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Biomedical subjects

Jane M Sullivan

Publications and source records attributed to Jane M Sullivan.

11 recordsLinked to original sources
PubMed2006

Amyloid precursor protein overexpression depresses excitatory transmission through both presynaptic and postsynaptic mechanisms.

Overexpression of the amyloid precursor protein (APP) in hippocampal neurons leads to elevated beta-amyloid peptide (Abeta) production and consequent depression of excitatory transmission. The precise mechanisms underlying APP-induced synaptic depression are poorly understood. Uncovering these mechanisms could provide insight into how neuronal function is compromised before cell death during the early stages of Alzheimer's disease. Here we verify that APP up-regulation leads to depression of transmission in cultured hippocampal autapses; and we perform whole-cell recording, FM imaging, and immunocytochemistry to identify the specific mechanisms accounting for this depression. We find that APP overexpression leads to postsynaptic silencing through a selective reduction of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated currents. This effect is likely mediated by Abeta because expression of mutant APP incapable of producing Abeta did not depress transmission. In addition, although we eliminate presynaptic silencing as a mechanism underlying APP-mediated inhibition of transmission, we did observe an Abeta-induced presynaptic deficit in vesicle recycling with sustained stimulation. These findings demonstrate that APP elevation disrupts both presynaptic and postsynaptic compartments.

Amyloid beta-Peptides↗
PubMed2006

A simple depletion model of the readily releasable pool of synaptic vesicles cannot account for paired-pulse depression.

Paired-pulse depression (PPD) is a form of short-term plasticity that plays a central role in processing of synaptic activity and is manifest as a decrease in the size of the response to the second of two closely timed stimuli. Despite mounting evidence to the contrary, PPD is still commonly thought to reflect depletion of the pool of synaptic vesicles available for release in response to the second stimulus. Here it is shown that PPD cannot be accounted for by depletion at excitatory synapses made by hippocampal neurons because PPD is unaffected by changes in the fraction of the readily releasable pool (RRP) released by the first of a pair of pulses.

Animals↗
PubMed2006

Synaptic vesicle protein 2 enhances release probability at quiescent synapses.

We report a thorough analysis of neurotransmission in cultured hippocampal neurons lacking synaptic vesicle protein 2 (SV2), a membrane glycoprotein present in all vesicles that undergo regulated secretion. We found that SV2 selectively enhances low-frequency neurotransmission by priming morphologically docked vesicles. Loss of SV2 reduced initial release probability during a train of action potentials but had no effect on steady-state responses. The amount and decay rate of asynchronous release, two measures sensitive to presynaptic calcium concentrations, are not altered in SV2 knock-outs, suggesting that SV2 does not act by modulating presynaptic calcium. Normal neurotransmission could be temporarily recovered by delivering an exhaustive stimulus train. Our results indicate that SV2 primes vesicles in quiescent neurons and that SV2 function can be bypassed by an activity-dependent priming mechanism. We propose that SV2 action modulates synaptic networks by ensuring that low-frequency neurotransmission is faithfully conveyed.

Action Potentials↗
PubMed2006

Synaptic vesicles caught kissing again.

In this issue of Neuron, Harata et al. use a novel quenching technique to provide compelling evidence that kiss-and-run is the dominant mode of vesicle fusion at hippocampal synapses and that the prevalence of kiss-and-run can be modulated by stimulus frequency. The increased incidence of kiss-and-run at lower frequencies may ensure that vesicles are available for use during periods of high demand.

Electrophysiology↗
PubMed2006

Synaptotagmin IV does not alter excitatory fast synaptic transmission or fusion pore kinetics in mammalian CNS neurons.

Synaptotagmin IV (Syt IV) is a brain-specific isoform of the synaptotagmin family, the levels of which are strongly elevated after seizure activity. The dominant hypothesis of Syt IV function states that Syt IV upregulation is a neuroprotective mechanism for reducing neurotransmitter release. To test this hypothesis in mammalian CNS synapses, Syt IV was overexpressed in cultured mouse hippocampal neurons, and acute effects on fast excitatory neurotransmission were assessed. We found neurotransmission unaltered with respect to basal release probability, Ca2+ dependence of release, short-term plasticity, and fusion pore kinetics. In contrast, expression of a mutant Syt I with diminished Ca2+ affinity (R233Q) reduced release probability and altered the Ca2+ dependence of release, thus demonstrating the sensitivity of the system to changes in neurotransmission resulting from changes to the Ca2+ sensor. Together, these data refute the dominant model that Syt IV functions as an inhibitor of neurotransmitter release in mammalian neurons.

Amino Acid Substitution↗
PubMed2006

Overexpression of proteins in neurons using replication-deficient virus.

Overexpression of proteins is a powerful way to determine their function. Until recently, the low efficiency of neuronal transfection has made it difficult to use overexpression and structure-function studies to investigate the role of neuronal proteins in their native environment. The development of neurotrophic viral systems has overcome the obstacle of low efficiency and allows for unprecedented opportunities to use biochemical and electrophysiological techniques to assess the effects of overexpressing wild-type or mutant proteins in neurons. Here, a general protocol for the production of replication-deficient Semliki Forest virus constructs directing the overexpression of proteins of interest in cultured mammalian neurons is described.

Animals↗
PubMed2005

Synaptotagmin mutants Y311N and K326/327A alter the calcium dependence of neurotransmission.

