Activity-dependent development of the vertebrate nervous system.
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Animals↗
Biomedical subjects
R D Fields
Publications and source records attributed to R D Fields.
Calcium, network activity, and the role of NMDA channels in synaptic plasticity in vitro.
Functionally effective neuronal circuits are constructed through a competitive process that requires patterned neuronal activity elicited by structured input from the environment. To explore the mechanisms of this activity-dependent synaptic restructuring, we have developed an in vitro preparation of mouse spinal cord neurons maintained in a 3-chambered cell-culture system. Sensory afferents that received chronic electrical stimulation for 3-5 d developed stronger synaptic connections than unstimulated afferents converging onto the same postsynaptic spinal cord neuron. Exposure to 100 microM DL-2-amino-5-phosphonovaleric acid (APV), an antagonist of the NMDA channel, during the stimulation period prevented the competitive advantage associated with electric stimulation. However, when APV was applied with a higher concentration of calcium (3 mM), activity-dependent synaptic plasticity was no longer inhibited by the NMDA receptor antagonist. This reversal of APV block of the plasticity was not impaired by reducing transmitter release with 3 mM magnesium (in addition to 3 mM calcium and APV). A suppressant effect of APV on spontaneous activity was observed, which was attributed to loss of the NMDA component of the EPSP. Activity-dependent plasticity was also blocked if spontaneous activity was suppressed with dilute tetrodotoxin (TTX; 5-10 nM), a dosage that reduces excitability of neurons but is insufficient to block sodium-dependent action potentials. These experiments bring into question how NMDA channel activation is involved in the processes of synaptic remodeling during development. The data suggest that postsynaptic activity is required for synaptic remodeling, but this activity need not involve NMDA receptor activation specifically for activity-evoked synaptic plasticity. Instead, the mechanism for plasticity appears to operate through calcium-dependent processes in general.
2-Amino-5-phosphonovalerate↗
Mechanisms involved in activity-dependent synapse formation in mammalian central nervous system cell cultures.
Differences in neuronal activity produced by electrical stimulation lead to competition between synapses from sensory afferents converging on a common spinal cord neuron. Studies were performed on neurons dissociated from the mouse spinal cord and grown in culture dishes with three compartments. Synaptic efficacy from stimulated afferents was increased compared with unstimulated convergents, and the number of functional connections was increased by stimulation compared with control cultures. Blocking NMDA channel activation with 100 microM APV in medium containing 1.8 mM calcium inhibited this synaptic plasticity, but plasticity was not blocked by APV in medium in which the calcium concentration was elevated to 3 mM. These experiments support the view that electrical activity differentially influences processes that cause a persistent decrease in synaptic efficacy or lead to synapse elimination and those that increase synaptic strength or lead to synapse augmentation. We interpret our results in terms of a model in which these antagonistic mechanisms are both regulated via changes in calcium levels and second messengers that are modulated by electrical activity. A significant portion of the activity-related calcium influx relevant to synaptic plasticity passes through the NMDA channel, but other sources of calcium are involved. In particular, competitive elimination of synapses appears to occur during blockade of NMDA channels if the extracellular concentration of calcium is elevated moderately. The outcome of competition between the two calcium-dependent but antagonistic processes may depend either on their differential sensitivity to intracellular calcium concentration or separate specificities to NMDA and non-NMDA receptor-linked mechanisms.
Action Potentials↗
Effects of patterned electrical activity on neurite outgrowth from mouse sensory neurons.
A noninvasive method of electric stimulation was used in cell culture preparations to determine the effects of patterned electrical activity on the morphology and motility of mammalian central nervous system growth cones. Neurites from dorsal root ganglion (DRG) neurons of fetal mice were allowed to grow under the barrier of an insert placed in culture dishes. The insert confined the cell bodies within separate experimental and control compartments, and provided a means of exciting action potentials in the growing neurites by extracellular current pulses delivered across the barrier. A phasic pattern of stimulation caused immediate retraction of the filopodia and lamellipodium. Further outgrowth was halted and in many cases retraction of the neurite ensued. No changes in morphology or growth cone motility were evoked by electric stimulation when action potentials were blocked with 1 microM tetrodotoxin (TTX). These effects depended on the rate, pattern, and duration of stimulation. Phasic stimulation was more effective than stimulation with the same number of impulses delivered at a constant frequency. An important new observation was that cultures exposed to phasic stimulation for several hours contained actively growing neurites with normal growth cones which were insensitive to the stimulus. This apparent accommodation in neurites exposed to chronic stimulation may involve processes that regulate calcium conductance or buffering. Cessation of neurite outgrowth by action potentials could represent one mechanism linking morphological and functional characteristics in the developing CNS of mammals, by stabilizing the outgrowth of neurites forming appropriate synaptic contacts and leading to the retraction of growth cones from collaterals that have not formed appropriate contacts at the time the neuron enters into a functionally active circuit.
