Plasticity in the Regenerating Nervous System of the Lamprey, a “Simple” Vertebrate
Summary and Keywords
Human spinal cord injury (SCI) results in long-lasting disabilities due to the failure of damaged neurons to regenerate. The barriers to axon regeneration in mammalian central nervous system (CNS) are so great, and the anatomy so complex that incremental changes in regeneration brought about by pharmacological or molecular manipulations can be difficult to demonstrate. By contrast, lampreys recover functionally after a complete spinal cord transection (TX), based on regeneration of severed axons, even though lampreys share the basic organization of the mammalian CNS, including many of the same molecular barriers to regeneration. And because the regeneration is incomplete, it can be studied by manipulations designed to either inhibit or enhance it. In the face of reduced descending input, recovery of swimming and other locomotor functions must be accompanied by compensatory remodeling throughout the CNS, as would be required for functional recovery in mammals. For such studies, lampreys have significant advantages. They have several large, identified reticulospinal (RS) neurons, whose regenerative abilities have been individually quantified. Other large neurons and axons are visible in the spinal cord and can be impaled with microelectrodes under direct microscopic vision. The central pattern generator for locomotion is exceptionally well-defined, and is subject to significant neuromodulation. Finally, the lamprey genome has been sequenced, so that molecular homologs of human genes can be identified and cloned. Because of these advantages, the lamprey spinal cord has been a fertile source of information about the biology of axon regeneration in the vertebrate CNS, and has the potential to serve as a test bed for the investigation of novel therapeutic approaches to SCI and other CNS injuries.
Following injury to the human CNS, recovery is limited by failure of severed axons to regenerate and restore synaptic connections with their normal target neurons. Patients are left with long-lasting disabilities. Neuronal injuries trigger regenerative responses, but in humans and other mammals, these are overwhelmed by inhibitory influences. Elucidating the molecular mechanisms underlying CNS axon regeneration, and its inhibition by environmental factors, is essential if we are to improve functional outcomes for persons affected by SCI and other CNS injuries, and ease the burden of injury to both patients and caregivers. Unlike mammals, lampreys experience functional recovery after complete SCI. Although they belong to one of the oldest extant vertebrate lineages, they share many of the structural, physiological, and molecular features of their mammalian cousins.
The lampreys are a heterogeneous group of jawless fishes (superclass Agnatha) from the order Petromyzontifores, consisting of about 38 distinct species. Along with hagfishes, lampreys are among the most primitive extant vertebrates, and diverged during the Cambrian Period, over 500 million years ago, from the line of ancestors that would become the jawed vertebrates (Gnathostomata). Their populations span most of the world’s coastal and fresh waters (Hardisty & Potter, 1971).
Physically, adult lampreys are defined by a toothed sucking mouth and cartilaginous skeleton that supports their elongated bodies, which range in length from approximately 13 to over 100 cm. Lampreys have 7 paired gills, a large eye on either side of the head, and a single nostril on the dorsal surface of the head. Although lampreys lack paired lateral fins, they are efficient swimmers, propelling themselves through the water using an undulating wave motion that propagates rostro-caudally (Grillner, McClellan, Sigvardt, Wallen, & Wilen, 1981).
Lampreys have a complicated life cycle (Wald, 1957). They spend most of their lives as larvae, or ammocoetes, (Figure 1A) living in the sediment of fresh water streams and filter-feeding on the surrounding particulate matter. During this time, their eyes and mouths are poorly developed. They remain larvae, in a relatively stable state, for several years (approximately 5 years for the sea lamprey P. marinus) before metamorphosing into adults (Figure 2A, B) and migrating to the open sea. There, depending on the species of lamprey, they will parasitize local fauna for 1–3 years, before returning to streams to spawn and die (Hardisty & Potter, 1971). For reasons largely relating to availability, much of the research on anatomical neuroplasticity employs the large larval form of sea lampreys (Figure 1), although functional recovery and axonal regeneration also occur in the post-metamorphic adult (Cohen, Baker, & Dobrov, 1989; Lurie & Selzer, 1991) (Figure 2D), and much of the work on central pattern generation and physiological plasticity has employed adults of other lamprey species.
Lamprey Motor Control System
The reticulospinal (RS; Figure 1B) system, consisting of approximately 2,000 cells, is the primary source of spinal-projecting neurons in the lamprey brain and mediates descending motor control (Deliagina, Zelenin, Fagerstedt, Grillner, & Orlovsky, 2000; Deliagina, Zelenin, & Orlovsky, 2002; Swain, Snedeker, Ayers, & Selzer, 1993). Many of the RS neurons project the length of the spinal cord forming en passent mixed glutamatergic and electrical synapses with oscillators of the central pattern generating circuits to facilitate fictive swimming (Buchanan, Brodin, Dale, & Grillner, 1987; Buchanan & Cohen, 1982; McClellan, 1992, 1994a; Ohta & Grillner, 1989; Rovainen, 1974b; Smetana, Juvin, Dubuc, & Alford, 2010; Takahashi & Alford, 2002).
Movement is initiated by projections onto RS neurons from several brain regions. Goal-directed locomotion is controlled by glutamatergic and cholinergic projections from the mesencephalic locomotor region (Le Ray et al., 2003; Sirota, Di Prisco, & Dubuc, 2000). The RS system can also be activated through sensory inputs originating from the dorsal column nuclei or the trigeminal nerve relay (Deliagina et al., 2000; Dubuc, Bongianni, Ohta, & Grillner, 1993a, 1993b; Finger & Rovainen, 1982; Viana Di Prisco, Ohta, Bongianni, Grillner, & Dubuc, 1995). In addition to locomotion, the RS system is also responsible for maintaining posture and equilibrium via connections with vestibular neurons, although there are some vestibular projections directly to the rostral spinal cord (Alford & Dubuc, 1993; Bussieres & Dubuc, 1992a, 1992b; Bussieres, Pflieger, & Dubuc, 1999; Orlovsky, Deliagina, & Wallen, 1992; Pflieger & Dubuc, 2004; Rovainen, 1979; Zelenin, Pavlova, Grillner, Orlovsky, & Deliagina, 2003).
