Electrophysiologic evidence of regeneration of lamprey spinal neurons

doi:10.1016/0014-4886(84)90128-6

Experimental Neurology

Volume 83, Issue 3, March 1984, Pages 618-628

https://doi.org/10.1016/0014-4886(84)90128-6 Get rights and content

Abstract

Morphologic evidence has shown that the anteriorly projecting axons of giant interneurons (GIs) can regenerate after spinal transection in larval sea lampreys (19). In the present study, we showed that the regenerating neurites of GIs were electrically exciteable. We also showed evidence for regeneration of descending afferent connections to GIs. Spinal cords were transected at the level of the cloaca. After at least 70 days recovery, GIs located 1.5 to 17.0 mm below the scar were impaled with microelectrodes. Stimulating electrodes were placed at various distances above the scar. Six of 13 GIs located 4 to 17 mm below the scar could be activated antidromically. For 1 GI, the rostralmost point of stimulation which elicited these responses was 13.5 mm above the scar. For the others, the range was 0.5 to 4.5 mm. Estimated average conduction velocity in regenerated neurites was 0.50 m/s compared with 1.94 m/s for the parent axon. Twelve GIs could be orthodromically activated by fixed-latency EPSPs. The most rostal point of stimulation that could elicit such responses was 0.5 to 8.5 mm above the scar. There was an inverse relationship between the farthest distance of stimulation and the distance of the GI from the scar. These findings are consistent with the hypothesis that regeneration of axons across a spinal transection is limited to neurons whose cell bodies are situated within 1 to 2 cm from the transection, and that regenerating neurites grow only a few millimeters beyond the scar.

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Cited by (14)

