Full PapersEmbryonic Expression of Motoneuron Topography in the Rat Diaphragm Muscle
Abstract
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.
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Identification of the neural pathway underlying spontaneous crossed phrenic activity in neonatal rats
2009, NeuroscienceCitation Excerpt :Furthermore, the anatomy of the pathway at this time point is not the same as the early neonatal crossed phrenic pathway, but is closer to the adult neural pathway (Moreno et al., 1992; Boulenguez et al., 2007). The phrenic motor neuron pool establishes a rostrocaudal somatotopic organization on the diaphragm muscle (Greer et al., 1999; Laskowski and Owens, 1994; Laskowski and Sanes, 1987). Specifically, the rostral phrenic motor neurons tend to innervate the ventral part of the diaphragm while caudal phrenic motor neurons tend to innervate the dorsal part of the diaphragm.
Cervical spinal cord hemisection at C2 leads to paralysis of the ipsilateral hemidiaphragm in rats. Respiratory function of the paralyzed hemidiaphragm can be restored by activating a latent respiratory motor pathway in adult rats. This pathway is called the crossed phrenic pathway and the restored activity in the paralyzed hemidiaphragm is referred to as crossed phrenic activity. The latent neural pathway is not latent in neonatal rats as shown by the spontaneous expression of crossed phrenic activity. However, the anatomy of the pathway in neonatal rats is still unknown. In the present study, we hypothesized that the crossed phrenic pathway may be different anatomically in neonatal and adult rats. To delineate this neural pathway in neonates, we injected wheat germ agglutinin conjugated to horseradish peroxidase (WGA–HRP), a retrograde transsynaptic tracer, into the phrenic nerve ipsilateral to hemisection. We also injected cholera toxin subunit B–horseradish peroxidase (BHRP) into the ipsilateral hemidiaphragm following hemisection in other animals to determine if there are midline-crossing phrenic dendrites involved in the crossed phrenic pathway in neonatal rats. The WGA–HRP labeling was observed only in the ipsilateral phrenic nucleus and ipsilateral rostral ventral respiratory group (rVRG) in the postnatal day (P) 2, P7, and P28 hemisected rats. Bilateral labeling of rVRG neurons was shown in P35 rats. The BHRP study showed that many phrenic dendrites cross the midline in P2 neonatal rats at both rostral and caudal parts of the phrenic nucleus. There was a marked reduction of crossing dendrites observed in P7 and P28 animals and no crossing dendrites observed in P35 rats. The present results suggest that the crossed phrenic pathway in neonatal rats involves the parent axons from ipsilateral rVRG premotor neurons that cross at the level of obex as well as decussating axon collaterals that cross over the spinal cord midline to innervate ipsilateral phrenic motoneurons following C2 hemisection. In addition, midline-crossing dendrites of the ipsilateral phrenic motoneurons may also contribute to the crossed phrenic pathway in neonates.
Postnatal conversion of cross phrenic activity from an active to latent state
2009, Experimental NeurologySpinal cord hemisection rostral to the phrenic nucleus leads to paralysis of the ipsilateral hemidiaphragm and respiratory insufficiency. Recovery of the paralyzed hemidiaphragm may be induced by activating a latent respiratory motor pathway in adult rats. Although the pathway is latent in adults, it may not be latent in neonatal rats as shown by the spontaneous expression of activity over this pathway in an earlier in vitro study. Activity mediated over the latent pathway is known as “crossed phrenic activity”. Whether crossed phrenic activity following C2 spinal cord hemisection occurs spontaneously in the neonatal rat in vivo is still unknown. We hypothesized that crossed phrenic activity may be spontaneously expressed in neonates in vivo and may be converted from a spontaneously active state to a latent and nonfunctional state during postnatal development. Thus, a time course study was designed to analyze this activity in rat pups at different ages. The functional status of the ipsilateral and contralateral hemidiaphragms was tested by EMG analysis following hemisection. Crossed phrenic activity was expressed in ventral, lateral, and dorsal parts of the ipsilateral hemidiaphragm in P2 and some P3 and P4 neonatal rats. During postnatal development, the activity was observed only in the ventral area of the ipsilateral hemidiaphragm in P7, P14, P21 and P28 animals. Significant decreases in the extent of ventral crossed phrenic activity were observed from P2 to P28. The pathway generating this activity becomes latent by postnatal day 35. The present results suggest that spontaneous crossed phrenic activity occurs in vivo following C2 hemisection and the activity gradually decreases during the first four postnatal weeks.
Perinatal development of respiratory motoneurons
2005, Respiratory Physiology and NeurobiologyBreathing movements require the coordinated recruitment of cranial and spinal motoneurons innervating muscles of the upper airway and ribcage. A significant part of respiratory motoneuron development and maturation occurs prenatally to support the generation of fetal breathing movements in utero and sustained breathing at birth. Postnatally, motoneuron properties are further refined and match changes in the maturing respiratory musculoskeletal system. In this review, we outline developmental changes in key respiratory motoneuronal populations occurring from the time of motoneuron birth in the embryo through the postnatal period. We will also bring attention to major deficiencies in the current knowledge of perinatal respiratory motoneuron development. To date, our understanding of processes occurring during the prenatal period comes primarily from analysis of phrenic motoneurons (PMNs), whereas information about postnatal development derives largely from studies of PMN and hypoglossal motoneuron properties.
A morphological technique for exploring neuromuscular topography expressed in the mouse gluteus maximus muscle
2004, Journal of Neuroscience MethodsMotor 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.
Roles for ephrins in positionally selective synaptogenesis between motor neurons and muscle fibers
2000, NeuronMotor 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.
The Eph kinase ligand AL-1 is expressed by rostral muscles and inhibits outgrowth from caudal neurons
1996, Molecular and Cellular NeurosciencesIn the peripheral nervous system, neurons derived from specific rostrocaudal levels of the neuraxis selectively synapse on targets that arise from corresponding body positions. To identify molecules involved in such position-dependent connectivity, we used subtractive hybridization to isolate genes selectively expressed in rostral or caudal skeletal muscle. One mRNA that was more abundant in neck than in hindlimb muscles encoded the mouse ortholog of human AL-1 and chick RAGS, membrane-associated ligands of Eph tyrosine kinases that have recently been implicated in cortical axon fasciculation and retinotectal connectivity, respectively. We show here that mouse AL-1 is expressed in discrete regions of the central and peripheral nervous systems and in a subset of developing skeletal muscles. The abundance of AL-1 RNA in immortalized myogenic cell lines derived from rostral muscles is higher than in caudally derived lines, suggesting that levels are heritably maintained. Growth of neurites from cultured sensory ganglia and spinal cords is specifically inhibited by cells expressing AL-1, suggesting that this molecule could serve to guide peripheral axons. The inhibitory effects of AL-1 are position dependent, such that axons derived from caudal (lumbar) ganglia are more affected than those derived from rostral (cervical) ganglia. Together, these results support the notion that Eph kinases and their ligands regulate topographically appropriate neural connectivity in the peripheral nervous system, as well as in the central nervous system.