At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

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Free Radicals in the Physiological Control of Cell Function

Excitability. Excitability of cell membranes is crucial for signaling in many types of cell. Excitation in the physiological sense means that the cell membrane potential undergoes characteristic changes which, in most cases, go in the depolarizing direction. Single depolarization from the resting potential to potentials near 0 mV has generally been called an action potential. A schematic representation of a neuronal action potential is given in Fig. 12.1 A. The action potential is triggered when the membrane potential, which was at the resting level, depolarizes and reaches the threshold of excitation. This depolarization, which triggers the action potential, is generated by depolarizing synaptic currents, or depolarizing current coming from a membrane region that is already excited (propagation of an action potential), or by pacemaker currents mediated by pacemaker channels, or by current injected externally by an electrode. The duration of different types of action potential varies from seconds to less than 1 ms.

Ion Channels in Excitable Membranes

We describe the formation, maturation, elimination, maintenance, and regeneration of vertebrate neuromuscular junctions (NMJs), the best studied of all synapses. The NMJ forms in a series of steps that involve the exchange of signals among its three cellular components--nerve terminal, muscle fiber, and Schwann cell. Although essentially any motor axon can form NMJs with any muscle fiber, an additional set of cues biases synapse formation in favor of appropriate partners. The NMJ is functional at birth but undergoes numerous alterations postnatally. One step in maturation is the elimination of excess inputs, a competitive process in which the muscle is an intermediary. Once elimination is complete, the NMJ is maintained stably in a dynamic equilibrium that can be perturbed to initiate remodeling. NMJs regenerate following damage to nerve or muscle, but this process differs in fundamental ways from embryonic synaptogenesis. Finally, we consider the extent to which the NMJ is a suitable model for development of neuron-neuron synapses.

Development of the vertebrate neuromuscular junction.

Synaptic structure and development: the neuromuscular junction.

Little is known about genes that govern the development of the definitive endoderm in mammals; this germ layer gives rise to the intestinal epithelium and various other cell types, such as hepatocytes, derived from the gut. The discovery that the rat hepatocyte transcription factor HNF3 is similar to the Drosophila forkhead gene, which plays a critical role in gut development in the fly, led us to isolate genes containing the HNF3/forkhead (HFH) domain that are expressed in mouse endoderm development. We recovered mouse HNF3 beta from an embryo cDNA library and found that the gene is first expressed in the anterior portion of the primitive streak at the onset of gastrulation, in a region where definitive endoderm first arises. Its expression persists in axial structures derived from the mouse equivalent of Hensen's node, namely definitive endoderm and notochord, and in the ventral region of the developing neural tube. Expression of the highly related gene, HNF3 alpha, appears to initiate later than HNF3 beta and is first seen in midline endoderm cells. Expression subsequently appears in notochord, ventral neural tube, and gut endoderm in patterns similar to HNF3 beta. Microscale DNA binding assays show that HNF3 proteins are detectable in the midgut at 9.5 days p.c. At later stages HNF3 mRNAs and protein are expressed strongly in endoderm-derived tissues such as the liver. HNF3 is also the only known hepatocyte-enriched transcription factor present in a highly de-differentiated liver cell line that retains the capacity to redifferentiate to the hepatic phenotype. Taken together, these studies suggest that HNF3 alpha and HNF3 beta are involved in both the initiation and maintenance of the endodermal lineage. We also discovered a novel HFH-containing gene, HFH-E5.1, that is expressed transiently in posterior ectoderm and mesoderm at the primitive streak stage, and later predominantly in the neural tube. HFH-E5.1 is highly similar in structure and expression profile to the Drosophila HFH gene FD4, suggesting that HFH family members have different, evolutionarily conserved roles in development.

The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins.

Previous morphological studies of the adult rat soleus neuromuscular junction (NMJ) indicate that its ultrastructure changes with age, a consequence of a dynamic, ongoing remodeling of pre- and postsynaptic elements. With aging, the balance between axonal sprouting and withdrawal appears to shift in favor of denervation. Here we have examined the extent and direction of the remodeling process during the adult life of the rat (5–111 weeks) by quantificatation and morphometry of NMJ structure as seen in electron microscopic composites and in cholinesterase (ChE) stained whole mounts.



We have found that the net effect of axonal sprouting and withdrawal on the entire NMJ region varies with age: at 5–12 weeks there is rapid growth and maturation; at 12–48 weeks slower growth occurs, and at 48–82 weeks there is a gradual shift in direction to result in progressive loss of synaptic contact in individual NMJs; finally, at 82–111 weeks, there is loss of entire NMJs with some continued reinnervation at others. The diaphragm NMJ also exhibits continuous structural remodeling; however, between 82–111 weeks many changes in the ChE staining pattern appear abruptly.

Aging rat neuromuscular junnctions: A morphometric study of cholinesterase-stained whole mounts and ultrastructure

Ultrastructural remodeling, with evidence of focal deafferentation and reinnervation, occurs within normal young adult rat soleus neuromuscular junctions (Cardasis and Padykula, 1981). This may be related to normal variations in function. Recognition of this plasticity provides a basis for analysis of aging changes in junctional ultrastructure. Thirty soleus junctions were studied between 11 and 26 months of life. In these junctions, compared to younger ones (3-5 months) synaptic sites with the conventional ultrastructure become increasingly sparse. There is an increase in extent and frequency of exposed junctional folds, of intervention of Schwann cell cytoplasm between axon and junctional folds, and of numbers of lysosomes in all cytoplasmic profiles. Often primary clefts are shallow or missing, and secondary folds are widened and contain collagen. Features limited largely to these older junctions include highly pleomorphic myonuclei, deeply invaginated by myofibrils, and an increase in cellular profiles between basal lamina and sarcolemma. The identity of these profiles is unknown. At other locations within many of the same endplates, small intact terminals are associated with larger expanses of junctional folds, and several small terminals occur within the same primary cleft. Such terminals frequently contain dense-cored vesicles. These observations suggest continuation of some terminal axonal regeneration. Thus, the ultrastructure of these aging neuromuscular junctions reveals the same degenerative and regenerative events suggested by the ultrastructure of younger junctions, but suggests a shift in the balance between them.

Ultrastructural evidence of continued reorganization at the aging (11-26 months) rat soleus neuromuscular junction.

The ultrastructural organization of 40 soleus neuromuscular junctions from ten normal young adult male and female Sprague-Dawley (SD)-derived rats (Charles River Breeders, CD-Crl:COBS (SD)BR) has been studied. A smaller sample of motor endplates from the gastrocnemius, diaphragm, and extensor digitorum longus muscles of these rats as well as from the soleus muscles of two adult Wistar (W) rats (Crl:COBS(WI)BR) was included. Widespread ultrastructural reorganization was evident at the soleus neuromuscular junction during the growth period from three to five months of age. A major characteristic of reorganization is the presence of junctional folds not associated with axonal terminals; such sites occur within a single endplate adjacent to areas with typical intact synaptic associations. Additional features possibly related to remodelling are: (1) spatial separation of axonal terminals from the myofiber, (2) intervention of Schwann cell cytoplasm between an axon terminal and myofiber, (3) aggregates of satellite cells, and (4) folded or multilayered basal lamina. These features are most pronounced in the soleus muscle but occur to varying degrees in the neuromuscular junctions of other muscles of SD-derived rats.



Distinctive characteristics of the rat soleus postjunctional sarcoplasm include the widespread occurrence of myofibrillar components, abundant free and membrane-associated polysomes, and triads oriented in various planes. Away from such discrete sites, myofibers possess the usual highly oriented organization of myofibrils, T tubules, sarcoplasmic reticulum, and mitochondria.



The soleus muscle is a postural muscle that responds directly to rising workload imposed by continuous body growth during young adulthood by steady myofiber hypertrophy and conversion of motor units (Kugelberg, '76). This changing structural-functional relationship may be reflected also by ultrastructural remodelling of the neuromuscular junctions reported here.

Ultrastructural evidence indicating reorganization at the neuromuscular junction in the normal rat soleus muscle.

The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering

The effects of estradiol and nafoxidine on nuclear estrogen receptor binding, RNA polymerase activities, and uterine ultrastructure were studied. Animals were either injected with estradiol, implanted with estradiol/paraffin pellets, or injected with nafoxidine. Animals treated with nafoxidine or estradiol implants showed sustained long-term nuclear retention of estrogen receptor and increased nuclear RNA polymerase activities for up to 72 hr. A single injection of estradiol caused initial increases in these variables which returned to control levels by 24 hr after hormone treatment. Uterine tissue was examined by light and electron microscopy 72 hr after hormone treatments. Uteri from eith estradiol-implanted or nafoxidine-treated animals showed markedly increased hypertrophy of the luminal epithelial cells. Nuclei in sections of the uteri of these hyperestrogenized animals displayed a large number and wide array of nuclear bodies composed of a filamentous capsule and granular cores. We conclude that hyperestrogenization, a condition that eventually results in abnormal cell growth, is correlated with increased and sustained nuclear binding of the estrogen receptor, increased and sustained RNA polymerase activity, and the appearance of nuclear bodies.

Constance A. Cardasis

Papers

Aging rat neuromuscular junnctions: A morphometric study of cholinesterase-stained whole mounts and ultrastructure

Ultrastructural evidence of continued reorganization at the aging (11-26 months) rat soleus neuromuscular junction.

Ultrastructural evidence indicating reorganization at the neuromuscular junction in the normal rat soleus muscle.

The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering

Role of estrogen receptor binding and transcriptional activity in the stimulation of hyperestrogenism and nuclear bodies