Regenerating motor bridge axons refine connections and synapse on lumbar motoneurons to bypass chronic spinal cord injury.

To restore motor control after spinal cord injury requires reconnecting the brain with spinal motor circuits below the lesion. A bridge around the injury is an important alternative to promoting axon regeneration through the injury. Previously, we reported a novel motor bridge in rats. The thirteenth thoracic nerve was detached from the muscle it innervates and the cut end implanted caudally into the lumbar gray matter where motor bridge axons regenerate. In this study, we first determined that regenerating bridge axons project to spinal motor circuits. Stable projections were present in ventral motor laminae of the cord, including putative synapses directly on motoneurons, 2 months after insertion in the intact cord. At this time, earlier-forming dorsal horn projections were mostly eliminated. Regenerating axons were effective in evoking leg motor activity as early as 2 weeks. We next determined that bridge axons could regenerate caudal to a chronic injury. We hemisected the spinal cord at L2 and inserted the bridge nerve 1 month later at L5 and found ventral laminae projections similar to those in intact animals, including onto motoneurons directly. Finally, we determined that the bridge circuit could be activated by neural pathways rostral to its origin. For spinally hemisected animals, we electrically stimulated the rostral spinal cord and recorded evoked potentials from the bridge and, in turn, motor responses in the sciatic nerve. Our findings suggests that bridge motoneurons could be used by descending motor pathways as premotor interneurons to transmit neural signals to bypass a chronic spinal injury.

Mouse colon sensory neurons detect extracellular acidosis via TRPV1.

Extracellular acidification contributes to pain by activating or modulating nociceptor activity. To evaluate acidic signaling from the colon, we characterized acid-elicited currents in thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglion (DRG) neurons identified by content of a fluorescent dye (DiI) previously injected into the colon wall. In 13% of unidentified LS DRG neurons (not labeled with DiI) and 69% of LS colon neurons labeled with DiI, protons activated a sustained current that was significantly and reversibly attenuated by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine. In contrast, 63% of unidentified LS DRG neurons and 4% of LS colon neurons exhibited transient amiloride-sensitive acid-sensing ion channel (ASIC) currents. The peak current density of acid-elicited currents was significantly reduced in colon sensory neurons from TRPV1-null mice, supporting predominant expression of TRPV1 in LS colon sensory neurons, which was also confirmed immunohistochemically. Similar to LS colon DRG neurons, acid-elicited currents in TL colon DRG neurons were mediated predominantly by TRPV1. However, the pH producing half-activation of responses significantly differed between TL and LS colon DRG neurons. The properties of acid-elicited currents in colon DRG neurons suggest differential contributions of ASICs and TRPV1 to colon sensation and likely nociception.

Temporal and spatial expression of homeotic genes is important for segment-specific neuroblast 6-4 lineage formation in Drosophila.

Different proliferation of neuroblast 6-4 (NB6-4) in the thorax and abdomen produces segmental specific expression pattern of several neuroblast marker genes. NB6-4 is divided to form four medialmost cell body glia (MM-CBG) per segment in thorax and two MM-CBG per segment in abdomen. As homeotic genes determine the identities of embryonic segments along theA/P axis, we investigated if temporal and specific expression of homeotic genes affects MM-CBG patterns in thorax and abdomen. A Ubx loss-of-function mutation was found to hardly affect MM-CBG formation, whereas abd-A and Abd-B caused the transformation of abdominal MM-CBG to their thoracic counterparts. On the other hand, gain-of-function mutants of Ubx, abd-A and Abd-B genes reduced the number of thoracic MM-CBG, indicating that thoracic MM-CBG resembled abdominal MM-CBG. However, mutations in Polycomb group (PcG) genes, which are negative transregulators of homeotic genes, did not cause the thoracic to abdominal MM-CBG pattern transformation although the number of MM-CBG in a few per-cent of embryos were partially reduced or abnormally patterned. Our results indicate that temporal and spa-tial expression of the homeotic genes is important to determine segmental-specificity of NB6-4 daughter cells along the anterior-posterior (A/P) axis.

