Physiological and morphological evidence for coupling in mouse salivary gland acinar cells.

Three experimental techniques were employed to examine coupling between acinar cells of the mouse salivary gland. Passage of DC current pulses via intracellular microelectrodes between neighboring cells showed that small ions could be directly passed from one cell to another. Intracellular iontophoresis of the dye Lucifer Yellow CH into a single cell indicated that small molecules could spread by means of intercellular cytoplasmic bridges througout an acinus and, occasionally, into cells of adjacent acini. Freeze-fracture replicas of acinar cell membranes indicated the presence of gap junctions which were correlated with both electrical and dye coupling experiments. Suggestions are made for the function of direct intercellular exchange in salivary secretory cells. The role of electrical coupling in coordination of the activity of different secretory cell types is discussed as one possible function.

Unique responses of differentiating neuronal growth cones to inhibitory cues presented by oligodendrocytes.

During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since they develop from minor processes whose growth cones are inhibited (nearly 70% collapse) by contact with oligodendrocytes. Recombinant MAG(rMAG)-coated beads caused collapse of 72% of axonal growth cones but only 29% of differentiating dendritic growth cones. Unlike their response to contact with oligodendrocytes, few growth cones of minor processes were inhibited by rMAG-coated beads (20% collapsed). These results reveal the capability of differentiating growth cones of the same neuron to partition the complex molecular terrain they navigate by generating unique responses to particular inhibitory environmental cues.

Cardioaccelerator release in Periplaneta americana (L).

Focal electrical stimulation of the brain of Periplaneta americana evokes the release of a factor with high cardioaccelerator activity. Results of extirpation of components of the retrocerebral complex from an isolated head preparation imply that the corpus cardiacum is the critical component of this system. The cardioaccelerator is a heat-stable proteinaceous compound.

Suppression of neurite elongation and growth cone motility by electrical activity.

Electrical activity may regulate a number of neuronal functions in addition to its role in transmitting signals along nerve cells. The hypothesis that electrical activity affects neurite elongation in sprouting neurons was tested by stimulating individual snail neurons isolated in cell culture. The findings demonstrated that growth cone advance, and thus neurite elongation, is reversibly stopped during periods when action potentials are experimentally evoked. A decrease in filopodial number and growth cone area was also observed. Thus, action potentials can mediate the cessation of neurite outgrowth and thereby may influence structure and connectivity within the nervous system.

Selection of a novel connection by adult molluscan neurons.

Predictable change in neuronal connectivity can be induced in the buccal ganglia of adult Helisoma snails when neuritic growth is evoked by axotomy. Both transient and stable novel electrical connections are established between identified neurons. The breaking of inappropriate, normally transient connections is contingent on the formation of an appropriate connection.

Serotonin selectively inhibits growth cone motility and synaptogenesis of specific identified neurons.

The motile activity of growth cones of specific identified neurons is inhibited by the neurotransmitter serotonin, although other identified neurons are unaffected. As a consequence, affected neurons are unable to form electrical synapses, whereas other neurons whose growth is unaffected can still interconnect. This result demonstrates that neurotransmitters can play a prominent role in regulating neuronal architecture and connectivity in addition to their classical role in neurotransmission.

Electrical synapse formation depends on interaction of mutually growing neurites.

A neuron's competence to form electrical synapses depends on its growth status. Experiments in situ and in cell culture with identified neurons of the snail Helisoma demonstrate that active neurite outgrowth from both potential partners must be spatially and temporally coincident for electrical synapse formation to occur.

Role of intracellular calcium in NI-35-evoked collapse of neuronal growth cones.

A myelin-associated protein from the central nervous system, the neurite growth inhibitor NI-35, inhibits regeneration of lesioned neuronal fiber tracts in vivo and growth of neurites in vitro. Growth cones of cultured rat dorsal root ganglion neurons arrested their growth and collapsed when exposed to liposomes containing NI-35. Before morphological changes, the concentration of free intracellular calcium ([Ca2+]i) showed a rapid and large increase in growth cones exposed to liposomes containing NI-35. Neither an increase in [Ca2+]i nor collapse of growth cones was detected in the presence of antibodies to NI-35. Dantrolene, an inhibitor of calcium release from caffeine-sensitive intracellular calcium stores, protected growth cones from collapse evoked by NI-35. Depletion of these caffeine-sensitive intracellular calcium stores prevented the increase in [Ca2+]i evoked by NI-35. The NI-35-evoked cascade of intracellular messengers that mediates collapse of growth cones includes the crucial step of calcium release from intracellular stores.

Local increases in intracellular calcium elicit local filopodial responses in Helisoma neuronal growth cones.

Highly localized changes in intracellular Ca2+ concentration ([Ca2+]i) can be evoked in neuronal growth cones; these are followed by local changes in filopodia. Focally applied electric fields evoked spatially restricted, high magnitude increases in growth cone [Ca2+]i. The earliest and greatest increases were localized to small regions within a growth cone. Such fields also produced characteristic changes in the disposition of filopodia: both filopodial length and number were significantly increased on the cathode side of growth cones. The requirement for extracellular Ca2+ and the strong correlation between the evoked rise in [Ca2+]i and the changes in filopodia (r = 0.98) indicate that cathode stimulation results in local Ca2+ influx, leading to locally increased [Ca2+]i and local changes in filopodial behavior.

