Optical analysis of glutamate spread in the neuropil.

Fast synaptic communication uses diffusible transmitters whose spread is limited by uptake mechanisms. However, on the submicron-scale, the distance between two synapses, the extent of glutamate spread has so far remained difficult to measure. Here, we show that quantal glutamate release from individual hippocampal synapses activates extracellular iGluSnFr molecules at a distance of >1.5 µm. 2P-glutamate uncaging near spines further showed that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-Rs and N-methyl-D-aspartate (NMDA)-Rs respond to distant uncaging spots at approximately 800 and 2000 nm, respectively, when releasing the amount of glutamate contained in approximately five synaptic vesicles. The uncaging-induced remote activation of AMPA-Rs was facilitated by blocking glutamate transporters but only modestly decreased by elevating the recording temperature. When mimicking release from neighboring synapses by three simultaneous uncaging spots in the microenvironment of a spine, AMPA-R-mediated responses increased supra-additively. Interfering with extracellular glutamate diffusion through a glutamate scavenger system weakly reduced field synaptic responses but not the quantal amplitude. Together, our data suggest that the neuropil is more permissive to short-range spread of transmitter than suggested by theory, that multivesicular release could regularly coactivate nearest neighbor synapses and that on this scale glutamate buffering by transporters primarily limits the spread of transmitter and allows for cooperative glutamate signaling in extracellular microdomains.

Antibodies raised against aldehyde-fixed antigens improve sensitivity for postembedding electron microscopy.

BACKGROUND: Antibodies are one of the most important tools in biological research. High specificity and sensitivity of antibodies are crucial to obtain reliable results. Tissue fixed with glutaraldehyde (GA) is commonly used in electron microscopical investigations. The fixation and embedding routine in preparation of tissue for post-embedding electron microscopy (EM) will mask and structurally alter epitopes, making antibody-antigen interaction inefficient, with low labeling intensities. One of the main factors in this regard is the use of GA as fixative. NEW METHOD: To alleviate these technical challenges, we immunized rabbits with antigen pre-fixed with GA. We hypothesized that the resulting antibodies would have stronger affinity to antigens that have been conformationally changed and denatured by GA, the way they are in fixed tissue. COMPARISON WITH EXISTING METHOD AND RESULTS: An initial screening with western blotting (WB) showed results consistent with our hypothesis. In-house antibodies raised against GA-fixed SNARE proteins SNAP-25 and VAMP2, binds more strongly to fixed proteins compared to non-fixed proteins, while the pattern is opposite with the commercially available antibodies raised against non-fixed antigens (standard antibodies). Quantitative post-embedding EM of hippocampal synapses gave higher labeling intensities with anti-GA-SNAP-25 and anti-GA-VAMP2 compared to standard antibodies. Importantly, light microscopy (LM) and EM with our antibodies revealed stronger labeling of GA-fixed than formaldehyde (FH) treated brains. CONCLUSION: Our results highlight the experimental potential of raising antibodies against GA-treated antigen to improve sensitivity of the antibodies for postembedding immunogold EM.

Leaky synapses: regulation of spontaneous neurotransmission in central synapses.

The mechanisms underlying spontaneous neurotransmitter release are not well understood. Under physiological as well as pathophysiological circumstances, spontaneous fusion events can set the concentration of ambient levels of neurotransmitter within the synaptic cleft and in the extracellular milieu. In the brain, unregulated release of excitatory neurotransmitters, exacerbated during pathological conditions such as stroke, can lead to neuronal damage and death. In addition, recent findings suggest that under physiological circumstances spontaneous release events can trigger postsynaptic signaling events independent of evoked neurotransmitter release. Therefore, elucidation of mechanisms underlying spontaneous neurotransmission may help us better understand the functional significance of this form of release and provide tools for its selective manipulation. For instance, our recent investigations indicate that the level of cholesterol in the synapse plays a critical role in limiting spontaneous synaptic vesicle fusion. Therefore, alterations in synaptic cholesterol metabolism can be a critical determinant of glutamatergic neurotransmission at rest. This article aims to provide a closer look into our current understanding of the mechanisms underlying spontaneous neurotransmission and the signaling triggered by these unitary release events.

SNARE function analyzed in synaptobrevin/VAMP knockout mice.

SNAREs (soluble NSF-attachment protein receptors) are generally acknowledged as central components of membrane fusion reactions, but their precise function has remained enigmatic. Competing hypotheses suggest roles for SNAREs in mediating the specificity of fusion, catalyzing fusion, or actually executing fusion. We generated knockout mice lacking synaptobrevin/VAMP 2, the vesicular SNARE protein responsible for synaptic vesicle fusion in forebrain synapses, to make use of the exquisite temporal resolution of electrophysiology in measuring fusion. In the absence of synaptobrevin 2, spontaneous synaptic vesicle fusion and fusion induced by hypertonic sucrose were decreased approximately 10-fold, but fast Ca2+-triggered fusion was decreased more than 100-fold. Thus, synaptobrevin 2 may function in catalyzing fusion reactions and stabilizing fusion intermediates but is not absolutely required for synaptic fusion.

