Roles for ephrins in positionally selective synaptogenesis between motor neurons and muscle fibers.

Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.

Positionally selective growth of embryonic spinal cord neurites on muscle membranes.

Motor neurons from distinct positions along the rostrocaudal axis generally innervate muscles or muscle fibers from corresponding axial levels. These topographic maps of connectivity are partially restored after denervation or transplantation under conditions in which factors of timing and proximity are eliminated. It is therefore likely that motor neurons and some intramuscular structures bear cues that bias synapse formation in favor of positionally matched partners. To localize these cues, we studied outgrowth of neurites from embryonic spinal cord explants on carpets of membranes isolated from perinatal rat muscles. Neurites from rostral (cervical) and caudal (lumbar) spinal cord slices exhibit distinct growth preferences. In many instances, rostrally derived neurites grew selectively on membranes from forelimb muscles or from a single thoracic muscle (the serratus anterior) when given a choice between these membranes and membranes from hindlimb muscles or laminin. Caudally derived neurites almost never exhibited such rostral preferences, but instead preferred membranes from hindlimb muscles or a single hindlimb muscle (the gluteus) to rostral muscles or laminin. Likewise, spinal neurites exhibited distinct position-related preferences for outgrowth on membranes of clonal myogenic cell lines derived from specific rostral and caudal muscles. Taken together these results suggest that the membranes of motor axons and myotubes bear complementary labels that vary with rostrocaudal position and regulate neuromuscular connectivity.

Imidazole myopathy. Production of the myopathy and its dependence on acetylcholine.

An acetylcholine-mediated myopathy has been produced in the soleus muscle of the rat by the daily injection of imidazole, a compound that accelerates the metabolism of adenosine 3':5' cyclic phosphate by activating the enzyme phosphodiesterase. The imidazole-treated muscles were found to have a lowered resting membrane potential. This study suggests that a decrease in resting membrane potential may make skeletal muscle more vulnerable to necrosis by acetylcholine released during normal activity.

Biochemical and ultrastructural changes in skeletal muscle induced by a creatine antagonist.

To evaluate the essentiality of creatine and phosphocreatine for the maintenance of the ultrastructure of skeletal muscle, chicks were fed a creatine antagonist, beta-guanidinobutyric acid (beta-GBA), as 2% of a Chow diet. Chicks fed beta-GBA exhibited growth retardation and weakness, and they accumulated large amounts of a monosubstituted guanidino compound, presumably beta-GBA, in their skeletal muscles. After 2 wk, there was a 74% decrease in the uptake of [14C]-1-creatine into pectoralis muscles of chicks fed beta-GBA. After 2 wk there as a significant decrease in phosphocreatine concentrations in pectoralis muscles from 20.1 +/- 2.8 mumoles per g wet weight (mean +/- S.D.) for 8 control chicks to 16.5 +/- 2.5 for 7 chicks fed beta-GBA. Selected fibers of the pectoralis and gastrocnemius muscles of chicks fed beta-GBA exhibited ultrastructural abnormalities including loss of thick and thin filaments, disruption of the Z band, dilated mitochondria, and dilated and displaced sarcoplasmic reticulum. The pectoralis muscles of chicks given 6% creatine in addition to 2% beta-GBA in the diet accumulated little beta-GBA, maintained normal phosphocreatine concentrations, and exhibited no significant ultrastructural abnormalities. These findings are the first experimental evidence that high concentrations of phosphocreatine are essential for the maintenance of the ultrastructural integrity of skeletal muscle.

A morphological technique for exploring neuromuscular topography expressed in the mouse gluteus maximus muscle.

Motor neuron pools innervate muscle fibers forming an ordered topographic map. In the gluteus maximus (GM) muscle, as well as additional muscles, we and others have demonstrated electrophysiologically that there exists a rostrocaudal distribution of axon terminals on the muscle surface. The role of muscle fiber type in determining this topography is unknown. A morphological approach was designed to investigate this question directly. We combined three different methods in the same muscle preparation: (1) the uptake of activity-dependent dyes into selected axon terminals to define the spinal segmental origin of a peripheral nerve terminal; (2) the fluorescent labeling of nicotinic acetylcholine receptors to determine motor endplate size; (3) the immunocytochemical staining of skeletal muscle to determine fiber subtype. We applied these methods to the mouse GM muscle to determine the relationship between muscle fiber type and the topographic map of the inferior gluteal nerve (IGN). Results from this unique combination of techniques in the same preparation showed that axon terminals from more rostral spinal nerve segments of origin are larger on rostral muscle fibers expressing myosin heavy chain (MyHC) IIB epitope than caudal type IIB fibers. Because type IIB fibers dominate the GM, this suggests that for these rostral axons terminal size is independent of fiber type. How this axon terminal size is related to the topographic map is the next question to be answered.

