A Developmental Handshake: Neuronal Control of Ionic Currents and Their Control of Neuronal Differentiation Nicholas C. Spitrer Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093

INTRODUCTION Electrical excitability has been recognized for two centuries to be important for rapid signalling (Galvani, 179 l ), but the roles of channels and receptors in guiding fundamental processes of development have begun to be appreciated only more recently. The functional activity of ion channels and transmitter receptors can trigger molecular events of transcription and posttranslational modification that are central to the process of differentiation. The purpose of this brief review is to focus on the roles of channel activity in controlling neuronal differentiation, and particularly on the increasing number of instances in which endogenous physiological activity has been shown to be important. It is now feasible to make experiments increasingly less invasive, while simultaneously examining events at molecular levels of resolution. These control mechanisms are reciprocal: Cells regulate ion flux, and ion flux regulates neuronal differentiation, including ion flux. While some phenotypes are switched on, others are switched off. Negative feedback ensures a form of self-limiting process necessary for precise orchestration of developmental events. The fact that these loops operate over short spans of time at early stages of development has facilitated their detection. They may also be operative over longer periods in the adult, for Received June 24, 1991; accepted June 25. 199 1. Journal of Neurobiology, Vol. 22. No. 7. pp. 659-673 (199 1) 8 1991 John Wiley & Sons, Inc. CCC 0022-303419 11070659- I S$04.00

the direction of changes associated with memory and learning or for homeostasis. The dual function of ion channels for both short- and long-term signalling is developmentally parsimonious. It has many parallels in embryogenesis, because the complexity of development requires efficient use and reuse of a limited number of genes. Various segmentation genes in Drosophila illustrate such multiple deployment, as they are active first in the process of segmentation itself and later in neurogenesis. Several observations set the stage for recent progress. First, many ion channels and receptors were found to appear rather early in development; in a number of cases, they have achieved their mature form by the time of hatching or birth. Second, neurons were shown to exhibit a fixed temporal sequence in which various membrane properties are expressed. Third, membrane properties were often found to be expressed in an immature form initially, and assume their mature character later. A fascinating aspect of this process is the transient expression of some channels and receptors, followed by their disappearance at a subsequent stage. Observations of early and sequential expression, and of changes in early phenotypes, prompted investigations of the possibility that their signal transduction activity triggers later steps of development. ACTION POTENTIALS Two patterns of development of action potential mechanisms can be discerned. The first pattern is 659

660

Spitzer

one in which the ionic dependence of the impulse changes during development, and is illustrated by primary sensory neurons of Xenopus embryos. Rohon-Beard neurons are accessible for recordings at early stages of development and exhibit long-duration, calcium-dependent action potentials at the time of closure of the neural tube. The action potential is rapidly converted to a brief sodium-dependent spike, largely during the next day of development (Baccaglini and Spitzer, 1977). A similar sequence has been reported for amphibian motor neurons and interneurons (Spitzer and Lamborghini, 1976; Willard, 1980), chick motor neurons ( McCobb, Best, and Beam, 1990), amphibian and murine dorsal root ganglion neurons (Baccaglini, 1978; Matsuda, Yoshida, and Yonezawa, 1978), rat sympathetic ganglion cells (Nerbonne and Gurney, 1989), rat and chick cortical and brain nuclear neurons ( Ahmed, Walker, and Fellows, 1983; Mori-Okamoto, Ashida, Maru, and Tatsuno, 1983; Pettigrew, Crepel, and Krupa, 1988), and murine neuroblastoma and rat pheochromocytoma (PC 12) cells (Dichter, Tischler, and Green, 1977; Miyake, 1978; OLague and Huttner, 1980; Ritchie, 1979). The maturation of the action potential is a cell autonomous process in amphibian spinal neurons once primary induction has occurred, since it can occur in cultures of single cells (Henderson and Spitzer, 1986). The second pattern of action potential development is one in which the ionic basis of the impulse is relatively constant during development, and is illustrated by neurons from the mesencephalic neural crest of quail embryos (Bader, Bertrand, and Dupin, 1985). These neurons express brief sodium-dependent action potentials from the beginning, and long-duration, calcium-dependent action potentials are not seen unless potassium current is blocked. Similar findings have been obtained for mesencephalic neural crest and chick ciliary ganglion neurons (Bader, Bertrand, Dupin, and Kato, 1983a,b), grasshopper interneurons (Goodman and Spitzer, 1981 ), rat motoneurons (Ziskind-Conhaim, 1988), murine spinal neurons (Krieger and Sears, 1988), and for amphibian myocytes (DeCino and Kidokoro, 1985; Henderson and Spitzer, 1986). Developmental Changes in Currents The difference between these two patterns is due largely to the difference in development of outward potassium currents, which modulate the amplitude of inward currents. Calcium and sodium currents are present in most of the cells that have

been studied, irrespective of their mode of development. After they appear, net calcium currents are generally stable or increase; decreases in low voltage-activated T currents that are present at very early stages appear to be compensated by increases in high voltage-activated N and L currents (Barish, 1986; Gottmann, Dietzel, Lux, Huck, and Rohrer, 1988; Lovinger and White, 1989; McCobb, Best, and Beam, 1989; Yaari, Hamon, and Lux, 1987). Although sodium currents increase in density and undergo small changes in kinetics (Huguenard, Hamill, and Prince, 1988; McCobb et al., 1990; O’Dowd, Ribera. and Spitzer, 1988), their roles in intracellular signalling may entail depolarization and activation of calcium currents (see below). Howcver proton-activated sodium currents appear prior to calcium and sodium currents in some systems (Gottmann, Dietzel, Lux, and Ruedel, 1989; Grantyn, Perouansky, Rodnguez-Tkbar, and Lux, 1989), and their developmental functions are as yet uncharacterized. The first pattern arises as the result of late expression of potassium currents that are initially small and slowly activated, allowing extended activation of sustained inward calcium currents. The action potential duration is established by the balance between calcium and potassium currents. Potassium currents increase during development and truncate sustained inward calcium currents by repolarizing the cells. The second pattern occurs through the action of substantial potassium currents combined with relatively modest calcium currents early in development, preventing the expression of sustained calcium influx at the earliest times of impulse generation. Interestingly, excitability of striated and cardiac muscle cells also follows one of these two patterns of development (see Spitzer, 1985, for review). The identity of the controlling potassium current varies from one system to another. In some instances, for both patterns of differentiation, sustained potassium currents govern the degree to which calcium currents dominate action potentials at early stages (Krieger and Sears, 1988; McCobb et al., 1990; Nerbonne and Gurney, 1989; Ribera and Spitzer, 1989, 1990). In others, the A current precedes the delayed rectifier (Aguayo, 1989; Bader, Bertrand, and Dupin, 1985). However, in some cases potassium currents are initially larger than calcium currents (Ribera and Spitzer, 1991 ) or precede the expression of calcium currents altogether (Salkoff, 1985). In these cases the action potential is correspondingly brief, and the calciumdependent component is small. These insights into the process of maturation

Neuronic Control of Ionic Currents

were provided by whole cell voltage clamp recording of macroscopic currents from cells at different stages of development. For amphibian spinal neurons in culture that exhibit the first developmental pattern, the largest changes are seen in the delayed rectifier potassium current during an 18-h period shortly after closure of the neural tube ( O’Dowd et al., 1988). Initially this current is small and matches the calcium current rather closely. Its density increases 3-fold, and its rate of activation increases 2-fold during this period; it changes little thereafter. Changes in sodium, calcium, and calcium-dependent potassium current are less substantial. An inactivating potassium A current is expressed in all neurons somewhat later (Ribera and Spitzer, 1990). Computer reconstruction of the action potential from whole-cell, voltage-clamped currents provides definitive evidence for the predominant role of the delayed rectifier in maturation of the action potential in amphibian spinal neurons (Barish, 1986; Lockery and Spitzer, 1991). The currents recorded experimentally are sufficient to account for the full extent of shortening of the action potential and its change in ionic dependence (Lockery and Spitzer, 1991). The contributions from any other currents (Hussy, 1991 j are therefore likely to be small. Furthermore, the normal developmental changes of the delayed rectifier can accomplish 94% of the decrease in duration in the absence of changes in other currents. The delayed rectifier is not unique in its ability to accomplish the maturation of the action potential because increases in amplitude of the calcium-dependent potassium current would also be effective in promoting maturation. Although decreases in calcium current would be most efficient in reducing calcium influx, the utility of calcium influx as a second messenger may be the basis of its retention during neuronal differentiation in systems studied thus far. Changes in single channels in Xenopus spinal neurons underlie the changes in whole-cell potassium currents. Two channel classes, of 15 and 30 pS conductance, give rise to the macroscopic delayed rectifier of amphibian spinal neurons (Harris, Henderson, and Spitzer, 1988). During development, increases in the number of channels of both classes account for the increase in whole-cell current density, but the increase in rate of activation of the whole-cell current is accounted for by changes in the 30-pS channel class alone. Channels underlying the macroscopic calcium-dependent potassium current in these cells initially exhibit little sensitivity to internal calcium, which is then acquired during further differentiation (Blair and

