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Logo of jnPublished ArticleArchivesSubscriptionsSubmissionsContact UsJournal of NeurophysiologyAmerican Physiological Society
J Neurophysiol. Oct 2009; 102(4): 2477–2484.
Published online Aug 19, 2009. doi:  10.1152/jn.00446.2009
PMCID: PMC2775388

Zebrafish Motor Neuron Subtypes Differ Electrically Prior to Axonal Outgrowth

Abstract

Different muscle targets and transcription factor expression patterns reveal the presence of motor neuron subtypes. However, it is not known whether these subtypes also differ with respect to electrical membrane properties. To address this question, we studied primary motor neurons (PMNs) in the spinal cord of zebrafish embryos. PMN genesis occurs during gastrulation and gives rise to a heterogeneous set of motor neurons that differ with respect to transcription factor expression, muscle targets, and soma location within each spinal cord segment. The unique subtype-specific soma locations and axonal trajectories of two PMNs—MiP (middle) and CaP (caudal)—allowed their identification in situ as early as 17 h postfertilization (hpf), prior to axon genesis. Between 17 and 48 hpf, CaPs and MiPs displayed subtype-specific electrical membrane properties. Voltage-dependent inward and outward currents differed significantly between MiPs and CaPs. Moreover, by 48 hpf, CaPs and MiPs displayed subtype-specific firing behaviors. Our results demonstrate that motor neurons that differ with respect to muscle targets and transcription factor expression acquire subtype-specific electrical membrane properties. Moreover, the differences are evident prior to axon genesis and persist to the latest stage studied, 2 days postfertilization.

INTRODUCTION

During initial stages of postmitotic differentiation, substantial changes in voltage-gated ion currents occur in vertebrate motor neurons, even prior to synapse formation. In many vertebrate spinal motor neurons, inward and outward current amplitudes progressively increase, reducing action potential durations and enabling repetitive firing (Gao and Ziskind-Conhaim 1998; McCobb et al. 1990; O'Dowd et al. 1988; Spitzer and Lamborghini 1976; Ziskind-Conhaim 1988). Even though transcription factor expression and innervation patterns reveal different motor neuron subtypes, previous studies of motor neuron electrical differentiation have considered motor neurons as one population. This is primarily explained by the fact that traditional indicators of neuronal subtypes (e.g., expression of marker mRNAs or protein) require fixation protocols that preempt physiological study, especially in situ (Gao and Ziskind-Conhaim 1998; Krieger and Sears 1988; McCobb et al. 1990; Spitzer and Lamborghini 1976). In contrast, dye-labeling of neurons allows in situ identification of neuronal subtypes, on the basis of morphology and innervation targets, and is compatible with physiological recording methods (Buss et al. 2003; Saint-Amant and Drapeau 2000). However, this method is not useful to identify neuronal subtypes on the basis of morphology at stages prior to axon genesis. Thus previous work has provided little insight into whether motor neuron subtypes differ from or resemble each other with respect to electrical membrane properties at initial stages of postmitotic differentiation.

In the embryonic zebrafish spinal cord, each hemisegment has three to four early-born primary motor neurons (PMNs). PMNs differ with respect to soma location within each hemisegment of the embryonic spinal cord. In rostral–caudal order of the positions of their soma in each hemisegment, the PMN subtypes are RoP (rostral), MiP (middle), and CaP (caudal); occasionally, a fourth variable PMN, VaP, is present in the vicinity of CaP (Eisen 1991; Eisen et al. 1986; Myers et al. 1986; Westerfield et al. 1986). PMN axons follow a common initial pathway to exit the spinal cord but project to different muscle targets soon after they reach the periphery.

We have taken advantage of the stereotypic locations of PMN subtypes to identify them in situ for physiological study. In combination with the Tg(hb9:GFP) transgenic line that expresses green fluorescent protein (GFP) in motor neurons, we were able to identify different motor neuron subtypes in situ prior to axon genesis as well as after synapse formation (Flanagan-Steet et al. 2005). Thus the zebrafish model system overcomes many previous obstacles that prevented subtype-specific analysis of differentiation of motor neuron electrical excitability.

