The sea lamprey tyrosine hydroxylase: cDNA cloning and in situ hybridization study in the brain.

Lampreys belong to the oldest group of extant vertebrates, the agnathans or cyclostomes. Thus, they occupy a key phylogenetic position near the root of the vertebrate tree, which makes them important to the study of nervous system evolution. Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis and is considered a marker of catecholaminergic neurons. In the present study, we report partial cloning of the sea lamprey tyrosine hydroxylase (TH) cDNA and the pattern of TH transcript expression in the adult brain by means of in situ hybridization. Sea lamprey TH mRNA is characterized by the presence of a large untranslated sequence in the 3' end that contains a typical polyadenylation signal (ATTAAA). The deduced partial TH protein sequence presents a conserved domain with two His residues coordinating Fe(2+) binding and a conserved cofactor binding site. Neurons expressing the TH transcript were observed in the preoptic, postoptic commissure, dorsal hypothalamic, ventral hypothalamic, mammillary and paratubercular nuclei of the prosencephalon. In situ hybridization experiments also confirmed the existence of a catecholaminergic (dopaminergic) striatal population in the brain of the adult sea lamprey. A few granule-like cells in the olfactory bulbs also showed weak TH transcript expression. No cells showing TH transcript expression were observed in the rostral rhombencephalon, which suggests the absence of a locus coeruleus in the sea lamprey. Comparison of the pattern of TH mRNA expression in the prosencephalon between lampreys and teleost fishes revealed both similarities and differences. Our results suggest that the duplication of the TH gene might have occurred before the separation of agnathans and gnathostomes.

Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.

There is controversy about whether axotomized neurons undergo death or only severe atrophy after spinal cord injury (SCI) in mammals. Lampreys recover from complete spinal transection, but only about half of the severed spinal-projecting axons regenerate through the site of injury. The fates of the unregenerated neurons remain unknown, and until now death of axotomized spinal-projecting neurons has not been described in the lamprey brain. We now report that in animals allowed to survive for 12 or more weeks after spinal cord transection, several identified reticulospinal (RS) neurons were missing in Nissl-stained or neurofilament-immunostained brain whole mounts. At earlier times, these neurons were swollen and pale in Nissl-stained preparations. Retrograde fluorescent labeling from the site of transection combined with TUNEL histochemistry suggested that neuronal death, including that of the identified RS neurons, began in animals 4 weeks posttransection, reaching a peak at 12-16 weeks. This was not seen in untransected animals. The TUNEL positivity suggests that some cells were dying by apoptosis. Of special interest, among the identified neurons, this delayed cell death was restricted to neurons that at earlier posttransection times have a low probability of regeneration. These data show that SCI induces delayed cell death in lamprey spinal-projecting neurons and suggest that the reason why some neurons are "bad regenerators" is that they are already undergoing apoptotic cell death. Thus protection from apoptosis may be necessary in order to enhance axonal regeneration after SCI.

In vivo transfection of lamprey brain neurons by gene gun delivery of DNA.

The lamprey has been used extensively in studies of CNS axon regeneration. Progress in determining molecular mechanisms involved in regeneration will require the ability to manipulate expression of target genes or to introduce new genes, but in vivo neuronal transfection has posed difficulties in the mature intact nervous system of vertebrates, including the lamprey. In this paper we report successful transfection of neurons in the brain of living lampreys by means of a hand-held Helios Gene Gun. Particle-mediated ("gene gun") gene transfer has been applied to a variety of cell and tissue types but although it has been used in brain slices and dissociated cultured neurons, to our knowledge it has not been reported as a method for transfection of brain cells in a living animal. Gold particles coated with plasmids containing the gene for the reporter beta-galactosidase were propelled by helium at 150--200 psi toward the exposed floor of the 4th ventricle. Transfected animals were examined by X-gal histochemistry at various recovery times. beta-glactosidase activity was detected as early as 2 days after gene transfer and lasted for at least 6 weeks, the longest time studied. Transgene expression lasted longer in neurons than in glia. The expression product was transported anterogradely into reticulospinal axons and by 6 weeks could be traced into the spinal cord for 8--10 mm caudal to the obex. This raises the possibility of identifying the growth cones of developing or regenerating axons belonging to transfected neurons in functional studies of manipulated genes.

In situ hybridization in wholemounted lamprey spinal cord: localization of netrin mRNA expression.