661

Dionne, 1985) . In several systems, developmental increases in mean-channel open time can lead to augmentation of whole-cell current ( Bregestovski et al., 1988; Yool, Dionne, and Gruol, 1988). Spontaneous Activity

The developmental significance of calcium-dependent action potentials is indicated by the presence of spontaneous influx at early stages of differentiation of amphibian spinal neurons in vitro, and perturbation of later differentiation by prevention of influx of calcium (Holliday and Spitzer, 1990). The use of fura-2 and time-lapse video microscopy has allowed noninvasive detection of transient elevations of intracellular calcium, present at early stages when cells are capable of calcium-dependent impulses. Cells display spontaneous elevations of intracellular calcium roughly 10 times per hour, and intracellular calcium may remain elevated for 5-20 s each time. Spontaneous activity is eliminated by removal of extracellular calcium or by specific pharmacological blockers, indicating the involvement of voltage-dependent calcium channels. The trigger for these spontaneous events may be the low voltage-activated (LVA) calcium T current (Gu and Spitzer, 1991 ) . Confocal microscopy using fluo-3 reveals that depolarization causes calcium to rise rapidly to pm levels, both in the nucleus and in the cytoplasm (Holliday, Adams, Sejnowski,and Spitzer, 1991;see also Hernandez-Cruz, Sala, and Adams, 1990; Pryzwara, Bhave, Bhave, Wakade, and Wakade, 1991). These elevations result from calcium-induced calcium release from the endoplasmic reticulum. The levels of intracellular calcium are strongly buffered, because steady state levels are constant throughout development ( Holliday and Spitzer, 1990). Removal of calcium from culture medium during an early period alone alters neurite outgrowth and the acquisition of neurotransmitter phenotype (Holliday and Spitzer, 1990). The disruption of development is greatest during the period when spontaneous elevations of intracellular calcium are most frequent. Furthermore, depletion of intracellular stores of calcium is equally effective, even in the presence of spontaneous influx, demonstrating the importance of calcium-dependent calcium release (Holliday et al., 1991 j. Critical Periods

Intriguing features of developing systemsare particular restricted periods during which aspects of dif-

662

Spitzer

ferentiation are consolidated, within which they exhibit an irreversible sensitivity to perturbation. Maturation of electrical excitability of amphibian spinal neurons in vitro is sensitive to blockers of RNA and protein synthesis when applied at early stages. and subsequently becomes insensitive (Blair, 1983; O’Dowd, 1983). Actinomycin D, cycloheximide, and puromycin are effective in blocking the normal shortening of the action potential that leads to reduction of calcium influx. The use of reversible inhibitors of mRNA synthesis has demonstrated the existence of a critical period for transcription for the differentiation of the delayed rectifier potassium current (Ribera and Spitzer, 1989). Application of dichlororibobenzimidazole during an early period appears to block its development in an irreversible manner. The increases in both density and activation rates are halted. Removal of inhibition allows general recovery of RNA synthesis. The transcriptionally sensitive potassium A current develops during the next day, delayed only by the interval of blockade, while the delayed rectifier remains arrested in its immature form. The result is consistent with a model whereby a transcription factor required for the functional maturation of the delayed rectifier is present only during the critical period and absent thereafter. Clones for Xenopus potassium channel genes encoding a delayed rectifier may enable testing this hypothesis (Ribera, 1990). A sensitive period for processes dependent on calcium influx is demonstrated in the same system. Roles for spontaneous activity involving transient elevations of intracellular calcium are apparent, and the requirement for calcium influx is restricted to a particular period (Holliday and Spitzer, 1990; see earlier). The absence of calcium during this interval enhances neurite outgrowth and suppresses neurotransmitter expression.Analysis ofdevelopmental regulation of the delayed rectifier potassium current has demonstrated a role for calcium ions (Desarmenien and Spitzer, 199l ) . The normal increase in rate of activation of this current requires calcium influx during the critical period for transcription, mediated by release of calcium from the endoplasmic reticulum. Calcium acts via protein kinase C (PKC), because its effect is mimicked by stimulation of the enzyme in the absence of calcium, and suppressed by its depletion in the presence of calcium. Global suppression of kinases has no greater effect than the specific suppression of PKC, suggesting that this enzyme may be the only one involved. Although PKC may act directly to phosphorylate potassium channels, it is perhaps more likely that it acts on a transcription factor to

effect the synthesis of a different class of channels. Calcium-induced calcium release raises intranuclear levels of calcium more than those in the cytosol (Holliday, Adams, Sejnowski, and Spitzer, 1991), consistent with a role of calcium in regulating activity of transcription factors. Functional Significance

The functional significance of calcium-dependent action potentials that are present only transiently at early stages of neuronal differentiation is beginning to be understood. There is now evidence for spontaneous activity (Holliday and Spitzer, 1990), although the normal firing frequency in vivo has not yet been established. Calcium influx, triggering the release of calcium from intracellular stores, has been shown to influence neurite outgrowth, neurotransmitter levels, and the properties of potassium channels (Bixby and Spitzer, 1984a; Desarmenien and Spitzer, 1991; Holliday and Spitzer, 1990; Holliday et al., 1991 ). Calcium influx elicited by depolarization has been shown to influence the choice of neurotransmitter phenotype in cultured sympathetic neurons by stabilizing adrenergic differentiation ( Walicke and Patterson, 1981 ) . Tyrosine hydroxylase activity is increased by electrical activity in vivo (Black, Chikaraishi, and Lewis, 1985) . The increase in enzyme activity in culture is the result of increases in transcripts for the enzyme (Raynaud, Faucon-Biguet. Vidal, Mallet, and Weber, 1987; Vidal, Raynaud, and Weber, 1989). In contrast, cholinergic differentiation of chick ciliary ganglion and mouse spinal neurons is promoted by calcium influx stimulated by depolarization that increases choline acetyltransferase activity (Ishida and Deguchi, 1983; Nishi and Berg, 1981 ) . Normal increases in glutaminase activity in developing cerebellar granule cells can be further stimulated by depolarization that entails calcium channel activity (Moran and Patel, 1989). Levels of dopamine and substance P precursor in mouse substantia nigra and mouse superior cervical ganglion are also influenced by depolarization ( Friedman, Dreyfus, McEwen, and Black, 1988; Roach, Adler, and Black, 1987). Mechanisms of gene regulation by elevation of intracellular calcium are being intensively investigated using rat pheochromocytoma (PC 12) cells as a model system. When induced to undergo differentiation, they develop much like sympathetic ganglion neurons (Greene and Tischler, 1982). Calcium influx elicited by depolarization causes rapid induction of immediate early genes, the best studied of which is c-fus,encoding a transcription fac-