Synaptotagmin I, a calcium-binding synaptic vesicle protein, is thought to act as the calcium sensor for fast neurotransmission, but what synaptotagmin I does, upon binding calcium, to trigger exocytosis is still unknown. To begin to examine the role of synaptotagmin I's interactions with calcium-dependent binding partners, three mutant versions of synaptotagmin I reported to affect calcium-dependent self-oligomerization (Y311N, K327A, and K326/327A) were expressed in cultured mouse hippocampal neurons lacking endogenous synaptotagmin I, and effects on neurotransmission were evaluated by comparison with transmission rescued by wild-type synaptotagmin I. All three mutants reduced transmitter release. To separate effects on calcium binding from effects on calcium-dependent oligomerization, we measured the calcium dependence of exocytosis for two of the mutants. Both showed apparent calcium affinity much lower than wild-type, a reduction sufficient to account for the neurotransmission defects. We conclude that self-oligomerization is unlikely to play any significant role in triggering synaptic vesicle exocytosis.

Amino Acid Substitution↗
PubMed2003

The synaptotagmin C2A domain is part of the calcium sensor controlling fast synaptic transmission.

Synaptotagmin is a synaptic vesicle protein that has been proposed to be the calcium sensor responsible for fast neurotransmitter release at synapses. Synaptotagmin's two C2 domains, C2A and C2B, each provide a calcium binding pocket lined with negative charges contributed by five conserved aspartates. We find that even when all of C2A's conserved aspartates are neutralized by replacement with asparagines, neurotransmitter release still occurs at hippocampal synapses in culture. Because exocytosis continues to be dependent on extracellular calcium concentration, the C2A domain cannot represent the entire calcium sensor. C2A does appear to be part of the calcium sensor, however, because substitution of D232 alters the calcium dependence of release, perhaps by reducing the number of calcium ions that must bind to trigger exocytosis. We conclude that neutralization of the negative charge at D232 by coordination of a calcium ion is necessary--but not sufficient--for fast neurotransmission at mammalian CNS synapses.

Alcohol Oxidoreductases↗
PubMed2003

Cannabinoid receptor activation differentially modulates ion channels in photoreceptors of the tiger salamander.

Cannabinoid CB1 receptors have been detected in retinas of numerous species, with prominent labeling in photoreceptor terminals of the chick and monkey. CB1 labeling is well-conserved across species, suggesting that CB1 receptors might also be present in photoreceptors of the tiger salamander. Synaptic transmission in vertebrate photoreceptors is mediated by L-type calcium currents-currents that are modulated by CB1 receptors in bipolar cells of the tiger salamander. Presence of CB1 receptors in photoreceptor terminals would therefore be consistent with presynaptic modulation of synaptic transmission, a role seen for cannabinoids in other parts of the brain. Here we report immunohistochemical and electrophysiological evidence for the presence of functional CB1 receptors in rod and cone photoreceptors of the tiger salamander. The cannabinoid receptor agonist WIN 55212-2 enhances calcium currents of rod photoreceptors by 39% but decreases calcium currents of large single cones by 50%. In addition, WIN 55212-2 suppresses potassium currents of rods and large single cones by 44 and 48%, respectively. Thus functional CB1 receptors, present in the terminals of rod and cone photoreceptors, differentially modulate calcium and potassium currents in rods and large single cones. CB1 receptors are therefore well positioned to modulate neurotransmitter release at the first synapse of the visual system.

Ambystoma↗
PubMed2002

G-protein alpha subunit isoforms couple differentially to receptors that mediate presynaptic inhibition at rat hippocampal synapses.

Presynaptic receptors that are coupled to heterotrimeric G-proteins are found throughout the brain and are responsible for modulating synaptic transmission. At least 10 G-protein-coupled receptors (GPCRs) reduce transmission in hippocampal neurons. Additionally, hippocampal neurons express up to 17 different Galpha, Gbeta, and Ggamma subunits, making for a striking array of possible heterotrimer compositions and GPCR-heterotrimer interactions. The identity of the Galpha subunit is likely a critical determinant in coupling specificity between GPCRs and their molecular effectors mediating presynaptic inhibition. We studied the role of four Galpha(i/o) subunits (Galpha(o1), Galpha(i1,) Galpha(i2), and Galpha(i3)) in mediating presynaptic inhibition in hippocampal neurons by expressing pertussis toxin-insensitive (PTx-ins) Galpha(i/o) mutants. PTx treatment of these cells disrupts coupling of endogenous subunits, leaving only the mutant Galpha subunits to couple with native GPCRs and betagamma subunits. Successful rescue of presynaptic inhibition indicates that the expressed mutant Galpha subunit can couple to the GPCR of interest. All four PTx-ins Galpha subunits rescued presynaptic inhibition by adenosine A1 receptors. A PTx-ins Galpha subunit also rescued adenosine A1-mediated inhibition of spontaneous vesicle fusion frequency. Of the remaining GPCRs tested, cannabinoid CB1, somatostatin, and GABA(B) receptors displayed an alpha subunit-dependent selectivity in binding to G-protein heterotrimers, whereas group III metabotropic glutamate receptor-mediated inhibition was not rescued by expression of any of the four PTx-ins Galpha subunits. Differential coupling of G-protein alpha subunits may be a means of achieving specificity between different GPCRs and their molecular targets for mediating presynaptic inhibition.

Animals↗