The lamprey has been used as a model for the study of vertebrate neuronal circuitry and spinal cord regeneration. One of the advantages of this preparation is the ability to view the entire CNS in wholemount, including several identified neurons and neuron groups. However, because of difficulties in penetration of molecular reagents past the dense meninx primitiva and glia limitans, there has been no reliable method for in situ hybridization in spinal cord wholemounts. We now describe a protocol that accomplishes this while preserving the structural integrity of the cord. In order to enhance penetration of probes and antibodies, the m. primitiva was surgically stripped from the spinal cord after incubation of the fresh tissue in 0.1% collagenase I. Additional modifications that enhanced hybridization signal include (a) increasing the amount of Tween-20 in the hybridization mix to 2%, instead of the typical 0.2%; (b) carrying out the hybridization for 36 h and applying the anti-digoxigenin antibody to the tissue for 48 h. Using this protocol, we showed that netrin mRNA is expressed in dorsal cells, in medium sized neurons of the lateral gray matter and in the glial/ependymal cells of the spinal cords of lampreys. This method will help to study the expression of molecules of interest in identified neurons and neuronal groups without the need for serial section reconstruction.

Expression of the netrin receptor UNC-5 in lamprey brain: modulation by spinal cord transection.

The sea lamprey recovers from spinal cord transection by a process that involves directionally specific regeneration of axons. The mechanisms underlying this specificity are not known, but they may involve molecular cues similar to those that guide the growth of spinal cord axons during development, such as netrins and semaphorins. To test the role of guidance cues in regeneration, we cloned netrin and its receptor UNC-5 from lamprey central nervous system (CNS) and studied their expression after spinal cord transection. In situ hybridization showed that (1) mRNA for netrin is expressed in the spinal cord, primarily in neurons of the lateral gray matter and in dorsal cells; (2) mRNA for UNC-5 is expressed in lamprey reticulospinal neurons; (3) following spinal cord transection, UNC-5 message was dramatically downregulated at two weeks, during the period of axon dieback; (4) UNC-5 message was upregulated at three weeks, when many axons are beginning to regenerate; and (5) axotomy-induced expression of UNC-5 occurred primarily in neurons whose axons regenerate poorly. Because the UNC-5 receptor is thought to mediate the chemorepellent effects of netrins, netrin signaling may play a role in limiting or channeling the regeneration of certain neurons. These data strengthen the rationale for studying the role of developmental guidance molecules in CNS regeneration.

Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.

During regeneration of lamprey spinal axons, growth cones lack filopodia and lamellipodia, contain little actin, and elongate much more slowly than do typical growth cones of embryonic neurons. Moreover, these regenerating growth cones are densely packed with neurofilaments (NFs). Therefore, after spinal hemisection the time course of changes in NF mRNA expression was correlated with the probability of regeneration for each of 18 identified pairs of reticulospinal neurons and 12 cytoarchitectonic groups of spinal projecting neurons. During the first 4 weeks after operation, NF message levels were reduced dramatically in all axotomized reticulospinal neurons, on the basis of semiquantitative in situ hybridization for the single lamprey NF subunit (NF-180). Thereafter, NF expression returned toward normal in neurons whose axons normally regenerate beyond the transection but remained depressed in poorly regenerating neurons. The recovery of NF expression in good regenerators was independent of axon growth across the lesion, because excision of a segment of spinal cord caudal to the transection site blocked regeneration but did not prevent the return of NF-180 mRNA. The early decrease in NF mRNA expression was not accompanied by a reduction in NF protein content. Thus the axotomy-induced loss of most of the axonal volume resulted in a reduced demand for NF rather than a reduction in volume-specific NF synthesis. We conclude that the secondary upregulation of NF message during axonal regeneration in the lamprey CNS may be part of an intrinsic growth program executed only in neurons with a strong propensity for regeneration.

Cytoskeletal changes correlated with the loss of neuronal polarity in axotomized lamprey central neurons.

Axotomy within 500 microm of the soma (close axotomy) causes identified neurons (anterior bulbar cells or ABCs) in the lamprey hindbrain to lose their normal polarity and regenerate axons ectopically from dendritic tips, while axotomy at more distal sites (distant axotomy) results in orthotopic axonal regeneration from the axon stump. We performed immunocytochemical, electron microscopic and in situ hybridization analyses comparing ABCs subjected to close and distant axotomy to elucidate the mechanism by which neuronal polarity is lost. We show that polarity loss in ABCs is selectively and invariably preceded and accompanied by the following cellular changes: (1) a loss of many dendritic microtubules and their replacement with neurofilaments, (2) a loss of immunostaining for acetylated tubulin in the soma and proximal dendrites, and (3) an increase of immunostaining for phosphorylated neurofilaments in the distal dendrites. We also show that these changes do not depend on either the upregulation or spatial redistribution of neurofilament message, and thus must involve changes in the routing of neurofilament protein within axotomized ABCs. We conclude that close axotomy causes dendrites to undergo axonlike changes in the mechanisms that govern the somatofugal transport of neurofilament protein, and suggest that these changes require the reorganization of dendritic microtubules. We also suggest that the bulbous morphology and lack of f-actin in the tips of all regenerating sprouts supports the possibility that axonal regeneration in the lamprey CNS does not involve actin-mediated "pulling" of growth cones, but depends instead on the generation of internal extrusive forces.