Neuronic Control Gf'Ionic Currents

tor ( Greenberg, Ziff, and Greene, 1986; Kruijer, Schubert, and Verma, 1985; Milbrandt, 1986; Morgan and Curran, 1986; see Morgan and Curran, 1991, for review). Some of these changes in gene cxpression are also produced by treatment with NGF. However, c-fos is normally expressed during the development of the central nervous system (CNS) (Caubert, 1989; Curran and Franza, 1988). The elements governing the regulation of its expression include motifs similar to the serum response element (SRE) and CAMP-regulatoryelement (CRE) (Montminy, Sevarino, Wagner, Mandel, and Goodman, 1986; Treisman, 1986); the former responds to protein kinase C-dependent signals (Gilman, 1988) while the latter confers calcium sensitivity (Sheng, McFadden, and Greenberg, 1990). Phosphorylation of the CRE binding protein by Ca/calmodulin kinase appears to enhance its ability to activate transcription (Dash, Karl, Colicos, Prywes, and Kandel, 1991; Sheng, Thompson, and Greenberg, 1991 ) . Several other early genes regulated by calcium influx encode other transcription factors (e.g., Milbrandt, 1987, 1988). Although many functions may be served by clustering of voltage-dependent channels, the focal delivery of calcium ions to specific regions of the cell is likely to be of significance for neuronal differentiation. The spatial localization of channels in single cells has long been identified in mature neurons (Poo, 1985). Calcium currents have been shown to be further restricted to limited regions of their membrane (Roberts, Jacobs, and Hudspeth, 1990;Thompson and Coombs, 1988). Segregation of channels is seen within single cells at early stages ofdevelopment (Catterall, 1981 ) ,and the excitability of processes seems to differentiate prior to that of the cell body (Goodman and Spitzer, 1979, 1981; Willard, 1980). In individual neurons, calcium channels can be localized to cell bodies, dendrites and growth cones but excluded from axons (Anglister, Farber, Shahar, and Grinvald, 1982; Mourre, Cervera, and Lazdunski, 1987; Streit and Lux, 1989; Westenbroek, Ahlijanian, and Catterall, 1990). Moreover, the distribution of ion channels can change during development ( Angelides, Elmer, Loftus, and Elson, 1988; Kocsis, Ruiz, and Waxman, 1983; Ritchie, 1982; Waxman and Foster, 1980). The long duration of calcium-dependent action potentials could also exert electrical effects as a consequence of the prolonged reversal of the sign of the membrane potential. Sustained depolarization has been suggested to facilitate insertion of membrane proteins, such as receptors for neuro-

663

transmitters ( Ohmori and Sasaki, 1977) . Depolarization of oocytes can block polyspermic fertilization in a reversible manner (Jaffc, 1976; Shen and Steinhardt, 1984). Moreover, the orientation of proteins in artificial membranes can be voltage-dependent (Blumenthal, Kempf, Van Renswoude, Weinstein, and Klausner, 1983). Sodium-dependent impulses have a substantial impact on later aspects of development, as demonstrated by the action of TTX on synapse elimination (Thompson, Kuffler, and Jansen, 1979; VanEssen, 1982) and establishment of connectivity patterns (Archer, Dubin, and Stark, 1982; Boss and Schmidt, 1984; Meyer, 1982). At these stages the role of impulse generation may involve synaptic activity and postsynaptic influx of calcium (see later). In a few cases, application of TTX illustrates an effect on earlier aspects of differentiation (Bergey, Fitzgerald. Schrier, and Nelson, 1981; Jackson, Lecar, Brcnneman, Fitzgerald, and Nelson, 1982; Offord and Catterall, 1989; Sherman and Catterall, 1984; Westbrook and Brenneman, 1984). However, it is clear that sodium-dependent action potentials cannot play a role at the earliest stages of neuronal development when they are absent (Goodman and Spitzer, 1979), or in instances where blockade by local anesthetic or TTX can be shown to have no detectable effect on aspects of later development (Bixby and Spitzer, 1984a; Harris, 1980, 198 1 ; Harrison, 1904; Obata, 1977). NEUROTRANSMITTER SENSITIVITY

Developmental changesin responsesto neurotransmitters occur through changes in channel properties, allowing regulation of calcium influx in some cases. This is formally similar to developmental changes in action potentials that involve sequential activation of different channel classes in varying proportions. The process can result from altered subunit composition. A developmental change in conductance and open time of the nicotinic acetylcholine receptor of striated muscle arises through the switch of subunits of this heteroligomeric channel, as y is replaced by t (Gu and Hall, 1988; Mishina et al., 1986). Glycine receptors exhibit low strychnine affinity in neonatal rat spinal cord, and the sensitivity to glycine increases as the result of the disappearance of the a2* subunit (Becker, Hoch, and Betz, 1988; Kuhse, Schmieden, and Bctz, 1990). One of two coding sequences in transcripts of the AMPA-selective glutamate receptor (termed flop) increases during development, while anothcr (called flip) , is expressed in an invariant

664

Spitzer

pattern ( Monyer, Seeburg, and Wisden, 1991 ). Glutamate activation of flip versions produces more current than those composed of flop (Sammer et al., 1990). Although the flip/flop channels are not calcium permeant, the calcium permeability of kainic acid-gated receptors is governed by their subunit composition ( Hollmann, Hartley, and Heinemann, 1991 ) and may be developmentally regulated. There are also examples of changes in receptor properties for which transcriptionally distinct subunit compositions have not been identified. GABA, receptors of rat superior cervical ganglion exhibit a developmental increase in resistance to blockade by zinc (Smart and Constanti, 1990). Interestingly, NMDA receptors in rat hippocampus can be less voltage sensitive ( Ben-Ari, Cherubini, and Krnjevic, 1988) and magnesium sensitive (Bowe and Nadler, 1990) at early stages of development, allowing greater influx of calcium than in the adult. However, developmental increases in glycine binding sites associated with the NMDA receptor (McDonald, Johnston, and Young, 1990; Tremblay, Roisin-Lallemand, and Ben-An, 1990) can enhance calcium influx in mature cells. Further opportunities for developmental regulation are afforded by G protein coupled receptors. At early stages of expression, the muscarinic ACh receptor appears to be incompletely coupled to a functional response. Newly synthesized receptors in embryonic chick heart, detected by QNB (quinuclidinol benzoate) binding, have not yet acquired full capacity to slow the beating rate and inhibit adenylate cyclase; cyclase activity can later increase even in the absence of protein synthesis (Galper, Klein, and Catterall, 1977; Hunter and Nathanson, 1984; Renaud, Barhanin, Cavey, Fosset. and Lazdunski, 1980). Similarly,cardiac myocytes from chick atria are more sensitive to carbachol than those from ventricular muscle, although the density of receptors detected in binding experiments and autoradiography is the same (Siege1and Fischbach, 1984). Because muscarinic receptor subtypes can evoke different patterns of calcium release ( Lechleiter, Girard, Clapham, and Peralta, 1 99 1 ), their expression during development will be of interest. Changes in the response to neurotransmitters can also arise through serial acquisition of sensitivities to more than one transmitter during the course of differentiation, leading to a change in the aggregate response. This process can be regulated postsynaptically, by serial appearance of receptors for different ligands (Clendening and Hume, 1990a), and influenced by the presence of nonneural cells

as well as other neurons (Clendening and Hume, 1990b). It may also be regulated presynaptically, by sequential appearance of two or more cotransmitters in other cases. However, in several instances, receptors for several different transmitters seem to appear at the same time (Bixby and Spitzer, 1984b; Goodman and Spitzer, 1979). The localization of neurotransmitter-activated channels in developing neurons has been mapped by iontophoretic application of neurotransmitters in vivo. Neurons initially become sensitive over their soma and processes; sensitivity later increases at sites of synaptogenesis and decreases elsewhere (Blagburn, Beadle, and Sattelle, 1985; Goodman and Spitzer, 1980). However increases in receptors can occur prior to synaptogenesis and even in the absence of afferent innervation (Hildebrand, Hall, and Osmond, 1979). Clear visualization of all regions of a neuron in dissociated cell culture has facilitated studies of localization of transmitter sensitivity. Differences in sensitivity suggest distinctions between axon and dendrites (O’Lague, Potter, and Furshpan, 1978). The topography of a neuron’s sensitivity to one transmitter can be different from that to another (Barker and Ransom, 1978). The mechanisms by which classes of receptors are targeted to specific regions of surface membrane remains to be defined. Transient Sensitivities

