Differentiation of electrical excitability in motoneurons

Brain Research Bulletin, Vol. 53, No. 5, pp. 547–552, 2000 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter

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Differentiation of electrical excitability in motoneurons Nicholas C. Spitzer,* Anne Vincent† and Nathan J. Lautermilch‡ Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA [Received 19 June 2000; Revised 14 August 2000; Accepted 17 August 2000] ABSTRACT: Investigation of the differentiation of electrical properties of motoneurons has been stimulated by the importance of these neurons for embryonic behavior and facilitated by their experimental accessibility. In this review, we examine the development of different patterns of excitability and their functions, and discuss the emergence of repetitive firing and localization of ion channels in axons and dendrites. Finally, we summarize studies of the role of extrinsic factors in differentiation. These changes associated with differentiation of young motoneurons may presage those occurring later in the context of plasticity in the mature nervous system. © 2001 Elsevier Science Inc.

action potential is brief and principally sodium-dependent from the time of its initial appearance. These neurons are unable to generate action potentials in the absence of sodium or presence of TTX. These observations raise a number of questions: What is the evidence for the existence of these two patterns of differentiation? What is the mechanism by which these calcium-dependent action potentials are converted to sodium-dependent impulses? What are the functions of long duration calcium-dependent action potentials during the brief period of development in which they are expressed? This issue assumes particular interest since these calcium-dependent action potentials are often expressed prior to synapse formation and thus are unlikely to be involved in propagation of impulses in neuronal networks. How do functions of sodiumdependent action potentials differ? What governs whether neurons express one of these forms of excitability rather than the other?

KEY WORDS: Action potentials, Calcium influx, Repetitive firing, Channel localization, Extrinsic regulation.

INTRODUCTION

PATTERNS OF MATURATION

The ability to fire action potentials is critical to the function of motoneurons. Local interneurons, which constitute a large fraction of the neuronal population in the central nervous system (CNS), have processes that are often short enough to allow electrotonic spread of depolarization or hyperpolarization from sites of synaptic activation to sites of transmitter release. In these cases, action potentials would not be required for synaptic transmission, and in a number of instances it appears that these cells do not normally fire action potentials. In contrast, because motoneurons are projection neurons with long axons that extend from the CNS to innervate muscles in the periphery, electrotonic spread of depolarization following excitatory synaptic input is generally insufficient. Regenerative electrical signals are required to relay information reliably. Studies of the ontogeny of excitability have revealed what at first appear to be two general patterns of differentiation. In the first, the initial form of the action potential is long in duration and becomes briefer with further development. Typically the duration reflects the ionic dependence of the impulse, which is initially largely calcium-dependent and enables substantial calcium influx. These neurons generate action potentials in the absence of extracellular sodium or in the presence of tetrodotoxin (TTX) to block voltage-dependent sodium channels. The action potential subsequently becomes sodium-dependent. In the second pattern, the

We have studied spinal cord neurons of Xenopus embryos during the period in which excitability matures. Seventy percent of neurons in cultures prepared from the neural plate are somatic motoneurons, by the criterion of cholinergic synapse formation with striated muscle [30,61]. These neurons as well as spinal sensory neurons and interneurons illustrate the first pattern of maturation of excitability. Action potentials are first elicited at the time of closure of the neural tube [56,75], and are typically on the order of 70 ms in duration. Ion substitution experiments and application of pharmacological blockers demonstrate that the inward current is carried chiefly by calcium, with a modest sodium contribution (Fig. 1A). Over the course of a further day of development, the ionic dependence of the action potential changes from calcium to sodium, and by tailbud stages the impulse is ⬃3 ms in duration (Fig. 1B). Whole cell recordings of voltage-clamped current reveal the presence of high-voltage-activated (HVA) and low-voltage-activated (LVA) calcium currents at the early stages of differentiation [50]. Strikingly, the peak amplitude of the large HVA current remains constant although the extent of its inactivation increases; the small LVA current disappears with further development [27]. Thus the change in ionic dependence of the action potential does not depend on a decrease in magnitude of the HVA currents. While the amplitude of sodium current increases, the most dramatic change in excitability is an increase in expres-

* Address for correspondence: Nicholas C. Spitzer, Department of Biology 0357, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0357, USA. Fax: ⫹1-(858)-534-7309; E-mail: [email protected] † Present address: Laboratoire de Neurochimie Fonctionnelle et Neuropharmacologie, Universite´ Henri Poincare´ Nancy 1, Vandoeuvre-le`s-Nancy, France. ‡ Present address: Department of Pharmacology, University of Washington, Seattle, WA, USA.