Microsomal opiate receptors differ from synaptic membrane receptors in proteolytic sensitivity.

We have found that opiate receptors in smooth microsomal fractions differ from synaptic membrane-associated receptors in proteolytic sensitivity. With 3 proteases of different substrate specificities (trypsin, chymotrypsin and S. griseus protease) smooth microsomal opiate receptors from rat brain were consistently less sensitive to limited proteolysis than were synaptic membrane receptors. Thiamine pyrophosphatase, a luminal Golgi membrane marker enzyme, exhibited a similar resistance to S. griseus protease in microsomal preparations, while microsomal Na+/K+-ATPase (ouabain-sensitive) was readily destroyed by trypsin. We also discovered that smooth microsomal opiate receptors co-migrate with both Golgi membrane and endoplasmic reticulum marker proteins on equilibrium density gradients under isopycnic conditions. Electron microscopic examination of the Golgi-enriched fraction showed the typical cisternae frequently associated with isolated Golgi membranes. Synaptic junctions, presynaptic membranes, myelin and mitochondria were conspicuously absent from this fraction. Since the microsomes isolated in vitro showed similar topography to those in vivo, the binding sites for opiates could be localized on the luminal surface membranes of the microsomal fractions. The exquisite sensitivity of synaptic membrane opiate receptors to proteolysis suggests that these receptors are found on the extracellular surface of the synaptic junction.

Ephrin-A5 overexpression degrades topographic specificity in the mouse gluteus maximus muscle.

Motor neurons project onto specific muscles with a distinct positional bias. We have previously shown using electrophysiological techniques that overexpression of ephrin-A5 degrades this topographic map. Here, we show that positional differences in axon terminal areas, an entirely different parameter of neuromuscular topography, are also eliminated with ephrin-A5 overexpression. Therefore, we now have both morphological and electrophysiological approaches to explore the mechanisms of neuromuscular topography.

Morphometric analysis of neuromuscular topography in the serratus anterior muscle.

Groups of neurons form ordered topographic maps on their targets, and defining the mechanisms that develop such maps, and re-connect them after disruption, has biological as well as clinical importance. The neuromuscular system is an accessible and well-studied model for defining the principles that guide map formation, both during its development and its reformation after motor nerve damage. We present evidence for the expression of this map at the level of nerve terminal morphology and muscle fiber type in the serratus anterior muscle. Morphometric analyses indicate, first, a rostrocaudal difference in nerve terminal size depending on the ventral root of origin of the axons. Second, motor endplates are larger on type IIB than type IIA muscle fibers. Third, whereas IIB muscle fibers are distributed rather evenly along the rostrocaudal axis of the muscle, the more rostral type IIB fibers are preferentially innervated by anteriorly derived (C6) motor neurons, and more caudal IIB fibers are preferentially innervated by posteriorly derived (C7) motor neurons. This inference is supported by analysis of the size of nerve terminals formed in each muscle sector by rostral and caudal roots, and by evidence that the larger terminals are on IIB fibers. These results demonstrate a subcellular expression of neuromuscular topography in the serratus anterior muscle (SA) muscle in the form of differences in nerve terminal size. These results provide deeper insights into the organization of a neuromuscular system. They also offer a rationale for a topographic map, that is, to allow spinal motor centers to activate selectively different compartments within a muscle.

Selective reinnervation of the rat serratus anterior muscle following denervation and partial target removal.

The rat serratus anterior (SA) muscle is reinnervated by cervical roots C6 and C7 with a topographic bias following transection or freezing of the long thoracic nerve. The study reported here was undertaken to determine the specificity of regenerating motoneurons when the size of the target SA muscle was reduced. After crush and removal of the caudal half of the muscle, a rostrocaudal map was reestablished that was no different from control or crush alone. Rostral (C6) axons dominated the innervation of muscles deprived of their caudal sectors. Input from caudal (C7) axons was significantly reduced. These results may be explained by preferential pathways or a competitive advantage held by rostral axons reinnervating rostral sectors to the detriment of caudal axons.

Embryonic expression of motoneuron topography in the rat diaphragm muscle.