Some cells exhibit particular neurotransmitter sensitivities at an early stage that disappear later. These transiently expressed sensitivities are likely to be developmentally significant, because they are not only being introduced but also removed at particular times. This phenomenon of transient transmitter sensitivity was first described in the development of a clonal line of rat skeletal muscle (L6) cells ( Steinbach, 1975 ). Myoblasts can produce slow hyperpolarizing responses to ACh that are insensitive to nicotinic antagonists. After fusion, myotubes show a fast depolarizing response mediated by nicotinic receptors. During a transition period, some cells show both responses. The process presumably reflects the disappearance of one class of receptor and the acquisition of another. Some primary sensory neurons exhibit a transient sensitivity to glycine at early stages of development in the amphibian embryo that disappears during the first few days of development, although GABA receptors persist (Bixby and Spitzer, 1982). Because no cells are added to or removed during this time, the response has been lost from the existing population. This general form of developmental la-

Xeuronic Control of Ionic Currents

bility has been observed for the epinephrine sensitivity of neurons of the murine locus coeruleus (Finlayson and Marshall, 1984), the glycine and GABA receptors of kitten retinal ganglion cells (lkeda and Robbins, 1984), and may be a feature of the dopamine response of chick retina neurons (Ventura, Klein, and DeMello, 1984). The transient increase in NMDA receptors during development is of particular interest, because antagonists block experience-dependent plasticity in the adult (Cline, Debski, and ConstantinePeron, 1987: Kleinschmidt, Bear, and Singer, 1987; Tsumoto, Hagiwara, Saton, and Hata, 1987). Autoradiographic labeling of receptors has revealed dense but transient expression of binding sites on populations of embryonic cortical neurons (Represa, Tremblay, and Ben-An, 1989; Tremblay, Roisin, Represa, Charriaut-Marlangue, and Ben-Ari, 1988). Receptors are spontaneously activated by endogenous neurotransmitter at early stages (Blanton, LoTurco, and Kriegstein, 1990; Jones and Heinemann, 1989). These NMDA receptors are normally lost during development (Fox, Sato, and Daw, 1989); this process appears to be activity-dependent, because the absence of sensory experience delays the loss of function (Fox, Daw, Sato, and Czepita, 1991 ). Moreover, this sensory deprivation leads to increased levels of transcripts for Ca/calmodulin protein kinase, glutamic acid decarboxylase and GAP43 (Neve and Bear, 1989), consistent with their regulation by calcium influx. Functional Significance

Several clues suggest roles for the early expression of neurotransmitter receptors. The localization of greatest sensitivity (Blagburn et al., 1985) and density of receptors (Hildebrand et al., 1979) in regions of future synaptic inputs prior to their arrival raises the possibility that they play a role in synaptogenesis. The presence of receptors at growth cones and their absence from neurites of cells developing in isolation in culture (Harris and Dennis, 1970; Haydon, McCobb, and Kater, 1984) indicates reorganization of membrane components, because new membrane is added at the growth cone (Bray and Bunge, 1973); the change in sensitivity prior to the formation of synaptic connections could play a role in determining where synapses form. The concentration of receptors at the base of neurites could specify a different localization of synaptic inputs (Pellegrino and Simoneau, 1984), while soma1 receptors reflect the overflow of receptors localized elsewhere. The establishment

66.5

of neuromuscular synapses when ACh receptors are blocked with curare or neurotoxins (Cohen, 1972; Jansen and VanEssen, 1975; Steinbach, Harris, Patrick, Schubert, and Heinemann, 1973) may reflect residual capability of the cells when one of several overlapping mechanisms is eliminated. However, there are instances in which synapses are formed at sites laclung receptor clusters (Frank and Fischbach, 1979). Neurotransmitters are secreted by growth cones at early stages (Hume, Role, and Fischbach, 1983; Young and Poo, 1983). The effect of transmitters on growth cone activity suggests their role in shaping the developing pattern of neuronal connectivity; application of serotonin or electrical stimulation elicits increases in intracellular calcium in growth cones of molluscan neurons in vitru that are associated with inhibition of motility (Cohan, Connor, and Kater, 1987) . Reduction of calcium influx accelerates neurite elongation ( Mattson and Kater, 1987; see also Bixby and Spitzer, 1984a). Different neurotransmitters can have interactive effects on neurite elongation (McCobb, Cohan, Connor, and Kater, 1988; Cohen-Cory, Dreyfus, and Black, 1991 ). It is significant that perturbation of the normal developmental expression of serotonin is associated with aberrations in neuronal morphology and synaptic connections in viva (Goldberg and Kater, 1984). Dopamine inhibits the motility of neuronal growth cones of embryonic chick retinal cells in culture (Lankford, DeMello, and Klein, 1988). Among other transmitters, NMDA raises postsynaptic intracellular calcium levels in developingneocortex ( Yuste and Katz, 1991 ) ,and can regulate neuronal morphology and synaptic strengths of CNS neurons, although other calciumdependent pathways may be involved (Fields, Yu, and Nelson, 1991). Activity of sodium-dependent action potentials influences the pattern of synaptic connections (Shatz, 1990), and these patterns are altered by specific blockade of postsynaptic NMDA receptors ( Constantine-Paton, Cline, and Debski, 1990). Observations that neurotransmitters can specifically inhibit growth cone motility are consistent with roles in synaptogenesis, terminating dendritic outgrowth at appropriate targets. More broadly, they may play a role in shaping neuronal architectures, specifying when and in what directions neurite extension can occur. A developmental role for neurotransmitter sensitivity is indicated by the finding that blockade of muscle ACh receptors reduces the extent of normally occurring cell death in the innervating population of motoneurons. Moreover, blockers of presynaptic input can increase cell death of postsynap-

666

Spitzer

tic neurons. The mechanisms involved are a subject of continuing analysis (Oppenheim, 1991 ). The concentration of intracellular calcium may regulate neuronal survival (see Lipton and Kater, 1989; Brenneman, Yu, and Nelson, 1990, for reviews).

DEVELOPMENTAL REGULATION BY ION FLUX The mechanisms involved in the control of cellular differentiation are diverse and complex. The relatively simple sequential activation of gene expression by transcription factors is central to the process. The experimental evidence for this view originates largely in early analyses of prokaryotic systems. However, this mechanism by itself does not fully satisfy several constraints relevant to eukaryotic differentiation. For excitable cells, including those of early embryos (see review by Moody, Simoncini, Coombs-Hahn, Spruce, and Villaz, this issue), the resolution of these constraints may be achieved in part by the functional expression ofion channels during development. The recent data provide support for the theoretical model of Britten and Davidson ( 1969), whereby the developmental sequence of gene expression is established by the activity of structural gene products in regulating the transcription of further structural genes. The first developmental constraint is the limitation imposed by the number of genes in the organism. This number is inadequate if transcription at every step of development must be specified by a different gene. The problem is at least partly relieved when specificityis achieved by the combinatorial actions of several transcription factors, for which there is substantial precedent. Heterodimers of leucine Lipper, zinc finger. homeobox, and helix-loop-helix proteins provide examples of both positive and negative transcriptional regulation (Benezra, Dans, Lockshon, Turner, and Weintraub, 1990; Gentz, Rauscher, Abate, and Curran, 1989; Glass, Lipkin, Devary, and Rosenfeld, 1989: Landschulz, Johnson, and McKnight, 1989; Treacy, He, and Rosenfeld. 1991 ). A binaiy mechanism whereby transcription factors can act in combinations promotes specific expression of a larger number of genes. Interestingly, several DNA-binding homeobox proteins have multimodal and transient patterns of expression. Some segmentation genes of Drosophila are activated in a striped pattern early in differentiation and briefly again in cells of the nervous system at a later stage, suggesting an economical, developmentally regulated