Within the RS system there are 32 large identifiable neurons (16 pairs) with ipsilateral spinal projections. These are often referred to as “Müller cells,” although Johanes Müller identified only the 8 largest pairs in the 19th century. There also are 4 neurons with decussating axons - 2 large Mauthner neurons (Mth) and 2 smaller auxiliary Mauthner cells (mth’). The Müller cells cluster into 3 groups: the mesencephalic (M1-4), the anterior rhombencephalic (also called “isthmic”; I1-6), and the middle rhombencephalic (B1-6). The middle rhombencephalic nucleus also contains the Mauthner (Mth) and auxiliary Mauthner (mth’) cells (Figure 1B, C). In addition to the large identifiable neurons, there are 4 bilateral clusters of smaller RS neurons: the mesencephalic reticular nucleus (MRN), and the anterior (ARRN), middle (MRRN), and posterior (PRRN) rhombencephalic nuclei. As a general rule, neurons in the medial aspect of the brain project ipsilaterally while those positioned laterally project contralaterally.
Remaining clusters of small RS neurons can be grouped according to cytoarchitectural landmarks (Swain et al., 1993). Notably, the isthmic RS neurons in the anterior rhombencephalon are responsible for propagating coordinated locomotion. Stimulation of the large identified neurons can elicit movement but, by themselves, are insufficient to initiate locomotion (Rovainen, 1967a). RS projections in the spinal cord decrease gradually along the rostral-caudal axis. Horseradish peroxidase labeling showed a 77% drop-off in RS axon numbers between the level of the 7th gill (10% body length) and the level of the cloaca (75% body length) (Swain et al., 1993).
Although many CNS elements are well conserved between lampreys and mammals, the lamprey nervous system lacks myelin (Bullock, Moore, & Fields, 1984). Consequently conduction velocity is determined only by axon diameter. In the uninjured animal, conduction velocities vary by cell but are in the range of 1–6 meters per second for the large RS axons and 1.5 meters per second for the ascending axons of the giant interneurons of the caudal spinal cord (Mackler & Selzer, 1987).
Physiological Synaptic Plasticity
A key feature of chemical synaptic transmission in the CNS is the ability of patterns of use and disuse to alter the strength of information transfer through a neural pathway. Such synaptic plasticity is commonly viewed as the physiological substrate for learning and memory. In this respect, lampreys have many commonalities with other vertebrates, and this includes the presence of reflex habituation (Birnberger & Rovainen, 1971), facilitation and post-tetanic potentiation (Adler, Yaari, David, & Selzer, 1986; Parker, 2000; Parker & Grillner, 2000) in the spinal cord, and long-term potentiation (LTP) in the brainstem (Alford, Zompa, & Dubuc, 1995). Using an in vitro preparation, potentiation of vestibular inputs onto RS neurons was shown to be calcium dependent, and included NMDA receptors, although these were dispensable. Instead, the LTP required activation of metabotropic glutamate receptors (Alford et al., 1995). NMDA receptors also are integral features of the central pattern generator (CPG) for locomotion in the lamprey spinal cord (Grillner et al., 1981), and NMDA-mediated CPG activity is subject to neuromodulation by serotonin (W. Zhang, Pombal, el Manira, & Grillner, 1996) and substance P (Parker & Grillner, 1998), among other influences. Such pharmacological neuromodulation has been termed “metaplasticity” (Parker & Grillner, 1999) to distinguish it from use-dependent changes in synaptic efficacy. Behavioral studies are limited largely to responses to odorants and other stereotyped reflexive activities, and there are few studies of learning or memory in the lamprey (Choi, Jeon, Johnson, Brant, & Li, 2013). Whether this is because lampreys have relatively inflexible behavior, or the behavior is just difficult to study is not clear.
The lamprey is remarkable for the presence of mixed electrical–chemical synapses in both the brain (Stefanelli & Caravita, 1970) and spinal cord (Rovainen, 1974a, 1974b). Indeed, the majority of the connections from Müller and Mauthner axons onto local neurons in the spinal cord feature this type of composite synapse, in which unbranched presynaptic axons form en passant synapses onto postsynaptic dendrites, with vesicle-containing junctions adjacent to gap junctions located along the axon’s length. The function of the electrical component has yet to be fully understood, but because electrical synapses show very little fluctuation in strength, they may serve to set a basal level of postsynaptic excitability. Thus synchronous bilateral activation of motor neurons by Müller and other RS axons could stiffen the body, thereby increasing the force of propulsion resulting from CPG outputs. In any case, this electrical component may place some limits on the plasticity of synaptic transmission in the lamprey spinal cord.
Regeneration of Supraspinal Motor Command Axons
Like mammals, lampreys are paralyzed below a complete SCI. Unlike mammals, lampreys will gradually recover coordinated movements below the injury. Experimental lesions most often consist of complete spinal cord TX. As a cartilaginous fish, the lamprey spinal cord is easy to access, using a longitudinal incision along the dorsal midline and gentle retraction of the musculature. The exposed spinal cord can then be easily severed with iridectomy scissors (Barreiro-Iglesias, Zhang, Selzer, & Shifman, 2014). Alternative approaches, including hemisections or crushes, also are occasionally employed.
The lamprey was of interest to 19th- and early 20th-century physiologists, including Sigmund Freud, who determined that the dorsal cells of the spinal cord are primary sensory neurons because they sent one axon out the dorsal roots (Freud, 1877). Early attempts were made to reproduce in lampreys the work on spinal cord regeneration after tail amputation in amphibians, but results were inconsistent. The first description of lamprey neural regeneration after spinal cord TX was published by Dr. Kazimierz Maroń at the Polish Academy of Sciences in Kraków using larvae of the European river lamprey, Lampetra fluviatilis (Maroń, 1959). Proliferation of ependymal cells was observed within one week of injury and fusion of the severed cord at two weeks, with some thin, unidentified axons traversing the lesion. Full recovery was observed approximately 40 days after injury. These basic findings have held up over the years, and were confirmed in the sea lamprey Petromyzon marinus by Emerson Hibbard at NIH, who also showed restoration of continuity in the giant RS axons (Hibbard, 1963).