Mechanism of glia-neuron cell-fate switch in the Drosophila thoracic neuroblast 6-4 lineage.

During development of the Drosophila central nervous system, neuroblast 6-4 in the thoracic segment (NB6-4T) divides asymmetrically into a medially located glial precursor cell and a laterally located neuronal precursor cell. In this study, to understand the molecular basis for this glia-neuron cell-fate decision, we examined the effects of some known mutations on the NB6-4T lineage. First, we found that prospero (pros) mutations led to a loss of expression of Glial cells missing, which is essential to trigger glial differentiation, in the NB6-4T lineage. In wild-type embryos, Pros protein was localized at the medial cell cortex of dividing NB6-4T and segregated to the nucleus of the glial precursor cell. miranda and inscuteable mutations altered the behavior of Pros, resulting in failure to correctly switch the glial and neuronal fates. Our results suggested that NB6-4T used the same molecular machinery in the asymmetric cell division as other neuroblasts in cell divisions producing ganglion mother cells. Furthermore, we showed that outside the NB6-4T lineage most glial cells appeared independently of Pros.

Sacral neural crest cell migration to the gut is dependent upon the migratory environment and not cell-autonomous migratory properties.

Avian neural crest cells from the vagal (somite level 1-7) and the sacral (somite level 28 and posterior) axial levels migrate into the gut and differentiate into the neurons and glial cells of the enteric nervous system. Neural crest cells that emigrate from the cervical and thoracic levels stop short of the dorsal mesentery and do not enter the gut. In this study we tested the hypothesis that neural crest cells derived from the sacral level have cell-autonomous migratory properties that allow them to reach and invade the gut mesenchyme. We heterotopically grafted neural crest cells from the sacral axial level to the thoracic level and vice versa and observed that the neural crest cells behaved according to their new position, rather than their site of origin. Our results show that the environment at the sacral level is sufficient to allow neural crest cells from other axial levels to enter the mesentery and gut mesenchyme. Our study further suggests that at least two environmental conditions at the sacral level enhance ventral migration. First, sacral neural crest cells take a ventral rather than a medial-to-lateral path through the somites and consequently arrive near the gut mesenchyme many hours earlier than their counterparts at the thoracic level. Our experimental evidence reveals only a narrow window of opportunity to invade the mesenchyme of the mesentery and the gut, so that earlier arrival assures the sacral neural crest of gaining access to the gut. Second, the gut endoderm is more dorsally situated at the sacral level than at the thoracic level. Thus, sacral neural crest cells take a more direct path to the gut than the thoracic neural crest, and also their target is closer to the site from which they initiate migration. In addition, there appears to be a barrier to migration at the thoracic level that prevents neural crest cells at that axial level from migrating ventral to the dorsal aorta and into the mesentery, which is the portal to the gut.

The incidence of initial doublets in the discharges of motoneurones of two different inspiratory muscles in the cat.

1. Trains of action potentials in motoneurones frequently commence with an initial doublet; i.e. a uniquely short interspike interval. Previous authors have speculated on the functional importance of initial doublets. Here we test the hypotheses that these doublets are associated with particular classes of motoneurones or particular physiological conditions. 2. Discharges of inspiratory motoneurones were recorded extracellularly in the thoracic ventral horn of anaesthetized, paralysed cats. Seventy units (35 each with axons in the internal and external intercostal nerves) were classified on the basis of their maximum firing rates, start times in the respiratory cycle and axonal destination. 3. Initial doublets were defined by an interspike interval < 14 ms. Of seventeen units firing initial doublets, fifteen had axons in the external intercostal nerve and two had axons in the internal intercostal nerve. Neither maximum firing rate nor start time during the respiratory cycle predicted the occurrence of doublets. 4. The chemical drive to breathe was manipulated by altering the CO2 content of the inspired gas or by briefly stopping the respiratory pump. Varying the chemical drive to breathe had no consistent effect on the occurrence of initial doublets. 5. These results support the view that initial doublets are part of the normal pattern of discharge of motoneurones. However, because the incidence of doublets does not consistently support previous functional hypotheses, we argue that the occurrence of doublets may not necessarily be dictated by the CNS, but in some circumstances it is an epiphenomenon dependent on the state of the motoneurone, in particular on the statistical properties of its synaptic inputs.