combinatorial process (Bier, Jan, and Jan, 1990; DiNardo, Kuner, Theis, and O’Farrell, 1985: Doe. Hiromi, Gehring, and Goodman, 1988a; Doe, Smouse, and Goodman, 1988b; Frasch, Hoey, Rushlow, Doyle, and Levine, 1987). Although many transcription factors bind directly to DNA, some exert their activity indirectly, by binding to other transcription factors (Treacy et a]., 1991 ). In a similar manner, modulation of calcium influx at particular stages of development may modify the specific binding of transcription factors to allow stage-specificactivation of genes. In amphibian spinal neurons, spontaneous calcium influx and resultant release from internal stores appear to elevate calcium levels in the nucleus. Calcium may interact with kinases or calcium-binding proteins to alter the specificities of transcription factors and direct novel gene expression (Dash et al., 1991; Gilman, 1988; Jensen, Ohmstede, Fisher, O h , and Sahyoun, 1991;Kapiloff. Mathis, Nelson, Lin, and Rosenfeld. 199I ; Sheng et al., 1991 ). Because calcium channels, stores, and binding proteins are components already required for other basic processes, the ensuing specificity of regulation is achieved with a parsimony of genes. Calcium influx mediated by ligand-activated receptors appears to be developmentally significant for specification of neurotransmi tter levels, neurite extension, and synapse formation, and may be implicated in elevating intracellular calcium at earlier stages of differentiation. Mobilization of intracellular calcium by inositol trisphosphate and diacylglycerol, via G protein coupled receptors, could also provide a form of functional regulation. Activation of nucleotidyl cyclasesprovides another potential mechanism by which transcriptional control is likely to be achieved. These mechanisms may be central to normal maturation in cases in which cells do not exhibit long duration action potentials but express transmitter receptors at early stages of development. Electrical excitability and calcium influx have been shown to be influenced by products of intracellular metabolism, in addition to activation of voltage and ligand gated channels at the cell surface. In pancreatic p cells, the synthesis of ATP from glucose suppresses ATP-sensitive potassium channels and leads to depolarization. The ensuing production of calcium-dependent action potentials and calcium influx then contributes to the exocytotic secretion of insulin (Petersen and Findlay, 1987). There is also evidence for a direct action of metabolites on calcium channels, lowering the threshold for voltage activation (Rojas, Hidalgo, Carroll, Li, and Atwater, 1990; Smith, Rorsman,

Neuronic Control of Ionic Currents

and Ashcroft, 1989: Velasco, Petersen, and Petersen, 1988). Such processes in rapidly growing and differentiating cells may trigger calcium influx during development. A second boundary condition of development is the appropriate level of expression of genes whose products must act in concert to be effective. This condition is satisfied if the early expression of one or more genes regulates and is regulated by the activity of those expressed later. In amphibian spinal neurons, the production of calcium-dependent action potentials is essential for development, and conversion to brief sodium-dependent events may be critical for survival and further maturation of the cells. Calcium influx and intracellular release at early stages of differentiation regulate the functional expression of potassium current in a transcription-dependent manner. The resulting changes promote the shortening of the action potential and the reduction of calcium influx, although calcium channels are still present. This control mechanism is paralleled by those that have been well characterized in mature tissues, such as the pathways for aromatic amino acid biosynthesis. A third requirement of development is the appropriate clocking of events. In addition to levels of expression. the sequence in which genes are transcribed must be tightly regulated. The existence of such controls is intimated by the uniformity of expression of the pattern of differentiation in cells of a specifictype. The activation of one set ofgenes by the expression of function of others builds in a delay that permits the appearance of a phenotype after some interval. Of equal importance is the role of functional expression in the creation of transient patterns of expression. By developmentally limiting the presence of a phenotype whose prolonged action could be deleterious to the cell, survival and further differentiation are enabled. These processes are illustrated in the differentiation of amphibian spinal neurons by the action of calcium influx and release on the subsequent expression of potassium current hours later. During this time, transient elevations of calcium are stimulated by calcium influx through voltage-dependent channels, and trigger events that are critical for standard differentiation. Moreover, this system exhibits the characteristic of negative feedback, as the potassium current enhances repolarization of action potentials and reduces calcium influx. This process limits the impact of intracellular calcium on developmental modulation of potassium current. One may expect substantial roles for voltagegated channels, as well as ligand-gated channels in

667

addition to other pores and pumps, in promoting ion flux that regulates transcriptional and posttranslational events of development. Regulation by functional activity is a well-known phenomenon in the nervous system, and the roles of sodium-dependent impulse activity and activation of neurotransmitter receptors have important consequences in shaping patterns of synaptic connections. The effects of functional activity and calcium flux also have significant impact on the early differentiation of single neurons. I thank Beverly Clendening, Michel Desarmenien, Uwe Ernsberger, Xiaonan Gu, Jaime Renart, and Angeles Rihera for comments on the manuscript. My research is supported by NlH grants NS15918 and NS25916.

REFERENCES AGUAYO,L. G. ( 1989 j . Post-natal development of K + currents studied in isolated rat pineal cells. J. Physiol. 41 4:283-300. AHMED, Z., WALKER,P. S., and FELLOWS, R. E. ( 1983). Properties of neurons from dissociated fetal rat brain in serum-free culture. J. Neurosci. 332448-2462. ANGELIDES, K. J., ELMER,L. W., LOFTUS,D., and ELSON,E. (1988). Distribution and lateral mobility of voltage-dependent sodium channels in neurons. .I. Cell B i d . 106:1911-25. ANGLISTER. L., FARBER, I. C.. SHAHAR,A., and GRINVALD. A. ( 1982). Localization ofvoltagesensitive calcium channels along developing neuritcs: their possible role in regulating neurite elongation. Dev. Bio/. 94:35 1-365. ARCHER,S. M., DUBIN, M. W., and STARK, L. A. ( 1982).Abnormal development ofkitten retino-genicd a t e connectivity in the absence of action potentials. Science 217:743-745. BACCAGLINI, P. I. ( 1078). Action potentials of embryonic dorsal root ganglion neurones in Xenopus tadpoles. .I. Phj”;io/. 283.585-604. BACCAGLINI, P. 1. and SPITZER:N. C. ( 1977). Developmental changes in the inward current of the action potential of Rohon-Beard neurones. J . Physiol. 271:93-117. BADER,C. R., BERTRAND, D., DUPIN. E., and KATO, A. C. ( 1983a). Membrane currents in a developing parasympathetic ganglion. Dev. B i d . 985 15-5 19. BADER,C. K., BERTRAND, D., DUPIN,E., and &TO, A. C. ( 1983b).Development ofelectrical properties of cultured avian neural crest. Nature 305808-8 10. BADER,C. R., BERTRAND, D., and DUPIN,E. (1985). Voltage-dependent potassium currents in developing neurones from quail mesencephalic neural crest. J. Physiol. 366: 129- 15 I. BARISH,M. E. ( 1986 j . Differentiation of voltage-gated

668

Spitzer

potassium current and modulation of excitability in cultured amphibian spinal neurones. J. Physiol. 375229-250. BARKER, J. L. and RANSOM,B. R. ( 1978). Amino acid pharmacology of mammalian central neurones grown in tissue culture. J. Physiol. 280:33 1-354. BECKER, C.-M., HOCH,W., and BETZ,H. (1988). Glycine receptor heterogeneity in rat spinal cord during postnatal development. EMBO J. 7:37 17-3726. E., and KRNJEVIC, K. ( 1988). BEN-ARI,Y., CHERUBINI, Changes in voltage dependence of NMDA currents during development. Neurosci. Lett. 94538-92. R., DANS,R. L., LOCKSHON,D., TURNER,D., BENEZRA, and WEINTRAUB, H. ( 1990). Thc protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61:49-59. G. K., FITZGERALD, S. C., SCHRIER, B. K., and BERGEY, NELSON,P. G. ( I98 I ). Neuronal maturation in mammalian cell culture is dependent on spontaneous electrical activity. Br. Res. 207:49-58. BEST,P. M., MCCOBB,D. P., and BEAM,K. G. ( 1988). Developmental changes in whole-cell Na+ and K + currents from identified chick motoneurons. Biophys. J. 53:430. BIER,E., JAN,L. Y.,and JAN,Y. N. ( 1990).rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophilu melunoguster. Genes Dev. 4: 190-203. BtxBY, J. L. and SPITZER, N. C. ( 1982).The appearance and development of chemosensitivityin Rohon-Beard neurones of the Xenopus spinal cord. J. Physiol. 3305 13-536. BIXBY,J. L. and SPITZER,N. C. ( 1984a). Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca++ and Na' influx. Dev. Biol. 106539-96. BIXBY,J. L. and SPITZER,N. C. (1984b). The appearance and development of neurotransmitter sensitivity in Xenopus embryonic spinal neurones in vitro. J. Physiol. 353: 143- 15 5 . BLACK,1. B., CHIKARAISHI, D. M., and LEWIS,E. J. ( 1985). Trans-synaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion. Bruin Rex 339:lSl-153. BLAGBURN, J. M., BEADLE,D. J., and SATTELLE, D. B. ( 1985). Development of chemosensitivity of an identified insect interneurone. J. Neurosci. 5: 1 167- 1 175. BLAIR,L. A. C. ( 1983). The timing of protein synthesis required for the development of the sodium action potential in embryonic spinal neurons. J. Neurosci. 3:1430-1436. BLAIR,L. A. C. and DIONNE,V. E. ( 1985). Developmental acquisition of Ca*+-sensitivityby K + channels in spinal neurones. Nature 315329-33 1. BLANTON,M. G.: Lo TURCO,J. J., and KRIEGSTEIN, A. R. ( 1990). Endogenous neurotransmitter activates N-methyl-D-aspartatereceptorson differentiatingneurons in embryonic cortex. Proc. Nut. Acud. Sci. 879027-8030.

