Propriospinal Fibers Research Articles

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays.

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Reorganization of spinal neural circuitry and functional recovery after spinal cord injury

Reorganization of spinal neural circuitry and functional recovery after spinal cord injury

Lack of Axonal Sprouting of Spared Propriospinal Fibers Caudal to Spinal Contusion Injury Is Attributed to Chronic Axonopathy

We have previously shown that a small percentage of long descending propriospinal tract (LDPT) axons are spared, whereas few short thoracic propriospinal (TPS) fibers survive 2 weeks following severe (50 mm weight drop) low thoracic spinal cord contusion injury (SCI). Here, we extended those findings to a moderate (25 mm weight drop) T9 SCI and assessed the effects of this lesion severity on propriospinal tract fibers at different time periods after injury. We anterogradely labeled fibers with fluororuby (FR) or WGA-HRP to determine their location and number 2, 4, 6, and 16 weeks post-SCI. Findings were compared with non-injured controls. At chronic time points, surviving FR-labeled LDPT fibers rostral to the injury remained as reactive endings or as putative regenerative sprouts. Caudal to the injury, spared LDPT fibers ran along a rim of lateral and ventral white matter, and ended as small abnormal-appearing putative terminal boutons or reactive endings within the intermediate gray matter of lumbosacral cord, with little axonal arborization and no evidence of injury-induced sprouting. One striking difference in the WGA-HRP experimental operates was the increased density of labeling of spared axons within the white matter caudal to the injury compared to controls. This labeling pattern was reminiscent of the labeling found after axotomy in studies by others, and raises a question as to contusion injury-induced impaired axonal transport. We hypothesize that axonal sprouting of axons after partial spinal cord injury seen in previous investigations was not found in the present investigation because of the additional pathological effects of contusion injury, similar to what is observed after traumatic brain injury.

Location of Spinal Cord Pathways That Control Hindlimb Movement Amplitude and Interlimb Coordination During Voluntary Swimming in Turtles

We performed mechanical lesions of the midbody (D2-D3; second to third postcervical spinal segments) spinal cord in otherwise intact turtles to locate spinal cord pathways that 1) activate and control the amplitude of voluntary hindlimb swimming movements and 2) coordinate hindlimb swimming with the movement of other limbs. Pre- and postlesion turtles were held by a band clamp around the carapace just beneath the water surface in a clear Plexiglas tank and videotaped from below so that kinematic measurements could be made of voluntary forward swimming with motion analysis software. Movements of the forelimbs (wrists) and hindlimbs (knees and ankles) were tracked relative to stationary reference points on the plastron to obtain bilateral measurements of hip and forelimb angles as functions of time along with foot trajectories. We measured changes in limb movement amplitude, cycle period, and interlimb phase before and after spinal lesions. Our results indicate that locomotor command signals that activate and regulate the amplitude of voluntary hindlimb swimming travel primarily in the dorsolateral funiculus (DLF) at the D2-D3 level and cross over to drive contralateral hindlimb movements. This suggests that electrical stimulation of the D3 DLF, which was previously shown to evoke swimming movements in the contralateral hindlimb of low-spinal turtles, activated the same locomotor command pathways that the animal uses during voluntary behavior. We also show that forelimb-hindlimb coordination is maintained by longitudinal spinal pathways that are largely confined to the ventrolateral funiculus (VLF) and mediate phase coupling of ipsilateral limbs, presumably by interenlargement propriospinal fibers.

Magnetically evoked inter-enlargement response: An assessment of ascending propriospinal fibers following spinal cord injury

