The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.

Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.

Extracellular matrix molecule chondroitin sulfate proteoglycans (CSPGs) are highly upregulated in scar tissues and form a potent chemical barrier for CNS axon regeneration. Recent studies support that the receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member leukocyte common antigen related phosphatase (LAR) act as transmembrane receptors to mediate CSPG inhibition. PTPσ deficiency increased regrowth of ascending axons into scar tissues and descending corticospinal tract (CST) axons into the caudal spinal cord after spinal cord injury (SCI). Pharmacological LAR inhibition enhanced serotonergic axon growth in SCI mice. However, transgenic LAR deletion on axon growth in vivo and the role of LAR in regulating regrowth of other fiber tracts have not been studied. Here, we studied the role of LAR in restricting regrowth of injured descending CNS axons in deficient mice. LAR deletion increased regrowth of serotonergic axons into scar tissues and caudal spinal cord after dorsal over-hemitransection. LAR deletion also stimulated regrowth of CST fibers into the caudal spinal cord. LAR protein was upregulated days to weeks after injury and co-localized to serotonergic and CST axons. Moreover, LAR deletion improved functional recovery by increasing BMS locomotor scores and stride length and reducing grid walk errors. This is the first transgenic study that demonstrates the crucial role of LAR in restricting regrowth of injured CNS axons.

Receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member LAR act as transmembrane receptors that mediate growth inhibition of chondroitin sulfate proteoglycans (CSPGs). Inhibition of either receptor increases axon growth into and beyond scar tissues after CNS injury. However, it is unclear why neurons express two similar CSPG receptors, nor whether they use the same or different intracellular pathways. We have now studied the signaling pathways of these two receptors using N2A cells and primary neurons derived from knockout mice. We demonstrate that both receptors share certain signaling pathways (RhoA, Akt and Erk), but also use distinct signals to mediate CSPG actions. Activation of PTPσ by CSPGs selectively inactivated CRMP2, APC, S6 kinase and CREB. By contrast LAR activation inactivated PKCζ, cofilin and LKB1. For the first time, we propose a model of the signaling pathways downstream of these two CSPG receptors. We also demonstrate that deleting both receptors exhibits additive enhancement of axon growth in adult neuronal cultures in vitro. Our findings elucidate the novel downstream pathways of CSPGs and suggest potential synergy of blocking their two PTP receptors.

The large, conserved semaphorin gene family encodes axon guidance molecules in both invertebrates and vertebrates. The primitive vertebrate lamprey diverged near the time of vertebrate origins and is useful for understanding the gene duplication events that led to the increased complexity of the vertebrate genome. We characterized the sequence and expression pattern of semaphorins and their receptors genes in the sea lamprey, Petromyzon marinus. We uncovered two members of the semaphorin family in sea lamprey. The first encodes a diffusible class 3 type semaphorin protein that is most similar to the human and mouse Sema3F (71% amino acid identity). The second encodes a transmembrane class 4 type semaphorin that is most similar to mouse Sema4D and human Sema4G, with 38% amino acid identity within the Sema domain. We also identified in lamprey two members of the semaphorin receptor family, lamprey Plexin A1 and Plexin A2. Phylogenetic analysis indicates that lamprey Sema3 and Sema4 represent precursor genes existing prior to the origin of the vertebrate Sema3A-G and Sema4A-G subfamilies. Therefore, the gene duplication event that gave rise to those subfamilies must have occurred after the divergence of jawed vertebrates from jawless fish. These semaphorins and plexins are expressed in unique and dynamic patterns in lamprey spinal cord and brain during development.