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FIG. 1. Action potentials (V) recorded from Xenopus spinal neurons at early (A) and late (B) stages of differentiation in culture in response to injected currents (I). A. The action potential is of long duration in standard saline; it is largely unaffected by removal of Na⫹ but appears to be blocked by addition of 10 mM Co2⫹. (B) Action potentials are brief in standard saline; addition of Co2⫹ has little effect, but tetrodotoxin (TTX; 1 ␮g/ml) abolishes them (in another neuron). 0 indicates zero potential. After [56].

sion of delayed rectifier current that triples in amplitude and increases in its rate of inactivation during this period of development [9,50]. A calcium-dependent potassium current undergoes relatively little change during this time, and an inactivating potassium A current develops later and appears to limit repetitive firing [53]. A rapidly activating calcium-dependent potassium current expressed at larval stages contributes to termination of ventral root bursts during fictive swimming [60]. Computer reconstruction of the impulse shows that the increase in amplitude of delayed rectifier current is the major contributor to the shortening of action potential duration and change in ionic dependence [38]. The increase in density of potassium channels, in combination with greater depolarization resulting from an increased density of sodium current, causes rapid repolarization that suppresses the contribution of sustained inward calcium current. This type of maturation is not unique to Xenopus spinal cord neurons, as calciumdependent action potentials are also transiently expressed in the rat dorsal motor nucleus of the vagus nerve [41], suggesting that this may be a shared developmental mechanism across species. The process by which excitability matures and potassium current is upregulated in amphibian spinal neurons appears to be cell autonomous, since it occurs normally in cultures of single, isolated neurons grown in a fully defined medium [31]. RNA and protein synthesis are required [11,49]. Although not yet demonstrated for motoneurons, which express xKv2.2 potassium channel gene transcripts [12], the requirement for RNA synthesis in other Xenopus spinal neurons reflects the transcription of mRNA encoding potassium channels (xKv1, [52]; xKv3.1, [70]). Changes in expression of potassium channels and resulting changes in excitability can be rapid, occurring within hours [37], and may involve the activities of auxiliary ␤ subunits [36,62]. In contrast, chick ciliary ganglion neurons [5,24] and rat spinal motoneurons [2,21,71,77] including phrenic motoneurons [39,40] develop according to the second pattern, exhibiting action potentials that are brief and largely sodium-dependent at the earliest stages at which they are recorded (Fig. 2). In these neurons, the contribution of sustained inward calcium current is suppressed by coincident expression of sustained outward potassium current. Inward calcium current is small in chick embryo ciliary ganglion neurons, and blocking sodium entry prevents generation of action potentials. Developing rat spinal motoneurons do not generate impulses in the absence of sodium current in some cases [39,77]

but are able to do so in others [71]. Chick hindlimb motoneurons develop in a similar manner [44]. Calcium currents are detected in mouse spinal neurons at later stages of development [34]. Both forms of excitability have also been observed in developing sensory neurons. Largely calcium-dependent action potentials are first recorded in amphibian and murine dorsal root ganglion cells [4,42] and in chick dorsal root ganglion neurons [24]. Largely sodium-dependent action potentials are first recorded in quail mesencephalic neurons that give rise to sensory as well as autonomic neurons [6,7] and in primary afferent neurons of the chick cochlear ganglion [66]. Differentiating interneurons also display these different forms of excitability. Action potentials in rat and chick cortical and brain nuclear neurons [1,47,51] are largely calcium-dependent. In contrast, action potentials in cat retinal ganglion cells [55] are brief and sodium-dependent at early

FIG. 2. Action potentials recorded from 7-day postnatal rat spinal motoneurons in response to antidromic stimulation. (A) Control. (B) Addition of Cd2⫹. (C) Superposition of (A) and (B) demonstrates a decrease in the afterdepolarization and elimination of the afterhyperpolarization. (D) Expansion of the time scale for C reveals the small decrease in action potential duration by Cd2⫹ (arrows). After [71].

