Regenerating crayfish motor axons assimilate glial cells and sprout in cultured explants

Phasic and tonic motor nerves originating from crayfish abdominal ganglia, in 2-3-day-old cultured explants, display at their transected distal ends growth zones from which axonal sprouts arise. The subcellular morphology of this regenerative response was examined with thin serial-section electron microscopy and reveals two major remodeling features. First, the external sprouts that exit the nerve are a very small part of a much more massive sprouting response by individual axons comprising several orders of internal sprouts confined to the nerve. Both internal and external sprouts have a simple construction: a cytoskeleton of microtubules and populations of mitochondria, clear synaptic vesicles, membranous sacs, and extrasynaptic active zone dense bars, features reminiscent of motor nerve terminals. Close intermingling of the sprouts of several axons give rise to a neuropil-like arbor within the nerve. Thus, extensive sprouting is an intrinsic response of crayfish motor axons to transection. Second, an equally dramatic remodeling feature is the appearance of nuclei, which resemble those of adjacent glial cells, within the motor axons. These nuclei often appear where the adjoining membranes of the axon and glial cell are disrupted and where free-standing lengths of the double membrane are present. These images signify a breakdown of the dividing membranes and assimilation of the glial cell by the axon, the nucleus being the most visible sign of such assimilation. Thus, crayfish motor axons respond to transection by assimilating glial cells that may provide regulatory and trophic support for the sprouting response.     

Morphological transformation of synaptic terminals of a phasic motoneuron by long-term tonic stimulation

In vivo stimulation of a relatively "silent" phasic crayfish motoneuron changes the ultrastructure of its synaptic terminals to a more tonic phenotype. The closer muscle of the crayfish claw is supplied by only 2 excitatory motoneurons, one of which is phasic and the other tonic. The ultrastructures of conditioned phasic, unconditioned phasic, and tonic motor terminals were compared. The terminals of the tonic motor axon were larger in cross-sectional area, had larger mitochondria, greater synaptic contact area, and were more varicose than unconditioned phasic terminals. Following long-term tonic stimulation of the phasic axon, its terminals became more varicose, mitochondrial cross-sectional area more than doubled, and synapses and mitochondria came into closer proximity, although mean terminal cross-sectional area did not change. Thus, the conditioned phasic terminals became more similar to those of the tonic motor axon. These changes in ultrastructure correlate with, and may be causally linked to, previously reported changes in neuromuscular synaptic physiology produced by in vivo tonic stimulation of this motoneuron. We conclude that the ongoing level of impulse activity can affect the ultrastructural differentiation of synaptic terminals and synapses of the phasic motoneuron.     

New mutations in the PKD1 gene in Czech population with autosomal dominant polycystic kidney disease

Background  

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease. The disease is caused by mutations of the PKD1 (affecting roughly 85% of ADPKD patients) and PKD2 (affecting roughly 14% of ADPKD patients) genes, although in several ADPKD families, the PKD1 and/or PKD2 linkage was not found. Mutation analysis of the PKD1 gene is complicated by the presence of highly homologous genomic duplications of the first two thirds of the gene.     

Long-term changes in neuromuscular synapses with altered sensory input to a crayfish motoneuron

Prolonged changes in crayfish motoneuron electrical activity result in adaptations in neuromuscular synapses which are consistent with findings at other synapses. In this study we establish that this long-term adaptation (LTA) of crayfish neuromuscular synapses to increased activation of the motoneuron does not require the activation of any other neurons. Selectively increasing the impulse activity of the relatively inactive fast closer excitor motoneuron (FCE) over a period of 7 days results in a 41% reduction in initial amplitude of the excitatory postsynaptic potential (EPSP), and a 42% decrease in synaptic fatigue. These changes in EPSP properties have been previously shown to be due to decreased initial transmitter release and greater sustained release of transmitter during prolonged stimulation. Chronic stimulation of sensory receptors known to produce subthreshold synaptic potentials in the central processes of the FCE elicits LTA of its neuromuscular synapses. The initial EPSP is decreased by 21%, and the synaptic fatigue is reduced by 17%. These results lead to the hypothesis that the primary event leading to LTA of neuromuscular synapses is depolarization of the motoneuron.     

Ca2+ clearance at growth cones produced by crayfish motor axons in an explant culture