BLUMENTHAL, R., KEMPF, C., VAN RENSWOLJDE, J., WEINSTEIN, J. N., ~ ~ ~ K L A U S R. NE , D.R(1983). Voltage-dependent orientation of membrane proteins. J. Cell. Biochem. 2255-67. J. T. ( 1984). Activity and the Boss, V. C. and SCHMIDT, formation of ocular dominance patches in dually innervated tectum ofthe goldfish. J. Neurosci. 12:289 12905. J. V. ( 1990). Developmental BOW, M. A. and NADLER, increase in the sensitivity to magnesium of NMDA receptors on CA 1 hippocampal pyramidal cells. Dev. Br. Res. 5655-61. BRAY,D. and BUNGE,M. B. ( 1973). The growth cone in neurite extension. In: Locomotion of' Tissue Cells, CIBA Symposium 14: 195-209. Elsevier, Amsterdam. BREGESTOVSKI, P. D., PRINTSEVA, 0. Y., SEREBRYAKOV, V., STINNAKRE, J., TURMIN,A., and ZAMOYSKI, V. ( 1988). Comparison of Ca2+-dependentK + channels in the membrane of smooth muscle cells isolated from adult and foetal human aorta. Pfluegers Archiv. 413% 13. BKENNEMAN, D. E., Yu, C., and NELSON,P. G. ( 1990). Multi-determinate regulation of neuronal survival: neuropeptides, excitatory amino acids and bioelectric activity. Int. J. Dev. Neurosci. 8:37 1-378. BRITTEN, R. J. and DAVIDSON, E. H. ( 1969).Gene regulation for higher cells: a theory. Science 165349-357. CATTERALL, W. A. ( 198 1 ). Localization of sodium channels in cultured neural cells. J . Neurosci. 1:777783. J. ( 1989). c-,fosproto-oncogene expressionin CAUBERT, the nervous system during mouse development. Mol. Cel Biol. 9~2269-2272 . CLENDENING, B. and HUME,R. I. ( 1990a). Expression of multiple neurotransmitter receptors by sympathetic preganglionic neurons in vitro. J. h'eurosci. 10:39773991. B. and HUME,R. 1. ( 1990b). Cell interacCLENDENING, tions regulate dendritic morphology and responses to neurotransmitters in embryonic chick sympathetic preganglionic neurons in vitro. J. Neurosci. 10:39924005. M. CLINE,H., DEBSKI,E. A., and CONSTANTINE-PATON, ( 1987). N-methyl-D-aspartate receptor antagonist desegregates eye-specific stripes. Proc. Nut. Acud. Sci, 84:4342-4345. COHAN,C. S., CONNOR:J. A., and KATER,S. B. ( 1987). Electrically and chemically mediated increases in intracellular calcium in neuronal growth cones. J . Neurosci. 7:3588-3599. COHEN,M. W. (1972). The development ofneuromuscular connexions in the presence of D-tubocurarine. Br. Res. 41:4 57-46 3. COHEN-CORY, S., DREYFUS,C. F., and BLACK,I. B. ( 199 1 ) . NGF and excitatory neurotransmitters regulate survival and morphogenesis of cultured cerebellar Purkinje cells. J. Neurosci. 11:462-47 1. CONSTANTINE-PATON, M., CLINE,H. T., and DEBSKI,E. ( 1990). Patterned activity, synaptic convergence,and

Neuronic Control of Ionic Currents the NMDA receptor in developing visual pathways. Ann. Rev. Neurosci. 13:129-154. CURRAN, T. and FRANZA,B. R., JR. (1988). Fos and Jun: the AP- 1 connection. Cell 55395-397. DASH,P. K., KARL,K. A., COLICOS,M. A., PRYWES, R., and KANDEL,E. R. ( 1991 ) . CAMPresponse elementbinding protein is activated by Ca” lcalmodulin-as well as CAMP-dependent protein kinase. Proc. Nut. Acud. Sci. 88506 1-5065. DECINO,P. and KIDOKORO, Y . ( 1985). Development and subsequent neural tube effects on the excitability of cultured Xenopus myocytes. J . Neuro.sci. 5: 147 I 1482. DESARMENIEN, M. G. and SPITZER,N. C. ( 199 1 ). Role of calcium and protein kinase C in development of the delayed rectifier potassium current in Xenopus spinal neurons. Neuron. To appear. DICHTER,M. A., TISHCLER, A. S., and GREENE,L. A. ( 1977). Nerve growth factor-induced increase in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line. Nuture 268501-504. DOE, C. Q., HIROMI,Y., GEHRING,W. J., and GOODMAN,C. S. (1988a). Expression and function of the segmentation gene fushi turuzu during Drosophilu neurogenesis. Science 239: 170- I 75. DOE,C. Q., SMOUSE,D., and GOODMAN, C. S. ( 1988b). Control of neuronal fate by the Drosophilu segmcntation gene even-skipped. Nature 333:376-378. DINARDO,S., KUNER,J. M., THEIS,J., and OFARRELL, P. H. ( 1985). Development of embryonic pattern in D.melunogaster as revealed by accumulation of the nuclear engrailed protein. Cell 43: 5 9-6 9. FIELDS,R. D., Yu, C., and NELSON,P. G. ( 199 1 ). Calcium, network activity, and the role of NMDA channels in synaptic plasticity in vitro. J. Neurosci. 11:134136. FINLAYSON, P. G. and MARSHALL, K. C. ( 1984). Hyperpolarizing and age-dependent depolarizing responses of cultured locus coeruleus neurons to noradrenaline. Dev. Br. Rex 15165-175. Fox, K., DAW,N., SATO,H., and CZEPITA,D. ( 1991). Dark-rearing delays the loss of NMDA-receptor function in kitten visual cortex. Nature 350:342-344. FOX,K., SATO,H., and DAW,N. ( 1989). The location and function of NMDA receptors in cat and kitten visual cortex. J. Neurosci. 9:2443-2454. FRANK.E. and FISCHBACH, G. D. (1979). Early events in neuromuscular junction formation in vitro. J. Cell Biol. 83:143- 158. FRASCH, M., HOEY,T., RUSHLOW, C., DOYLE,H., and LEVINE,M. ( 1987). Characterization and localization of the even-skipped protein of Drosophila. EMBO J . 6~749-759. FRIEDMAN, W. J., DREYFUS,C. F., MCEWEN,B., and BLACK,I. B. ( 1988). Presynaptic transmitters and depolarizing influencesregulate development of the substantia nigra in culture. J. Neurosci. 8:36 16-3623. W. A. GALPER,J. B., KLEIN, W., and CATTERALL,