The aim of the present study was to characterize a novel electrophysiological assessment, the magnetically evoked interenlargement response (MIER), by defining the anatomical location of the fast conducting (large myelinated) ascending axons that mediate the response and the relationship between the response and locomotor function following experimental spinal cord injury. Electromyographic (EMG) responses were recorded from the triceps muscles following magnetic stimulation of one hip. Short-latency (∼ 6 ms) EMGs were recorded from triceps muscles in normal controls and following different laceration injuries (dorsal, lateral or dorsal and lateral hemisections) or a 150-kilodyne (kd) contusion injury at the T9 level. The amplitude of the triceps MIER was significantly correlated to the area of spared white matter in the lateral funiculus and to hindlimb function during open field locomotion ( r 2 = 0.55). Following a complete lateral hemisection, MIERs were present in the triceps bilaterally following stimulation of either hip. Responses could also be recorded from the masseter muscles indicating that the influence of this pathway extends beyond the spinal cord. Anatomical evidence of a bilaterally distributed propriospinal pathway was found when biotinylated dextran amine (BDA) was injected into the lateral white matter on one side of the spinal cord at T9. BDA-labeled axons with varicosities were found bilaterally in the intermediate and ventral gray matter of the caudal region of the cervical enlargement. These observations suggest that MIERs may be useful to quantitatively assess neurotransmission and functional recovery over time after experimental spinal cord injury.

Origin of phasic synaptic inhibition in myotomal motoneurons during fictive locomotion in the lamprey.

The periodic membrane potential fluctuations in motoneurons during fictive locomotion in the lamprey, a primitive vertebrate, involve phasic synaptic excitation and inhibition. This paper investigates the origin of the phasic synaptic input to lamprey myotomal motoneurons in the in vitro spinal cord preparation with regard to the relative contribution of descending propriospinal input from interneurons in the local segment. The synaptic drive to myotomal motoneurons in the most rostral and the most caudal part of the spinal cord preparation are compared before and after selective spinal cord lesions. Current clamp recordings of the same cell before and after lesion showed that neither the excitatory phase nor the inhibitory phase was abolished after interruption of the descending or the ascending ipsilateral input, or after interrupting crossing segmental input by a local longitudinal midline incision. None of these sources thus appears to be alone responsible for the phasic synaptic drive. To quantitatively evaluate these effects, and in particular the contribution from the descending propriospinal fibres to the inhibitory phase, voltage clamp recordings were made in combination with a spinal cord hemisection just rostral to the motoneuron. The input from propriospinal interneurons in approximately 15 rostral segments may be responsible for as much as 70% of the phase of inhibitory current during the locomotor cycle. In accordance with these findings, a similar voltage clamp analysis of rostrally and caudally located motoneurons showed that the average peak-to-peak amplitude of the current fluctuations in rostral cells was approximately 50% of that in caudal cells.

Outgrowth of the pyramidal tract in the rat cervical spinal cord: Growth cone ultrastructure and guidance

In order to examine the mode of outgrowth of the pyramidal tract in the rat, the ultrastructure of its pathway in the dorsal funiculus of the spinal cord was analysed. The analysis was performed by means of serial sections of the third cervical segment before and during the arrival of pyramidal tract axons, and focussed on the morphology and microenvironment of the growth cones. Growth cones appear as elongated terminal enlargements without side branches. Two zones could be discerned: the distal, usually lamellipodial fine granular zone, containing no organelles, except for an occasional clear vesicle; and the proximal organelle-rich zone, which contains various organelles, such as agranular reticulum and vesicular structures. In addition, the proximal organelle-rich zone contains round or elliptic structures, limited by two concentric membranes, that enclose reticular and vesicular elements. The electron density of these structures varied from as low as the surrounding growth cone matrix to as dark as lysosomal structures, suggesting their involvement in turnover processes. At embryonic day 20, the most ventral part of the dorsal funiculus, where the first pyramidal tract axons are due to arrive within two days, is populated by axons that are relatively small compared to those in the rest of the dorsal funiculus. At birth, the arrival of the first pyramidal tract axons is marked by the presence of numerous large growth cone profiles in between small axons in the most ventral part of the dorsal funiculus; no circumscript bundle separated from the ascending sensory fiber tracts is present yet. The growth cones descend, club-shaped and 1 to 2 microns in diameter, without lamellipodia or filopodia. Within the same area a second growth cone type is present, which contains dense-core vesicles and has spread-out lamellipodia. Most of these growth cones are ascending and they probably belong to primary afferent or propriospinal fibers. At postnatal day 2, the pyramidal tract can be readily delineated from the adjacent fasciculus cuneatus where myelination has already started, but no glial boundary is present. The abundant growth cones are 1-2 microns wide and extend single unbranched lamellipodia, up to 15 microns long, which often enfold parallel axons or other growth cones. At postnatal day 4, growth cones are scarce in the tract. They measure 1 micron or less in diameter and each extends a single, straight lamellipodium or filopodium over 1 to 7 microns in the caudal direction.(ABSTRACT TRUNCATED AT 400 WORDS)

Propriospinal fibers in the white matter of the cat sacral spinal cord.

