Showing papers by "Timothy H. Murphy published in 1999"

P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A.

Abstract: Spatial and temporal changes in intracellular calcium concentrations are critical for controlling gene expression in neurons. In many neurons, activity-dependent calcium influx through L-type channels stimulates transcription that depends on the transcription factor CREB by activating a calmodulin-dependent pathway. Here we show that selective influx of calcium through P/Q-type channels is responsible for activating expression of syntaxin-1A, a presynaptic protein that mediates vesicle docking, fusion and neurotransmitter release. The initial P/Q-type calcium signal is amplified by release of calcium from intracellular stores and acts through phosphorylation that is dependent on the calmodulin-dependent kinase CaM K II/IV, protein kinase A and mitogen-activated protein kinase kinase. Initiation of syntaxin-1A expression is rapid and short-lived, with syntaxin-1A ultimately interacting with the P/Q-type calcium channel to decrease channel availability. Our results define an activity-dependent feedback pathway that may regulate synaptic efficacy and function in the nervous system.

Correlation of Miniature Synaptic Activity and Evoked Release Probability in Cultures of Cortical Neurons

Abstract: Spontaneous miniature synaptic activity is caused by action potential (AP)-independent release of transmitter vesicles and is regulated at the level of single synapses. In cultured cortical neurons we have used this spontaneous vesicle turnover to load the styryl dye FM1–43 into synapses with high rates of miniature synaptic activity. Automated selection procedures restricted analysis to synapses with sufficient levels of miniature activity-mediated FM1–43 uptake. After FM1–43 loading, vesicular FM1–43 release in response to AP stimulation was recorded at single synapses as a measure of release probability. We find that synapses with high rates of miniature activity possess significantly enhanced evoked release rates compared with a control population. Because the difference in release rates between the two populations is [Ca2+]o-dependent, it is most likely caused by a difference in release probability. Within the subpopulation of synapses with high miniature activity, we find that the probabilities for miniature and AP-evoked release are correlated at single synaptic sites. Furthermore, the degree of miniature synaptic activity is correlated with the vesicle pool size. These findings suggest that both evoked and miniature vesicular release are regulated in parallel and that the frequency of miniature synaptic activity can be used as an indicator for evoked release efficacy.

Behaviour of NMDA and AMPA receptor-mediated miniature EPSCs at rat cortical neuron synapses identified by calcium imaging.