669

( 1977). Muscarinic acetylcholinereceptorsin developing chick heart. J. Biol. Chem. 25253692-8699. GALVANI, L. ( 1791 ). Commentary on the effect of electricity on muscular motion. Green, R. M., transl., 1953. Elizabeth Licht, Cambridge, MA. GENTZ,R., UUSCHER,F. J., ABATE,C., and CURRAN, T. ( 1989). Parallel association of fos and jun domains. Science 243: 1695- 1699. GILMAN,M. J. (1988). The c-.fus serum response element responds to protein kinase C-dependent and -independent signals but not to cyclic AMP. Genes Dev. 2:394-402. GLASS,C. K., LIPKIN,S. M., DEVARY, 0. V., and RoSENFELD. M. G. ( 1Y89). Positive and negative regulation of gene transcription by a retinoic acid-thyroid hormone receptor heterodimer. Cell 59:697-708. GOLDBERG. J. I. and KATER,S. B. (1989). Expression and function of the neurotransmitter serotonin during development of the Helisoma nervous system. Dev. Bid. 131:483-495. GOODMAN, C. S. and SPITZER, N. C. ( 1979). Embryonic development of identified neurones: Differentiation from neuroblast to neurone. Naturr 280:208-2 14. C. S. and SPITZER, N. C. ( 1980). Embryonic GOODMAN, development of neurotransmitter receptors in grasshoppers. In: Receptors ,for Neurotransmitters, Hormones, and Pheromones in Insects, D. B. Sattelle and L. M. Hall, Eds., Elsevier, Amsterdam, pp. 195-207. GOODMAN, C. S. and SPITZER, N. C. ( 1981 ). The development of electrical properties of identified neurones in grasshopper embryos. J. Physiol. 313385-403. GOTTMANN, K., DIETZEL,I. D., Lux, H. D., HUCK,S., and ROHRER,H. (1988). Development of inward currents in chick sensory and autonomic neuronal precursor cells in culture. J. Neurosci. 8:3722-3732. K., DIETZEL,I. D., Lux, H. D., and RUEGOTTMANN, DEL, C. ( 1989). Proton-induced Na+ current develops prior to voltage-dependent Nai and Ca2+currents in neuronal precursor cells from chick dorsal root ganglion. Neurosci. Lett. 99:90-94. M., RODR~CUEZ-T~BAR, GRANTYN,R., PEROUANSKY, A., and Lux, H. D. ( 1989). Expression of depolarizing voltage- and transmitter-activated currents in neuronal precursor cells from the rat brain is preceded by a proton-activated sodium current. Dev. Br. Rex 49: 150- 155. GREENBERG, M. E., ZIFF, E. B.. and GREENE,L. A. ( 1986). Stimulation of neuronal acetylcholine receptors induces rapid gene transcription. Science 23453083. A. ( 1982). PC12 cultures GREENE,L. A. and TISCHLER, in neurobiological research. Adv. Cell. Neurohiol. 3~373-414. Gu, Y . and HALL,Z. W. (1988). Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated muscle. Neuron 1:117-125. Gu, X. and SPITZER,N. C. ( 1991), T-type Ca++current

670

Spitzer

and its function in triggering Cat+ influx in embryonic Xenopus spinal neurons. Soc. Neurosci. Ahstr. 17: HARRIS,A. J. and DENNIS, M. J. ( 1970). Acetylcholine sensitivity and distribution on mouse neuroblastoma cells. Science 167:1253-1255. HARRIS,W. A. ( 1980). The effect of eliminating impulse activity on the development of the retinotectal projection in salamanders. J. C o i p . Neurol. 194:303-3 17. HARRIS,W. A. (1981). Neural activity and development. ilnn. Rev.Physiol. 43689-7 10. HARRIS,G. L.,HENDERSON,L. P., and SPITZER,N. C. ( 1988). Changes in densities and kinetics of delayed rectifier potassium channels during neuronal differentiation. Neuron 1:739-750. HARRISON, R. G. ( 1904). An experimental study of the relation of the nervous system to the developing musculature in the embryo ofthe frog. Am. J. dnat. 3:197220. HAYDON,P. G . , MCCOBB,D. P., and KATER, S. B. ( 1984). Serotonin selectivelyinhibitsgrowth cone motility and synaptogenesis of specific identified neurons, Science 226561-564. HENDERSON, L. P. and SPITZER, N . C. ( 1986). Autonomous early differentiation of neurons and muscle cells in single cell cultures. Dev. Biol. 113:381-387. HERNANDEZ-CRUZ, A., SALA, F., and ADAMS,P. R. ( 1990). Subcellular calcium transients visualized by confocal microscopy in a voltage-clamped vertebrate neuron. Science 2472358-862. HILDEBRAND, J. G., HALL,L. M., and OSMOND,B. C. (1979). Distribution of binding sites for 1251-labeled a-bungarotoxin in normal and deafferented antennal lobes of Manduca sexta. Proc. Nut. Acad. Sci. 76:499503. HOLLIDAY,J., ADAMS, R. J., SEJNOWSKI, T. J.: and SPITZER. N. C. ( 199 I). Calcium-induced release of calcium regulates differentiation of spinal neurons. Neuron. To appcar. HOLLIDAY, J. and SPITZER,N. C. (1990). Spontaneous calcium influx and its roles in differentiation of spinal neurons in culture. Dcv. Bid. 141:13-23. HOLLMANN.M., HARTLEY,M.: and HEINEMANN. S. ( 199 1 ). Ca2+permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition. Science 252235 1-853. HUGUENARD,J. R., HAMILL,0. P.. and PRINCE,D. A. ( 1988). Developmental changes in Na conductances in rat neocortical neurons: appearance of a slowly inactivating component. J. Neurophysiol. 59:778-795. HUME?R. I., ROLE,I. W., and FISCHBACH, G. D. ( 1983). Acetylcholine release from growth cones detected with patches of acetylcholine receptor-rich membranes. Nature 305:632-634. HUNTER,D. D. and NATHANSON, N. M. (1984). Decreased physiological sensitivity mediated by newly synthesized muscarinic acetylcholine receptors in embryonic chicken heart. Proc. Nal. ilcacl. Sci. 81:35823586. HUSSY,N. ( 199 1 ). Developmental change in calcium+

activated chloride current during the differentiation of Xenopus spinal neurons in culture. Dev. Biol.To appear. IKEDA, H. and ROBBINS,J. (1984). Are some disused transmitter receptors on kitten retinal ganglion cells “lost” during development? J. Physiol. 357: 12P. ISHIDA, I. and DEGUCHI, T. ( 1983). Effect of depolarizing agents on choline acetyltransferase and acetylcholinesterase activities in primary cell cultures of spinal cord. J. Neurosci. 3: 18 18- 1823. JACKSON, M. B., LECAR,H., BRENNEMAN, D. E., FITZGERALD, S., and NELSON, P. G. ( 1982). Electrical development in spinal cord cell culture. J. Neurosci. 2: 1052-106 1. JAFFE, L. A. ( 1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature 261:6871. JANSEN, J. K. S. and VANESSEN,D. C. ( 1975). Re-innervation of rat skeletal muscle in the presence of n-bungarotoxin. J. Ph~~siol. 250:65 1-667. JENSEN, K. F., OHMSTEDE,C. A., FISHER, R. s., OLIN, J. K., and SAHYOUN. N. ( 199 1 ) . Acquisition and loss of a neuronal Ca”/calmodulin-dependent protein kinase during neuronal diff‘erentiation.Proc. fiat. Acad. Sci. 88:4050-4053. JONES,R. S. and HEINEMANN, U. ( 1989). Spontaneous activity mediated by NMDA receptors in immature rat entorhinal cortex in vitro. Neurosci. Lett. lW:9398. KAPILOFF, M. S., MATHIS,J. M., NELSON,C. A., LIN, C. R.. and ROSEWELD.M. G. ( 1991 ). Calcium/calmodulin-dependent protein kinase mediates a pathway for transcriptional regulation. Proc. Nut. Acad. Sci. 88:3 7 10-3 7 14. KLEINSCHMIDT. A., BEAR?M. F., and SINGER,W. ( 1987). Blockade of ‘NMDA’ receptors disrupts experience-dependent plasticity of kitten striate cortex. Science 238:3 55 -3 5 8. KOCSIS,J. D., RUIZ.J. A,, and WAXMAN,S. G. ( 1983). Maturation of mammalian myelinated fibers: changes in action-potential characteristics following 4-aminopyridine application. J. Neurophysiol. 50:449-463. KRIEGER, C. and SEARS,T. A. ( 1988). The development of voltage-dependent ionic conductances in murine spinal cord neurones in culture. Can. J. Physiol. Pharm. 66:1328-1336. KRUIJER,W., SCHUBERT, D., and VERMA,I. M. ( 1985). Induction of proto-oncogene fix by nerve growth factor. Proc. Nut. Acad. Sci. 82:7330-7334. KUHSE,J., SCHMIEDEN, V., and BETZ,H. ( 1990). A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit. Neuron 5867873. LANDSCHULZ, W. H.. JOHNSON, P. F.. and MCKMGHT, S. L.( 1989).TheDNAbindingdomainoftheratliver nuclear protein C/EBP is bipartite. Science 243: 168 11687. LANKFORD,K. L., DEMELLO,F. G., and KLEIN,W. L. ( 1988). D,-type dopamine receptors inhibit growth