The propriospinal system, which consists of those neurons completely contained within the spinal cord, is important because it underlies much spinal behavior. To provide quantitative data on this system, the present study determines numbers of axons in the isolated S2 cat spinal cord and compares these figures with the normal. The conclusion is that 60% of the fibers in the spinal cord at this location are propriospinal. Findings of particular interest are that the great majority of unmyelinated propriospinal axons are found in the dorsal part of the lateral funiculus, and that there are large numbers of descending myelinated fibers in the dorsal funiculi. These data will serve as a basis for evaluating axon numbers that follow various experimental regimens purporting to result in neural sprouting.

Primary afferent and propriospinal fibers in the rat dorsal and dorsolateral funiculi.

The purpose of this study is to determine the numbers of primary afferent and propriospinal fibers in the dorsal and dorsolateral funiculi of the rat. The reason for concentrating on these areas is that they contain large numbers of unmyelinated axons. Our data are axonal numbers from the S2 segment of spinal cord in animals that had unilateral dorsal rhizotomies or spinal cord isolations. The major conclusions are 1) that 23% of the primary afferent fibers in the dorsal funiculus are unmyelinated; 2) that there are approximately 12,500 unmyelinated primary afferent fibers in the dorsolateral funiculus, which is more than the number of primary afferent fibers in the dorsal funiculus and tract of Lissauer combined, and 3) that approximately 25% of the axons in the dorsal funiculus and 44% of the axons in the dorsolateral funiculus are propriospinal. These data modify and extend previous ideas of the organization of spinal white matter.

Propriospinal fibers reaching the lumbar enlargement in the rat

Proprisopinal fibers reaching the lumbar enlargement were investigated in rat by means of retrograde transport of wheat germ agglutinin-horseradish peroxidase conjugate coupled or not coupled with gold particles. Unilateral or medial bilateral injections were done. Identification of projection cells was done by tetramethylbenzidine histochemistry or gold-silver intensification procedures. Unilateral injections resulted in bilateral labeling, with patterns and density related to the spinal segments of origin. Sacral, lumbar and thoracic afferents showed identical patterns. Ipsilateral connections originated laterally from dorsal, intermediate and ventral horns. Contralateral connections originated medially from laminae VII and VIII and laterally from the reticular extension of the neck of the dorsal horn. Cervical afferents were symmetrical, arising from both lamina VIII and the reticular extension of the neck of the dorsal horn. Lamina X projection cells were seen at all levels when injection sites involved this area. Laminae III and IV were almost totally devoid of projection cells. Superficial layer cells (laminae I and II) showed some labeling when injections were situated dorsally. The organization of these tracts in rat is similar to that in cat and monkey. Their origin is discussed in relation to those of long ascending pathways reaching supraspinal levels.

The differential effects of baclofen on segmental and descending excitation of spinal interneurones in the cat.

Intravenous baclofen (1-6.25 mg kg-1) substantially reduced the monosynaptic excitation of neurones in the intermediate nucleus of the cat spinal cord by impulses in group I extensor muscle primary afferent fibres, but had little or no effect on excitation by stimulating fibres of the ipsilateral dorsolateral funiculus or the contralateral red nucleus. Relatively low concentrations of baclofen thus appear not to influence the release of excitatory transmitter from the terminals of rubrospinal, corticospinal and long descending propriospinal fibres, in contrast to the reduction of the release of primary afferent transmitters.

Propriospinal fibers in the rat.