These studies were complicated because the spinal cords were TXed through the skin and muscle without exposing the cord, so that completeness of the SCI could not be assured. This was corrected at Washington University in St. Louis by Carl Rovainen, who TXed the spinal cord under direct microscopic vision and documented regeneration of individual giant RS axons across the lesion in serial transverse sections at 11 weeks post-TX (Rovainen, 1976). The giant axons had progressed only a few millimeters beyond the injury, although recovered swimming and other behaviors was extensive. Indeed, the lampreys, which were initially paralyzed below the lesion, slowly regained significant movement and coordination over the course of four weeks. Similar anatomical and behavioral observations were made at the University of Pennsylvania by Michael Selzer, who also performed electrophysiological studies (Selzer, 1978). After spinal cord TX in the gill region, electrical stimulation of the head or tail elicited body movements only rostral or caudal to the lesion, respectively. Stimulation began to elicit movement across the lesion by 5 weeks after injury and coordinated swimming was recovered around 10 to 13 weeks after injury. At these times, head stimulation evoked tail movements even when the skin, viscera and body wall musculature surrounding the lesion were removed—demonstrating that motor recovery was mediated through the spinal cord, and not through the lateral line nerves or reflexively in response to contraction of surrounding body musculature. However, direct electrical stimulation of the rostral spinal cord, elicited recordable electrical activity only as far as 5–10 millimeters below the injury. The finding that functional recovery was almost complete before the large RS axons had regenerated and even though the axon regeneration would ultimately extended only a few millimeters, suggested that the regenerated fibers innervated local spinal networks or relays to restore locomotion through the caudal cord.
The nature of these local networks has been elucidated by Sten Grillner and his colleagues at the Karolinska Institute in Stockholm, and their scientific heirs, who used the lamprey as their premier model to study the central pattern generator (CPGs) for locomotion (Grillner, McClellan, Sigvardt, & Wallen, 1983). Evidence that the CPGs became reconnected across the TX was found by Avis Cohen and colleagues (Cohen, Mackler, & Selzer, 1986), who showed that when previously transected spinal cords were removed from the animal, and ventral root bursts induced by bath application of L-DOPA, segments caudal to the lesion discharged with fixed time lags after rostral segments. The implication was that regeneration of propriospinal axons could mediate functional recovery even before supraspinal axons had regenerated. Evidence for rapid regeneration of smaller unidentified axons long before the earliest regeneration of the large RS axons was found by Diana Lurie and colleagues at the University of Pennsylvania (Lurie, Pijak, & Selzer, 1994).
A prominent feature of regeneration in the Müller and Mauthner neurons was the attenuation of their regenerating axons to much narrower daughter neurites, and this almost surely meant that many axons were lost in the serial sections before their termination, as suggested by the lesser distances of regeneration measured histologically than electrophysiologically. The development of intracellular tracer injection made it possible to follow a regenerated axon much further, often to its termination, as done by Malcolm Wood and Melvin Cohen at Yale University, who were able to use intracellular injection of horseradish peroxidase to demonstrate that regenerating axons of Müller and Mauthner neurons formed synapse-like terminals distal to a spinal cord lesion (Wood & Cohen, 1979). Using light and electron microscopy, they identified vesicle-containing structures near dendrites a few millimeters distal to the lesion. Interestingly, these terminations lacked the gap junction densities of electrical synapses that are characteristic of the composite electrical–chemical synapses made by these axons in uninjured animals. This observation received electrophysiological confirmation in intracellular recordings by Mackler and Selzer from postsynaptic spinal cord neurons upon intracellular stimulation of identified RS neurons (Mackler & Selzer, 1985). These investigators also found that within the limited distances of regeneration, the axons grew selectively in their correct paths (Mackler, Yin, & Selzer, 1986), and formed synapses specifically with correct types of neurons distal to the TX (Mackler & Selzer, 1987).
Together, these early studies demonstrated that following SCI, supraspinal axons in the lamprey undergo spontaneous regeneration, which although limited in distance, is sufficiently specific to mediate functional recovery (Cohen, Mackler, & Selzer, 1988). The findings by different laboratories were remarkably consistent. Modest differences in the reported time courses of regeneration and functional recovery were due largely to variations in the ages of the lampreys (younger, smaller larvae regenerating faster than older larvae or post-metamorphic specimens) and the temperature of the water in which the animals recovered (faster at room temperature than in colder water). Whether the RS axons have to regenerate across the lesion or only reform synapses with regenerating propriospinal relay neurons is still not clear. Building on these findings, later studies explored molecular mechanisms of axon regeneration after injury, and demonstrated additional remodeling of local and supraspinal circuits to facilitate the behavioral recovery.
Regeneration of Descending Axons Mediating Locomotor Activation
Lamprey behavioral recovery after SCI follows stereotypic stages of increasing motor coordination. Immediately after injury, only vigorous head oscillations above the level of injury can be observed (stage 1); this is followed by isolated and then repetitive caudal flexion waves (stages 2 and 3). At this point, swimming begins to recover. Further recovery consists of coordinated rostral-caudal movement (stage 4) and ability to maintain correct dorsal-ventral posture (stage 5). Stimulating the “isthmic” RS neurons in the anterior rhombencephalon of stage 5 recovered lampreys was sufficient to produce locomotion of the tail, suggesting regeneration of descending drive (Currie & Ayers, 1983).
More convincing evidence that regeneration could mediate coordinated locomotion was obtained using isolated preparations of the CNS. Under these conditions, the spinal cord could be stimulated and the resulting discharge patters recorded in the ventral roots. One main advantage of this system compared to the intact animal is the elimination of mechanosensory or other pathways that could potentially activate movements in segments across the lesion, in the absence of regenerated axons. Using this approach, recovery of fictive swimming was confirmed 2 to 6 months after complete spinal cord TX in isolated lengths of spinal cord, in which ventral root discharges above and below the lesion showed fixed phase lags (Cohen et al., 1986). This was true even after bath application of curare, a potent paralyzing nicotinic acetylcholine receptor antagonist, which ruled out mechanosensory feedback by any muscle fibers accidently left in the preparation (Cohen, 1988).