Traumatic spinal cord injury (SCI) results in persistent functional deficits due to the lack of axon regeneration within the mammalian CNS. After SCI, chondroitin sulfate proteoglycans (CSPGs) inhibit axon regrowth via putative interactions with the LAR-family protein tyrosine phosphatases, PTPσ and LAR, localized on the injured axon tips. Unlike mammals, the sea lamprey, Petromyzon marinus, robustly recovers locomotion after complete spinal cord transection (TX). Behavioral recovery is accompanied by heterogeneous yet predictable anatomical regeneration of the lamprey's reticulospinal (RS) system. The identified RS neurons can be categorized as "good" or "bad" regenerators based on the likelihood that their axons will regenerate. Those neurons that fail to regenerate their axons undergo a delayed form of caspase-mediated cell death. Previously, this lab reported that lamprey PTPσ mRNA is selectively expressed in "bad regenerator" RS neurons, preceding SCI-induced caspase activation. Consequently, we hypothesized that PTPσ deletion would reduce retrograde cell death and promote axon regeneration. Using antisense morpholino oligomers (MOs), we knocked down PTPσ expression after TX and assessed the effects on axon regeneration, caspase activation, intracellular signaling, and behavioral recovery. Unexpectedly, PTPσ knockdown significantly impaired RS axon regeneration at 10 weeks post-TX, primarily due to reduced long-term neuron survival. Interestingly, cell loss was not preceded by an increase in caspase or p53 activation. Behavioral recovery was largely unaffected, although PTPσ knockdowns showed mild deficits in the recovery of swimming distance and latency to immobility during open field swim assays. Although the mechanism underlying the cell death following TX and PTPσ knockdown remains unknown, this study suggests that PTPσ is not a net negative regulator of long tract axon regeneration in lampreys.

Mitochondrial dysregulation is a key event in HIV-1 infection. Recent studies have suggested that age-related neurodegenerative disorders are associated with increased mitochondrial DNA (mtDNA) damage. As accelerated ageing was found in HIV-1 patients, we hypothesized that HIV-1 infection or HIV-1 proteins can lead to mtDNA damage. Unrepaired mtDNA impairs mitochondrial function, which can lead to oxidative stress and cell death. Investigations of mechanisms of mtDNA damage are limited by the lack of available human models.

Novel plant DING proteins (full-length 38 kDa p38SJ, and 27 kDa p27SJ) exhibit phosphatase activity and modulate HIV-1 gene transcription. Previously, we demonstrated that DING regulates HIV-1 gene transcription by dephosphorylation and inactivation of CTD RNA polymerase II, the major elongating factor of HIV-1 Long Terminal Repeats (LTR). Because the transcription of HIV-1 is controlled by several viral and cellular factors, including p65/p50 subunits of NF-κB, we hypothesized that DING phosphatase can also affect the phosphorylation and activity of p65 NF-κB, in addition to C-terminal Domain (CTD) of RNA Polymerase II (RNAPII), to suppress HIV-1 gene transcription and inhibit HIV-1 infection.

The direct visualization of axons within their native tissue environment by magnetic resonance (MR) microscopy is presented for the first time in the excised larval sea lamprey spinal cord. A home-built transverse radio frequency coil of 1.5 mm diameter was used in conjunction with a commercial 400 MHz MR microscopy system, implementing both 2-D and 3-D imaging pulse sequences. Images having nominal voxel sizes of 9x9x500 and 9x9x125 microm(3), respectively, in the lamprey spinal cord were obtained, resolving individual Mauthner and Müller axons. Furthermore, architectural changes associated with axonal degeneration were visualized in the spinal cord of one animal, excised 8 weeks after hemisection of the cord. Although the lamprey previously has not been the subject of MR microscopy investigations, these results demonstrate the method's potential for imaging this axon system, which is an important model of spinal cord injury and regeneration.