Action Potentials↗
Synaptic connections in vitro: modulation of number and efficacy by electrical activity.
The functional architecture of synaptic circuits is determined to a crucial degree by the patterns of electrical activity that occur during development. Studies with an in vitro preparation of mammalian sensory neurons projecting to ventral spinal cord neurons slow that electrical activity induces competitive processes that regulate synaptic efficacy so as to favor activated pathways over inactive convergent pathways. At the same time, electrical activity initiates noncompetitive processes that increase the number of axonal connections between these sensory and spinal cord neurons.
Action Potentials↗
Nerve regeneration through artificial tubular implants.
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Animals↗
Dense-cored vesicle-containing components of the terminal nerve of sharks and rays.
This paper describes electron microscopic observations of dense-cored vesicle-containing axons, cell bodies, and endings of the terminal nerve in several elasmobranchs. The vesicles are found in two apparent cell types, one with a polymorphic nucleus and another with an oval nucleus. The types may correspond to cells producing one each of two neuropeptides (LHRH and FMRF-amide) that have previously been localized in the nerve. Dense-cored vesicles are found in many unmyelinated fibers in both the terminal nerve proper and its major ganglia. Some of these form complicated structures with interdigitation and wrapping of membranes. Vesicle-containing fibers branch from the nerve, run along nearby blood vessels, and appear to end adjacent to endothelial cells which demonstrate vesicular activity. The observations suggest terminal nerve neurosecretion into the cerebral circulation. Synapses are found in and near the ganglia where they appear to be axodendritic, with multiple contacts in some cases.
Animals↗
Regional membrane heterogeneity in premyelinated CNS axons: factors influencing the binding of sterol-specific probes.
Binding of the sterol-specific probe filipin to developing optic nerve axonal membrane is spatially heterogeneous prior to association of glial cells with the axons. Experiments were performed using different sterol binding probes (filipin, tomatin, and saponin), at different temperatures (4 degrees C, 23 degrees C, and 37 degrees C), after incubation in different ionic conditions (10 mM Ca2+, 10 mM EGTA, and 20 mM Mg2+), to examine factors that may be responsible for this membrane heterogeneity in rat optic nerve. The patchy pattern of filipin binding is apparent with each sterol-specific probe, even prior to glial ensheathment, and is retained when membrane fluidity is increased at higher temperatures. Increased Ca2+ concentration increased membrane stability, and increased Mg2+ reduced the patchiness of filipin binding. After tannic acid staining, regions of the cytoskeleton are seen associated with the membrane via filaments extending from microtubules to the membrane, preferentially in regions where filipin interaction with the membrane is inhibited. The non-uniform interaction of filipin with the axolemma suggests an underlying heterogeneity in the sterol composition and stability of the membrane. Heterogeneity of premyelinated axonal membrane may provide an important formative influence in the differentiation of axons to their mature morphology and function.
Animals↗
Diminished dorsal root GABA sensitivity following chronic peripheral nerve injury.
The depolarizing effect of gamma-aminobutyric acid (GABA) on rat lumbar dorsal roots was studied in a sucrose gap chamber following axotomy or crush injury of the sciatic nerve or dorsal root. The mean depolarization elicited by GABA on normal dorsal roots (3.96 +/- 0.71 mV, N = 14) was significantly reduced following chronic sciatic axotomy (2.02 +/- 0.99 mV, N = 15). Chronic sciatic crush injury had no significant effect on dorsal root GABA sensitivity. The amplitudes of the dorsal root compound action potentials were the same from rats with normal and injured sciatic nerves, indicating that axons proximal to the sciatic nerve lesion did not undergo appreciable degeneration. A marked loss of dorsal root GABA sensitivity was also seen following dorsal root axotomy or crush injury (1.02 +/- 0.98 mV (N = 10) and 0.69 +/- 0.70 mV (N = 9), respectively). These results indicate that GABA sensitivity of dorsal roots is attenuated following peripheral nerve lesions in which regeneration and functional reconnection with peripheral targets are prevented. Previous work indicates that the primary afferent depolarization is reduced under similar conditions. The reduction in GABA sensitivity of dorsal root fibers described here may have a contributory role in the reduced primary afferent depolarization that follows peripheral nerve transection, which has pathophysiologic implications in chronic pain syndromes.