The restoration of phase locking between CPGs rostral and caudal to a TX could be an adequate explanation of recovered swimming coordination, and does not require regeneration of descending connections. Nor does it not imply a role for the large RS axons whose regeneration had been documented histologically. By including the brain in the in vitro preparation, descending neurons could be activated by pharmacological or microelectrical stimulation, and their effects on motor discharges could be measured directly. By bathing a length of spinal cord that included the lesion in low calcium Ringer’s solution, the effect of removing most chemical transmission over long distances could be tested, thus eliminating the likelihood of local propriospinal reflexes. Under these conditions, it was shown that descending motor command pathways from the brain regenerated after SCI and directly activated spinal motor networks caudal to the TX (McClellan, 1988, 1990). However, severing the giant Müller axons in vitro by making a medial spinal cord cut did not disrupt locomotor patterns in the regenerated spinal cords, suggesting that this cell population is not critical for restoring locomotor coordination between rostral and caudal spinal segments (McClellan, 1990).
Early studies based on serial histological sections or intracellular tracer injections had shown that RS axons regenerated only a few millimeters past the lesion at times when behavioral recovery appeared to be complete (Hibbard, 1963; Maroń, 1959; Rovainen, 1976; Selzer, 1978; Wood & Cohen, 1979), and that with time, some of these axons partially retract (Yin & Selzer, 1983, 1984). Other studies suggested that descending fibers continue to regenerate slowly over long periods of time. Thirty-two weeks following a complete TX, ventral root activity, examined in vitro in the caudal spinal cord showed relatively normal phase lags compared to much more rostral segments, suggesting direct RS fiber innervation of caudal CPGs (Davis & McClellan, 1993). Retrograde labeling of regenerated descending fibers with horseradish peroxidase showed that some axons had regenerated as long as 56 mm (Davis & McClellan, 1994a). Nevertheless, most axons regenerated only short distances, and in this there is general agreement. In uninjured lampreys, retrograde labeling revealed approximately 1250 RS neurons whose axons project 20%, 900 RS neurons 40%, and 825 neurons 60% of body length. Thirty-two weeks after complete spinal cord TX at approximately 10% body length, in animals 72–142 mm long, retrograde labeling revealed 1086 RS neurons at 10%, 430 RS neurons at 40%, and 49 RS neurons at 60% body length. Although the number of regenerated spinal-projecting axons was smaller than the descending projection in uninjured animals, the regeneration appeared sufficient to restore direct activation of motor networks caudal to the original lesion. However, kinematic and electromyographic analysis suggested that relatively normal locomotor activity in caudal spinal cord regions can occur even in the absence of regenerated supraspinal fibers (Davis, Troxel, Kohler, Grossmann, & McClellan, 1993). These findings confirmed that compensatory mechanisms such as regeneration of short propriospinal relays and mechanosensory feedback may play important, complementary roles in restoring function to the spinal cord-injured animal (McClellan, 1994b).
Good- and Bad-Regenerating Neurons
Although the lamprey RS system, as a whole, regenerates after a SCI, regeneration is highly variable among individual neurons, and in any given animal, only approximately half of the identifiable RS neurons regenerate axons beyond an injury (Davis & McClellan, 1994b; Jacobs et al., 1997). By retrograde labeling with horseradish peroxidase from across the TX, two groups obtained similar regeneration profiles despite small differences in protocol (Davis & McClellan, 1994b; Jacobs et al., 1997; Swain, 1989). The best regenerators were the Müller cells I3, I5, and B5, along with the auxiliary Mauthner cell (mth’). Poor regenerators included the Mauthner neuron (Mth) and the Müller cells I1, B3, and B4. Regeneration probabilities also were heterogeneous among the large number of smaller RS neurons. Ten weeks after spinal cord TX at the level of the 5th gill, horseradish peroxidase was introduced into a second lesion 5 mm caudal to the original injury to label the RS neurons whose axons had regenerated. Regeneration probabilities were calculated for 12 cytoarchitectonic groups. Probabilities above 50% were observed in the middle RS, isthmic RS, medial superior RS, and posterior mesencephalic cell groups. The regeneration probabilities for neurons in the medial diencephalic and dorsolateral inferior RS nuclei were less than 20%.
Retrograde Cell Death of Bad-Regenerating Neurons
One of the major questions surrounding the observation of “good” and “bad” -regenerating neurons is the ultimate fate of those cells that fail to regenerate their axons. A 2008 study demonstrated that many neurons undergo a delayed form of apoptosis (Shifman, Zhang, & Selzer, 2008). In this study, larval lampreys were followed for up to 1 year post-TX. Among the identifiable neurons, cell death was observed almost exclusively in those with low likelihood of regeneration (Hu, Zhang, & Selzer, 2013). A strong correlation was observed between the probability of an axon’s regenerating, and survival of its neuron at 1 year. A combination of retrograde labeling with fluorescent dextran amines, neurofilament immunohistochemistry, and toluidine blue O staining were used to examine neuron morphology. The earliest degenerative changes were observed at 2 weeks but cells did not begin to disappear until approximately 16 weeks post-SCI. These findings are unlikely to be artifacts due to toxicity of the retrograde tracers. Careful comparisons of retrograde labeling by fluoro-gold and horseradish peroxidase indicated that at times up to 4 months after spinal cord TX, these labels did not promote cell death, although over time fluoro-gold labeling may degrade (McClellan, Zhang, & Palmer, 2006). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity was observed faintly beginning at 1 month after injury. Additional changes in dying neurons before disappearance included a reduction in the Nissl substance, pyknosis, and nuclear fragmentation. Together, these changes suggest the cells were dying through a delayed form of apoptosis. Apoptotic signaling was observed even earlier by assaying for caspase activity. In brain wholemounts, staining with fluorchrome-labeled inhibitors of caspases (FLICA) co-localized with TUNEL staining, but was noted as early as 1–2 weeks post-TX (Hu et al., 2013). This is significant because the large RS axons do not regenerate beyond the TX until at least five weeks. Thus it is unlikely that the delayed apoptosis is a consequence of the failure of its axon to regenerate, but presumably reflects an intrinsic property of the neuron that makes it vulnerable to axotomy.