Neurofilament spacing, phosphorylation, and axon diameter in regenerating and uninjured lamprey axons.

It has been postulated that phosphorylation of the carboxy terminus sidearms of neurofilaments (NFs) increases axon diameter through repulsive electrostatic forces that increase sidearm extension and interfilament spacing. To evaluate this hypothesis, the relationships among NF phosphorylation, NF spacing, and axon diameter were examined in uninjured and spinal cord-transected larval sea lampreys (Petromyzon marinus). In untransected animals, axon diameters in the spinal cord varied from 0.5 to 50 microns. Antibodies specific for highly phosphorylated NFs labeled only large axons (> 10 microns), whereas antibodies for lightly phosphorylated NFs labeled medium-sized and small axons more darkly than large axons. For most axons in untransected animals, diameter was inversely related to NF packing density, but the interfilament distances of the largest axons were only 1.5 times those of the smallest axons. In addition, the lightly phosphorylated NFs of the small axons in the dorsal columns were widely spaced, suggesting that phosphorylation of NFs does not rigidly determine their spacing and that NF spacing does not rigidly determine axon diameter. Regenerating neurites of giant reticulospinal axons (GRAs) have diameters only 5-10% of those of their parent axons. If axon caliber is controlled by NF phosphorylation via mutual electrostatic repulsion, then NFs in the slender regenerating neurites should be lightly phosphorylated and densely packed (similar to NFs in uninjured small caliber axons), whereas NFs in the parent GRAs should be highly phosphorylated and loosely packed. However, although linear density of NFs (the number of NFs per micrometer) in these slender regenerating neurites was twice that in their parent axons, they were highly phosphorylated. Following sectioning of these same axons close to the cell body, axon-like neurites regenerated ectopically from dendritic tips. These ectopically regenerating neurites had NF linear densities 2.5 times those of uncut GRAs but were also highly phosphorylated. Thus, in the lamprey, NF phosphorylation may not control axon diameter directly through electrorepulsive charges that increase NF sidearm extension and NF spacing. It is possible that phosphorylation of NFs normally influences axon diameter through indirect mechanisms, such as the slowing of NF transport and the formation of a stationary cytoskeletal lattice, as has been proposed by others. Such a mechanism could be overridden during regeneration, when a more compact, phosphorylated NF backbone might add mechanical stiffness that promotes the advance of the neurite tip within a restricted central nervous system environment.

Developmental increases in expression of neurofilament mRNA selectively in projection neurons of the lamprey CNS.

Neurofilaments of the sea lamprey are unique in being homopolymers of a single subunit (NF-180). Digoxigenin-labeled RNA probes complementary to NF-180 were used to determine the distribution and timing of expression of neurofilament message in the brain and spinal cord of the lamprey. In the brainstem, detection of NF-180 mRNA was restricted to neurons with axons projecting to the spinal cord or the periphery. The majority of brainstem neurons, whose axons project locally, did not express NF-180 within the detection limits of this technique. NF-180-positive neurons included cells with a wide range of axon diameters, suggesting neurofilament mRNA expression was linked to axon length rather than caliber. To further evaluate this hypothesis, expression was studied in animals of different developmental stages between larvae and adults. In younger (shorter) larvae, the large Mauthner and rhombencephalic Müller cells did not express NF-180 mRNA, even though their axons are among the largest caliber in the animal and extend the entire length of the spinal cord. In contrast, many other reticulospinal neurons, whose axons are smaller in diameter than those of the Müller and Mauthner cells, expressed NF-180 message throughout larval development. Furthermore, neurons of the cranial motor nuclei did not express NF-180 until later developmental stages and the extraocular motor neurons did not label until metamorphosis. Therefore, while detectable neurofilament mRNA expression in the lamprey is restricted to neurons with long axons, its expression in this population of neurons appears to be developmentally regulated by factors still not determined. It is postulated that need for NF message is determined by a balance between the volume of axon to be filled and the rate of turnover of NF in that axon.