Here, we test whether and when electrical excitability differs among motor neuron subtypes that are identifiable in situ. We focus on the MiP and CaP PMN subtypes and characterize their electrical membrane properties between 17 and 48 h postfertilization (hpf), a period that extends from stages just prior to axon genesis to well after synapse formation. Similar to other vertebrate motor neurons, MiP and CaP electrical membrane properties change significantly during this developmental period. However, by being able to distinguish MiP from CaP, we found that each PMN subtype has a stereotypic phenotype for electrical excitability. Moreover, the differences were evident prior to axon genesis even before the PMNs first fire action potentials. Overall, the results indicate that electrical excitability serves as an early read-out of subtype-specific differentiation of motor neurons.

METHODS

Animals

Transgenic Tg(hb9:GFP) zebrafish (Danio rerio) embryos were bred according to guidelines outlined in The Zebrafish Book (Westerfield 1995). Embryos were incubated at 28.5°C in embryo media (EM, in mM: 130 NaCl, 0.5 KCl, 0.02 Na2HPO4, 0.04 KH2PO4, 1.3 CaCl2, 1.0 MgSO4, and 0.4 NaH2CO3) and staged according to external morphology (Kimmel et al. 1995). Embryos were studied at 17, 22–24 (24), and 44–48 (48) hpf.

Embryo preparation

Zebrafish embryos were mounted using veterinarian suture glue (3M Vetbond, Revival Animal Health, Orange City, IA) onto a Sylgard-coated recording chamber (Dow Corning, Midland, MI) and then killed in the presence of 0.01% tricaine (ethyl 3-aminobenzoate methanesulfonate salt) prior to trunk skin removal. After washing with Ringer solution (in mM: 145 NaCl, 3 KCl, 1.8 CaCl2, and 10 HEPES; pH 7.4), embryos were transferred to external recording solution. Blunt dissection with polished borosilicate glass electrodes removed muscle and meninges and exposed PMNs.

Electrophysiology

Whole cell current- and voltage-clamp recordings were obtained from PMNs using patch electrodes (2.5- to 3.5-MΩ resistance) and an Axopatch-200B amplifier (Axon Instruments, Molecular Devices, Sunnyvale, CA). Electrodes were made using a P-97 microelectrode puller (Sutter Instruments, Novato, CA) and filled with intracellular pipet solution (in mM: 135 KCl, 10 EGTA, and 10 HEPES; pH 7.4).

In the majority of cases, the capacitative transient had two components, a large fast one and a smaller slow one, representing the proximal somal and distal axonal compartments. It was not possible to obtain good space clamp of the axonal compartment and, consequently, analysis of voltage-clamp data was limited to peak current densities.

In the Tg(hb9:GFP) line, not only later-born motor neurons and interneurons but also PMNs express GFP at stages subsequent to MiP and CaP axon genesis. Accordingly, for 24- to 48-hpf recordings, we included a fluorescent dye (60–100 μM Alexa Fluor 594; Invitrogen, Eugene, OR) in the pipet solution to identify MiP and CaP on the basis of their stereotypical dorsal and ventral axonal projections.

Current-clamp experiments were performed to determine evoked electrical responses to depolarizing current injections at room temperature, using a bath solution containing (in mM): 125 NaCl, 2 KCl, 10 CaCl2, and 5 HEPES; pH 7.4. We also performed current-clamp recordings in the absence of current stimulation to evaluate the properties of spontaneous action potentials. To immobilize the fish during recordings, we added the muscle sodium channel blocker μ-conotoxin GIIIA (~30 to 120 nM; Bachem, Torrance, CA) to the bath. Steady injection of current set the PMN membrane potential to (−60 to −75 mV) and transient current injections (1 or 100 ms) ranging between 0.05 and 0.5 nA were applied to elicit single or repeated firing. Current injections were applied in 0.05-nA increments, with a 1-s recovery period following each stimulation. The rise time and rate of rise were calculated for the interval between threshold and peak amplitude of the action potential. Decay time and slope were calculated from the peak of the action potential to the time of 50% decay. The duration of the action potential was calculated as the time between threshold and 50% decay.