Differentiation of alpha and gamma motoneurons by the retrograde uptake of horseradish peroxidase.

The classification of motoneurons based on size alone may not be an absolute morphological criterion. There appears to be a fair difference in the pattern of horseradish peroxidase uptake between the phrenic and the intercostal motoneurons. Hence we would like to suggest that the gamma and the alpha motoneurons differ in the horseradish peroxidase uptake.

Neuromuscular organization of the pectoralis (pars thoracicus) of the pigeon (Columba livia): implications for motor control.

The pectoralis (pars thoracicus) of the domestic pigeon (Columba livia) is divisible into two anatomical parts, the pars sternobrachialis (SB) and the pars thoracobrachialis (TB). Innervation to this complex is from rostral and caudal branches of the brachial ventral cord. In four anesthetized pigeons, the distribution of muscle units associated with each nerve branch was mapped after prolonged stimulation of each nerve and subsequent analysis for muscle fiber glycogen. An additional three animals were used to analyze the morphology, distribution, and histochemical profiles of the muscle fibers in the SB and TB subregions. Fibers were characterized on the basis of their reactions for myofibrillar adenosine triphosphates (alkaline and acid preincubation) and reduced nicotinamide adenine dinucleotide diaphorase (NADH-D). The SB is primarily innervated by the rostral nerve branch and the TB by the caudal nerve branch. For two-thirds of the muscle's length, the SB is separated from the TB by an aponeurosis, the membrana intermuscularis (MI). SB and TB fibers located posteroventral to the caudal margin of the MI are innervated variously by both nerves. Two populations of fibers were recognized, distinguishable primarily by 1) fiber diameter and 2) density of the NADH-D reaction product. Compared to the TB, the SB possesses a higher average percentage of large fibers. Within the SB but not the TB the percentage of large fibers increases from deep to superficial. These data support our previous findings that the pars thoracicus of the pigeon is partitioned into at least two functional subunits, each with a potential for independent action on the wing during flight.

Morphometrical study of the cat thoracic dorsal root ganglion cells in relation to muscular and cutaneous afferent innervation.

The sizes of neuronal somata in cat dorsal root ganglia were determined at the different thoracic segmental levels (T1-T13). The intersegmental variations in the average value and class distribution of diameters were analysed. The maximal and minimal average mean cell diameters were 51.1 and 43.3 microns at the T1 and T2 levels, respectively. Caudally, this value gradually increased from T2 to T8 (47.8 microns) and thereafter decreased progressively to T12 (44.7 microns). At T1, large cells (greater than 50 microns in diameter) were 3.3-fold in excess compared to small ones (less than 35 microns in diameter). The proportion of large to small cells strongly decreased to a 0.9 ratio from T1 to T2, then increased again from T2 (0.9) to T8 (2.3). The size distributions of the overall cell populations were compared to those of neurones supplying muscular targets via the external intercostal nerves or cutaneous targets via the lateral branch of the internal intercostal nerves, identified following the retrograde transport of horseradish peroxidase. The size distribution of cells serving cutaneous nerves was similar to that exhibited by the overall population of ganglion cells. In contrast, the size distributions of cells giving rise to muscle afferents tended towards smaller values. In the thoracic dorsal root ganglia, the cell body sizes of the muscular primary afferents were close to those previously reported for the visceral primary afferents.

Morphological properties of respiratory intercostal motoneurons in cats as revealed by intracellular injection of horseradish peroxidase.