Abstract: Simultaneous recording of intracellular calcium concentration at a synapse and synaptic currents from the cell body allows mapping of miniature excitatory postsynaptic currents (mEPSCs) to single synapses. In the absence of extracellular Mg2+, 77% of synapses had mEPSCs with fast and slow components, attributed to AMPA- and NMDA-type glutamate receptors, respectively. The remainder of synapses (23%) had mEPSCs that lacked a fast component; these responses were attributed to NMDA receptors. A strong positive correlation between the amplitude of the calcium transient and the NMDA receptor-mediated mEPSC was observed, indicating that the mEPSCs originate from an identified synapse. At synapses that had both mEPSC components, the AMPA receptor component was positively correlated with charge influx mediated by NMDA receptors during repeated synaptic events. No periodic failure in the AMPA receptor mEPSC was observed at synapses expressing both receptor components. A significant positive correlation between the mean amplitudes of NMDA and AMPA receptor components of mEPSCs is observed across different synapses. We suggest that factors effecting both receptor classes, such as the amount of transmitter in synaptic vesicles, might contribute to the variation in mEPSC amplitude during repeated miniature events at a single synapse. Although the average postsynaptic response at different synapses can vary in amplitude, there appears to be a mechanism to keep the ratio of each receptor subtype within a narrow range. Fast excitatory synaptic transmission in the central nervous system is primarily mediated by two major classes of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) (Bekkers & Stevens, 1989; Jonas & Spruston, 1994). It has been shown that both receptor subtypes can be co-localized at synapses, but at some synapses NMDARs dominate over AMPARs, and other synapses exist that lack AMPARs altogether (Bekkers & Stevens, 1989; Silver et al. 1992; Wu et al. 1996; Malenka & Nicoll, 1997; O'Brien et al. 1998; Petralia et al. 1999). Processes that regulate the distribution of functional AMPARs have been proposed as important regulators of activity-dependent synaptic modulation and development (Wu et al. 1996; Malenka & Nicoll, 1997; Kamboj & Huganir, 1998; O'Brien et al. 1998; Petralia et al. 1999). Regarding the mechanism of differential receptor distribution, it has been shown that anchoring proteins are required for the clustering of AMPARs and NMDARs (Ehlers et al. 1996; Muller et al. 1996; Dong et al. 1997; O'Brien et al. 1998). A number of studies suggest that the amplitude of AMPAR-mediated mEPSCs varies during repeated events at single synapses (Liu & Tsien, 1995; Forti et al. 1997; Liu et al. 1999). If AMPARs are not saturated by glutamate from single synaptic vesicles, presynaptic factors, such as the amount of neurotransmitter in a synaptic vesicle, would contribute to the response amplitude variability (Liu & Tsien, 1995; Forti et al. 1997; Liu et al. 1999). In the case of NMDARs, as they have a high affinity for glutamate, it was suggested that they may be saturated by glutamate from single synaptic vesicles (Patneau & Mayer, 1990) but more recent evidence suggests that NMDARs are not saturated by the quantal transmitter release and that presynaptic factors are thus capable of controlling synaptic responses mediated by NMDARs (Min et al. 1998; Mainen et al. 1999). The mechanism of quantal response variation has been intensely studied, since it may provide insight into how synaptic efficacy is changed during neuronal plasticity (Bekkers & Stevens, 1989; Liu & Tsien, 1995; Murphy et al. 1995; Forti et al. 1997; Gomperts et al. 1998). To determine the variability in synaptic events at single synapses and between synapses, it is necessary to map events to a single synapse because neurons receive inputs from multiple synapses. Calcium imaging techniques take advantage of the high Ca2+ permeability of NMDARs to identify synaptic events at a single synapse (Muller & Connor, 1991; Malinow et al. 1994; Murphy et al. 1994, 1995; Schiller et al. 1998). Here, we have recorded mEPSCs from identified synapses of cultured cortical neurons by simultaneously recording synaptic currents from the cell body and intracellular calcium concentration at the synapse (Murphy et al. 1994, 1995). At synapses that expressed both NMDAR and AMPAR components we found significant positive correlation between the amplitude of AMPAR- and NMDAR-mediated mEPSCs during repeated synaptic events. This correlation suggests that presynaptic factors could contribute to the variation in mEPSC amplitude. In addition, we found significant positive correlation between the synaptic responses mediated by AMPARs and NMDARs across synapses. It is suggested that synapses have regulatory mechanisms to maintain a relatively constant ratio of each receptor type.

Analysis of multiquantal transmitter release from single cultured cortical neuron terminals.

Abstract: Analysis of multiquantal transmitter release from single cultured cortical neuron terminals. Application of single synapse recording methods indicates that the amplitude of postsynaptic responses o...

Ultrastructural correlates of quantal synaptic function at single CNS synapses.

Abstract: We have tested the hypothesis that functional differences between synapses are associated with ultrastructure in cultured cortical neurons. Using Ca 21 imaging, we measured NMDA receptor-mediated miniature synaptic calcium transients attributed to the spontaneous release of single transmitter quanta. After imaging, the identified neurons were processed for serial transmission electron microscopy. At sites of quantal NMDA receptor-dependent Ca 21 transients, we confirmed the presence of excitatory synapses and measured spine size and synaptic contact area. Our results demonstrate that synapse size correlates positively with the amplitude of the NMDA receptor-mediated postsynaptic response, suggesting that larger synapses express a greater number of NMDA receptors. Therefore, regulation of quantal amplitude may involve processes that alter synapse size.

Amplification of calcium signals at dendritic spines provides a method for CNS quantal analysis.

Abstract: It has been proposed that the small volume of a dendritic spine can amplify Ca2+ signals during synaptic transmission. Accordingly, we have performed calculations to determine whether the activation of N-methyl-D-aspartate (NMDA) type glutamate receptors during synaptic transmission results in significant elevation in intracellular Ca2+ levels, permitting optical detection of synaptic signals within a single spine. Simple calculations suggest that the opening of even a single NMDA receptor would result in the influx of approximately 310 000 Ca2+ ions into the small volume of a spine, producing changes in Ca2+ levels that are readily detectable using high affinity Ca2+ indicators such as fura-2 or fluo-3. Using fluorescent Ca2+ indicators, we have imaged local Ca2+ transients mediated by NMDA receptors in spines and dendritic shafts attributed to spontaneous miniature synaptic activity. Detailed analysis of these quantal events suggests that the current triggering these transients is attributed to the activation of 10-fold higher frequency of transients than others. As expected for events mediated by NMDA receptors, miniature synaptic Ca2+ transients were suppressed by extracellular Mg2+ at negative membrane potentials; however, the Mg2+ block could be removed by depolarization.