Voltage-clamp recordings were performed to study total whole cell outward and inward currents underlying CaP and MiP firing properties. The same bath and pipet solutions used for current clamp, as described earlier, were used for these recordings. Under these conditions, the outward currents reflect the net outward currents [voltage-dependent (IKv) as well as calcium-dependent (IKC) currents], referred to as IKv/Ca, and inward currents [the net sodium (INa) and calcium (ICa) currents], referred to as INa/Ca. Voltage steps from −40 to 110 mV in 10-mV increments were applied from a holding potential of −80 mV. Current amplitudes were measured as either the steady state for outward IKv/Ca or the peak for inward INa/Ca. To allow comparison of data among PMN subtypes, current amplitudes were normalized to cell surface area, based on cell capacitance. Data are presented as current densities (pA/μm2).

To isolate IKv, calcium current was blocked by substitution of calcium by cobalt in the external bath solution (in mM: 80 NaCl, 3 KCl, 5 MgCl2, 10 CoCl2, and 5 HEPES; pH 7.4). Additionally, to block voltage-gated sodium currents about 500 nM tetrodotoxin (TTX; Calbiochem, Gibbstown, NJ) was added to the bath solution. The intracellular pipet solution and voltage step protocol described earlier were used for these recordings.

Sodium currents were isolated by blocking calcium and potassium currents with appropriate blockers in the external bath solution (in mM: 125 NaCl, 3 KCl, 20 TEA-Cl, 10 CoCl2, and 10 HEPES; pH 7.2). The pipet solution contained (in mM): 125 CsCl, 10 NaCl, 10 EGTA, and 10 HEPES; pH 7.2. Sodium currents were activated by a series of voltage steps ranging between −60 and 90 mV following brief hyperpolarization to −100 mV. Following each test pulse the holding potential was returned to −80 mV.

A P/8 protocol was used for subtraction of passive leak and capacitative transients. Clampex 9.2 (Molecular Devices, Sunnyvale, CA) was used for data acquisition and analysis was performed with Clampfit 9.2 (Molecular Devices) and Axograph X (AxoGraph Scientific, Sydney, Australia). Series resistance was compensated to 70–80%.

Data presentation and statistical analysis

Data are presented as means ± SE. Statistical analysis was done using the Student's t-test unless otherwise indicated (Instat 3 software, GraphPad Software, La Jolla, CA). For data showing a non-Gaussian distribution, the nonparametric Mann–Whitney test was used. Values of P ≤ 0.05 were considered to be indicative of statistical significance.

RESULTS

Dye labeling and the Tg(hb9:GFP) line allowed reliable in situ identification of CaPs and MiPs

The PMN subtypes CaP and MiP differ at the molecular and morphological levels (Appel et al. 1995; Myers et al. 1986; Tokumoto et al. 1995). However, it is not known whether CaPs and MiPs also show subtype-specific excitability properties. We tested whether membrane excitability serves as a read-out of motor neuron subtype-specific differentiation.

Our electrophysiological studies required unambiguous identification of CaPs and MiPs in 17- to 48-hpf zebrafish embryos. For this purpose, we took advantage of the Tg(hb9:GFP) transgenic line that uses the hb9 promoter to drive expression of the reporter transgene, GFP, in motor neurons and an interneuron subset (Flanagan-Steet et al. 2005). We were able to unambiguously identify spinal neurons as CaP or MiP at 17, 24, and 48 hpf by fluorescent dye labeling cells while recording from Tg(hb9:GFP) embryos (Fig. 1).

Fig. 1.
Reliable identification of primary motor neuron (PMN) subtypes allows in situ characterization of caudal and middle primary motor neuron (CaP and MiP, respectively) electrical properties at early embryonic stages. A: PMN electrical properties were studied ...

Between 17 and 48 hpf, PMN axonal outgrowth differs along the rostrocaudal gradient, being most advanced rostrally (Flanagan-Steet et al. 2005; Myers et al. 1986). Accordingly, for each stage studied, we restricted our recordings to limited regions of the developing spinal cord. At 17 hpf, PMN axons initiate outgrowth and begin to exit the spinal cord, a process that is pioneered by the CaP axon (Myers et al. 1986). We limited recordings at this stage to hemisegments, with PMNs that either had no axons or those that did not yet exit the spinal cord, as demonstrated by GFP and fluorescent dye labeling.