This study was undertaken to describe details of the location and cellular morphology of functionally identified (inspiratory or expiratory) external and internal intercostal motoneurons on the basis of intracellular injection of horseradish peroxidase (HRP). Sixty HRP-labeled motoneurons were examined; 44 in transverse, 16 in sagittal sections. In the upper thoracic segments (T3-T4), there was only a small overlap in the location of inspiratory external and internal intercostal motoneurons; the inspiratory external motoneurons were generally found more ventromedially within the ventral horn than either inspiratory or expiratory internal intercostal motoneurons. No major morphological differences were observed between the types of motoneurons studied. The number of primary dendrites ranged from 6 to 10. The dendrites projected mainly along the medial or the lateral border of the ventral horn, and rostrocaudally up to 1,760 micron from the cell body. The paths taken by dendrites to fill the territory occupied by the dendritic trees appeared to depend upon location of the cell body. Few dendrites penetrated the white matter. Axon diameters varied from 1.1 to 6.7 micron (mean 3.6 +/- 1.3 micron, n = 55). Collateral branches were identified in 78% of axons. The number of branches arising from a given axon varied from 1 to 4. It is concluded that the respiratory intercostal motoneurons form a morphologically homogeneous population, in spite of their functional differences.

Collateral sprouting of sensory axons in the hairy skin of the trunk: a morphological study in adult rats.

The capacity of sensory axons in the hairy skin of adult rats to extend branches into adjacent denervated skin has been studied by anterograde tracing with wheat germ agglutinin-horseradish peroxidase conjugate. In one series of experiments the Th10 intercostal nerve area was isolated and the distribution of sensory axons from the lateral cutaneous nerve was examined in control experiments and at various times after the denervation. In a second series of experiments, the entire Th10 was isolated, and the caudal extension of sensory axons from the Th10 spinal nerve was examined in control experiments and at various times after the denervation. The findings indicate that thin as well as coarse cutaneous sensory nerve axons can extend collateral sprouts within their 'own' dermatome as well as outside their normal segmental territory. Thus the dermatomal border does not seem to be a limit for collateral sprouting of coarse sensory nerve axons.

Somato-visceral convergence in cat dorsal root ganglion neurones demonstrated by double-labelling with fluorescent tracers.

We have investigated the possibility of somato-visceral convergence in thoracic sensory dorsal root ganglia (DRG) of cats, using the technique of double-labelling with fluorescent tracers. Combinations of propidium iodide (PI) and Fast Blue (FB) or bisbenzimide (Bb) were found to be the most appropriate. FB or Bb were injected into the splanchnic nerve, and PI into the intercostal nerves. The 9th-11th thoracic cord segments and ganglia were removed after perfusal and frozen sections cut. Single- and double-labelled neurones were observed in the DRG and single-labelled neurones only in the ventral horn (PI) and lateral horn (FB, Bb) of the thoracic spinal cord. The double-labelled neurones accounted for approximately 1% of all labelled neurones, although this may be an underestimate due to the techniques used to prevent migration of the tracer from labelled cells. The double-labelled neurones provide evidence for somato-visceral convergence and indicate that the pre-spinal convergence hypothesis could explain the phenomenon of referred pain.

Somatic and visceral primary afferents in the lower thoracic dorsal root ganglia of the cat.

Anterograde transport of horseradish peroxidase (HRP) through somatic and visceral nerves was used to estimate the proportions of somatic and visceral dorsal root ganglion (DRG) cells of the lower thoracic ganglia of the cat. A concentrated solution of HRP was applied for at least 5 hours to the central end of the right greater splanchnic nerve and of the left T9-intercostal nerve of adult cats. Some animals remained under chloralose anaesthesia for the duration of the HRP transport time (up to 53 hours) whereas longer HRP application and transport times (4-5 days) were allowed in animals that recovered from barbiturate anaesthesia. Visceral DRG cells were found in approximately equal numbers in all ganglia examined (T7-T11). Population estimates were obtained for the T8 and T9 ganglia where visceral DRG cells were found to be 6.2% (T8) and 5.2% (T9) of the total cell population. In contrast, somatic DRG cells were found in large numbers in the ganglia examined (T8 and T9) where they amounted to over 90% of the cell population. Measurement of cross-sectional areas and estimates of cell diameters of the DRG cells showed greater proportions of large somatic cells (diameter greater than 40 micron) than of large visceral cells. Similar distributions of cell size were found for both somatic and visceral DRG cells with diameters less than 40 micron. These results show that the proportion of visceral afferent fibres in the dorsal roots that mediate the spinal cord projection of the splanchnic nerve is very small. Since viscerosomatic convergence in the thoracic spinal cord is very extensive, the present results suggest considerable divergence of the visceral afferent input to the central nervous system.