The phrenic motoneuron pool of the rat projects onto the diaphragm muscle with a distinct rostrocaudal bias. This bias is detectable at birth and is reestablished following denervation. In an effort to define the mechanisms underlying this topographic bias, we asked whether growing phrenic motoneurons select their muscle contacts initially upon first contact or whether the initial neuromuscular distribution is random, to be specified later through synaptic rearrangement. The onset of neurotransmission in embryonic diaphragm muscles aged E-14 to E-18 was studied using focal extracellular microelectrodes. Two important phenomena were observed. First, motoneurons from all three cervical ventral roots (C4, C5, and C6) establish functional innervation at the same time. Second, already at E-15, when the earliest synaptic potentials could be recorded, a distinct rostrocaudal bias was detected. This bias was amplified as innervation progressed to rostral and caudal sectors during E-16 to E-18. These results suggest that growing phrenic motoneurons make topographic choices as they navigate toward their muscle targets. Moreover, the results indicate that further research into the mechanisms for topographic selectivity should focus on initial nerve-muscle contacts in the embryo, rather than secondary processes of error correction.

Presynaptic effects of neuromuscular cholinesterase inhibition.

Paraoxon, an irreversible organophosphorus inhibitor of cholinesterase, produces a myopathy beginning at the neuromuscular junction in rat diaphragm muscles. Thirty minutes after paraoxon was injected i.p. (0.5 mg/kg), neuromuscular cholinesterase activity was reduced to 36% of control. The frequency of miniature end-plate potentials (MEPPs) in diaphragms from paraoxon-treated rats was 109/sec compared with 2.9/sec in saline-injected controls. The faster frequency was seen after paraoxon gradually declined to control rates within 6 hours after injection. The quantum content of end-plate potentials was reduced to 66% of that seen in saline controls. Spontaneous and impulse-related antidromic activity was observed along the phrenic nerve after paraoxon which also gradually diminished with time. When the phosphorylated acetylcholinesterase was reactivated with 10(-3) pyridine-2-aldoxime methiodide, MEPP frequency was reduced significantly and antidromic activity was abolished. Block of axonal excitability with 10(-6) M tetrodotoxin reduces the effects of paraoxon on MEPP frequency and antidromic activity, while acetylcholinesterase remains inhibited. In vitro perfusion with 6 times 10(-8) M paraoxon increases MEPP frequency and initiates antidromic activity. It is concluded that inhibition of neuromuscular cholinesterase by paraoxon leads to an alteration of transmitter release, and this may be associated with ultrastructural abnormalities observed at the motor endplate.

Denervation and age modify neuromuscular positional selectivity.

The rostrocaudal position of neurons within the spinal motor pool maps systematically onto the surface of several muscles in mammals. In an effort to understand the mechanisms that generate such maps, we have been studying choices made by embryonic spinal cord neurons on muscle membrane substrates in the in vitro stripe assay. In this report we explore the effects of postnatal age of the muscle on neurite choice, and how prior denervation modifies this choice. Our results further differentiate rostral from caudal motor neurons in preferring one substrate to another. First, caudal neurites prefer to grow on P6 neonatal caudal over rostral membranes, but lose this ability to distinguish axial position of origin in older muscles. Rostral neurites prefer growth on rostral membranes, but this preference also diminishes with age. Second, when adult muscles have been denervated, both rostral and caudal neurites regain their positional growth selectivity. Third, caudal neurites are particularly sensitive to substrate choice. When growing on a preferred substrate (gluteus) caudal neurites prefer neonatal over adult membranes. These results support the concept of fundamental differences in the growth preferences of rostral and caudal spinal neurites. These differences will assist in the identification of molecular guidance cues that determine the formation of neuromuscular positional maps.

Topographic mapping of motor pools onto skeletal muscles.

We have studied the segmental innervation of 2 rat skeletal muscles, the diaphragm and the serratus anterior. Both muscles are thin, flat, and composed of several sectors that form a clear rostrocaudal progression. Each is innervated through a single nerve, which is in turn supplied by motor neurons from several cervical spinal segments. Using intracellular recording, we found that in both cases, the rostrocaudal axis of the motor pool is systematically mapped onto the rostrocaudal axis of the muscle's surface. For the diaphragm, electrophysiological results were confirmed by immunohistochemical identification of denervated fibers following section of single ventral roots and by retrograde labeling of motoneurons following localized application of fluorescent dyes. In addition, an immunohistochemical method was used to study the arrangement of motor axons in the phrenic nerve, which supplies the diaphragm, and to show that contributions from individual ventral roots are compartmentalized within this nerve. We suggest that segmental ordering of axons in the nerve, axonal guidance at branch points in the nerve, and positional labels within the muscle may all contribute to the rostrocaudal mapping of motor pools onto muscle.