At 24 hpf, the region of interest comprised the eight hemisegments rostral to the most caudal portion of the yolk extension. In 48 hpf embryos, we focused on the eight hemisegments immediately caudal to the end of the yolk extension. Compared with 24-hpf embryos, a greater number of GFP-expressing neurons were present in every hemisegment of 48-hpf embryos, presumably due to the later genesis of secondary motor neurons (Myers et al. 1986).

As early as 17 hpf, CaPs and MiPs displayed different membrane excitability properties

Between 15 and 17 hpf, PMNs initiate terminal differentiation and, by 17 hpf, CaPs and MiPs express distinguishing sets of transcription factors and have stereotypic somal locations within each spinal cord hemisegment (Appel et al. 1995; Eisen 1991; Tokumoto et al. 1995). At 17 hpf, depolarizing current injections did not elicit action potentials in either CaPs or MiPs (not shown). Further, at 17 hpf, net-inward currents (INa/Ca) were not detected in either CaPs or MiPs, consistent with their inability to fire action potentials (not shown). These findings raised the possibility that CaPs and MiPs are initially similar in their electrical membrane properties. However, at 17 hpf, net-outward currents (IKv/Ca) of small amplitude could be detected in both PMNs. Further, CaP IKv/Ca density was sevenfold greater than that of MiPs (Fig. 2 A), suggesting that as early as 17 hpf, CaPs and MiPs display different electrical membrane properties.

Fig. 2.
CaP and MiP show subtype-specific excitability properties during early stages of differentiation. A: At 17 hpf, the electrical membrane properties of CaPs and MiPs already differ. Net-outward current density was greater for CaPs than that for MiPs (* ...

At 24 hpf, CaPs and MiPs fired action potentials with different properties

At 24 hpf, CaP and MiP axons have innervated their ventral and dorsal muscle targets, respectively. In contrast to 17 hpf, CaPs and MiPs fired single action potentials at 24 hpf. However, the waveform of action potentials fired by CaPs versus MiPs differed significantly (Fig. 2B). For evoked action potentials, the rates of rise and decay of CaP action potentials were 2.3- and 2.8-fold greater, respectively, than those evoked from MiPs (Fig. 2C). Consequently, at 24 hpf, durations of CaP action potentials were briefer than those recorded from MiPs.

The differences in CaP and MiP firing behaviors most likely reflect subtype-specific isoforms and/or number of ion channels underlying the inward and outward conductances. To identify the molecular basis for PMN subtype-specific firing properties, we characterized their whole cell outward and inward currents (INa/Ca and IKv/Ca, respectively).

Depolarization-activated inward currents underlie the upstroke of action potentials. For both CaPs and MiPs, inward currents developed soon after 17 hpf. At 24 hpf, the amplitude of inward currents differed between PMNs (Fig. 2D) with CaP INa/Ca density being 2.6-fold greater than that of MiPs (Fig. 2E).

Similar to the net-inward currents, at 24 hpf, CaPs had larger net-outward current amplitudes than those of MiPs (Fig. 2D). The differences in magnitudes of net-outward current density between CaPs and MiPs were in the same range as those observed for net-inward currents (Fig. 2E). For example, at 24 hpf, CaP IKv/Ca density was 2.8-fold greater than that of MiPs.

Inactivation of outward currents may influence membrane repolarization, thereby affecting action potentials and repetitive firing properties. We compared the extent of time-dependent inactivation of CaP and MiP outward currents. Both PMN subtypes displayed time-dependent decreases in outward current amplitude, or inactivation, during a sustained depolarization. Although the extent of inactivation observed differed between the two PMN subtypes (Fig. 2F), the significance is not clear due to voltage-clamp errors (see methods).

PMN electrical properties differ at 48 hpf

At 48 hpf, brief current injections continued to evoke single action potentials from both CaPs and MiPs (Fig. 3 A). However, in contrast to waveforms recorded at 24 hpf, durations of CaP and MiP action potentials at 48 hpf were 3.3- (*P = 0.006) and 6.5-fold (*P = 0.005) briefer, respectively.