Restoration of function in external intercostal motoneurones of the cat following partial central deafferentation.

The activity of external intercostal motoneurones in the cat was studied under anaesthesia and paralysis before and after partial central deafferentation caused by single or double ipsilateral hemisections of the thoracic spinal cord. The normal efferent inspiratory discharges recorded from external intercostal nerve filaments caudal to the upper lesion were greatly reduced acutely, but activity of approximately normal intensity and phase at eupneoic levels of CO2 was restored within a few days and remained at similar levels for up to two years. The patterns of the restored activity were abnormal, with more discharges of alpha-motoneutrones during expiration than normal and a stronger modulation of the discharges by the respiratory pump than normal. A common abnormal component of the restored activity was a tonic discharge in hypocapnic apnoea, often modulated by the respiratory pump. This activity was never seen in normal animals or in those with acute lesions under similar conditions of anaesthesia. Synchronization of the discharges of alpha-motoneurones caudal to the upper lesion was studied by constructing cross-correlation histograms between paired groups of motoneurones, each group being represented by the discharges in one filament. Synchronization was stronger than normal, usually extending over a time course of +/- 20 to +/- 50 ms (broad-peak synchronization). This synchronization was particularly strong for the discharges in hypocapnic apnoea. We conclude that the restored activity was derived in large part from abnormal tonic (non-respiratory-phased) inputs, partly proprioceptive in origin, probably involving spinal cord interneurones with abnormally synchronized discharges. This conclusion is supported by intracellular measurements including respiratory drive potentials, synaptic noise and average common excitation potentials.

The development and postnatal organization of motor nuclei in the rat thoracic spinal cord.

The topographic organization of the motor nuclei supplying individual thoracic muscles in postnatal rats was investigated by horseradish peroxidase (HRP) labelling from the periphery. To determine whether aspects of the same organization are apparent at early developmental stages, we used HRP to label neurons contributing axons to the two primary rami of thoracic nerves in rat fetuses ranging in age from the 13th day of gestation to birth. This developmental period includes stages during which peripheral nerves and muscles are forming. The results show that motoneurons in the appropriate positions in the ventral horn have axons in each primary ramus even at stages prior to the innervation of muscle. However, in fetuses at certain developmental stages, there are also neurons located outside the motor nuclei which send axons into the peripheral nerves.

Quantitative morphological changes in phrenic and intercostal motor columns and their respective spinal cord segments during postnatal development in the kitten.

In newborn kittens, the nervous control of breathing appears more mature than that of motricity which follows a cephalo-caudal evolution. In order to determine if the different postnatal evolutions of the respiratory and the motor function have an anatomical support at the spinal cord level, we made morphometric comparisons of the postnatal development of the spinal segments including motor columns sustaining both limb and respiratory movements (cervical and thoracic segments), with the postnatal development of segments containing only motoneurones involved in locomotion (lumbar segments). Furthermore, we used horseradish peroxidase to label cervical and thoracic groups of inspiratory motoneurones, i.e. the phrenic and the intercartilaginous motor nuclei at several postnatal ages. The present study suggests that the development of the white matter is the same at every spinal level and that it is delayed compared to the maturation of the grey matter. Overall evaluations of grey matter areas showed that the thoracic grey matter is more mature at birth but, further, has a slower rate of growth than the cervical and lumbar ones. This observation may be related to the maturity of the respiratory phasic activities within the early postnatal life. The phrenic and intercartilaginous motor nuclei have different patterns of development. These results suggest that the spinal postnatal functional maturation is not strictly related to its quantitative macroscopic changes.