Expression of nerve-muscle topography during development.

Previous studies have indicated that in 2 muscles of the adult rat, the anterior serratus and the diaphragm, the rostrocaudal axis of the motoneuron pool projects topographically onto the rostrocaudal axis of the muscle. In the present work we have asked whether this orderly topography emerges as a function of postnatal synaptic rearrangement or whether this pattern is already established at birth. The anterior serratus muscle was studied over the period ranging from embryonic day 17 through postnatal day 30. Using 2 criteria of topography, average segmental innervation and average target field of cervical roots C6 and C7, we found that a topographic distribution of the motoneuron pool is already present prior to birth and maintained throughout the postnatal period. Moreover, both C6 and C7 form an orderly map over the surface of the serratus in the embryo, and the topography is sharpened during postnatal periods. The diaphragm also is topographically innervated at birth and undergoes a comparable sharpening of the projection map postnatally. We conclude that the topographic projection of motoneurons is established prior to birth in these muscles, and postnatal synaptic rearrangement serves to sharpen the topographic map toward the adult pattern. These results also suggest that the pursuit of basic mechanisms underlying topography should be directed toward initial embryonic nerve-muscle contacts.

Topographically selective reinnervation of adult mammalian skeletal muscles.

In 2 rat muscles, serratus anterior and the diaphragm, the rostrocaudal axis of the motor pool is mapped onto the rostrocaudal axis of the muscle's surface (Laskowski and Sanes, 1987a). One possible basis for this orderly topography is that motor axons and intramuscular structures bear labels that favor connectivity among positionally matched partners. To test for the existence of such labels, we asked whether axons would selectively reinnervate appropriate portions of the muscles following nerve transection. We found that, on average, rostral and caudal halves of each muscle were preferentially reinnervated by axons from the rostral and caudal halves of its motor pool, respectively. In the serratus anterior, reinnervation was more selective following denervation in neonates than following denervation in adults, although in neither case was the normal pattern of innervation reestablished completely. These results show that motor axons can selectively reinnervate adult rat muscles, and support the idea that positional cues play a role in organizing neuromuscular topography.

Branching patterns of the rat phrenic nerve during development and reinnervation.

Previous studies have shown that the phrenic motor nucleus in the rat projects onto the diaphragm muscle, forming an orderly topographic map. Moreover, this topography is partially restored upon reinnervation. This orderly map is expressed prior to birth, suggesting that early contacts between nerve and muscle are topographically appropriate. The phrenic divides during embryonic development into rostral and caudal branches, and motor axons preferentially enter the appropriate branch. In an effort to understand the mechanisms that underlie the choices growing phrenic neurons make in selecting their appropriate muscle targets, we examined the patterns of branching displayed by the phrenic nerve during development and reinnervation. In all muscles studied the phrenic nerve splits into three primary branches, rostral, caudal, and crural. At a coarse level the pattern of branching of the phrenic is remarkably consistent from animal to animal and at all ages of development. At a finer level of resolution, however, there is an asymmetry between right and left hemidiaphragms. Moreover, the precise emergence of any particular branch is unpredictable, resulting in an overall incongruence in branching architecture from animal to animal. The hemidiaphragm muscle grows unevenly, particularly on the right side, resulting in greater muscle fiber elongation medially. Upon reinnervation, the same coarse pattern of branching is reestablished, but the higher order pattern is much simpler and muscle growth is slower than in controls. These results suggest that very early in development primary branches of the phrenic funnel axons into three well-defined zones in the muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

An early preceptorship and medical students' beliefs, values, and career choices.

BACKGROUND AND OBJECTIVES: Curriculum influence on career choice is difficult to determine. In this study we explored the impact of a summer rural/underserved preceptorship on the residency choices of participants and on the beliefs and attitudes of participating students about rural underserved primary care practices. METHODS: Two data sets are used to examine the Rural/Underserved Opportunities Program (R/UOP). Matriculation and residency selection information is analyzed to compare R/UOP participants with nonparticipants. Second, a survey eliciting beliefs and attitudes about various career choices was given to participants before and after the experience and to a sample of non-participating classmates matched for age, race, and ethnicity. RESULTS: At matriculation, R/UOP participants gave higher rankings to primary care specialties as possible career choices. They were more likely to be matched in a primary care residency than nonparticipants. R/UOP participants expressed belief in more differences between urban and rural practice than did nonparticipants. They maintained their higher attitudes towards rural practice. CONCLUSIONS: R/UOP supports preexisting beliefs and positive attitudes towards rural underserved primary care careers. Participating students do not have large differences at entry into medical school. They are more likely to select primary care residencies, compared with nonparticipants.