Action Potentials↗
Functionally significant plasticity of synaptic morphology: studies on the ribbon synapse of the ampullae of Lorenzini.
Changes in electrophysiological properties measured in vitro were correlated with ultrastructural differences at synapses between sense cells and the primary afferent neurons in electrosensory organs of the thornback ray (the ampullae of Lorenzini). Variation in synaptic structure was classified into four synaptic morphotypes, which appear to represent stages in a cyclic pattern of ultrastructural modification associated with changes in synaptic efficacy. Synapses with deeper postsynaptic troughs, and active zone regions located at the "narrow point" of the presynaptic evagination, and other morphological differences, were associated with greater sensitivity and spontaneous activity. Furthermore, the morphology of synapses was different in organs that had shown increasing, decreasing or stable trends in sensitivity prior to fixation, suggesting that changes in synaptic physiology and morphology are interrelated, and providing evidence for the sequence of ultrastructural modifications represented by the four synaptic morphotypes. These results support the conclusion that synaptic morphology is plastic and that this plasticity has functional significance in terms of the threshold sensitivity and spontaneous activity monitored from the afferent nerves. Plasticity of synaptic morphology which is associated with changes in the efficacy of transmitter release at chemically mediated synapses could be important in relatively long-term phenomena.
Animals↗
Macromolecular structure of axonal membrane in the optic nerve of the jimpy mouse.
The macromolecular structure of the axon membrane in 26-28-day-old Jimpy mice and control optic nerve were examined with quantitative freeze-fracture electron microscopy. Premyelinated and myelinated axons were observed in control optic nerves, with axonal diameters of premyelinated axons being generally smaller than that of myelinated axons (approximately 0.2-0.4 micron vs approximately 0.5-1.5 micron, respectively). Axon membrane from control optic nerves exhibited an asymmetrical partitioning of intramembranous particles (IMP). P-faces of internodal membrane displayed nearly twice as many IMP as the premyelinated axolemma (1,731 vs 893 micron-2, respectively). E-faces of internodal and premyelinated axolemma exhibited IMP densities of 124 and 157 micron-2, respectively. Few myelinated axons were apparent in optic nerves from Jimpy mice. The amyelinated axons of Jimpy mice displayed a spectrum of axonal diameters, ranging from approximately 0.2 to 1.5 micron. P-face densities of amyelinated axons, considered as a group, exhibited a wide range (600-2,100 micron-2). However, large diameter (greater than or equal to 0.5 micron) axons exhibited a significantly greater P-face IMP density than that of small caliber (greater than 0.5 micron) axons (1,525 vs 1,032 micron-2, respectively). Aggregations of E-face IMP were not observed along amyelinated axons of Jimpy optic nerves. The results demonstrate that the changes in P-face IMP density that occur during development of normal myelinated axons also occur in developing axons of Jimpy optic nerve, irrespective of a lack of normal glial cell association, and provide further evidence that the primary defect of hypomyelination within Jimpy mice is not attributed to the neuron.
Animals↗
Filipin-cholesterol binding in CNS axons prior to myelination: evidence for microheterogeneity in premyelinated axolemma.
The distribution of cholesterol in axonal membrane of developing rat optic nerves prior to myelination was studied by freeze-fracture cytochemistry. Binding of the cholesterol-specific probe, filipin, to the axolemma of premyelinated axons was heterogeneous; this suggests the presence of microdomains of axolemma with different membrane composition and/or cytoskeletal/extracellular matrix association. Although the reasons for this binding pattern have not yet been determined, heterogeneity occurs prior to association of glia with the axon, and may reflect regional differences in lipid/sterol composition of the axonal membrane bilayer, or distribution of membrane-associated cytoskeleton. The distribution of intramembranous particles was not obviously associated with the pattern of filipin binding in early developing axons, however, as might have been expected from the attending differences in fluidity of the membrane microdomains. Microheterogeneity in axonal membranes of developing axons could have an influence on several membrane properties, and may be associated with processes important for growth and differentiation of axons.