As with regeneration, retrograde neuronal death had molecular correlates, and because the bad regenerators also were bad survivors, the correlations were inverse. Thus using methods developed to multiply-label brainstem wholemounts for apoptotic signaling (FLICA), regeneration (retrograde labeling), immunohistochemistry, and in situ hybridization, neurons that labeled for activated caspases also expressed PTPσ and LAR (Hu et al., 2013; G. Zhang, Hu, Li, Huang, & Selzer, 2014). Although RhoA expression was not specific to bad-regenerator RS neurons, knockdown of RhoA with a retrogradely delivered, translation-blocking morpholino antisense oligonucleotide reduced caspase activation after spinal cord TX, just as it enhanced axon regeneration in these same neurons (Hu et al., 2017). Similarly, after axotomy, bad-surviving neurons accumulated high levels of synuclein (Busch & Morgan, 2012), a protein associated with synaptic vesicles that accumulates abnormally in Parkinson disease. Inhibiting synuclein synthesis with CLR01, or with a translation-blocking morpholino, improved neuronal survival (Fogerson et al., 2016).
These observations suggest a push-pull relationship between cell death and regeneration, with potentially shared underlying signaling mechanisms.
Regeneration of the Monoaminergic System
In mammals, small monoaminergic axons frequently have been noted to be the most responsive to regenerative therapies. Thus it is of interest to review the regenerative potential of these neurons in the lamprey. The organization of the serotonergic system is well conserved among lampreys and mammals (Barreiro-Iglesias, Villar-Cervino, Anadon, & Rodicio, 2008). Descending lamprey serotonergic fibers originate from the caudal rhombencephalon and help optimize central pattern generators in the spinal cord (Brodin, Buchanan, Hokfelt, Grillner, & Verhofstad, 1986; Harris-Warrick & Cohen, 1985). Spinal intrinsic sources of serotonin include the neurons along the ventral plexus (midline neurons just ventral to the central canal) and dorsal root ganglia (Harris-Warrick, McPhee, & Filler, 1985; W. Zhang et al., 1996). To investigate the regenerative ability of descending 5-HT fibers, investigators assessed axon regeneration after complete TX by making a second TX 1 mm caudal to the first and retrogradely labeling regenerating neurons with Neurobiotin (Cornide-Petronio, Ruiz, Barreiro-Iglesias, & Rodicio, 2011). At 5 months after the initial SCI, 20–40% of the axotomized 5-HT immunoreactive neurons in the rhombencephalon had reinnervated the spinal cord distal to the injury. This is not particularly better than other spinal-projecting neurons. Interestingly, the cell bodies of the regenerated 5-HT fibers were significantly smaller than 5-HT neurons in uninjured lampreys. It is possible that 5-HT neurons atrophy or undergo retrograde cell death after injury similarly to other RS neurons. Additional plastic changes within the brain include transient upregulation of mRNA encoding the 5-HT1a G protein-coupled receptor, which returns to baseline within 3 to 7 weeks (Cornide-Petronio, Fernandez-Lopez, Barreiro-Iglesias, & Rodicio, 2014). Using high-performance liquid chromatography, another study found that serotonin levels distal to the injury are restored only to approximately 39% of control values at 26 weeks after lesioning. Serotonin levels proximal to injury returned to normal (Cohen, Abdelnabi, Guan, Ottinger, & Chakrabarti, 2005). The incomplete regeneration of the serotonergic system following acute injury does not appear to preclude substantial behavioral recovery, suggesting that other compensatory plasticity is at play.
Like serotonin, dopamine elicits modulatory effects on swimming (McPherson & Kemnitz, 1994; Schotland et al., 1995; Svensson, Woolley, Wikström, & Grillner, 2003). In the gill region of the spinal cord there are two intrinsic populations of dopaminergic neurons—the CSF contacting cells (CSFc) along the central canal and cells in the ventromedial region of the cord (VM cells). Within 2 weeks of complete SCI, the numbers of both cell types were markedly reduced both distal and proximal to the lesion (Fernandez-Lopez et al., 2015). Similarly, the numbers of dopamine immunoreactive fibers in the same regions were reduced by 75%. However, over the course of 6 months, both dopaminergic innervation and numbers of CSFc and VM cells gradually returned to normal levels. Surprisingly, there were no changes in D2 mRNA expression after injury as assessed by in situ hybridization. The mechanisms mediating regeneration of the dopaminergic system are still unknown, but an examination of cell proliferation in the lamprey spinal cord after complete TX identified a population of BrdU positive cells in the ependymal zone (G. Zhang, Vidal Pizarro, Swain, Kang, & Selzer, 2014). Labeling appeared within 3 weeks of injury and cells could be found at least 0.5 millimeters on either side of the spinal cord lesion. Because the cells never left the ependymal region, the investigators speculated that they may be newly formed CSFc cells. Consequently, it is possible that after injury, lost dopaminergic neurons are replaced by local progenitors.
Sprouting in Regenerating Fibers
Unlike axons in the CNS of mammals, in lamprey, spared axons do not appear to form collateral sprouts after acute injury. For example, unilateral crush injuries of the trigeminal nerve did not result in sprouting of contralateral trigeminal sensory fibers across the midline (Calton, Philbrick, & McClellan, 1998; G. Zhang, Jin, Hu, Rodemer, & Selzer, 2015). Hemisection of the spinal cord, interrupting RS axons on the same side, was followed by regeneration of many of the cut axons for short distances (a few millimeters), but did not result in sprouting of contralateral spared axons across the midline, even at levels caudal to the level of maximal axon regeneration (G. Zhang et al., 2015). Thus, lamprey axons regenerate when they are injured but so far, do not sprout collaterals when their neighbors are injured.
However, it has long been known that regenerating RS fibers can branch profusely, especially as they approach the injury site (Rovainen, 1976; Selzer, 1978; Wood & Cohen, 1979; Yin & Selzer, 1983). Long neurites have be shown to sprout from the caudal dendrites of giant interneurons after their axons have been interrupted by a spinal cord TX placed within a cm rostral to their perikaryon (Mackler et al., 1986; Yin, Mackler, & Selzer, 1984). In these cases, the original axon retracted all the way back to the cell body, but the sprouted neurites decussated and regenerated rostralward in paths that mimicked those of the original axon. Depending on the location of the lesion, axotomy also induced sprouting from the dendrites of the large RS neurons (Hall & Cohen, 1983). While axotomy 1 mm or more from the perikaryon resulted in axon regeneration, often with the sprouting of more than one terminal branch, severing the fiber close to the cell body, usually within 500μm, induced extensive dendritic sprouting. Long neurites grew for approximately 3 months, reaching lengths of up to 3 millimeters before beginning to retract (Hall & Cohen, 1988a, 1988b). These sprouts were only induced with axotomy. Lesions limited to the dendrites or adjacent brain tissue did not induce sprouting (Hall & Cohen, 1988b). The neurites that sprouted from the dendrites were axon-like in structure, including a neurofilament-dominated cytoskeleton (Hall, Poulos, & Cohen, 1989). Immunohistological and ultrastructural examination revealed that these sprouts were enriched in phosphorylated neurofilaments and largely depleted of tubulin. The dendrites producing the sprouts showed structural abnormalities as well, including a loss of acetylated microtubules. The investigators hypothesized that dendritic microtubules were destabilized following invasion by neurofilaments (Hall, Lee, & Kosik, 1991; Hall, Yao, Selzer, & Kosik, 1997).
Lamprey brains also undergo remodeling when SCI is more remote than the close axotomy that elicits neuritic sprouting from the dendrites of axotomized neurons. Following spinal cord TX at the level of the 5th gill, whole brain synapsin I mRNA and protein levels were increased in the rhombencephalon (Lau et al., 2011). At 11 weeks after injury, phospho-synapsin (serine 9) immunoreactivity increased in the hindbrain, consistent with neurite sprouting or increased transmitter release within the brain. At the same time, ultrastructural examination showed increased numbers of neurites and a shift to smaller sizes of neurites in sample electron micrographs through the hindbrain neuropil. The identities of the neurons of origin of these neurites are unknown, as are their possible role in compensatory mechanisms to promote functional recovery.
Intracellular electrophysiological recording and stimulation after animals had recovered functionally from spinal cord TX demonstrated that regenerating axons formed synapses on appropriate targets distal to the lesion (Mackler & Selzer, 1985). These regenerated synapses appeared to be specific in that inappropriate neuronal pairs were not synaptically connected (Mackler & Selzer, 1987). The newly formed synapses were found within 8 mm of the TX site, consistent with the known distances of regeneration for identified neuron types. Regenerated fibers were smaller than uninjured axons with slower conduction velocities—approximately 1 meter per second compared to 1.6 meters per second in controls. The monosynaptic potentials recorded across a recovered injury had larger chemical components and smaller electrical components than the normal synaptic connections observed between the same cell types in uninjured animals.
These electrophysiological observations were consistent with ultrastructural studies, in which regenerated synapses from descending RS neurons lacked gap junctions (Oliphint et al., 2010; Wood & Cohen, 1979), the structural correlates of electrical excitatory synaptic potentials. At 10 to 12 weeks after SCI, histological and ultrastructural examination in fluorescent-phalloidin labeled RS axons showed that the regenerated axons formed vesicle-containing chemical synapses that were much less abundant (75% of uninjured controls, not statistically significant) but had regained almost their normal synapse density when normalized to the reduced surface area of the attenuated circumference of regenerated neurites. However, they appeared less mature than normal because they had fewer vesicles, and contained simplified, shorter presynaptic densities (Oliphint et al., 2010). The electrical component of the synapse did not regenerate. In the area immediately distal to the lesion, the investigators calculated RS synapse numbers of 9–11.5% of control values. When extrapolated for the full length of the spinal cord, the authors estimated that the spinal cord distal to the TX contains only 1–2% of the normal total number of RS synapses. The ability of the lamprey to achieve functional recovery despite such limited reinnervation of the distal spinal cord, highlights the importance of short-range synaptic relays, intersegmental connections between CPGs, and additional compensatory mechanisms.
Characterization of Regenerating Axon Tips
The translucency of the lamprey CNS, and the presence of large identified RS neurons and their axons, provide an opportunity to compare the mechanisms of embryonic axon outgrowth in vivo or in vitro with the mechanisms that drive regeneration of CNS axons in vivo. The evidence suggests that these two processes may be quite different. Severed axons of the descending RS neurons can regenerate at rates up to 5 micrometers per hour. The regeneration process is discontinuous and alternates between periods of growth, pause, and retraction (Jin et al., 2009). As the regenerating axon tips progress caudally, they form close associations with glial processes (Lurie et al., 1994). The morphology of the axon tip reflects its regeneration status. In a two-photon imaging study, static or retracting tips appeared rounded with some retracting tips showing signs of breakdown of structural integrity. Growing tips, on the other hand, formed long conical shapes, often with a finger-like protrusion in direction of regeneration (Jin et al., 2009). In any case, the tips of regenerating axons are unlike the growth cones of mammalian neurons in culture (Lurie et al., 1994). The lamprey tips lack lamellipodia and filopodia, are densely packed with neurofilaments (Pijak et al., 1996), but contain little F-actin (Hall et al., 1997; Jin et al., 2009). This suggests that actin-myosin molecular motors, which are essential for growth cone motility, may not drive axon regeneration in the CNS. It has been postulated that an internal protrusive force, perhaps generated by neurofilament transport or assembly may be involved in the mechanism of growth. Neurofilament mRNA expression decreased during the first 4 weeks after SCI, but recovered selectively in good-regenerating neurons (Jacobs et al., 1997). Furthermore, blocking the formation of neurofilaments by inhibiting the NF-180 subunit, prevented axon regeneration when assessed at 4 and 9 weeks after spinal cord TX (G. Zhang et al., 2015).