Fig. 3.
At 48 hpf, CaP and MiP firing properties and underlying currents continue to differ. A: at 48 hpf, brief current injections elicited single action potentials in CaPs (black trace) and MiPs (gray trace). B: during an evoked action potential, CaP membranes ...

Developmental changes in both CaP and MiP inward and outward currents likely underlie the shortening of action potential duration that occurs between 24 and 48 hpf. For example, INa/Ca density increased 2.9-fold in CaPs (*P = 0.0001) and fourfold in MiPs (*P = 0.003; Mann–Whitney test). Similarly, between 24 and 48 hpf, IKv/Ca density increased 3.4-fold in CaPs (*P = 0.0001) and 6.2-fold in MiPs (*P = 0.0001; Mann–Whitney test).

We also analyzed the properties of CaP and MiP action potentials that occurred during spontaneous bursts of activity. At 48 hpf, both PMN subtypes displayed spontaneous depolarizations (Fig. 3C). We found that the spontaneous CaP and MiP action potentials differed from each other with respect to rates of rise and rate of fall (Fig. 3D). Thus although spontaneous CaP and MiP action potentials had properties that overall resembled those of evoked action potentials (Fig. 3, A and B), differences in the CaP and MiP action potential waveforms and rates of rise and decay were much more evident (Fig. 3, C and D).

Consistent with differences between CaP and MiP action potentials, underlying currents also showed important differences (Fig. 3, EG). Notably, both inward and outward currents had larger densities in CaPs than those in MiPs (Fig. 3F). In addition, IKv/Ca inactivation was still more prominent in MiPs (Fig. 3G). MiP IKv/Ca showed 6.3% inactivation, whereas CaP IKv/Ca inactivated by only 3.3%.

In summary, at both 24 and 48 hpf, CaPs and MiPs displayed subtype-specific inward and outward current properties. Further, the different properties of CaP and MiP voltage-gated currents resulted in subtype-specific action potential properties for the PMN subtype, regardless of developmental stage.

IKv properties showed PMN subtype-specific differences

To look more directly at the underlying inward and outward currents, we recorded sodium- (INa) or voltage-dependent potassium current (IKv) in isolation (Fig. 4, A and B). At both 24 and 48 hpf, CaPs had significantly larger peak INa and IKv amplitudes (Fig. 4, C and D), suggesting that the sodium- and voltage-gated potassium currents account for many of the subtype-specific differences noted in the net inward and outward currents (Figs. 2 and and33).

Fig. 4.
At both 24 and 48 hpf, voltage-dependent sodium and potassium current amplitudes differed in CaPs vs. MiPs. A: CaP (black traces) and MiP (gray traces) INas differed in amplitude at both 24 (left) and 48 (right) hpf. B: CaP (black traces) and MiP (gray ...

Repetitive firing properties distinguished CaPs and MiPs

In addition to evoking single action potentials with brief current injections, we also recorded under conditions designed to evoke repetitive firing. At 24 hpf, long (100-ms) current injection did not evoke repetitive firing in either CaPs or MiPs. However, at 48 hpf, both CaPs and MiPs responded to 100-ms current injection by firing repetitively (Fig. 5).

Fig. 5.
At 48 hpf, CaPs and MiPs show different patterns of repetitive firing. At 48 hpf, both CaP and MiP fired multiple impulses in response to sustained (100-ms) depolarization. However, whereas both PMNs were able to fire repetitively, CaPs and MiPs differed ...

To compare the repetitive firing behaviors of CaPs and MiPs, we measured several properties (Table 1). CaPs and MiPs did not differ with respect to most properties analyzed. However, there was one notable exception: the degree of membrane potential repolarization achieved between the first and second action potentials. CaPs repolarized more efficiently and, on average, achieved a membrane potential of −40.4 ± 2.0 mV versus one of −29.8 ± 3.9 mV in MiPs (Table 1, Fig. 5). This difference is consistent with the subtype-specific outward current properties found for CaPs and MiPs and, in particular, with the smaller density and greater extent of inactivation for MiP IKv/Ca.