RhoA activation in axotomy-induced neuronal death
2018, Experimental Neurology
Citation Excerpt :
RhoA inactivation fosters axon growth and functional recovery following SCI (Lehmann et al., 1999; Dergham et al., 2002; Fournier et al., 2003), but because in mammalian SCI experiments it is difficult to distinguish regeneration of severed axons from collateral sprouting by spared axons, it is not clear whether RhoA is suppressing axon regeneration or only sprouting, two processes that appear to have different mechanisms (Lee et al., 2010; Koch et al., 2014). We have used the lamprey as an experimental model to get around this difficulty because its axons regenerate after complete spinal cord injury and regeneration is easily documented by histological and electrophysiological means (Rovainen, 1976; Selzer, 1978; Yin and Selzer, 1983, 1984; Cohen et al., 1988; Davis Jr and McClellan, 1994a; Banerjee et al., 2016). Although lamprey reticulospinal axons can regenerate after SCI, the regeneration is not complete.
After spinal cord injury (SCI) in mammals, severed axons fail to regenerate, due to both extrinsic inhibitory factors, e.g., the chondroitin sulfate proteoglycans (CSPGs) and myelin-associated growth inhibitors (MAIs), and a developmental loss of intrinsic growth capacity. The latter is suggested by findings in lamprey that the 18 pairs of individually identified reticulospinal neurons vary greatly in their ability to regenerate their axons through the same spinal cord environment. Moreover, those neurons that are poor regenerators undergo very delayed apoptosis, and express common molecular markers after SCI. Thus the signaling pathways for retrograde cell death might converge with those inhibiting axon regeneration. Many extrinsic growth-inhibitory molecules activate RhoA, whereas inhibiting RhoA enhances axon growth. Whether RhoA also is involved in retrograde neuronal death after axotomy is less clear. Therefore, we cloned lamprey RhoA and correlated its mRNA expression and activation state with apoptosis signaling in identified reticulospinal neurons. RhoA mRNA was expressed widely in normal lamprey brain, and only slightly more in poorly-regenerating neurons than in good regenerators. However, within a day after spinal cord transection, RhoA mRNA was found in severed axon tips. Beginning at 5 days post-SCI RhoA mRNA was upregulated selectively in pre-apoptotic neuronal perikarya, as indicated by labelling with fluorescently labeled inhibitors of caspase activation (FLICA). After 2 weeks post-transection, RhoA expression decreased in the perikarya, and was translocated anterogradely into the axons. More striking than changes in RhoA mRNA levels, RhoA was continuously active selectively in FLICA-positive neurons through 9 weeks post-SCI. At that time, almost no neurons whose axons had regenerated were FLICA-positive. These findings are consistent with a role for RhoA activation in triggering retrograde neuronal death after SCI, and suggest that RhoA may be a point of convergence for inhibition of both axon regeneration and neuronal survival after axotomy.
Axon Regeneration in the Lamprey Spinal Cord
2015, Neural Regeneration
The lamprey has been used to distinguish regeneration from collateral sprouting in a true vertebrate central nervous system (CNS). (1) Animals recover and can be studied after complete spinal transection (TX), so that regeneration past the lesion is unambiguous. (2) Identified neurons in brain and spinal cord can be visualized in vivo and in CNS whole mounts. Fluorescently labeled axons can be followed and the structure of their distal tips discerned during regeneration in living animals. (3) The spinal projecting neurons of the lamprey brain have been characterized with regard to their anatomical projections, their synaptic connections to neurons in the spinal cord, their roles in locomotion and their regenerative abilities. (4) Molecular expression patterns can be correlated with regenerative abilities in individually identified giant reticulospinal neurons and in clusters of smaller spinal projecting neurons. (5) Drugs and even large molecules can penetrate to the center of the cord after surface application. (6) Perhaps most importantly, axon regeneration is robust but not complete. About half of the spinal projecting axons regenerate beyond the lesion by 12 weeks post-TX and these regenerate only a few millimeters rather than all the way back to their previous caudalmost targets. Thus there is equal room for molecular and pharmacological manipulations to enhance or inhibit regeneration, i.e., experiments are not hindered by floor or ceiling effects. These advantages have been used to discover several important features of regeneration that may well apply to mammalian species as well. (a) Regeneration of the axons of large, identified neurons of the brain and spinal cord is specific with regard to pathfinding and synaptic reconnection, so that it can be assumed that sufficient specificity cues persist in the developed nervous system to underlie recovery if a sufficient amount of regeneration could be achieved. (b) The mechanism of axon elongation during regeneration does not involve the actin–myosin molecular motor that underlies growth cone pulling in early embryonic axon outgrowth. Instead the growing axon tips are simple in shape, lack filopodia, have relatively little F-actin, and are tightly packed with neurofilaments. This is important because much of the work on therapeutic approaches to promote regeneration has been predicated on the assumption that regeneration recapitulates the mechanisms of embryonic axonal development. (c) The heterogeneity in the abilities of neurons to regenerate their axons through the same environment means that axon regeneration is determined not only by environmental factors, but also by neuron-intrinsic differences. Some of these include differences in the expression of cytoskeleton, transmembrane receptors, and sensitivity to axotomy-induced retrograde apoptosis.
Non-mammalian model systems for studying neuro-immune interactions after spinal cord injury
2014, Experimental Neurology
Citation Excerpt :
In most studies of SCI in lamprey, the injury model is a complete transection. Full functional recovery is achieved over a stereotypical time course of 12 weeks, which corresponds to regeneration of reticulospinal axons through the lesion site (Cohen et al., 1986, 1989; Rovainen, 1967, 1976; Selzer, 1978; Wood and Cohen, 1979, 1981; Yin and Selzer, 1984). The lamprey spinal cord is non-myelinated and contains descending axons from reticulospinal neurons, as well as intraspinal motor neurons, sensory neurons and interneurons (Grillner and Jessell, 2009).
Mammals exhibit poor recovery after injury to the spinal cord, where the loss of neurons and neuronal connections can be functionally devastating. In contrast, it has long been appreciated that many non-mammalian vertebrate species exhibit significant spontaneous functional recovery after spinal cord injury (SCI). Identifying the biological responses that support an organism's inability or ability to recover function after SCI is an important scientific and medical question. While recent advances have been made in understanding the responses to SCI in mammals, we remain without an effective clinical therapy for SCI. A comparative biological approach to understanding responses to SCI in non-mammalian vertebrates will yield important insights into mechanisms that promote recovery after SCI. Presently, mechanistic studies aimed at elucidating responses, both intrinsic and extrinsic to neurons, that result in different regenerative capacities after SCI across vertebrates are just in their early stages. There are several inhibitory mechanisms proposed to impede recovery from SCI in mammals, including reactive gliosis and scarring, myelin associated proteins, and a suboptimal immune response. One hypothesis to explain the robust regenerative capacity of several non-mammalian vertebrates is a lack of some or all of these inhibitory signals. This review presents the current knowledge of immune responses to SCI in several non-mammalian species that achieve anatomical and functional recovery after SCI. This subject is of growing interest, as studies increasingly show both beneficial and detrimental roles of the immune response following SCI in mammals. A long-term goal of biomedical research in all experimental models of SCI is to understand how to promote functional recovery after SCI in humans. Therefore, understanding immune responses to SCI in non-mammalian vertebrates that achieve functional recovery spontaneously may identify novel strategies to modulate immune responses in less regenerative species and promote recovery after SCI.