Regenerative failure of spinal axons is commonly ascribed to signaling of F-actin depolymerization and growth cone collapse by molecules such as the myelin-associated growth inhibitors. cAMP is thought to promote regeneration at least in part by neutralizing this effect, either by direct action in the growth cone or indirectly by transcriptional mechanisms. In vivo evidence for this is based mainly on partial lesion studies in which it is sometimes difficult to distinguish regeneration of injured axons from collateral sprouting by uninjured axons. Moreover, previous observations on fixed lamprey central nervous system (CNS) suggested that regeneration may not involve growth cones. To distinguish actively growing axons from static or retracting ones, fluorescently labeled large reticulospinal axons were imaged in the living, transected lamprey cord with and without application of cAMP analogs and then studied by 2-photon microscopy. Axon tip movements over 2-48-hour intervals indicated: 1) regeneration was intermittent; 2) cAMP decreased initial axon retraction and increased subsequent regeneration up to 11-fold; 3) the increase in regeneration was due to an increase in velocity of axon growth, but not in the time spent in forward movement; 4) tips of actively regenerating axons were more sharply contoured than static tips but no filopodia or lamellipodia were observed, even in db-cAMP; and 5) during active growth, axon tips contained vesicle-like inclusions and were highly immunoreactive for neurofilaments. Staining for F-actin and microtubules was variable and F-actin was not concentrated at the leading edge. Thus, cAMP accelerates regeneration of lamprey spinal axons without inducing formation of growth cones.

In mammals, there are three neurofilament (NF) subunits (NF-L, NF-M, and NF-H), but it was thought that only a single NF, NF180, exists in lamprey. However, NF180 lacked the ability to self-assemble, suggesting that like mammalian NFs, lamprey NFs are heteropolymers, and that additional NF subunits may exist. The present study provides evidence for the existence of a lamprey NF-L homolog (L-NFL). Genes encoding two new NF-M isoforms (NF132 and NF95) also have been isolated and characterized. With NF180, this makes three NF-M-like isoforms. In situ hybridization showed that all three newly cloned NFs are expressed in spinal cord neurons and in spinal-projecting neurons of the brainstem. Like NF180, there were no KSP multiphosphorylation repeat motifs in the tail regions of NF132 or NF95. NF95 was highly identical to homologous parts of NF180, sharing 2 common pieces of DNA with it. Northern blots suggested that NF95 may be expressed at very low levels in older larvae. The presence of L-NFL in lamprey CNS may support the hypothesis that as in mammals, NFs in lamprey are obligate heteropolymers, in which NF-L is a required subunit.

Knockout studies suggest that PTEN limits the regenerative capacities of CNS axons as a dominant antagonist of PI3 kinase, but the transgenic approach is not feasible for treating patients. Although application of bisperoxovanadium may block PTEN function, it is a general inhibitor of phosphotyrosine phosphatases and may target enzymes other than PTEN, causing side effects and preventing firm conclusions about PTEN inhibition on regulating neuronal growth. A pharmacological method to selectively suppress PTEN post-injury could be a valuable strategy for promoting CNS axon regeneration. We identified PTEN antagonist peptides (PAPs) by targeting PTEN critical functional domains and evaluated their efficacy for promoting axon growth. Four PAPs (PAP 1-4) bound to PTEN protein expressed in COS7 cells and blocked PTEN signaling in vivo. Subcutaneous administration of PAPs initiated two days after dorsal over-hemisection injury significantly stimulated growth of descending serotonergic fibers in the caudal spinal cord of adult mice. Systemic PAPs induce significant sprouting of corticospinal fibers in the rostral spinal cord and limited growth of corticospinal axons in the caudal spinal cord. More importantly, PAP treatment enhanced recovery of locomotor function in adult rodents with spinal cord injury. This study may facilitate development of effective therapeutic agents for CNS injuries.

Using mouse optic nerve (ON) crush as a CNS injury model, we and others have found that activation of the mammalian target of rapamycin complex 1 (mTORC1) in mature retinal ganglion cells by deletion of the negative regulators, phosphatase and tensin homologue (PTEN), and tuberous sclerosis 1 promotes ON regeneration. mTORC1 activation inhibits eukaryotic translation initiation factor 4E-binding protein (4E-BP) and activates ribosomal protein S6 kinase 1 (S6K1), both of which stimulate translation. We reasoned that mTORC1's regeneration-promoting effects might be separable from its deleterious effects by differential manipulation of its downstream effectors. Here we show that S6K1 activation, but not 4E-BP inhibition, is sufficient to promote axon regeneration. However, inhibition of 4E-BP is required for PTEN deletion-induced axon regeneration. Both activation and inhibition of S6K1 decrease the effect of PTEN deletion on axon regeneration, implicating a dual role of S6K1 in regulating axon growth.