Table 1.
CaP and MiP repetitive firing properties at 48 hpf

DISCUSSION

The major findings presented here demonstrate that as soon as we could identify CaPs and MiPs on the basis of their stereotypic somal locations and hb9-driven GFP expression, they display subtype-specific excitability properties. Furthermore, the differences in CaP and MiP excitability properties become more extensive at subsequent developmental stages. Overall, the findings support the view that motor neuron subtypes follow different programs of differentiation resulting in several subtype-specific properties, including electrical excitability in addition to transcription factor expression and identity of innervation targets.

Developmental maturation of voltage-gated currents in CaPs and MiPs

The sequence of developmental changes in excitability in CaPs and MiPs parallel that described for neural plate cells of Ambystoma embryos in that delayed rectification precedes the development of inward conductances and the ability to fire action potentials (Warner 1973). In addition, in Ambystoma spinal neurons as well as chick and rat motor neurons, the amplitudes of inward and outward currents increase markedly during development (Barish 1986; Gao and Ziskind-Conhaim 1998; McCobb et al. 1990). Similarly, in Xenopus spinal neurons and zebrafish spinal sensory neurons marked increases in both INa and IKv amplitude occur that drive the developmental shortening of the action potential duration (Desarmenien et al. 1993; O'Dowd et al. 1988; Pineda et al. 2005; Ribera and Nüsslein-Volhard 1998).

We observed that the extent of time-dependent inactivation of outward current significantly decreased by 48 hpf. Interestingly, we did not observe similar time-dependent inactivation for isolated IKv, suggesting that this property marks calcium-dependent potassium current. Calcium-dependent potassium currents underlie the afterhyperpolarization that follows action potentials and regulates repetitive firing rates and durations (Bean 2007; el Manira et al. 1994; McLarnon 1995). In zebrafish motor neurons, apamin-sensitive channels regulate the duration of spontaneous bursts recorded at 24 hpf (Saint-Amant and Drapeau 2001). Thus developmental changes in the amplitude and kinetics of calcium-dependent potassium currents may additionally contribute to the maturation of firing behavior in PMNs.

In summary, early development of CaP and MiP electrical properties resembles the programs reported for other vertebrate spinal neurons.

Molecular basis for developmentally regulated electrical excitability

The developmental changes in ion currents of zebrafish motor neurons may reflect changes in the identities of the ion channel molecules expressed at different stages of differentiation. Such a mechanism appears to underlie developmental regulation of INa in mammalian motor neurons that predominantly express the voltage-gated sodium channel isotypes Nav1.2 and Nav1.3 during embryonic stages but Nav1.6 later during postnatal stages (Alessandri-Haber et al. 2002; Schaller and Caldwell 2000). Similarly, for potassium channels, expression of Kv1.1 and Kv1.2 is more prominent in adult than that in embryonic motor neurons (Alessandri-Haber et al. 2002; Rasband et al. 1998).

Alternatively, ion currents may change during development as the result of changes in the expression of modulating molecules. For example, beta subunits promote cell surface expression of the pore-forming alpha subunits for sodium and potassium channels (Chen et al. 2002; Hanlon and Wallace 2002; Isom et al. 1994; Pineda et al. 2008; Schmidt and Catterall 1986; Schmidt et al. 1985). In addition, beta subunits are known to modulate activation and inactivation of these currents and these effects can be isoform specific (Chen et al. 2002; Hanlon and Wallace 2002; Isom et al. 1994; Torres et al. 2007). Moreover, the expression of specific beta subunits for sodium and potassium channels varies both temporally and spatially within the CNS during development (Downen et al. 1999; Kazen-Gillespie et al. 2000; Lazaroff et al. 1999). Whether ion channel beta subunits contribute to developmental changes in CaP and MiP excitability is not yet known and warrants further study.

