Synaptic basis of a sub-second representation of time in a neural circuit model.

Temporal sequences of neural activity are essential for driving well-timed behaviors, but the underlying cellular and circuit mechanisms remain elusive. We leveraged the well-defined architecture of the cerebellum, a brain region known to support temporally precise actions, to explore theoretically whether the experimentally observed diversity of short-term synaptic plasticity (STP) at the input layer could generate neural dynamics sufficient for sub-second temporal learning. A cerebellar circuit model equipped with dynamic synapses produced a diverse set of transient granule cell firing patterns that provided a temporal basis set for learning precisely timed pauses in Purkinje cell activity during simulated delay eyelid conditioning and Bayesian interval estimation. The learning performance across time intervals was influenced by the temporal bandwidth of the temporal basis, which was determined by the input layer synaptic properties. The ubiquity of STP throughout the brain positions it as a general, tunable cellular mechanism for sculpting neural dynamics and fine-tuning behavior.

Contact-dependent aggregation of functional Ca2+ channels, synaptic vesicles and postsynaptic receptors in active zones of a neuromuscular junction.

To examine whether Ca2+ channels aggregate in a contact-dependent manner, we characterized the distribution of synaptic vesicles and postsynaptic receptors, and compared it to the location of Ca2+ entry sites, in a Xenopus laevis nerve-muscle coculture preparation using a localized Ca2+ detection method. The majority (75%) of Ca2+ entry sites at spontaneously formed nerve-muscle contacts were associated with enhanced immunofluorescence to the synaptic vesicle protein, SV2. In contrast, only 11% of recorded sites without Ca2+ transients exhibited significant SV2 immunofluorescence. When comparing the spatial distribution of synaptic markers with that of Ca2+ entry sites, we found that the majority of Ca2+ entry sites (61%) were associated with both enhanced SV2 immunofluorescence and R-BTX fluorescence, thereby identifying putative neurotransmitter release sites where Ca2+ channels, synaptic vesicles and postsynaptic receptors are colocalized. Using polystyrene beads coated with a heparin binding protein known to mediate in vitro postsynaptic receptor clustering, we show that the location of Ca2+ domains was associated with enhanced SV2 immunofluorescence at neurite-to-bead contacts. We conclude that the localization of functional Ca2+ channels to putative active zones follows a contact-dependent signalling mechanism similar to that known to mediate vesicle aggregation and AChR clustering.

Contribution of presynaptic calcium-activated potassium currents to transmitter release regulation in cultured Xenopus nerve-muscle synapses.

Using Xenopus nerve-muscle co-cultures, we have examined the contribution of calcium-activated potassium (K(Ca)) channels to the regulation of transmitter release evoked by single action potentials. The presynaptic varicosities that form on muscle cells in these cultures were studied directly using patch-clamp recording techniques. In these developing synapses, blockade of K(Ca) channels with iberiotoxin or charybdotoxin decreased transmitter release by an average of 35%. This effect would be expected to be caused by changes in the late phases of action potential repolarization. We hypothesize that these changes are due to a reduction in the driving force for calcium that is normally enhanced by the local hyperpolarization at the active zone caused by potassium current through the K(Ca) channels that co-localize with calcium channels. In support of this hypothesis, we have shown that when action potential waveforms were used as voltage-clamp commands to elicit calcium current in varicosities, peak calcium current was reduced only when these waveforms were broadened beginning when action potential repolarization was 20% complete. In contrast to peak calcium current, total calcium influx was consistently increased following action potential broadening. A model, based on previously reported properties of ion channels, faithfully reproduced predicted effects on action potential repolarization and calcium currents. From these data, we suggest that the large-conductance K(Ca) channels expressed at presynaptic varicosities regulate transmitter release magnitude during single action potentials by altering the rate of action potential repolarization, and thus the magnitude of peak calcium current.

Measurement of action potential-induced presynaptic calcium domains at a cultured neuromuscular junction.

Spatially localized Ca(2+) domains are thought to play a key role in action potential (AP)-evoked neurotransmitter release at fast synapses. We used a stage-scan confocal spot-detection method and the low-affinity Ca(2+) indicator Oregon Green 488 BAPTA-5N to study the spatiotemporal profile of presynaptic AP-induced Ca(2+) domains. Families of scanned AP-induced fluorescence transients were detected from spot locations separated by 200-300 nm, within the vicinity of Ca(2+) entry sites. Typically, the largest transient in a particular scan peaked within approximately 1 msec and decayed with rapid (tau(1) of 1.7 msec) and slow components (tau(2) of 16 msec, tau(3) of 78 msec). As the spot was incrementally displaced, transients progressively exhibited a slowing in their time-to-peak and a loss of the fast decay component. Three-dimensional graphs of fluorescence versus time and spot displacement revealed the presence of AP-induced fluorescence domains that dissipated within approximately 7 msec. The size of fluorescence domains were estimated from the full-width at half-maximum of gaussian fits to isochronal DeltaF/F plots and ranged from 0.6 to 3.0 micrometer, with a mean +/- SD of 1.6 +/- 0.6 micrometer. Model simulations of a localized Ca(2+) entry site predicted the major features of the fluorescence transients and suggested that, within approximately 1 msec of the initiation of the Ca(2+) current, both the fluorescence domain and the underlying Ca(2+) domain do not extend significantly beyond the site of entry. Consistent with this prediction, the intracellular addition of EGTA (up to 2 mM) accelerated the decay of the measured transients but did not affect the domain size.

Inverted double-gap isolation chamber for high-resolution calcium fluorimetry in skeletal muscle fibers.

Here we describe an improved inverted double-grease-gap isolation chamber that allows the formation of grease seals of high mechanical stability and high electrical resistance. We also provide a detailed description of the procedure used to mount the muscle fibers onto the apparatus. The new chamber permits the electrophysiological study of muscle fibers using an inverted microscope and high-resolution objectives, thus complying with the requirements of modern fluorescence confocal detection methods. The simplicity and reliability of the mounting procedure make this chamber preferable over other gap isolation chambers currently used for simultaneous electrophysiological and optical studies of calcium release. Experimental results obtained from amphibian muscle fibers are presented to illustrate the performance of the chamber when using global fluorescence detection, confocal spot detection, and laser confocal scanning imaging.

Direct measurements of presynaptic calcium and calcium-activated potassium currents regulating neurotransmitter release at cultured Xenopus nerve-muscle synapses.

The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. We used cultured embryonic Xenopus neuromuscular junctions and simultaneous pre- and postsynaptic patch-clamp current-recording procedures to identify the major presynaptic conductances underlying the initiation of neurotransmitter release. Step depolarizations and action potential waveforms elicited Na and K currents along with Ca and Ca-activated K (KCa) currents. The onset of KCa current preceded the peak of the action potential. The predominantly omega-CgTX GVIA-sensitive Ca current occurred primarily during the falling phase, but there was also significant Ca2+ entry during the rising phase of the action potential. The postsynaptic current began a mean of 0.7 msec after the time of maximum rate of rise of the Ca current. omega-CgTX also blocked KCa currents and transmitter release during an action potential, suggesting that Ca and KCa channels are colocalized at presynaptic active zones. In double-ramp voltage-clamp experiments, KCa channel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement with increasing interpulse interval may reflect the time course of either the deactivation of KCa channels or the diffusion/removal of Ca2+ from sites of neurotransmitter release after an action potential.

Localized detection of action potential-induced presynaptic calcium transients at a Xenopus neuromuscular junction.

1. Action potential (AP)-induced fluorescence transients were measured, using Ca2+ indicators and a spot-detection method, at single nerve terminals of a cultured Xenopus neuromuscular junction preparation with simultaneous measurement of neurotransmitter release. 2. Transients obtained using the low affinity Ca2+ indicator Oregon Green 488 BAPTA-5N (OGB-5N) exhibited rapid rising (t1/2 (time at which one-half of the peak fluorescence was attained) = 0.54 ms) and decaying (tau fast = 1.9 ms) phases. The higher affinity indicator Oregon Green 488 BAPTA-2 (OGB-2) produced transients with significantly slower kinetics (t1/2 = 2 ms; tau slow = 73 ms). 3. Tetanic stimulation elicited distinct increases in fluorescence in response to each AP. Each OGB-5N fluorescence increase was more rapid than those observed using OGB-2. Furthermore, a smaller proportion of residual fluorescence at the end of the train was observed using OGB-5N. 4. When OGB-5N was used, a significant [Ca2+] increase was observed prior to the release of neurotransmitter. This was not observed when OGB-2 was used. 5. We conclude that the use of localized optical detection coupled with low affinity Ca2+ indicators can help elucidate rapid changes in presynaptic [Ca2+] dynamics underlying evoked neurotransmitter release.

Growth factor modulation of insulin-like growth factor-binding proteins in rat osteoblast-like cells.

Insulin-like growth factors (IGFs) stimulate growth and differentiation of osteoblasts in culture, and these biological functions can be modulated by their binding proteins (IGFBPs). Previous studies have shown that IGFBP-2 is the major IGFBP synthesized by fetal rat osteoblast-like (ROB) cells, which also secret a minor 24-kilodalton IGFBP, presumably IGFBP-4. In this study we examined the modulation of the abundance of IGFBPs by various growth factors. By immunoprecipitation and ligand blotting, we have proved that the 24 kilodalton protein, which appeared at 25 kilodalton in the present study, is IGFBP-4, whose level was strongly enhanced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB homodimer. Induction of IGFBP-4 by these factors was detected at 1 nM (10- to 30-fold) and increased to 50- to 70-fold of control at 10 nM. The abundance of IGFBP-4 was also increased but to a lesser degree by transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF). As opposed to the actions of other growth factors, TGF-beta 1 substantially lowered the levels of IGFBP-2 and IGFBP-4. PTH and PTH-related peptide did not induce IGFBPs in ROB cells. This is in contrast to the findings from a malignant rat osteoblast-like cell line, UMR 106-01. Since the level of IGFBP-4 after bFGF treatment remained elevated in the presence of hydroxyurea, the induction was likely to be independent of the stimulatory effects of these factors on mitogenesis. To further examine the mechanisms by which IGFBPs were regulated, cultures were treated with actinomycin D and cycloheximide with and without bFGF. Although the synthesis of IGFBP-2 and IGFBP-4 were both inhibited by cycloheximide, actinomycin D blocked the synthesis of basal and bFGF-induced IGFBP-4 but not IGFBP-2. Total RNA extracted from ROB cells and hybridized with specific rat complementary DNAs for IGFBP-2 and IGFBP-4 showed single transcripts of 1.3 and 2.2 kilobases, respectively. Regardless of the changes at the protein level, the abundance of IGFBP-4 transcripts was not different from the vehicle-treated controls at 2, 8, and 24 h after bFGF treatment. Similarly, the levels of messenger RNA for IGFBP-2 and IGFBP-4 did not change during the same time course in the TGF-beta 1 treatment. These data demonstrate that in ROB cells, the abundance of IGFBPs is differentially regulated by various growth factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor.