DIFFERENTIATION OF ELECTRICAL EXCITABILITY stages of development. It should be noted that the development of excitability has been studied principally by recordings from the cell body of embryonic and postnatal neurons, and with few exceptions (e.g., [75]) little is known about the maturation of excitability in more remote regions of axons and in growth cones or incipient nerve terminals. What explains these different patterns of excitability? They are not particular to a specific class of neurons (e.g., motoneurons or sensory neurons), and they do not appear to be species-specific since both are observed in the same animal (e.g., the mouse nervous system: [34,42]). A clue may be provided by observation of an intermediate form of excitability in embryonic grasshopper interneurons [23]. In these neurons, the action potential of the cell body is not eliminated by blocking sodium or calcium influx; blockade of both simultaneously is required. This result, considered with the observations of rat spinal motoneurons noted above [71,77], raises the possibility that the contribution of sodium and calcium currents to the differentiation of excitability is a continuum and not represented by different discrete states. This view is consistent with the findings that the amount of each current expressed during an action potential can vary widely from one class of neuron to another, and within the same class of neuron from one species to another. Implications of this notion are developed further below. Calcium channels are retained in the neuronal membrane during differentiation of electrical excitability, and pharmacological blockade of potassium channels (e.g., with tetraethylammonium ions) converts brief sodium-dependent spikes to long duration calcium-dependent action potentials. Thus it seems likely that these calcium channels are normally utilized in some way, perhaps by suppressing their residual activity or by unmasking their function by suppressing outward potassium currents (see review by Perrier and Hounsgaard, in this issue). FUNCTIONS OF EMBRYONIC ACTION POTENTIALS To investigate potential roles of calcium-dependent action potentials in neuronal differentiation we imaged intracellular calcium in amphibian spinal neurons developing both in vitro and in vivo [26 –28]. Cultured amphibian spinal neurons exhibit spontaneous elevations of intracellular calcium both prior to and during neurite outgrowth. Spontaneous transient elevations of calcium ⬃10 s in duration are observed at frequencies of 1–10/h. These calcium transients, termed calcium spikes, are generated by calcium action potentials and the calcium influx that triggers calcium release from ryanodine receptor-activated calcium stores (calcium-induced calcium release). Remarkably, blocking and reimposing calcium transients on cultured neurons at these low frequencies demonstrate that they are necessary and sufficient for upregulation of delayed rectifier current in all neurons and for expression of the neurotransmitter ␥-aminobutyric acid (GABA) in a subset of interneurons. Regulation of the incidence of GABA expression occurs at the level of transcripts encoding xGAD 67, a GABA-synthetic enzyme [72]. The cholinergic phenotype may be similarly regulated in motoneurons [73]. What is the function of largely sodium-dependent action potentials? One possibility, apparently as yet untested, is that the more modest calcium entry associated with them remains sufficient to generate spontaneous calcium transients in the neurons in which they are generated. Such calcium transients could then have a range of developmental functions. On this view, whether neurons generate action potentials that are largely calcium- or sodiumdependent becomes irrelevant, and the key finding is that all differentiating neurons studied to date generate action potentials that enable calcium influx.

549 A prediction of this hypothesis is that neurons generating largely sodium-dependent action potentials have intracellular calcium stores that are more sensitive to release by calcium influx, perhaps by spatial localization of receptors or by combinations of ryanodine, IP3 and perhaps other receptors. Alternatively they may have larger low voltage-activated calcium T current, since this current is preferentially activated during brief action potentials [45], or experience higher frequencies of action potentials to facilitate calcium entry. Differences in the contributions of calcium entry across the plasma membrane and calcium release from stores would allow neurons to respond differentially to specific stimuli. However it remains possible that sodium-dependent action potentials do not lead to generation of calcium transients with developmental functions. DEVELOPMENT OF REPETITIVE FIRING Developmental changes in repetitive firing behavior have been studied in postnatal rat spinal motoneurons [20,29,71]. Action potentials are followed by a calcium-dependent afterdepolarization (ADP) and a subsequent afterhyperpolarization (AHP). The ADP can generate a burst of action potentials following the first spike [39]. Calcium-dependent potassium current prolongs the AHP and gives immature neurons a low firing rate, in comparison with that of mature motoneurons, which may be matched to other properties of the immature nervous system. Consistent with these findings, different types of K⫹ channels in embryonic and postnatal rat spinal neurons have opposite modulatory actions on action potential firing behavior: calcium-dependent potassium current produces a decrease in the firing rate while delayed rectifier and inactivating A current cause an increase [21]. Maturation of repetitive firing has also been intensively studied in postnatal rat hypoglossal motoneurons. The incidence of burst firing is highest in young neurons with large ADPs, coincident with high levels of expression of LVA calcium current that decline with age [63,67]. These neurons also generate an AHP produced by potassium currents, one of which is calcium-dependent [68]. Both young and adult neurons show a decline in firing rate in response to long duration current pulses, that is associated with summation of the AHP and prolongation of the action potential. However, during an intermediate stage of development many neurons exhibit an increase in firing rate associated with decrease in AHP and no change in the action potential, perhaps related to behavioral tasks of tongue muscles during this period. The relationship of steady state firing frequency to intensity of current injected is linear and highest in neonatal motoneurons, declining with age [69]. LOCALIZATION OF ION CHANNELS IN AXONS AND DENDRITES Localization of ion channels is critical to their function, and clustering of sodium channels in axons of the rat sciatic nerve has been followed immunocytochemically. Aggregates appear during the first 3 days postnatal, generally in the vicinity of Schwann cell processes; suppressing proliferation of Schwann cells with mitomycin C curtails formation of channel clusters [64]. These channels have been identified as sodium channel 6 (NaCh6; [33]). Potassium channel clusters do not appear until 1-week postnatal, in the nodal gap and paranodes, and shift to juxtaparanodal regions at 2– 4 weeks [65]. Myelination and oligodendrocyte development can proceed in the absence of action potentials in the mouse spinal cord [54]. The conduction velocity in axons of cat phrenic motoneurons ranges from 10 m/s at 2 weeks postnatal to 60 m/s at 14 weeks [15]. The excitability of dendrites of motoneurons has also been of interest in light of recent observations that back propagating action