Electrical development is PMN subtype specific

Our findings demonstrate that at three developmental stages, CaPs and MiPs show significant differences in electrical excitability properties. From the functional perspective, it is not clear why CaPs and MiPs have different electrical properties. The available evidence reveals no differences in the electrical properties of ventral muscle versus dorsal muscle, the targets of CaPs and MiPs, respectively (Buss and Drapeau 2000; Coutts et al. 2006). Moreover, studies of PMNs at later stages (e.g., >2 days postfertilization) raise the possibility that CaPs and MiPs eventually lose subtype-specific differences in electrical properties and display similar firing behaviors (Buss and Drapeau 2001; Buss et al. 2003).

An alternative possibility is that the subtype-specific differences in electrical properties play a developmental role. For example, activity plays an important role in selection of subtype-specific axonal trajectories and ultimate targets (Hanson and Landmesser 2004, 2006; Moody and Bosma 2005). In addition, the different activity patterns may lead to optimal release of retrograde factors from innervated muscles. In this regard, CaPs, but not MiPs, express the glial cell line–derived neurotrophic factor (GDNF) receptor GFRa1 (Shepherd et al. 2001). Further, ventral, but not dorsal axial, muscle expresses GDNF (Shepherd et al. 2001).

Mechanisms underlying subtype-specific differentiation

One interpretation of our results is that different genetic and molecular programs direct differentiation of electrical excitability in CaPs versus MiPs. Alternatively, it is possible that MiPs and CaPs follow the same program, although the differences noted reflect developmental delay in MiPs versus CaPs. For example, with respect to axonal development, MiP growth cones emerge about 2 h after those of CaPs (Eisen et al. 1989). Moreover, CaP axons always emerge from the spinal cord prior to MiPs (Myers et al. 1986). It is possible that the smaller current densities recorded from MiPs versus CaPs may in part be a sign of this small developmental delay. However, for several reasons, developmental delay cannot account for all differences noted in CaP and MiP electrical properties. First, our recording periods extended over 2-h windows, allowing us to compare currents recorded from MiPs that differed in age by the amount of time relevant to the developmental delay in question (~2 h). There were no obvious differences in MiP outward and inward current densities over the 2-h windows. Second, PMNs differ substantially in the extents to which total outward current (IKv/Ca) densities increase during the same developmental period. For example, between 17 and 24 hpf, IKv/Ca density increased 21-fold in MiPs but only eightfold in CaPs. Similarly, between 24 and 48 hpf, IKv/Ca density increased by sixfold in MiPs and only threefold in CaPs. Third, at both 24 and 48 hpf, IKv/Ca inactivates to greater extents in MiPs than in CaPs, suggesting that the molecular determinants of this current differ in the two PMN subtypes. Fourth, prior work has demonstrated that CaPs, but not MiPs, express the Nav1.6a sodium channel (Novak et al. 2006; Pineda et al. 2006). Thus substantial differences in electrical properties exist among CaPs and MiPs that cannot be fully explained by a developmental lag. Instead, our findings support the view that PMNs differ in molecular determinants of voltage-gated ion channels and, consequently, the genetic programs that direct differentiation of electrical excitability.

Are motor neuron subtypes electrically distinct in other systems? Studies of Drosophila motor neurons support this possibility (Pym et al. 2006). Ventrally (RP) and dorsally (aCC/RP2) projecting motor neurons display marked differences in both sodium and potassium current densities (Pym et al. 2006). Interestingly, RP and aCC/RP2 motor neurons differ in homeodomain (HD) gene expression (Landgraf et al. 1999; Thor and Thomas 1997). Moreover, even-skipped (eve), an HD gene expressed by aCC/RP2 but not RP motor neurons, plays a role in regulating potassium conductance in these cells (Pym et al. 2006).

Taken together, our findings raise the possibility that subtype-specific mechanisms orchestrate development of electrical properties differently in CaPs versus MiPs. Moreover, in consideration of studies done in Drosophila, genetic mechanisms, involving homeodomain transcription factors that are differentially expressed by MiPs and CaPs, might orchestrate the underlying events that specify motor neuron subtype-specific differentiation of electrical excitability.

GRANTS

This work was supported by Institute of Neurological Disorders and Stroke Grants F32NS-059120 to R. L. Moreno and R01NS-025217, R01NS-038937, and P30NS-048154 to A. B. Ribera.

ACKNOWLEDGMENTS

We thank members of the Ribera lab for discussion.

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