Another potential mechanism by which neurofilaments might be involved in regeneration is their local synthesis and assembly in the regenerating axon tip. Using immunofluorescence, in situ hybridization, and electron microscopy, the presence of protein synthetic machinery, including ribosomes, polyribosomes, and mRNA, was demonstrated in the severed tips of RS axons (Jin, Pennise, Rodemer, Jahn, & Selzer, 2016). The mRNA was more abundant in actively growing tips than in static tips, and mRNAs encoding cytoskeleton proteins, including β-Tubulin and the neurofilament subunit, NFL, were present, but β-Actin mRNA levels were very low, consistent with the hypothesis that long-distance axon regeneration is not mediated by actin-myosin molecular motors. These findings in the lamprey are important because it has been suggested that a relative inability of CNS axons to manufacture proteins locally might explain why axon regeneration is so limited in the mammalian CNS compared to peripheral nerves (Gumy, Tan, & Fawcett, 2010).
Axon Regeneration in Spinal Cord Neurons
Two populations of large intraspinal neurons that project ascending fibers to the brainstem are the dorsal cells (DCs) and giant interneurons (GIs). DCs are primary sensory neurons. These glutamatergic cells are found bilaterally in a dorsomedial position throughout the length of the spinal cord. Their processes extend out the dorsal roots and bidirectionally in the ipsilateral dorsal columns of the spinal cord (Fernandez-Lopez et al., 2012; Rovainen, 1967b). GIs are second order, glutamatergic sensory neurons present in the caudal spinal cord in the region of the dorsal fins. GI axons decussate in the spinal cord and project rostrally to the brain. Although their exact function is unknown, it has been hypothesized that they help modulate movement (Fernandez-Lopez et al., 2012; Rovainen, 1967b). Complete spinal cord TX severs ascending fibers, and after axotomy, DCs and GIs experience acute changes in both their morphological and electrophysiological characteristics (Yin, Mackler, & Selzer, 1987; Yin, Wellerstein, & Selzer, 1981). Axon tracing studies with horseradish peroxidase have shown that over time, both cell types can regenerate their fibers at least a few millimeters beyond the injury, following their normal trajectories (Armstrong, Zhang, & McClellan, 2003; Mackler et al., 1986; Yin et al., 1984; Yin & Selzer, 1983). Importantly, the regenerated GI neurites, are electrically excitable and form synapses only with appropriate targets (Mackler & Selzer, 1987; Yin & Selzer, 1984). As with other lamprey cell types, regeneration is variable. Following complete TX of the caudal spinal cord, intracellular injection of horseradish peroxidase revealed that 76% of GIs and 56% of DCs adjacent to a TX regenerated their axons past the lesion (Yin & Selzer, 1983). Retrograde tracing showed regeneration of GI and DC axons at least 9 millimeters rostral to a complete spinal cord TX, although the DCs showed less frequent regeneration than GIs and some other rostrally projecting neurons (Armstrong et al., 2003).
Peripheral Nervous System
Like central neurons, fibers in the peripheral nervous system of lampreys regenerate after acute injury (Calton et al., 1998). Following unilateral trigeminal root crush, larval lampreys recovered their normal escape response to ipsilateral oral hood stimulation over the course of 12 weeks. Horseradish peroxidase staining showed robust but incomplete regeneration of trigeminal motoneuron axons.
Molecular Determinants of Axon Regeneration
In mammals, the failure of axon regeneration after injury in the mature CNS is attributed to both a limited intrinsic growth capability and an extracellular environment that is nonpermissive for growth (for reviews, see He & Jin, 2016; Ohtake & Li, 2015). Given how unambiguously true axon regeneration can be assessed in the lamprey, and that the incompleteness of regeneration leaves room to either augment or inhibit regeneration experimentally, this model of SCI lends itself well to the examination of molecular influences on regeneration first identified in other species. Although it has not been possible to examine in the lamprey every molecule previously implicated in CNS axon regeneration, the sequencing of the lamprey genome (J. Smith et al., 2011; J. J. Smith et al., 2013) has made it possible to identify lamprey homologs of almost all of them, and several of the most prominent candidates have been cloned.
In a series of studies conducted over the past two decades, the bad-regenerator RS neurons in the lamprey expressed higher levels of mRNA for several putative growth-inhibiting receptors, or selectively upregulated them after spinal cord TX. For example, the chondroitin sulfate proteogylcans (CSPGs) are key inhibitory components of the reactive scar that forms around a spinal cord lesion. In rodent models, digesting CSPGS with the bacterial enzyme chondroitinase ABC (ChABC) or deleting the CSPG receptors protein tyrosine phosphatase σ (PTPσ) and leukocyte common antigen-related protein (LAR, also known as PTPRF), improves functional recovery after SCI (Bradbury et al., 2002; Fisher et al., 2011; Lang et al., 2015; Li et al., 2015; Ohtake, Wong, Abdul-Muneer, Selzer, & Li, 2016; Shen et al., 2009; Xu et al., 2015). Although lamprey CSPGs are abundantly expressed around the site of TX, mRNAs for PTPσ and LAR are selectively expressed in bad-regenerator RS neurons (G. Zhang, Hu, et al., 2014).
Downstream of the CSPG receptors and at a common point of convergence among numerous growth inhibitory pathways is the small GTPase, RhoA. In rodents, RhoA has been implicated in inhibiting axon regeneration after injury (Dubreuil, Winton, & McKerracher, 2003). Although lamprey RhoA mRNA expression was not restricted to bad-regenerating neurons (G. Zhang et al., 2017, in preparation), morpholino-mediated knockdown of RhoA enhanced axon regeneration after spinal cord TX (Hu et al., 2017).