Limited numbers of recycling vesicles in small CNS nerve terminals: implications for neural signaling and vesicular cycling.

The tiny nerve terminals of central synapses contain far fewer vesicles than preparations commonly used for analysis of neurosecretion. Photoconversion of vesicles rendered fluorescent with the dye FM1-43 directly identified vesicles capable of engaging in exo-endocytotic recycling following stimulated Ca(2+) entry. This recycling pool typically contained 30-45 vesicles, only a minority fraction (15-20% on average) of the total vesicle population. The smallness of the recycling pool would severely constrain rates of quantal neurotransmission if classical pathways were solely responsible for vesicle recycling. Fortunately, vesicles can undergo rapid retrieval and reuse in addition to conventional slow recycling, to the benefit of synaptic information flow and neuronal signaling.

Rapid reuse of readily releasable pool vesicles at hippocampal synapses.

Functional presynaptic vesicles have been subdivided into readily releasable (RRP) and reserve (RP) pools. We studied recycling properties of RRP vesicles through differential retention of FM1-43 and FM2-10 and by varying the time window for FM dye uptake. Both approaches indicated that vesicles residing in the RRP underwent rapid endocytosis (tau approximately 1s), whereas newly recruited RP vesicles were recycled slowly (tau approximately 30 s). With repeated challenges (hypertonic or electrical stimuli), the ability to release neurotransmitter recovered 10-fold more rapidly than restoration of FM2-10 destaining. Finding neurotransmission in the absence of destaining implied that rapidly endocytosed RRP vesicles were capable of reuse, a process distinct from repopulation from the RP. Reuse would greatly expand the functional capabilities of a limited number of vesicles in CNS terminals, particularly during intermittent bursts of activity.

Activity-dependent regulation of synaptic clustering in a hippocampal culture system.

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1-43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-D-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.

Properties of fast endocytosis at hippocampal synapses.

Regulation of synaptic transmission is a widespread means for dynamic alterations in nervous system function. In several cases, this regulation targets vesicular recycling in presynaptic terminals and may result in substantial changes in efficiency of synaptic transmission. Traditionally, experimental accessibility of the synaptic vesicle cycle in central neuronal synapses has been largely limited to the exocytotic side, which can be monitored with electrophysiological responses to neurotransmitter release. Recently, physiological measurements on the endocytotic portion of the cycle have been made possible by the introduction of styryl dyes such as FM1-43 as fluorescent markers for recycling synaptic vesicles. Here we demonstrate the existence of fast endocytosis in hippocampal nerve terminals and derive its kinetics from fluorescence measurements using dyes with varying rates of membrane departitioning. The rapid mode of vesicular retrieval was greatly speeded by exposure to staurosporine or elevated extracellular calcium. The effective time-constant for retrieval can be < 2 seconds under appropriate conditions. Thus, hippocampal synapses capitalize on efficient mechanisms for endocytosis and their vesicular retrieval is subject to modulatory control.

Visualization of synaptic activity in hippocampal slices with FM1-43 enabled by fluorescence quenching.

Fluorescence imaging of presynaptic uptake and release of styryl dyes such as FM1-43 has provided valuable insights into synaptic function. However, in studies of CNS neurons, the utility of these dyes has been severely limited by nonsynaptic background fluorescence. This has thwarted the use of FM dyes in systems more intact than dissociated neuronal cultures. Here, we describe an approach to selectively reduce undesired fluorescence through quenching of the surface-bound FM1-43 signal. The introduction of sulforhodamine, a fluorophore that is not taken up by synaptic vesicles, selectively reduced the nonsynaptic fluorescence in FM1-43-labeled hippocampal cultures. When applied to rat hippocampal slices, this procedure allowed us to observe activity-dependent staining and destaining of functional synapses. Extending the usefulness of styryl dyes to slice preparations may help make functional synaptic networks amenable to optical measurements.

Kinetics and regulation of fast endocytosis at hippocampal synapses.

Presynaptic nerve terminals often contain as few as a hundred vesicles and so must recycle them soon after exocytosis to preserve synaptic transmission and presynaptic morphology during repetitive firing. The kinetics and mechanisms of vesicular endocytosis and repriming have therefore been studied. Vesicles in hippocampal nerve terminals can become available to release their contents within approximately 40 s of the previous round of exocytosis. Studies using the styryl dye FM1-43 have estimated the time constant for endocytosis as approximately 20-30 s at least half of the total recycling time, which is much slower than endocytosis in other secretory systems. It seems paradoxical that the neurosecretory terminals that could benefit the most from rapid endocytosis do not use such a mechanism. Here we demonstrate the existence of fast endocytosis in hippocampal nerve terminals and derive its kinetics from fluorescence measurements using dyes with varying rates of membrane departitioning. The rapid mode of vesicular retrieval was much faster after exposure to staurosporine or elevated extracellular calcium. Thus hippocampal synapses take advantage of efficient mechanisms for endocytosis, and their vesicular retrieval is subject to modulatory control.

cAMP-dependent enhancement of dihydropyridine-sensitive calcium channel availability in hippocampal neurons.