Intracellular free Ca2+ concentration ([Ca2+]i) plays an important role in the regulation of growth cone (GC) motility; however, the mechanisms responsible for clearing Ca2+ from GCs have not been examined. We studied the Ca2+-clearance mechanisms in GCs produced by crayfish tonic and phasic motor axons by measuring the decay of [Ca2+]i after a high [K+] depolarizing pulse using fura-2AM. Tonic motor axons regenerating in explant cultures develop GCs with more rapid Ca2+ clearance than GCs from phasic axons. When Na/Ca exchange was blocked by replacing external Na+ with N-methyl-d-glucamine (NMG), [Ca2+]i decay was delayed in both tonic and phasic GCs. Tonic GCs appear to have higher Na/Ca exchange activity than phasic ones since reversal of Na/Ca exchange by lowering external Na+ caused a greater increase in [Ca2+]i for tonic than phasic GCs. Application of the mitochondrial inhibitors, Antimycin A1 (1 microM) and CCCP (10 microM), demonstrated that mitochondrial Ca2+ uptake/release was more prominent in phasic than tonic GCs. When both Na/Ca exchange and mitochondria were inhibited, the plasma membrane Ca2+ ATPase was effective in extruding Ca2+ from tonic, but not phasic GCs. We conclude that Na/Ca exchange plays a prominent role in extruding large Ca2+ loads from both tonic and phasic GCs. High Na/Ca exchange activity in tonic GCs contributes to the rapid decay of [Ca2+]i in these GCs; low rates of Ca2+ extrusion plus the release of Ca2+ from mitochondria prolongs the decay of [Ca2+]i in the phasic GCs.     

Chronic lead exposure alters presynaptic calcium regulation and synaptic facilitation in Drosophila larvae

Prolonged exposure to inorganic lead (Pb²⁺) during development has been shown to influence activity-dependent synaptic plasticity in the mammalian brain, possibly by altering the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). To explore this possibility, we studied the effect of Pb²⁺ exposure on [Ca²⁺]_i regulation and synaptic facilitation at the neuromuscular junction of larval Drosophila. Wild-type Drosophila (CS) were raised from egg stages through the third larval instar in media containing either 0 μM, 100 μM or 250 μM Pb²⁺ and identified motor terminals were examined in late third-instar larvae. To compare resting [Ca²⁺]_i and the changes in [Ca²⁺]_i produced by impulse activity, the motor terminals were loaded with a Ca²⁺ indicator, either Oregon Green 488 BAPTA-1 (OGB-1) or fura-2 conjugated to a dextran. We found that rearing in Pb²⁺ did not significantly change the resting [Ca²⁺]_i nor the Ca²⁺ transient produced in synaptic boutons by single action potentials (APs); however, the Ca²⁺ transients produced by 10 Hz and 20 Hz AP trains were larger in Pb²⁺-exposed boutons and decayed more slowly. For larvae raised in 250 μM Pb²⁺, the increase in [Ca²⁺]_i during an AP train (20 Hz) was 29% greater than in control larvae and the [Ca²⁺]_i decay τ was 69% greater. These differences appear to result from reduced activity of the plasma membrane Ca²⁺ ATPase (PMCA), which extrudes Ca²⁺ from these synaptic terminals. These findings are consistent with studies in mammals showing a Pb²⁺-dependent reduction in PMCA activity. We also observed a Pb²⁺-dependent enhancement of synaptic facilitation at these larval neuromuscular synapses. Facilitation of EPSP amplitude during AP trains (20 Hz) was 55% greater in Pb²⁺-reared larvae than in controls. These results showed that Pb²⁺ exposure produced changes in the regulation of [Ca²⁺]_i during impulse activity, which could affect various aspects of nervous system development. At the mature synapse, this altered [Ca²⁺]_i regulation produced changes in synaptic facilitation that are likely to influence the function of neural networks.     

Differences in Ca regulation for high-output Is and low-output Ib motor terminals in Drosophila larvae

We determined whether two classes of Drosophila larval motor terminals with known differences in structure and transmitter release also showed differences in Ca²⁺ regulation. Larval motor neurons can be separated into those producing large synaptic boutons (Ib) and those with small boutons (Is). Ib terminals release less transmitter during single action potentials (APs) than Is terminals, but show greater facilitation during high-frequency stimulation. We measured Ca²⁺ transients produced by single APs and AP trains after loading the terminals with the dextran-conjugated Ca²⁺ indicator Oregon Green 488 BAPTA-1 (OGB-1). The two pairs of Is and Ib terminals innervating muscle fiber 4 and fibers 6 and 7 were examined. The OGB-1 concentrations were measured in order to compare measurements from terminals with similar OGB-1 loading. For single APs, the change in OGB-1 fluorescence (ΔF/F) in Is boutons was significantly larger than in Ib boutons due to greater Ca²⁺ influx per bouton volume. The Is boutons had greater surface area and active zone number per bouton volume than Ib boutons; this could account for the differences in Ca²⁺ influx and argues for similar Ca²⁺ influx at Is and Ib active zones. As previously reported for the Ib boutons, the distal Is boutons had larger single-AP Ca²⁺ transients than proximal ones on muscle fibers 6 and 7, but not on fiber 4. This difference was not due to proximal–distal differences in surface area or active zones per bouton volume and may be due to greater Ca²⁺ influx at distal active zones. During AP trains, the Is Ca²⁺ transients were larger in amplitude and had longer decay time constants than Ib ones. This can be explained by a slower rate of Ca²⁺ extrusion from the Is boutons apparently due to lower plasma membrane Ca²⁺ ATPase activity at Is boutons compared to Ib boutons.