Segments of rat spinal cord were isolated by transecting in two places and sectioning all dorsal roots between the transections. Following this procedure, the areas of the gray and white matter are decreased by approximately 50% compared to normal. We feel, for reasons elaborated in the discussion, that the white matter of the isolated segments contains almost exclusively propriospinal axons. If this is accepted, then the axonal counts in this paper provide estimates of the numbers of propriospinal axons in rat spinal cord. In the isolated segments, the lateral funiculi contain 21,000 myelinated and 31,000 unmyelinated axons and the ventral funiculi 10,500 myelinated and 1,500 unmyelinated axons. The number of these fibers is approximately 33% of the number in unoperated spinal cords. Thus approximately one-third of the axons in rat sacral lateral and ventral funiculi are propriospinal, a lower figure than would have been predicted from classical work. The ratio of myelinated to unmyelinated fibers is higher for propriospinal fibers than for the other axons in these funiculi. Thus the propriospinal axons, as a group, are slightly larger than the other axons in these funiculi. This is against currently accepted thinking which generally regards the propriospinal fibers as the finest in the white matter of the cord. Finally, the quantification of propriospinal systems in these funiculi allows more precision in our thinking about the organization of the spinal cord.

Propriospinal control of interneurons in spinal reflex pathways from tendon organs in the cat

After chronic hemisection at C3, stimulation of propriospinal fibers in the dorsal quadrant at Th 10 facilitated disynaptic PSPs from Ib afferents in hindlimb motoneurons. Recording from interneurons monosynaptically activated from group I muscle afferents, and of extracellular focal synaptic potentials around them, revealed monosynaptic EPSPs from long propriospinal neurons. It is suggested that propriospinal neurons originating in the forelimb segments have direct excitatory connexions with interneurons of Ib reflex pathways to hindlimb motoneurons.

Quantitation of propriospinal fibers in the tract of Lissauer of the rat.

The present study uses a spinal cord isolation procedure to remove extrinsic axons but leave intrinsic axons intact. The isolation is done by sectioning the spinal cord in two places and then cutting all dorsal roots between the two sections. The axons that survive the isolation procedure are thought to be propriospinal axons. Following isolation, approximately one-third of the axons in sacral tracts of Lissauer in the rat survive. Thus approximately one-third of the axons in sacral tracts of Lissauer in the rat are propriospinal. Proportionately more myelinated than unmyelinated axons are lost. There are approximately equal numbers of surviving axons in the medial as opposed to the lateral part of the tract. This implies that there is, as yet, no morphological basis for dividing the tract into medial and lateral halves. The fact that the number of propriospinal axons in the tract of Lissauer has been quantitated offers more precision in our thinking about the organization of the dorsal horn.

Primary afferent axons in the tract of Lissauer in the monkey.

The main thrust of the present study was to determine the numbers of primary afferent fibers in the tract of Lissauer in the monkey. The findings are that approximately 40% of the axons in the tract of Lissauer are primary afferent axons from a single segment, and approximately another 40% are primary afferents from segments cranial and caudal to the segment under consideration. Presumably, the remaining 20% are propriospinal axons. There is relatively little difference in the proportions of primary afferents in medial as opposed to lateral parts of the tract, and in this respect the monkey differs somewhat from the rat and cat. Thus in the monkey the tract of Lissauer should probably be regarded as a primary afferent pathway with the propriospinal forming a distinct but relatively minor component of the tract.

Recovery of substance P in the cat spinal cord after unilateral lumbosacral deafferentation

Changes in substance P in the cat spinal cord after deafferentation of the hindlimb were investigated using the peroxidase-antiperoxidase technique. Unilateral lumbosacral dorsal root section (L 1-S 3) is followed by a decrease in dorsal horn (laminae I, II and V) substance P reaction product which is most marked at 10–11 days. There is no observable change in the ventral horn. The 13 or 15 day survivors demonstrate an increase over that seen at 10–11 days, and still greater amounts appear in the dorsal horn of 1 month survivors. After 1 month there is little further observable increase. The location of the returned reaction product resembles that of normals, but differs in its staining characteristics. Substance P containing cell bodies are observed in normal animals and on intact and deafferented sides of experimentals, suggesting that interneurons and propriospinal fibers nay be a source of the returning substance P reaction product. The decrease in dorsal horn substance P at short times after rhizotomy followed by an increase at longer times is consistent with axonal sprouting. If so, then the time course of sprouting parallels that of locomotor recovery and supports the hypothesis that the two phenomena are related.