Neuronic Control of Ionic Currents cone motility in cultured retina neurons: evidence that neurotransmitters act as morphogenic growth regulators in the developing nervous system. Proc. Nut. Acud. Sci. 854567-457 1 . LAUDER,J. M. (1990). Ontogeriy of the serotonergic system in the rat: serotonin as a developmental signal. -3nn. NY Acnd. Sci. 600:297-3 14. LECHLEITER,J., GIRARD,S., CLAPHAM,I).. and PERALTA, E. ( 199 1 ). Subcellular patterns o f calcium release determined by G protein-specific residues ofmuscarinic receptors. Nuture 350:505-508. LIPTON.S. A. and KATER,S. €3. ( 1989). Neurotransmitter regulation of neuronal outgrowth, plasticity and survival. Trends Neurosci. 12:265-270. LOCKERY? S. R. and SPITZER,N. C. ( 199 1 ). Reconstruction of action potential development from whole cell currents of differentiating spinal neurons. To appear. LOVINGER, D. M. and WHITE:G. ( 1989). Post-natal development of burst firing behavior and the lowthreshold transient calcium current examined using isolated neurons from rat dorsal root ganglia. Neurosci. Lett. 102:50-57. MATSUDA,Y., YSHIDA, S., and YONEZAWA. l'.( 1978). Tetrodotoxin sensitivity and Ca component of action potentials of mouse dorsal root ganglion cells cultured in vitro. Br. Re.s. 154:69-82. MATTSON,M. P. and KATER.S. B. ( 1987). Calcium regulation of neurite elongation and growth cone motility. .I. Neumsci. 7:4034-4043. MCCOBB,D. P., BEST,P. M., and BEAM,K. G. ( 1989). Development alters the expression of calcium currents in chick limb motoneurons. Neuron 2: 1633- 1643. MCCOBB,D. P., COHAN,C. S., CONNOR,J. A.: and KATER, S. B. (1988). Interactive effects of serotonin and acetylcholine on neurite elongation. Neuron 1:377-385. MCCOBB,D. P., BEST,P. M., and BEAM,K. G. ( 1990). The differentiation o f excitability in embryonic chick limb motoneurons. J. Neurosci. 10:2974-2984. MCDONALD:J. W.. JOHNSTON.M. V., and YOUNG, A. B. ( 1990). Differential ontogenic development of three receptors comprising the NMDA receptor/ channel complex in the rat hippocampus. Exp. Neurol. 110:237-247. MEYER,R. L. (1982). Tetrodotoxin blocks the formation of ocular dominance columns in goldfish. Science 218:589-591. MILBRANDT, J. (1986). Nerve growth factor rapidly induces c-fos in mRNA in PC12 rat pheochromocytoma cells. Pmc. Nut. Acud. Sci. 83:4789-4793. MILBRANDT, J. ( 1987). A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor. Science 238:797-799. MILBRANDT,J. ( 1988). Nerve growth factor induces a gcne homologous to the glueoeorticoid receptor gene. Neuron 1:1 83- 1 88. MISHINA,M., TAKAI,T., IMOTU,K., NODA,M., TAKAHASHI,T., NUMA, S., METHFESSEL,C., and SAKMA", B. ( 1986). Molecular distinction between fetal

671

and adult forms of muscle acetylcholine receptor. Nature 321:406-411. MIYAKE,M. ( 1978). The development of action potential mechanism in a mouse neuronal cell line in vitro. Br. Res. 143:349-354. MONTMINY,M. R., SEVARINO,K. A., WAGNER,J. A,, MANDEL,G., and GOODMAN,R. H. ( 1986). Identification ofa cyclic-AMP-responsive element within the rat somatostatin gene. Proc. Xut. Arud. Sci. 83:66826686. MOWER. H., SEEBURG:P. H., and WISDEN,W. ( 199I ). Glutamate-operated channels: developmentally early and mature forms arisc by alternative splicing. Neuron 6:799-8 10. MOODY, W. J., SIMONCINI,L., COOMBS-HAHN,J., SPRUCE,A. E., and VILLAZ,M. ( 199 1 ). The development of ion channels in early embryos. J. Neurohid. 22:674-684. MORAN.J. and PATEL,A. J. ( 1989). Effect ofpotassium depolariLation on phosphate-activated glutaminase activity in primary cultures of cerebellar granule neurons and astroglial cells during development. Dev. Bruin Re.4. 46:97-105. MORGAN.J. I. and CURRAN,T. ( 1986). The role of ion flux in the control of c-/us expression. Nature 322: 552-555. MORGAN,J. 1. and CURRAN,T. ( 199 I ). Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fus and j u n . Ann. Rev. Neurosci. 14:42 1-45 1. MORI-OKAMOTO, J., ASHIDA.H., MARU,E., and TATSUNO, J. ( 1983). The development of action potentials in cultures of explanted cortical neurons from chick embryos. Dev. B i d 97:408-416. MOURRE.C., CERVERA, P.: and LAZDUNSKI,M. ( 1987). Autoradiographic analysis in rat brain of the postnatal ontogeny of voltage-dependent Na channels, CaZ+dependent K + channels and slow Ca2+channels identified as receptors for tetrodotoxin, apamin and (-)desmethoxyverapamil. Bruin Res. 417:2 1-32. J. M. and GURNEY, A. M. ( 1989). DevelopNERBONNE, ment of excitable membrane properties in mammalian sympathetic neurons. J . Neurosci. 9:3272-3286. NEVE.R. L. and BEAR,M. F. ( 1989). Visual experience regulates gene expression in the developing striate cortex. Proc. Nut. Acud. S'ci 86:478 1-4784. NISHI, R. and BERG, D. K. ( 1981). Two components from eye tissue that differentially stimulate the growth and development of ciliary ganglion neurons in cell culture. J . Aeurosci. 1505-5 13. OBATA,K. ( 1977). Development of n e u r ~ m u ~ c u l a r transmission in culture with a variety of neurons and in the presence of cholinergic substances and TTX. Br. Res. 119:141-153. O'Dowu, D. K. ( 1983). RNA synthesis dependence of action potential development in spinal cord neurones. Nufure 303:6 19-62 1, O ' D o w , D. K., RIBERA,A. €3.. and SPITZER: N. C. ( 1988). Development of voltage-dependent calcium,

672

Spitzer

sodium and potassium currents inXenopus spinal neurons. J. Neurosci. 8:792-805. OFFORD,J. and CATTERALL, W. A. (1989). Electrical activity, CAMP, and cytosolic calcium regulate mRNA encoding sodium channel alpha subunits in rat muscle cells. Neuron 2: 1447- 1452. OHMORI,H. and SASAKI,S. (1977). Development of neuromuscular transmission in a larval tunicate. J. Physiol. 269:22 1-254. OLAGUE,P. H., POTTER,D. D., and FURSHPAN,E. J. ( 1978). Studies on rat sympathetic neurons developing in cell culture. 111. Cholinergic transmission. Dev. Biol. 67:424-443. O’LAGUE,P. H. and HUTTNER,S. L. ( 1980). Physiological and morphological studies of rat pheochromocytoma cells (PC 12 ) chemically fused and grown in culture. Proc. Nat. Acad. Sci. 77: l 70 l - 1705. OPPENHEIM, R. W. ( 199 1 ). Cell death during development of the nervous system. Ann. Rev. Neurosci. 14:453-501. PELLEGRINO,M. and SIMMONEAU, M. (1984). Distribution of receptors for acetylcholine and 5-hydroxytryptamine on identified leech neurones growing in culture. J. Physiol. 352:669-684. PETERSEN, 0. H. and FINDLAY,I. (1987). Electrophysiology of the pancreas. Physiol. Rev. 67:1054-1116. PETTIGREW, A. G . . CREPEL,F., and KRUPA.M. ( 1988). Development of ionic conductances in neurons of the inferior olive in the rat: an in vitro study. Proc. R . Soc. