Synaptic target contact enhances presynaptic calcium influx by activating cAMP-dependent protein kinase during synaptogenesis.

Individual dissociated supralateral radular tensor (SLT) muscle fibers were manipulated into contact with fura-2-filled neurites of presynaptic buccal motoneuron 19 from Helisoma in cell culture. Within 30 min of contact, action potential-evoked calcium accumulation was reversibly augmented from 228 +/- 82 nM to 803 +/- 212 nM, an action that was blocked by H-7 (40-100 microM). Calcium accumulation was not augmented when buccal motoneuron 19 contacted muscle or neuronal targets with which it does not form chemical synapses. Addition of pCPTcAMP (500 microM) to cultures reversibly enhanced calcium accumulation. Injection of IP20, a peptide inhibitor of cAMP-dependent protein kinase, prevented pCPTcAMP and SLT muscle from enhancing calcium accumulation. These data demonstrate that SLT muscle target retrogradely regulates calcium accumulation in presynaptic nerve terminals by locally activating presynaptic cAMP-dependent protein kinase.

Neuron-muscle contact changes presynaptic resting calcium set-point.

The role of retrograde signaling between target cells and their presynaptic partners during early cell-cell interactions was examined in cell culture. Using time-lapse video microscopy and fura-2 calcium analysis, we followed contacts between presynaptic neurites of Helisoma motoneurons and muscle fibers dissociated from identified partners in chemical synaptogenesis. Cytosolic calcium rose dramatically at the region of cell-cell contact and subsequently increased throughout the entire neuron. Changes in the calcium set-point of presynaptic neurons were maintained even when connections with muscle targets were severed. Changes in presynaptic calcium concentration were not detected following contact with partners in electrical synapse formation. These data, taken together with the fact that calcium changes coincide with the time course of chemical synaptogenesis in these neuron-muscle cultures, suggest that calcium changes may be involved in early stages of synapse formation.

Microtubule and chromatin dynamics during fertilization and early development in rhesus monkeys, and regulation by intracellular calcium ions.

To explore primate fertilization, oocytes and zygotes from fertile rhesus monkeys were imaged throughout fertilization, polyspermy, and artificial activation using confocal microscopy for microtubules and DNA, as well as ratiometric computer-enhanced video microscopy for intracellular calcium. Unfertilized oocytes displayed microtubules only in the radially oriented meiotic spindles. At insemination, a large calcium transient was followed by a series of smaller oscillations, and sperm astral microtubules had assembled from the sperm centrosome by 2.5 h after transient onset. This aster enlarged, and later duplicated, as the pronuclei converged near the cortex. Pronuclear apposition was prevented by microtubule inhibitors. At mitotic prophase, microtubules ensheathed both sets of condensing chromosomes. At metaphase, the spindle was barrel-shaped and eccentrically positioned with two small asters at the pole with the sperm tail. Microtubules emanating from the telophase spindle interacted with the adjacent cortex and displaced the spindle toward the cell center as first cytokinesis ensued. During polyspermy, each sperm nucleated an aster, and the frequency of calcium oscillations increased. Activation resulted initially in disarrayed microtubules that eventually organized into functional mitotic spindles. These kinetic results demonstrate that rhesus monkeys accomplish fertilization in a fashion nearly identical to that of humans and are, therefore, ideal models in which to investigate cytoskeletal events during human reproduction.

Activation of a peptidergic synapse locally modulates postsynaptic calcium influx.

We examined the synaptic connection between Phe-Met-Arg-Phe-NH2 (FMRFamide)-immunoreactive neurone VD4 and its target neurone P1, both found in the central nervous system of the pond snail Helisoma trivolvis. The major FMRFamide-like peak in neurone VD4 appears to be FMRFamide itself, based on its high performance liquid chromatography (HPLC) elution time and immunoreactivity before and after oxidation, but small peaks are also present at the elution times of Phe-Leu-Arg-Phe-NH2 (FLRFamide) and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide). The modulatory actions of the neuropeptides found in neurone VD4 were tested on the postsynaptic target cell P1. Bath application of both the tetrapeptides FMRFamide and FLRFamide at a concentration of 10(-5) mol l-1 reduced the macroscopic voltage-sensitive calcium current of neurone P1 in culture; FMRFamide by 45% and FLRFamide by 51%. Bath application of the heptapeptide GDPFLRFamide (10(-5) mol l-1) reduced the calcium current by only 8%. We reconstructed the synaptic connection between VD4 and P1 in culture. Action-potential-evoked calcium transients in neurites growing from P1 cells in culture were monitored using Fura-2. Addition of FMRFamide, FLRFamide or GDPFLRFamide reduced the magnitude of the calcium transient in P1. Stimulation of VD4 mimicked the effects of peptide application and caused localized reductions in the action-potential-evoked calcium transients in P1 at the points of contact between the neurites of neurones VD4 and P1. These results suggest that neurone VD4 modulates the calcium influx of neurone P1 through the release of endogenous FMRFamide-related peptides and that this modulatory action is restricted to sites of synaptic interaction.