Many axon guidance molecules that play a role in CNS development also are expressed in the mature nervous system and have been implicated in CNS axon regeneration. These include sempahorins, ephrins, netrins, repulsive guidance molecule (RGM), and slits (reviewed in Giger, Hollis, & Tuszynski, 2010; Yamashita, Mueller, & Hata, 2007). In lampreys, the chemorepulsive netrin receptor, UNC5 (Shifman & Selzer, 2000), and the receptor for Repulsive Guidance Molecule (RGM), neogenin (Shifman, Yumul, Laramore, & Selzer, 2009), were selectively upregulated after TX in bad-regenerating RS neurons. Although expression of the semaphorin receptor, plexin A, by itself did not correlate with regenerative ability, it added to the strength of the inverse correlation between regenerative probability and the co-expressions of UNC5 and Neogenin. Thus the coefficient of determination (r2) between regenerative probability and co-expression of all three receptors was 0.78 (Chen, Laramore, & Shifman, 2017).
Second messenger systems also influence regeneration. Upregulation of cyclic AMP (cAMP) is a major mediator of the pro-regenerative “conditioning lesion effect” in the peripheral nervous system (Neumann, Bradke, Tessier-Lavigne, & Basbaum, 2002; Qiu et al., 2002). Administration of cAMP analogues or inhibition of phosphodiesterase-4 (PDE4) improves neurite outgrowth after injury, likely via activation of protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) in rodents (Cai et al., 2002; Cai et al., 2001; Gao et al., 2004; Lu, Yang, Jones, Filbin, & Tuszynski, 2004; Murray & Shewan, 2008; Murray, Tucker, & Shewan, 2009; Nikulina, Tidwell, Dai, Bregman, & Filbin, 2004), and improved regeneration in the Mauthner axon of zebrafish embryos (Bhatt, Otto, Depoister, & Fetcho, 2004). In lampreys, application of cyclic AMP (cAMP) analogues reduced axon retraction after TX, and live imaging revealed increased elongation velocity up to 11-fold from approximately 23 to 269 µm per day at 2–3 weeks after injury (Jin et al., 2009). Interestingly, activating endogenous cAMP with forskolin did not alter the biophysical properties of regenerating RS neurons but did increase action potential durations of uninjured neurons (Pale, Frisch, & McClellan, 2013). Analogues of cAMP reduced cell death among both good- and bad-regenerator RS neurons and promoted regeneration of axons in straighter paths (Lau, Fogerson, Walsh, & Morgan, 2013).
Epigenetic modifications represent a potentially potent mechanism for controlling the genome-wide transcription program needed to sustain axon regeneration (reviewed in Trakhtenberg & Goldberg, 2012). Although there is evidence in mammals for epigenetic control of peripheral nerve regeneration, less information is available for this in the CNS. One important epigenetic modification is the acetylation of lysine residues in the histone tails, and its regulation by lysine acetyl transferases (KATs) and histone deacetylases (HDACs). Histones pack DNA into nucleosomes, the chromatin building blocks, generally making DNA less accessible for transcription. Histone acetylation unpacks the DNA and increases transcription. In the lamprey, mRNA expression of HDAC1 was found selectively in bad-regenerating neurons, which may serve as a mechanism to suppress the genetic program for axon regeneration (Chen, Laramore, & Shifman, 2016). This appeared to be quite specific, since several other KATs and HDACs were assessed, and although their expressions were modulated by spinal cord TX, they were not correlated with the regenerative probabilities of the individual RS neurons.
Lamprey-specific molecular correlates of regenerative ability have been described, but in most cases, their causal relationship to regeneration has not been established. A partial exception is the middle molecular weight neurofilament subunit, NF180. After axotomy, mRNAs for several neurofilmant subunits were downregulated within all of the identifiable RS neurons. The downregulation was transient in good regenerators but persistent in bad regenerators (Jacobs et al., 1997; G. Zhang, Jin, & Selzer, 2011). This pattern remained even if regeneration was blocked by removal of a length of spinal cord at the lesion site (Jacobs et al., 1997), suggesting that the recovery of NF180 expression was not a consequence of regeneration, but was an intrinsic property of neurons that were capable of regenerating their axons. Moreover, neurofilament expression directly contributed to axon regeneration as inhibition of NF180 synthesis by a translation-blocking morpholino inhibited regeneration (G. Zhang et al., 2015), which supported a role for neurofilaments in the mechanism of axon regeneration in the CNS. However, since the morpholino targets the translational start codon and was delivered retrogradely from the site of injury, it is not clear that the most important site of action is in the perikaryon. Some of the relevant synthesis of neurofilaments might be in the injured, regenerating axon tip, as described.
The lamprey CNS is remarkable for straddling the divide between the relatively fixed circuitry of primitive invertebrates and the more stochastic, plastic connectome of mammals. The presence of identifiable neurons and chemical synapses with a strong electrical component suggests a limited capacity for plasticity in the normal animal. Although LTP has been described in the lamprey, there have been few studies relating to more complex or long-lasting learning. Nor do lampreys show evidence for collateral sprouting by spared axons upon injury to nearby axons. However, after injury, many spinal cord axons regenerate. The regenerative abilities of these axons are heterogeneous. Within the RS system in any given animal, approximately half of the neurons regenerate their axons (Davis & McClellan, 1994b; Jacobs et al., 1997). By 2 months after SCI, descending axons extend only a few millimeters beyond the lesion, far short of their usual targets. Total innervation by the regenerated axons below the SCI is restored to only 1–2% of normal values (Oliphint et al., 2010). Reduced descending drive is compensated for, in part, by local remodeling and regeneration of propriospinal networks that may relay motor commends to distal targets (Benthall, Hough, & McClellan, 2017; Cooke & Parker, 2009).
Despite this limited amount of regeneration, motor function is restored to seemingly normal levels. This is especially relevant to human SCI because it suggests that even modest axon regeneration can produce major functional improvement. Although the complex mechanisms coordinating regeneration have yet to be fully elucidated, it is clear they involve both environmental cues and factors intrinsic to the neurons themselves. By studying regeneration in the lamprey, investigators learn how vertebrates can repair their nervous systems after injury. This may lead to identification of novel targets and approaches for therapeutic intervention to promote recovery in human SCI patients.
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