10 msec). These high-frequency bursts may play a critical role in the functional behavior of hippocampal neurons, but synaptic plasticity at such short times has not been carefully studied. To study synaptic modulation at very short time intervals, we applied pairs of stimuli with interpulse intervals ranging from 7 to 50 msec to CA1 synapses isolated by the method of minimal stimulation in hippocampal slices. We have identified three components of short-term paired-pulse modulation, including (i) a form of synaptic depression manifested after a prior exocytotic event, (ii) a form of synaptic depression that does not depend on a prior exocytotic event and that we postulate is based on inactivation of presynaptic N-type Ca2+ channels, and (iii) a dependence of paired-pulse facilitation on the exocytotic history of the synapse.

Animals↗
PubMed1997

The rate of aldehyde fixation of the exocytotic machinery in cultured hippocampal synapses.

The rate at which the transmitter release machinery is fixed by 2% glutaraldehyde at hippocampal synapses and the amount of release evoked by the fixative have been investigated. We recorded from hippocampal cells while fixative was applied with a rapid flow system. Release is blocked in less than a second and fixative-produced exocytosis is at most a few percent of what would be caused by a hypertonic stimulus that completely depletes the readily releasable pool of vesicles. The postsynaptic receptors for glutamate cease to respond to agonist with a time constant of approximately 3 s when fixative is applied. We conclude that some essential component of the exocytotic apparatus is fixed in less than a second and that the fixative does not significantly deplete the readily releasable pool.

Aldehydes↗
PubMed1997

Quantitative ultrastructural analysis of hippocampal excitatory synapses.

From three-dimensional reconstructions of CA1 excitatory synapses in the rodent hippocampus and in culture, we have estimated statistical distributions of active zone and postsynaptic density (PSD) sizes (average area approximately 0.04 micron2), the number of active zones per bouton (usually one), the number of docked vesicles per active zone (approximately 10), and the total number of vesicles per bouton (approximately 200), and we have determined relationships between these quantities, all of which vary from synapse to synapse but are highly correlated. These measurements have been related to synaptic physiology. In particular, we propose that the distribution of active zone areas can account for the distribution of synaptic release probabilities and that each active zone constitutes a release site as identified in the standard quantal theory attributable to Katz (1969).

Animals↗
PubMed1997

The small GTP-binding protein Rab3A regulates a late step in synaptic vesicle fusion.

The Rab family of low-molecular-mass GTP-binding proteins are thought to guide membrane fusion between a transport vesicle and the target membrane, and to determine the specificity of docking. The docking and fusion of vesicles is, however, a complex multistep reaction, and the precise point at which Rab proteins act in these sequential processes is unknown. In brain, the Rab protein Rab3A is specific to synaptic vesicles, whose exocytosis can be monitored with submillisecond resolution by following synaptic transmission. We have now determined the precise point at which Rab3A acts in the sequence of synaptic vesicle docking and fusion by using electrophysiological analysis of neurotransmitter release in Rab3A-deficient mice. Unexpectedly, the size of the readily releasable pool of vesicles is normal, whereas Ca2+-triggered fusion is altered in the absence of Rab3A in that a more-than-usual number of exocytic events occur within a brief time after arrival of the nerve impulse.

2-Amino-5-phosphonovalerate↗
PubMed1997

Heterogeneous release properties of visualized individual hippocampal synapses.

We have used endocytotic uptake of the styryl dye FM1-43 at synaptic terminals (Betz and Bewick, 1992) to study properties of individual synapses formed by axons of single hippocampal neurons in tissue culture. The distribution of values for probability of evoked transmitter release p estimated by dye uptake is continuous, with a preponderance of low p synapses and a broad spread of probabilities. We have validated this method by demonstrating that the optically estimated distribution of p at autapses in single-neuron microislands predicts, with no free parameters, the rate of blocking of NMDA responses by the noncompetitive antagonist MK-801 at the same synapses. Different synapses made by a single axon exhibited varying amounts of paired-pulse modulation; synapses with low p tended to be facilitated more than those with high p. The increment in release probability produced by increasing external calcium ion concentration also depended on a synapse's initial p value. The size of the recycling pool of vesicles was strongly correlated with p as well, suggesting that synapses with higher release probabilities had more vesicles. Finally, p values of neighboring synapses were correlated, indicating local interactions in the dendrite or axon, or both.

Action Potentials↗
PubMed1997

Heterogeneity of release probability, facilitation, and depletion at central synapses.

Previous studies of short-term plasticity in central nervous systems synapses have largely focused on average synaptic properties. In this study, we use recordings from putative single synaptic release sites in hippocampal slices to show that significant heterogeneity exists in facilitation and depletion among synapses. In particular, the amount of paired-pulse facilitation is inversely related to the initial release probability of the synapse. We also examined depletion at individual synapses using high frequency stimulation, and estimated the size of the readily releasable vesicle pool, which averaged 5.0 +/- 3.0 quanta (n = 13 synapses). In addition, these experiments demonstrate that the release probability at a synapse is directly correlated with the size of its readily releasable vesicle pool.

Animals↗
PubMed1997

Estimating the distribution of synaptic reliabilities.

Using whole cell recording from CA1 hippocampal pyramidal neurons in slices, we examined the progressive decrease of N-methyl-D-aspartate receptor-mediated synaptic responses in the presence of the open-channel blocker MK-801. Previous studies analyzing this decrease have proposed that hippocampal synapses fall into two distinct classes of release probabilities, whereas studies based on other methods indicate a broad distribution of synaptic reliabilities exists. Here we derive the theoretical relationship between the MK-801-mediated decrease in excitatory postsynaptic current amplitudes and the underlying distribution of synaptic reliabilities. We find that the MK-801 data are consistent with a continuous distribution of synaptic reliabilities, in agreement with studies examining individual synapses. In addition, changes in the MK-801-mediated decrease in response size as a consequence of altering release probability are consistent with this continuous distribution of synaptic reliabilities.

Animals↗