Metamorphosis of spinal-projecting neurons in the brain of the sea lamprey during transformation of the larva to adult: normal anatomy and response to axotomy.

The spinal projecting system of the sea lamprey (Petromyzon marinus) has been used extensively in studies of axonal regeneration in both larvae and adults. However, little is known about the changes that are undergone by this system during metamorphosis. In order to determine the developmental changes in the size of the descending spinal projection and in the morphology of its neurons, larval, transforming, and adult lamprey brains were labeled by retrograde transport of horseradish peroxidase (HRP) injected into the spinal cord at 25% of body length. Examination of brain wholemount preparations revealed that the total number of labeled neurons doubled during metamorphosis. Most of this increase could be explained by elongation of reticulospinal axons from the rostralmost segments of the spinal cord to locations caudal to the injection site. There were no additions or deletions of either identified reticulospinal neurons or of reticulospinal nuclear groups between the larval and the adult stages. The proportions of Müller and Mauthner cells that were labeled reached a maximum of 93% during the early stages of metamorphosis. Axons of these neurons are known to project almost the entire length of the cord, even in larvae. Therefore, the efficiency of retrograde transport appears to be greater during metamorphosis than during larval or adult stages. While changes in efficiency of retrograde transport could account for some of the apparent increase in reticulospinal neuron numbers between larvae and animals undergoing metamorphosis, this could not contribute to the further increase in the apparent size of the reticulospinal system in the adult, since efficiency of retrograde labeling in these animals was lower than that at earlier stages. With retrograde labeling, a significant increase was seen in the profusion of dendritic arborization of some Müller and Mauthner cells during the early stages of metamorphosis. This correlated with an increase in the incidence of extreme axonal die-back, as indicated by the presence of retraction bulbs within the brainstem. However, intracellular injection of Neurobiotin in untransected animals showed similar degrees of dendritic arborization at all examined stages of development. Therefore, the dendritic profusion did not reflect developmental changes in neuronal morphology but rather reflected an increased sensitivity to axotomy during metamorphosis. We conclude that, during the transformation of the lamprey from the large larval to the adult form, there is little change in either the size or the dendritic morphology of the identified giant reticulospinal neurons. With respect to the smaller reticulospinal neurons, the distance of projection of many of their axons increases during metamorphosis, but there is very little increase in the number of reticulospinal neurons.

Glial cells of the lamprey nervous system contain keratin-like proteins.

Lamprey axons regenerate following spinal cord transection despite the formation of a glial scar. As we were unable to detect a lamprey homologue of glial fibrillary acidic protein (GFAP), a major constituent of astrocytes, we studied the composition of intermediate filament (IF) proteins of lamprey glia. Monoclonal antibodies (mAbs) were raised to lamprey spinal cord cytoskeletal extracts and these mAbs were characterized by using Western blotting and immunocytochemistry. On two-dimensional (2-D) Western blots, five of the mAbs detected three major IF polypeptides in the molecular weight (MW) range of 45-56 kD. Further studies were conducted to determine the relationship between the lamprey glial-specific antigen and other mammalian IF proteins. Antikeratin 8 antibody recognized two of the three polypeptides. Several of the glial-specific mAbs reacted with human keratins 8 and 18 on Western blots. Keratin-like immunoreactivity was found in all parts of the central and peripheral nervous systems in both larval and adult lampreys. The immunocytochemical staining patterns of glial-specific mAbs were indistinguishable on lamprey spinal cord sections. However, on brain sections, two distinct patterns were observed. A subset of mAbs stained only a few glial fibers in the brain, whereas others stained many more brain glia, particularly the ependymal cells. The former group of mAbs recognized only the two lower MW polypeptides on 2-D Western blots, but the latter group of mAbs recognized all three major IF polypeptides. This correlation is supported by the observation that the highest MW IF polypeptide has an increased level of expression in the brain relative to the spinal cord. Thus, in the lamprey, the glial cells of both spinal cord and brain express molecules similar to simple epithelial cytokeratins, but their IFs may contain these keratins in different stoichiometric proportions. The widespread presence in the lamprey of primitive glial cells containing keratin-like intermediate filaments may have significance for the extraordinary ability of lamprey spinal axons to regenerate.

The single lamprey neurofilament subunit (NF-180) lacks multiphosphorylation repeats and is expressed selectively in projection neurons.