Neuron-specific and state-specific differences in calcium homeostasis regulate the generation and degeneration of neuronal architecture.

Many stimuli (e.g., neurotransmitters and electrical activity) regulate neuromorphogenesis by changing intracellular calcium. The ionophore A23187 was employed as a receptor-independent method to investigate neuronal calcium homeostasis. Distinctive neuron-specific (B5 versus B19) and state-specific (growing versus non-growing) differences in calcium homeostasis were observed in cultured identified Helisoma neurons. Fura-2 studies revealed that A23187 induced a transient rise in intracellular calcium in growing neurons B5 but a sustained rise in growing neurons B19. In stable-state (non-growing) cells A23187 evoked only a transient calcium rise. Both neuron-specific and state-specific differences in calcium homeostasis were dependent on extracellular sodium. Morphological studies also indicated that such differences in calcium-regulatory capacity can have profound consequences on the generation and degeneration of neuronal architecture.

Laminin and fibronectin guideposts signal sustained but opposite effects to passing growth cones.

Guidepost cells are known to alter the behavior of growth cones in vivo, yet the nature of communication and the type of signals employed are largely undefined. The present study demonstrates that model guideposts, composed of a single molecular species, are sufficient to change the navigation and the behavior of advancing growth cones well beyond the time of contact. Laminin on model guideposts caused a sustained increase in growth cone velocity, whereas fibronectin led to a sustained decrease. A spatially discrete array of multiple laminin-model guideposts maintained increased growth rates on fibronectin, as expected for homogeneous laminin, and also provided unambiguous directional guidance information. Laminin-evoked growth cone responses required activation of protein kinase C-dependent intracellular signalling mechanisms.

Interactive effects of serotonin and acetylcholine on neurite elongation.

Serotonin (5-HT) inhibits elongation of neurites of specific identified neurons. Here we report a novel, growth-enabling action of another neurotransmitter, acetylcholine (ACh). When applied simultaneously with serotonin, ACh prevents the inhibition of Helisoma neuron B19 neurite elongation that would occur in response to application of 5-HT alone. We also report that ACh prevents the rise in growth cone Ca2+ that would occur in response to application of 5-HT alone and that ACh blocks the electrical excitatory effect of 5-HT on neuron B19. These results support the hypothesis that growth cone motility and neurite elongation can be regulated by voltage-gated Ca2+ fluxes and suggest that the dynamics of neurite morphology may be complexly regulated by an array of neurotransmitters, as is functional electrical activity.

Interactions between entorhinal axons and target hippocampal neurons: a role for glutamate in the development of hippocampal circuitry.

A coculture system consisting of input axons from entorhinal cortex explants and target hippocampal pyramidal neurons was used to demonstrate that glutamate, released spontaneously from afferent axons, can influence both dendritic geometry of target neurons and formation of presumptive synaptic sites. Dendritic outgrowth was reduced in hippocampal neurons growing on entorhinal axons when compared with neurons growing off the axons. Presumptive presynaptic sites were observed in association with hippocampal neuron dendrites and somas. HPLC analysis showed that glutamate was released from the explants in an activity- and Ca2(+)-dependent manner. The general glutamate receptor antagonist D-glutamylglycine significantly increased dendritic outgrowth in pyramidal neurons associated with entorhinal axons and reduced presumptive presynaptic sites. Tetrodotoxin and reduction of extracellular Ca2+ also promoted dendritic outgrowth and reduced the formation of presumptive synaptic sites. The results suggest that the neurotransmitter glutamate may play important roles in the development of hippocampal circuitry.

Neurotransmitter regulation of neuronal outgrowth, plasticity and survival.

Molecules used for communication in mature nervous systems also play important roles in development, maintenance and plasticity of individual neurons. This paper reviews the evidence that neurotransmitters, in addition to their mediation of trans-synaptic information coding, can induce a spectrum of effects on neuronal cytoarchitecture, ranging from neurite sprouting to dendritic pruning and even cell death. Such profound alterations may well constitute a part of the normal functioning and structuring of the nervous system as well as contribute to severe pathological processes.

The sensory-motor role of growth cone filopodia.

Neuronal growth cones, the motile tips of elongating axons and dendrites, respond very precisely to cues encountered during pathfinding. During the past year, our knowledge about their sensory function, their integrative properties, and their motor function has advanced significantly. In particular, growth cone filopodia are currently being recognized for their prominent roles as sensors, transducers, and autonomous motor structures important for growth cone steering.

Developmental changes in the subcellular localization of calretinin.