The ultrastructure of the sinu-atrial node of the bat.

The sinu-atrial node (SAN) of the bat, Pipistrellus subflavus, is capable of generating a wide range of spontaneous activity varying from 20 bpm when hibernating to bursts of 800 bpm during active flight. Electrophysiological studies have shown an absence of arrhythmias even below 4 degrees C body temperature. In order to determine whether these physiological capabilities are based upon unique ultrastructural features of the bat SAN, the present study was conducted. We found that the structure of the SAN of the bat is typically mammalian. Diameters of all three cell types in the SAN (nodal, transitional, and atrial) are smaller than those observed in any other mammalian species. A morphometric analysis of cell junctions reveals that nodal-nodal and transitional-transitional cell contacts are primarily undifferentiated with few nexuses. Atrial-atrial cell contacts have a dominance of fasciae adherentes-type junctions with a small area left undifferentiated. Nexuses are much more prevalent in atrial-atrial cell contacts.

A mu-specific opioid peptide agonist increases excitability of pyramidal neurons in untreated and receptor up-regulated hippocampus.

The rat hippocampus contains the major types of opioid receptors, delta, mu, and kappa, as determined by autoradiographic and membrane binding analyses. Chronic exposure to excessive amounts of opioid antagonists results in a doubling of the number of binding sites. However, the direct electrophysiological significance of this increased number of opioid receptors in the central nervous system remains uncharacterized. We examined the effects of an opioid peptide with high affinity and high specificity for mu receptors, DAMGO (D-ala2-mePhe4-gly-ol5 enkephalin), under normal conditions and after 1 or 2 weeks of continuous infusion of the opiate antagonist naltrexone. Chronic infusion of naltrexone administered to the whole animal resulted in significant up-regulation (71%) of mu opioid receptors in the rat hippocampus. Slices of the hippocampus were perfused with artificial cerebrospinal fluid while recording population spikes in stratum pyramidale, excitatory postsynaptic potentials in stratum radiatum and while stimulating afferents in the Schaffer collaterals. Superfusion of slices with DAMGO produced a concentration-dependent increase in the amplitude of population spikes. No significant change was observed in the simultaneously recorded excitatory postsynaptic potential slope. This selective increase in population spike amplitude led to a leftward shift (19%) in the derived input-output curve. In addition, DAMGO superfusion produced extra spiking at higher stimulus intensities. Naltrexone reversed the DAMGO-induced increase in excitability, as well as prevented additional spikes. DAMGO superfusion of slices taken from chronically treated rats produced a much greater shift (42%) in the input-output curve than it did in untreated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential delay of reinnervating axons alters specificity in the rat serratus anterior muscle.

Previous studies have shown remarkable rostrocaudal selectivity by regenerating motoneurons to the rat serratus anterior (SA) muscle after freezing, crushing, or sectioning the long thoracic (LT) nerve. The LT nerve contains motoneurons from both the sixth and seventh cervical spinal nerves (C6 and C7), with C6 motoneurons as the major source of innervation throughout the muscle, and with C7 motoneurons innervating a larger percentage of muscle fibers caudally than rostrally. To determine if synaptic competition can play a role in neuromuscular topography, both the LT nerve and the branch carrying C6 (rostral) motoneurons to the LT nerve were crushed in newborn rats. This approach provides a temporal advantage to regenerating C7 (caudal) motoneurons. After an initial period during which C7 motoneurons reinnervated a larger proportion of muscle fibers than normal in all SA muscle sectors, C6 motoneurons regained their original proportion of rostral muscle fibers. Caudally, however, C7 motoneurons maintained an expanded territory. With this two-site crush method, the number of C6 motoneurons that reinnervate the SA muscle was significantly decreased from normal, whereas the number of C7 motoneurons remained the same. It is concluded that when C7 motoneurons are given a temporal advantage, synaptic specificity can be altered transiently in rostral muscle sectors and permanently in caudal sectors, and this is correlated with a disproportionate loss of C6 motoneurons. Moreover, this may be an important model for studies of synaptic competition, where terminals destined to be eliminated can be identified beforehand.