The immunoregulatory proteins C-C chemokines are potent chemoattractants of lymphocytes and monocytes, as well as activators and attractants of eosinophils and basophils. We have isolated a cDNA that encodes a seven transmembrane-spanning receptor, with homology to other chemoattractant receptors, that encodes a protein designated C-C CKR-1 that acts as a receptor for the C-C chemokines. Human and murine macrophage inflammatory protein 1 alpha (MIP-1 alpha), human human monocyte chemotactic protein 1 (MCP-1), and RANTES all bind to the C-C CKR-1 with varying affinities. Chemokine binding affinity does not predict how well the ligand will transmit a signal through the receptor: RANTES and human MIP-1 alpha induce a similar intracellular calcium flux while binding with disparate affinities, while MCP-1 and human MIP-1 beta induce calcium mobilization only at high concentrations. Finally, C-C chemokines were shown to bind a C-C CKR-1-related gene product encoded by cytomegalovirus, suggesting a role for C-C chemokines in viral immunity.

Arterial delivery of myoblasts to skeletal muscle.

One of the major limitations of myoblast implantation as a therapy for muscular disease is that multiple injections by intramuscular implantation may be required for widespread delivery of cells. Also, some sites (eg, the diaphragm) are relatively inaccessible to injection. As an alternative, we have undertaken intra-arterial administration of myoblasts. For these experiments, we used donor cell myoblasts from the immortal L6 cell line labeled with lacZ via the beta-gal-at-gal retrovirus. In our model, target rat skeletal muscle (tibialis anterior [TA]) was injured using 0.5 ml of 0.5% bupivacaine and 15 IU of hyaluronidase; saline was injected into the contralateral side as a control. We infused 3 x 10(6) lacZ-positive cells into the abdominal aorta of previously injured, immunosuppressed (cyclosporine A) rats. At 7, 14, and 28 days, TA, liver, heart, lung, and spleen were examined for lacZ staining. In both the injured and control muscles, a few differentiated, lacZ-positive muscle cells were present, both singly and in groups, at each time point. These studies demonstrate that genetically labeled, transformed myoblasts may migrate from the arterial circulation to muscle and fuse there to form differentiated muscle cells. It is conceivable that intra-arterial delivery of myoblasts may have a role in the therapy of selected diseases of skeletal muscle.

Irreversible reduction in potassium fluxes accompanies terminal differentiation of human myoblasts to myotubes.

Potassium and sodium fluxes believed to be important in the cellular response to serum and growth factors have not been widely investigated in cells which have undergone terminal differentiation. In this study we have analyzed two main K+ transport systems--the ouabain-sensitive Na+/K+ pump and the bumetanide-sensitive transporter--in human muscle in vitro at two developmental stages: proliferating myoblasts and differentiated myotubes. Myoblast differentiation to myotubes was accompanied by a marked decrease in both the ouabain-sensitive and the bumetanide-sensitive K+ (Rb+) influxes. The addition of serum to the terminally differentiated myotubes had no effect on these K+ transporters. However, serum addition to serum-deprived, undifferentiated myoblasts produced a marked stimulation of these K+ fluxes. The bumetanide-sensitive K+ transporter in human myoblasts and myotubes has the following properties: (1) It carries 30% and 40% of the total K+ influx in myoblasts and myotubes, respectively. (2) It performs net efflux of K+ in the undifferentiated myoblasts and zero net flux (self-exchange) in terminally differentiated myotubes. (3) It is dependent on extracellular Na+ and Cl- in addition to K+. (4) In myoblasts, the Km value for K+ is 1.36 mM, similar to the Km for K+ of the Na+/K+ pump. (5) It is resistant to ouabain (up to 2 mM) and sensitive to furosemide (K0.5 = 5 X 10(-6) M) and bumetanide (K0.5 = 10(-7) M). These data indicate that following terminal differentiation of proliferating myoblasts to mitotically inactive myotubes there is an irreversible reduction of K+ fluxes with a change in the net flux of K+ carried by the bumetanide-sensitive transporter.