Paralysis following spinal cord injury (SCI) is due to failure of axonal regeneration. It is believed that axon growth is inhibited by the presence of several types of inhibitory molecules in central nervous system (CNS), including the chondroitin sulfate proteoglycans (CSPGs). Many studies have shown that digestion of CSPGs with chondroitinase ABC (ChABC) can enhance axon growth and functional recovery after SCI. However, due to the complexity of the mammalian CNS, it is still unclear whether this involves true regeneration or only collateral sprouting by uninjured axons, whether it affects the expression of CSPG receptors such as protein tyrosine phosphatase sigma (PTPσ), and whether it influences retrograde neuronal apoptosis after SCI. In the present study, we assessed the roles of CSPGs in the regeneration of spinal-projecting axons from brainstem neurons, and in the process of retrograde neuronal apoptosis. Using the fluorochrome-labeled inhibitor of caspase activity (FLICA) method, apoptotic signaling was seen primarily in those large, individually identified reticulospinal (RS) neurons that are known to be "bad-regenerators." Compared to uninjured controls, the number of all RS neurons showing polycaspase activity increased significantly at 2, 4, 8, and 11 weeks post-transection (post-TX). ChABC application to a fresh TX site reduced the number of polycaspase-positive RS neurons at 2 and 11 weeks post-TX, and also reduced the number of active caspase 3-positive RS neurons at 4 weeks post-TX, which confirmed the beneficial role of ChABC treatment in retrograde apoptotic signaling. ChABC treatment also greatly promoted axonal regeneration at 10 weeks post-TX. Correspondingly, PTPσ mRNA expression was reduced in the perikaryon. Previously, PTPσ mRNA expression was shown to correlate with neuronal apoptotic signaling at 2 and 10 weeks post-TX. In the present study, this correlation persisted after ChABC treatment, which suggests that PTPσ may be involved more generally in signaling axotomy-induced retrograde neuronal apoptosis. Moreover, ChABC treatment caused Akt activation (pAkt-308) to be greatly enhanced in brain post-TX, which was further confirmed in individually identified RS neurons. Thus, CSPG digestion not only enhances axon regeneration after SCI, but also inhibits retrograde RS neuronal apoptosis signaling, possibly by reducing PTPσ expression and enhancing Akt activation.

The β-lactam antibiotic ceftriaxone (CTX) is a glutamate transporter subtype 1 (GLT-1) enhancer that reduces cocaine reinforcing efficacy and relapse in rats, but pharmacokinetic liabilities limit translational utility. An attractive alternative is clavulanic acid (CLAV), a structurally related β-lactamase inhibitor and component of FDA-approved Augmentin. CLAV retains the GLT-1 enhancing effects of CTX but displays greater oral bioavailability, brain penetrability and negligible antibacterial activity. CLAV reduces morphine conditioned place preference (CPP) and ethanol consumption in rats, but knowledge about the efficacy of CLAV in preclinical models of drug addiction remains sparse. Here, we investigated effects of CLAV (10 mg/kg, IP) on the acquisition, expression, and maintenance of cocaine CPP in rats, and on two glutamate biomarkers associated with cocaine dependence, GLT-1 and glutamate carboxypeptidase II (GCPII). CLAV administered during cocaine conditioning (10 mg/kg, IP x 4 d) did not affect the development of cocaine CPP. However, a single CLAV injection, administered after the conditioning phase, reduced the expression of cocaine CPP. In rats with established cocaine preference, repeated CLAV administration facilitated extinction of cocaine CPP. In the nucleus accumbens, acute CLAV exposure reduced GCPII protein levels and activity, and a 10-d CLAV treatment regimen enhanced GLT-1 levels. These results suggest that CLAV reduces expression and maintenance of cocaine CPP but lacks effect against development of CPP. Moreover, the ability of a single injection of CLAV to reduce both GCPII activity and protein levels, as well as expression of cocaine CPP, points toward studying GCPII as a therapeutic target of CLAV.