Mechanical properties of the lamprey spinal cord: Uniaxial loading and physiological strain
2013, Journal of Biomechanics
Citation Excerpt :
A significant amount of force experiments have been done in rodent models, which has allowed us to better understand the biomechanics of the spinal cord (1.2 MPa) and the physiological and in vitro effects of applied forces on nerves (e.g. stretching) (Maikos et al., 2008; Russell et al., 2012; Abe et al., 2002; Bora et al., 1980; Ichimura et al., 2005; Jou et al., 2000; Spiegel et al., 1993; Pfister et al., 2004; Pfister et al., 2006). Our animal model of choice, lamprey (Petromyzon Marinus), is a basal vertebrate model used to study spinal cord regeneration (Cohen et al., 1989; Cohen et al. 1988, 1986; Buchanan and Cohen, 1982; Lurie and Selzer, 1991a, 1991b; Yin and Selzer, 1984, 1983; Oliphint et al., 2010) and animal locomotion (Cohen et al., 1990; Cohen, 1988, 1987; Cohen et al., 1982; Cohen and Wallen, 1980; Tytell et al., 2010). We used the lamprey for its regenerative capabilities after SCI and thus their usefulness in understanding this process.
During spinal cord injury, nerves suffer a strain beyond their physiological limits which damages and disrupts their structure. Research has been done to measure the modulus of the spinal cord and surrounding tissue; however the relationship between strain and spinal cord fibers is still unclear. In this work, our objective is to measure the stress–strain response of the spinal cord in vivo and in vitro and model this response as a function of the number of fibers. We used the larvae lamprey (Petromyzon Marinus), a model for spinal cord regeneration and animal locomotion. We found that physiologically the spinal cord is pre-stressed to a longitudinal strain of 10% and this strain increases to 15% during swimming. Tensile measurements show that uniaxial, longitudinal loading is independent of the meninges. Stress values for uniaxial strains below 18%, are homogeneous through the length of the body. However, for higher uniaxial strains the Head section shows more resistance to longitudinal loading than the Tail. These data, together with the number of fibers obtained from histological sections were used in a composite-material model to obtain the properties of the spinal cord fibers (2.4 MPa) and matrix (0.017 MPa) to uniaxial longitudinal loading. This model allowed us to approximate the percentage of fibers in the spinal cord, establishing a relationship between uniaxial longitudinal strains and spinal cord composition. We showed that there is a proportional relationship between the number of fibers and the properties of the spinal cord at large uniaxial strains.
Axonal regeneration of descending and ascending spinal projection neurons in spinal cord-transected larval lamprey
2003, Experimental Neurology
The distributions of descending and ascending spinal projection neurons (i.e., spinal neurons with moderate to long axons) were compared in normal larval lamprey and in animals that had recovered for 8 weeks following a complete spinal cord transection at 50% body length (BL, normalized distance from the anterior head). In normal animals, application of HRP to the spinal cord at 60% BL (40% BL) labeled an average of 713.8 ± 143.2 descending spinal projection neurons (718.4 ± 108.0 ascending spinal projection neurons) along the rostral (caudal) spinal cord, most of which were unidentified neurons. Some of these neurons project for at least ∼50–60 spinal cord segments (∼36–47 mm in animals with an average length of ∼90 mm used in the present study). At 8 weeks posttransection, the numbers of HRP-labeled descending or ascending spinal neurons that extended their axons through the transection were about 40% of those in similar areas of the spinal cord in normal animals. Thus, in larval lamprey, axonal regeneration of descending and ascending spinal projection neurons is incomplete, similar to that found for descending brain neurons (Davis and McClellan, 1994a). The majority of restored projections were from unidentified spinal neurons that have not been documented previously. In contrast to results from several other lower vertebrates, in the lamprey ascending spinal neurons exhibited substantial axonal regeneration. Identified descending spinal neurons, such as lateral interneurons and crossed contralateral interneurons, and identified ascending spinal neurons, such as giant interneurons and edge cells, regenerated their axons at least 9 mm beyond the transection site in animals with an average length of ∼90 mm, which is appreciably farther than previously reported. In contrast, most dorsal cells, which are centrally located sensory neurons, exhibited very little axonal regeneration.
Locomotor recovery in spinal-transected lamprey: Regenerated spinal coordinating neurons and mechanosensory inputs couple locomotor activity across a spinal lesion
1990, Neuroscience
Larval lampreys recover locomotor function several weeks after receiving complete spinal transections. In behaviorally recovered whole-animals, the phase-coupling of locomotor activity across a lesion was similar to that observed along the body in normal, unlesioned lampreys. Two factors were found to contribute to recovery of locomotor coupling above and below a spinal transection. Firstly, under in vitro conditions regenerated spinal coordinating neurons could couple brainstem-evoked locomotor activity above and below a lesion in the absence of mechanosensory inputs. Secondly, in whole-animals mechanosensory inputs were capable of coupling locomotor activity across an acute, mid-body spinal transection in the absence of direct neural coupling through spinal coordinating neurons.
Since neither regenerated coordinating neurons nor mechanosensory inputs resulted in phase-lags that were as stable as those observed in recovered whole-animals, presumably both mechanisms contribute significantly to the restoration of locomotor coupling across a healed spinal lesion.

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¹: We thank Ms. Laurie Youngs for help in preparing figures. Supported by NIH grant NS 14387.

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Electrophysiologic evidence of regeneration of lamprey spinal neurons

Abstract

Exp. Neurol.

Brain Res.

Exp. Neurol.

Patterns of regeneration between individual nerve cells in the central nervous system of the leech

Nature (London)

The electrical activity of regenerating nerves in the cat

J. Neurophysiol.

Conduction velocity in myelinated fibers: computed dependence on internode distance

J. Neurol. Neurosurg. Psychiatry

Identification of interneurons with contralateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology

J. Neurophysiol.

The conduction velocity of regenerated peripheral nerve fibers

J. Physiol. (London)

Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats

Science