The lamprey is considered the most primitive living vertebrate and its neurofilaments (NFs) are unique in being homopolymers of a single 180 kDa subunit (NF-180). Previous immunologic studies have suggested that the sidearm of NF-180 is highly phosphorylated selectively in the largest diameter axons. We report in this study the isolation and characterization of cDNA clones encoding the NF-180 lamprey protein. In situ hybridization with digoxigenin-labeled cRNA revealed NF-180 message exclusively in neurons with long axons, such as reticulospinal neurons and cranial motor neurons. The core of NF-180 was similar in structure to those of mammalian neurofilaments, but surprisingly, the carboxy sidearm lacked the multiphosphorylation repeats characteristic of higher vertebrate and invertebrate neurofilaments. Overall there was a paucity of potential phosphorylation sites in the NF-180 carboxy-terminus compared to NF-M and NF-H of mammals, fish and squid. This, along with the highly acidic nature of the NF-180 sidearm, makes it unlikely that phosphorylation of sidearm residues regulates interfilament spacing and axon diameter through global electrostatic repulsion of the carboxy-terminus away from the filament backbone. Furthermore, the expression of a single neurofilament subunit in the lamprey that is most similar to the NF-M of higher vertebrates suggests that all three mammalian neurofilament subunits evolved from a single NF-M-like precursor.

Structure of reticulospinal axon growth cones and their cellular environment during regeneration in the lamprey spinal cord.

The large larval sea lamprey is a primitive vertebrate that recovers coordinated swimming following complete spinal transection. An ultrastructural study was performed in order to determine whether morphologic features of regenerating axons and their cellular environment would provide clues to their successful regeneration compared to their mammalian counterparts. Three larval sea lampreys were studied at 3, 4 and 11 weeks following complete spinal transection and compared with an untransected control. Müller and Mauthner cells or their giant reticulospinal axons (GRAs) were impaled and injected with horseradish peroxidase (HRP). Alternating thick and thin sections were collected for light and electron microscopy. A total of 9 neurites were examined. At all times, growth cones of GRAs differed from those of cultured mammalian neurons in being packed with neurofilaments and in lacking long filopodia, suggesting possible differences in the mechanisms of axon outgrowth. Morphometric analysis suggested that GRA growth cones contact glial fibers disproportionately compared to the representation of glial surface membranes in the immediate environment of these growth cones. No differences were found between glial cells in regenerating spinal cords and those of untransected control animals with regard to the size of the cell body and nucleus and the packing density of their intermediate filaments. Glial fibers in control animals and glial fibers located far from a transection were oriented transversely. Glial cells adjacent to the transection site sent thickened, longitudinally oriented processes into the blood clot at the transection site. These longitudinal glial processes preceded the regenerating axons. Desmosomes were observed on glia adjacent to the lesion but were scarce in the lesion during the first four weeks post-transection. These findings suggest that longitudinally oriented glial fibers may serve as a bridge along which axons can regenerate across the lesion. The presence of desmosomes might prevent migration of astrocytes near the transection, thus stabilizing the glial bridge.

A method for in situ hybridization in wholemounted lamprey brain: neurofilament expression in larvae and adults.

Nonisotopic in situ hybridization (NISH) using both cDNA and cRNA probes is rapidly gaining favor over autoradiographic methods. Typically, either biotinylated or digoxigenin-labeled probes are used to detect mRNAs in sectioned tissue or in cultured cells. With a few exceptions, most applications of NISH in wholemount preparations have been limited to Drosophila embryos. A protocol developed for NISH in whole adult Drosophila CNS was extended to wholemounted larval and adult lamprey brain preparations. Digoxigenin-labeled RNA probes were transcribed from cloned fragments of a lamprey neurofilament (NF180) cDNA. Hybridization with these probes, and comparisons with Nissl-stained wholemounts and wholemounts retrogradely labeled by injections of tracer into the spinal cord, demonstrated that NF180 mRNA was expressed in only a subset of neurons in the lamprey CNS. These included primarily neurons with long axons that project out of the brainstem, e.g., reticulospinal neurons and cranial motor neurons. Metamorphosis from the larval to the adult form was accompanied by an increase in the number of neurons expressing NF180 and in the apparent level of NF expression as judged by the intensity of labeling. For example, in the oculomotor and trochlear nuclei, expression of NF180 was seen in postmetamorphic young adult lampreys but not in larvae. In the trigeminal motor nucleus, both the number of neurons expressing NF180 and the intensity of the hybridization labeling increased with metamorphosis. The ability to do NISH in lamprey brain wholemounts eliminates the need for serial reconstructions and thus facilitates the study of selected gene expression during metamorphosis and regeneration.