Mitochondrial dysfunction is postulated to be a central event in fetal alcohol spectrum disorders (FASD). People with the most severe form of FASD, fetal alcohol syndrome (FAS) are estimated to live only 34 years (95% confidence interval, 31 to 37 years), and adults who were born with any form of FASD often develop early aging. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage, hallmarks of aging, are postulated central events in FASD. Ethanol (EtOH) can cause mtDNA damage, consequent increased oxidative stress, and changes in the mtDNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1). Studies of molecular mechanisms are limited by the absence of suitable human models and non-invasive tools.

Previously, we reported that RhoA knockdown by morpholino antisense oligonucleotides (MOs), and enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) at the site of injury with chondroitinase ABC (ChABC), each can reduce retrograde neuronal apoptosis after spinal cord transection in the lamprey. To elucidate the mechanisms in neuronal survival and axon regeneration, we have investigated whether these two effects are additive in vivo. We used lampreys as a spinal cord injury model. MOs were used to knockdown RhoA and Chondroitinase ABC (ChABC) was used to digest CSPGs in vivo. Retrograde labeling, fluorochrome-labeled inhibitor of caspase activity (FLICA), immunohistochemistry, and western blots were performed to assess axonal regeneration, neuronal apoptotic signaling and Akt activation. Four treatment combinations were evaluated at 2-, 4-, and 10-weeks post-transection: (1) Control MO plus enzyme buffer (Ctrl); (2) control MO plus ChABC; (3) RhoA MO plus enzyme buffer (RhoA MO); and (4) RhoA MO plus ChABC (RhoA MO + ChABC). Consistent with our previous findings, at 4-weeks post-transection, there was less caspase activation in the ChABC and RhoA MO groups than in the Ctrl group. Moreover, the RhoA MO plus ChABC group had the best protective effect on identified reticulospinal (RS) neurons among the four treatment combinations. At 2 weeks post-transection, when axons have retracted maximally in the rostral stump and are beginning to regenerate back toward the lesion, the axon tips in the three treatment groups each were closer to the transection than those in the Ctr MO plus enzyme buffer group. Long-term axon regeneration also was evaluated for the large, individually identified RS neurons at 10 weeks post-transection by retrograde labeling. The percent regenerated axons in the RhoA MO plus ChABC group was greater than that in any of the other groups. Akt phosphorylation levels at threonine 308 was quantified in the identified RS neurons by western blots and immunofluorescence. The RhoA MO plus ChABC treatment enhanced pAkt-308 phosphorylation more than any of the other treatment groups. Although some of the effects of CSPGs are mediated through RhoA activation, some growth-inhibiting mechanisms of RhoA and CSPGs are independent of each other, so combinatorial therapies may be warranted.