Brainstem auditory neurons in the chick nucleus magnocellularis (NM) express high levels of the neuron-specific calcium-binding protein calretinin (CR). CR has heretofore been considered a diffusible calcium buffer that is dispersed uniformly throughout the cytosol. Using high-resolution confocal microscopy and complementary biochemical analyses, we have found that during the development of NM neurons, CR changes from being expressed diffusely at low concentrations to being highly concentrated beneath the plasma membrane. This shift in CR localization occurs at the same time as the onset of spontaneous activity, synaptic transmission, and synapse refinement in NM. In the chick brainstem auditory pathway, this subcellular localization appears to occur only in NM neurons and only with respect to CR, because calmodulin remains diffusely expressed in NM. Biochemical analyses show the association of calretinin with the membrane is detergent-soluble and calcium-independent. Because these are highly active neurons with a large number of Ca2+-permeable synaptic AMPA receptors, we hypothesize that localization of CR beneath the plasma membrane is an adaptation to spatially restrict the calcium influxes.

Membrane recycling in the neuronal growth cone revealed by FM1-43 labeling.

Membrane dynamics within the chick ciliary neuronal growth cone were investigated by using the membrane-impermeant dye FM1-43. A depolarization-evoked endocytosis was observed that shared many properties with the synaptic vesicle recycling previously described at the presynaptic terminal. In addition, in the absence of depolarization a basal level of constitutive endocytotic activity was observed at approximately 30% of the rate of evoked endocytosis. This constitutive endocytosis accounted for large amounts of membrane retrieval: the equivalent of the entire growth cone surface area could be internalized within a 30 min period. Endosomes generated via constitutive and evoked processes were highly mobile and could move considerable distances both within the growth cone and out to the neurite. In addition to their different requirements for formation, evoked and constitutive endosomes displayed a significant difference in release properties. After a subsequent depolarization of labeled growth cones, evoked endosomes were released although constitutive endosomes were not released. Furthermore, treatment with latrotoxin released evoked endosomes, but not constitutive endosomes. Although the properties of evoked endosomes are highly reminiscent of synaptic vesicles, constitutive endosomes appear to be a separate pool resulting from a distinct and highly active process within the neuronal growth cone.

Spatial gradients of cytosolic calcium concentration in neurones during paradoxical activation by calcium.

4-Br-A23187 caused a calcium influx into chick sensory neurones and raised cytosolic calcium from a rest level of 97 +/- 7 nM to a peak of 296 +/- 30 nM. Despite the continued presence of ionophore, however, cytosolic calcium concentrations then fell. After 30 min in ionophore, cytosolic calcium concentration had returned to 105 +/- 5 nM, not significantly different from the value before ionophore addition. The permeability of the plasmalemma to divalent cations, as estimated by the manganese quench technique, was no lower at 30 min than at the peak of the cytosolic calcium transient. Thus the fall of calcium from its peak was not due to a slowing of calcium influx, but was due to an upregulation of mechanisms that remove calcium from the cytosol- an upregulation that persists even though cytosolic calcium has apparently returned to pre-stimulus levels. We used a novel fixed slit confocal microscope to examine the calcium concentration profile close to the plasmalemma. We found that after 25-30 min ionophore treatment, calcium concentration was elevated only in the cytoplasm within 1 micron of the plasmalemma. A maintained, elevated calcium under the plasmalemma can help explain the phenomenon of paradoxical activation seen in this and other cell types.

Ca2+ dynamics in neuronal growth cones: regulation and changing patterns of Ca2+ entry.

Digital ratio imaging of Fura-2 fluorescence was used to determine spatially resolved dynamics of Ca2+ changes in neuronal growth cones from the molluscs, Helisoma and Aplysia. Time resolution was approximately 1 s and spatial resolution a few mm depending upon the thickness of the cell region examined. Isolated growth cones of Helisoma were shown to recover from large Ca2+ loads over a time course of minutes, therefore demonstrating Ca2+ regulation mechanisms not dependent on the rest of the cell. Ca2+ changes monitored during action potential discharge showed sharply defined spatial gradients within the growth cones, probably arising from clustering of voltage-gated Ca-channels in the surface membrane. The regions of peak concentration change appeared to shift from central regions to the growth cone periphery as the growth cones matured. There was a marked difference in soma Ca2+ changes produced by action potentials depending on whether or not the soma had sprouted neurites. Neurite-free somata showed large Ca2+ changes, whereas in somata that had recently sprouted neurites there were almost no changes for similar electrical stimulation. Measurements on growth cones of N1E115 neuroblastoma cells showed static distributions of Ca2+ similar to those in the molluscan neurons.

Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells.

Indirect evidence supports a protective role of some EF-hand calcium-binding proteins against calcium-induced neurotoxicity. Little is known about how these proteins influence cytosolic calcium levels. After cloning the parvalbumin cDNA into an expression vector, teratocarcinoma cells (PCC7) were transfected. Parvalbumin-transfected and mock-transfected cells were loaded with the calcium indicator fura-2 and were exposed, in the same dish, to different concentrations of the calcium ionophore A23187 or to KCI. The results show that parvalbumin-transfected PCC7 cells had much better calcium buffering capacity than control cells.