550 potentials excite dendrites of neocortical and hippocampal neurons and promote calcium influx [57,58]. In rat embryo spinal cord cultures, motoneurons have nonuniformly distributed sodium and calcium conductances [35]. Patch clamp recordings reveal the presence of sodium and calcium currents in dendrites; the conduction velocity of propagating action potentials is estimated to be 0.5 m/s. Simultaneous imaging of voltage and calcium confirms active propagation and calcium elevation. Antibodies to specific calcium channel subtypes may enable structural localization of calcium channels as well [74]. Dendrites of adult cat motoneurons also appear to be able to generate action potentials, with a conduction velocity of 0.75 m/s [3,18]. Developmental enhancement of intracellular calcium increases induced by action potentials has been suggested to underlie maturation of calcium-dependent functions such as synaptic plasticity in hippocampal neurons [32], and could play a similar role in plasticity of motoneurons. EXTRINSIC REGULATION OF IONIC CURRENTS Although maturation of excitability may be the result of a cell autonomous program in some neurons, extrinsic factors have also been shown to be involved in regulating development. Differentiation of motoneurons is delayed in spinal cords cultured in the presence of TTX [76], demonstrating a role of electrical activity in their development, and this inhibitory action is reversed by incubation in high potassium that increases the frequency of spontaneous potentials. Application of brain-derived neurotrophic factor to rat muscle cells stimulates an increase in excitability of motoneurons innervating them [22], consistent with activity-dependent upregulation of neurotrophin-3 (NT-3). The full development of excitability in amphibian spinal motor neurons requires synaptic activation of postsynaptic muscle cells. Blockade of synaptic transmission causes broadening of action potentials and decreased density of potassium current; application of NT-3 specifically prevents these changes and enables increases in potassium current [48]. The development of electrical excitability can also be regulated by glia and other cell types ([10]; see also previous section). Continued expression of inactivating potassium A current in cultured rat superior sympathetic ganglion cells requires a factor secreted by non-neuronal cells [46]. Normal maturation of inactivating potassium A current and calcium-dependent potassium current in chick ciliary ganglion neurons in situ requires interactions with target tissue, and the calcium-dependent potassium current also requires normal innervation [16]. Expression of this current depends on posttranslational regulation by target-derived factors that include several forms of transforming growth factor-␤ [13,14, 59]. The ability to record from identified neurons in Drosophila should enable investigation of the role of specific cell contacts and secreted factors in regulating differentiation of electrogenesis [8]. Steroid hormones have prominent effects on the maturation of electrical excitability of motoneurons in several systems. Leg motoneurons of the pupa of the tobacco hornworm, Manduca sexta, demonstrate an increase in calcium current in response to 20-hydroxyecdysone; this effect is specific since it is not seen in larval leg motoneurons, and no effect on potassium current is observed [25]. Electrocytes that produce the electric organ discharge used in gender recognition in the electric fish, Sternopygus, are differentially affected by androgens and estrogens. Dihydrotestosterone and estradiol-17␤ slow and speed the inactivation time constants of sodium current, respectively, producing an electric organ discharge that is lower in frequency and longer in pulse duration in males than in females [17,19], and potassium current kinetics are co-regulated with those of sodium current [43].

SPITZER, VINCENT AND LAUTERMILCH CONCLUSIONS The differentiation of motoneurons involves changes in the ionic dependence of single action potentials and the ability of neurons to fire repetitively. Calcium influx associated with action potentials in young neurons appears to be important in regulating subsequent aspects of differentiation, and the firing frequency of these neurons seems to be tuned to the signaling requirements of the immature nervous system. The localization of ion channels in axons and dendrites has been characterized and appears to be dynamic during early stages of differentiation. Although some of the extrinsic factors influencing differentiation of excitability have been identified, the basis of initiation of the intrinsic program of development remains to be determined. ACKNOWLEDGEMENTS

We are grateful to Anna Marnick for invaluable library research. Work in our laboratory is supported by grants from the National Institute of Neurological Disorders and Stroke, National Institutes of Health and the National Science Foundation.

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