Aging↗
Changes in synaptic morphology associated with presynaptic and postsynaptic activity: an in vitro study of the electrosensory organ of the thornback ray.
The influence of synaptic activity on synaptic structure was studied by selectively stimulating the presynaptic or postsynaptic membranes of ribbon synapses in an in vitro preparation, and examining the ultrastructure of synapses with conventional electron microscopic methods. Functionally significant changes in synaptic morphology were observed after direct depolarization of the presynaptic membrane or incubation with the neurotransmitter glutamate to depolarize the postsynaptic membrane. After depolarizing the presynaptic membrane for 30 seconds, the depth of the postsynaptic trough was reduced, and other morphological changes correlated with decreased sensitivity and spontaneous activity were evident. Depolarizing the postsynaptic membrane by incubating synapses with the neurotransmitter glutamate, produced opposite effects. These results suggest that synapses can undergo functionally significant morphological changes in response to certain patterns of activity. The mechanism for these changes might include synaptic vesicle recycling processes, changes in ion concentration, or cytoskeletal alterations in the presynaptic, postsynaptic, or support cells. These mechanisms could operate in association with long-term changes in synaptic efficacy or account for some physiological phenomena such as synaptic fatigue or accommodation.
Action Potentials↗
Differences in intramembranous particle distribution in the paranodal axolemma are not associated with functional differences of dorsal and ventral roots.
Freeze-fracture methods were used to study intramembranous particles, which are believed to represent ectopic sodium channels in the paranodal axolemma, and their possible association with differences in action potential electrogenesis of maturing rat dorsal and ventral root fibers. Our results indicate that there is no significant association between paranodal axon membrane structure and the functional difference of dorsal and ventral root axons.
Action Potentials↗
Axons regenerated through silicone tube splices. I. Conduction properties.
Changes in conduction properties of axons regenerating across a 10-mm gap bridged by a silicone cuff were investigated from compound action potential responses. Compound action potentials were detected as early as 6 weeks after surgery, and were small and slowly conducted at maximum velocities of about 3 m/s. With longer regeneration time, the potentials increased in size, velocity, and complexity. Conduction velocities increased rapidly at first than slowly and asymptotically approached rates that were approximately 40% below normal after 10 months. One component of the compound action potential, the refractory period, decreased from 5 ms to near normal value after only 3 months. The time constant of excitation changed more rapidly, and after 2 months approximated values near those for controls. The properties of axons regenerated across an epineural suture with no gaps. The database for the time course of events established here will be useful in guiding studies using the silicone cuff technique as an in situ experimental chamber for studies of regeneration and remyelination.
Animals↗
Axons regenerated through silicone tube splices. II. Functional morphology.
The recovery of axons regenerated through silicone tube splices was studied with electron microscopic and morphometric methods. Regenerated nerves contained both myelinated and unmyelinated axons of near normal morphology. The number and diameter of axons increased with postoperative time, and size-frequency histograms demonstrated that regeneration occurred in all major fiber groups. Remyelination occurred between about 4 and 6 weeks. Some of the smallest regenerated axons had unusually thick myelin sheaths, but overall regenerated axons had a slightly thinner sheath compared with similar-size normal fibers, although the ratio of sheath thickness to axon size was within the normal limits of g = 0.65 to 0.8 by 6 weeks. Axons did not, however, regain their normal size within 10 months of surgery. This deficit was apparently the primary factor limiting conduction velocity in these regenerated axons.
Animals↗
Synaptic morphology and differences in sensitivity.
A relation between synaptic morphology and physiology was observed in an in vitro preparation of a sense organ (the ampulla of Lorenzini), in which activity was monitored from the primary afferent neurons before electron microscopic examination of the afferent synapses. The depth of the postsynaptic trough decreased as prefixation sensitivity of the sense organ decreased. This relation and other ultrastructural differences suggest that physiological properties of synapses are influenced by morphological features. Thus, synapses might be morphologically dynamic to modulate synaptic efficacy in relatively long-term phenomena.
Animals↗
Physical abilities of the mentally retarded child.
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Child↗