Paralysis following spinal cord injury (SCI) is due to interruption of axons and their failure to regenerate. It has been suggested that the small GTPase RhoA may be an intracellular signaling convergence point for several types of growth-inhibiting extracellular molecules. Even if this is true in vitro, it is not clear from studies in mammalian SCI, whether the effects of RhoA manipulations on axon growth in vivo are due to a RhoA-mediated inhibition of true regeneration or only of collateral sprouting from spared axons, since work on SCI generally is performed with partial injury models. RhoA also has been implicated in local neuronal apoptosis after SCI, but whether this reflects an effect on axotomy-induced cell death or an effect on other pathological mechanisms is not known. In order to resolve these ambiguities, we studied the effects of RhoA knockdown in the sea lamprey central nervous system (CNS), where after complete spinal cord transection (TX), robust but incomplete regeneration of large axons belonging to individually identified reticulospinal (RS) neurons occurs, and where some RS neurons show unambiguous delayed retrograde apoptosis after axotomy. RhoA protein was detected in neurons and axons of the lamprey brain and spinal cord, and its expression was increased post-TX. Knockdown of RhoA in vivo by retrogradely-delivered morpholino antisense oligonucleotides (MOs) to the RS neurons significantly reduced retrograde apoptosis signaling in identified RS neurons post-SCI, as indicated by Fluorochrome Labeled Inhibitor of Caspases (FLICA) in brain wholemounts. In individual RS neurons, the reduction of caspase activation by RhoA knockdown began at 2weeks post-TX and was still seen at 8weeks. RhoA knockdown slowed axon retraction and possibly increased early axon regeneration in the proximal stump. The number of axons regenerating beyond the lesion more than 5mm at 10weeks post-TX also was increased. Thus RhoA knockdown both enhanced true axon regeneration and inhibited retrograde apoptosis signaling after SCI.

In mammals neurofilaments (NF) are formed by coassembly of three subunits: NFL, NFM, and NFH (light, medium, and heavy). It had been believed that lampreys have only one subunit, NF180. However, a previous study showed that NF180 could not self-assemble but could coassemble with rat NFL, suggesting the existence of additional NF subunits in lamprey. More recently, we cloned three additional NF subunits. These new subunits and NF180 have now been transfected in combinations into SW13cl.2Vim(-) cells, which lack endogenous cytoplasmic intermediate filaments. None of the subunits could self-assemble. No combination of NF subunits could form filaments in the absence of lamprey NFL (L-NFL). Assembly occurred at 28°C, but not at 37°C. L-NFL could form thick NF bundles with NF180 but not with NF132 and NF95, which formed only fine filamentous arrays. To determine which parts of the NF subunits are required for filament or bundle formation, we constructed deletion mutants of NF180 and cotransfected them with L-NFL. As with mammalian NF, only constructs with intact head and core domains could form filaments with L-NFL. However, the full length of NF180 was required to form NF bundles. As with NF180, in situ hybridization indicated that mRNA for L-NFL and NF132 was downregulated in identified reticulospinal neurons by 5 weeks after spinal cord transection, but was reexpressed at 10 weeks selectively in those neurons whose axons have a high probability of regenerating. This is consistent with a possible role of NFs in the mechanism of axon regeneration.

In many mammalian neurons, dense clusters of ion channels at the axonal initial segment and nodes of Ranvier underlie action potential generation and rapid conduction. Axonal clustering of mammalian voltage-gated sodium and KCNQ (Kv7) potassium channels is based on linkage to the actin-spectrin cytoskeleton, which is mediated by the adaptor protein ankyrin-G. We identified key steps in the evolution of this axonal channel clustering. The anchor motif for sodium channel clustering evolved early in the chordate lineage before the divergence of the wormlike cephalochordate, amphioxus. Axons of the lamprey, a very primitive vertebrate, exhibited some invertebrate features (lack of myelin, use of giant diameter to hasten conduction), but possessed narrow initial segments bearing sodium channel clusters like in more recently evolved vertebrates. The KCNQ potassium channel anchor motif evolved after the divergence of lampreys from other vertebrates, in a common ancestor of shark and humans. Thus, clustering of voltage-gated sodium channels was a pivotal early innovation of the chordates. Sodium channel clusters at the axon initial segment serving the generation of action potentials evolved long before the node of Ranvier. KCNQ channels acquired anchors allowing their integration into pre-existing sodium channel complexes at about the same time that ancient vertebrates acquired myelin, saltatory conduction, and hinged jaws. The early chordate refinements in action potential mechanisms we have elucidated appear essential to the complex neural signaling, active behavior, and evolutionary success of vertebrates.