Cells of origin of propriospinal fibers and of fibers ascending to supraspinal levels. A HRP study in cat and rhesus monkey.

In the spinal cord of cat and rhesus monkey the cells of origin of long and short propriospinal fibers and those of the spinal fibers ascending to supraspinal levels were identified by means of retrograde HRP labeling after large unilateral HRP-injections, i.e. in the spinal white and grey matter at different levels, in the pons and in the dorsal column nuclei. The findings indicate the existence of the following arrangement. Long ascending supraspinal fibers arise mainly from neurons in the dorsal grey (laminae I-IV and the medial parts of laminae V and VI) as well as from neurons in the medial part of the ventral grey (lamina VIII), in Clarke's column and in the spinal border cell area. Some neurons in the dorsal grey projects to the dorsal column nuclei, which in turn distribute fibers back to the spinal cord. Long propriospinal fibers mainly derived from neurons in the medial part of the ventral grey (lamina VIII), while short propriospinal fibers are characteristically derived from neurons in the intermediate zone (lateral halves of laminae V and VI and lamina VII). The neurons located laterally in laminae V-VII distribute fibers mainly ipsilaterally, while those located medially in lamina VII distribute them to some degree bilaterally. The findings in cats with transections of either the dorsal or the ventral halves of the spinal white matter (both above and below the injected segment), show that the fibers from the dorsal grey and the lateral parts of laminae V-VII travel mainly through the dorsal half of the white matter, while those from the medial part of lamina VII and from lamina VIII travel mainly through the ventral half.

Experimental morphological study of endings of propriospinal fibers of the lateral funiculus in the cat cervical spinal cord

The distribution and ultrastructure of terminals of the propriospinal fibers of the lateral funiculus in the cervical segments of the cat spinal cord were studied by the experimental degeneration method. A preliminary lateral hemisection of the spinal cord was carried out 5–6 months earlier at the level of segments C2 or C3 to destroy all the long descending pathways; the lateral funiculus was then divided at the level of C4 or C5. It was shown by the method of Fink and Heimer that terminals of descending and ascending propriospinal pathways damaged by the second division are distributed in the gray matter ipsilaterally in the lateral zones of Rexed's laminase V–VII and also in the dorsolateral motor nuclei. An electron-microscopic study showed that the synapses of the degenerating terminals are mainly axo-dendritic in type and account for 14.5% of the total number of terminals counted. Residual synaptic vesicles in these terminals were spherical in shape. The mean diameter of the degenerating myelinated propriospinal fibers in the lateral funiculus was 10±3 µ. The results of this investigation were compared with those of electrophysiological investigations of the function of propriospinal neurons.

A long propriospinal system with direct effect on motoneurones and on interneurones in the cat lumbosacral cord.

Stimulation of the lateral funicle was performed at different segmenta levels in the cat in order to investigate the connecxions of long propriospinal neurones with ipsilateral lumbosacral motoneurones and interneurones projecting directly to them. In one series of experiments the propriospinal contribution was assessed from the difference between the effect of maximal stimulation in C 1 (activating supraspinal descending fibres) and in more caudal segments (activating supraspinal and propriospinal fibres). In another series on cats with chronic hemisection in C 3 pure propriospinal effects were evoked by stimulation of the thoracic spinal cord. In both series it was shown that volleys in long propriospinal neurones evoke monosynaptic EPSPs and disynaptic EPSP and/or IPSP in many flexor and extensor motoneurones; an extracellular monosynaptic focal synaptic potential was recorded in lamina VII of Rexed. The effects evoked by stimulation of the Th 11 segment after chronic C 3 hemisection were not found after chronic Th 10 hemisection. It is therefore tentatively suggested that they were due to stimulation of long descending propriospinal neurones originating in the lower cervical and upper thoracic segments, their axon trajectory being in the middle of the lateral funiculus and conduct at velocity 100 m/sec. Other effects evoked by stimulation of Th 11 after chronic Th 10 hemisection are ascribed to antidromic activation of axons of ascending neurones.