Dihydropyridine-sensitive calcium channels can be strongly modulated by cAMP-dependent phosphorylation. This modulation takes the form of increased channel availability in cardiac myocytes (for review, see McDonald et al., 1994) and has been suggested to be essential for voltage-dependent facilitation in adrenal chromaffin cells (Artalejo et al., 1992) and skeletal muscle (Sculptoreanu et al., 1993b). To determine the role of cAMP-dependent phosphorylation on dihydropyridine-sensitive calcium channels in hippocampal neurons, we have used both single-channel and whole-cell recording techniques and have examined the effects of the membrane-permeable cAMP analog 8-(4-chlorophenylthio) (CPT)-cAMP and the protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide (H-89). Hippocampal neurons contain two kinds of dihydropyridine-sensitive calcium channel activity: Ls and Lp (Kavalali and Plummer, 1994). The Ls channel closely resembles the cardiac L-type channel, whereas the Lp channel shows a novel low-voltage form of voltage-dependent potentiation (). 8-CPT-cAMP increased the availability of both the Ls and Lp channels and caused a parallel increase in Lp channel reopenings at the repolarization potential that result from voltage-dependent potentiation. This effect was completely blocked by the broad spectrum kinase inhibitor H-7 and by the protein kinase A-specific inhibitor H-89. The two inhibitors, however, did not disrupt baseline potentiation of the Lp channel, suggesting that cAMP-dependent protein kinase activity can enhance Ls and Lp channel activity but is not required for voltage-dependent potentiation in hippocampal neurons.

Dendritic Ca2+ channels characterized by recordings from isolated hippocampal dendritic segments.

Dendritic arbors are critical for the information processing capability of central neurons, but quantitative analysis of their membrane properties has been hampered by their geometrical complexity. Here, we have focused on an important source of Ca2+ entry in dendrites, the voltage-gated Ca2+ channels, by applying the whole-cell voltage-clamp technique to isolated dendritic segments ("dendrosomes") from rat hippocampal neurons. We found that low voltage-activated T-type Ca2+ channels provide a significantly larger fraction of the Ca2+ influx in dendrites than their counterparts in cell bodies. Surprisingly, 60%-70% of the high voltage-activated Ca2+ current in dendrosomes was N and P/Q type, and these channels were susceptible to neurotransmitter inhibition, suggesting a novel physiological role for G protein-regulated Ca2+ channel modulation in controlling dendritic excitability and Ca2+ signaling.

Multiple voltage-dependent mechanisms potentiate calcium channel activity in hippocampal neurons.

Neuronal voltage-gated calcium channels provide a pathway for calcium influx that is required for processes ranging from intracellular signaling to alterations in cellular excitability. In hippocampal neurons, we have characterized a subtype of dihydropyridine-sensitive L-type calcium channels (Lp channel) that shows multiple kinds of voltage-dependent potentiation of its activity. One type of potentiation is elicited by low-voltage stimuli (-10 mV) and can be seen in dual-pulse protocols in which a transient hyperpolarization is interposed between conditioning and test pulses. The second type of potentiation is elicited by much higher voltages (+60 mV) and is selectively deactivated at hyperpolarized voltages. We have compared these types of potentiation in the Lp channel, the "standard" L-type channel, and the cardiac L-type channel. Our results show that the high-voltage potentiation is common to all three channel types. The low-voltage form of potentiation, however, is unique to the Lp channel. Thus, the Lp channel shows two kinds of potentiation that differ in their voltage dependence and rate of decay. Therefore, calcium channel plasticity in the hippocampus has a variety of forms distinguished by their stimulus requirements and duration.

Selective potentiation of a novel calcium channel in rat hippocampal neurones.

1. Calcium channel activity in cultured embryonic hippocampal neurones was studied with the cell-attached configuration of the patch clamp technique. Single-channel recordings revealed the presence of a novel kind of calcium channel activity characterized by marked bursts of re-openings following voltage pulses to +20 mV from a holding potential of -40 mV. 2. The re-openings were greatly prolonged by the dihydropyridine (DHP) agonist (+)-(S)-202-791, thus ruling out the possibility that they arose from T-, N- or P-type channels. Furthermore, the novel gating pattern could be readily distinguished from that of the L-type channel which showed only conventional tail currents. 3. Since the novel gating pattern was stable over many minutes, we provisionally referred to it as a novel kind of calcium channel that showed voltage-dependent potentiation (Lp channel) to distinguish it from the 'standard' L-type channel (Ls channel). 4. Lp channels could also be distinguished from Ls channels on the basis of slope conductance (24.3 vs. 26.9 pS for Lp and Ls, respectively) and mean DHP-induced long open time (2.7 vs 11 ms at +20 mV for Lp and Ls, respectively). 5. Voltage-dependent potentiation of Lp channel activity was studied using a dual-pulse protocol. When preceded by conditioning prepulses, Lp responses to test pulses were greatly increased. Ls- and N-type calcium channels showed no such enhancement of their activity. 6. Long-duration recordings revealed no clear evidence for transitions from Ls to Lp gating (or vice versa), suggesting that Ls and Lp activities arose from different kinds of calcium channels or that Lp gating is an unusually long-lived mode of Ls channel gating.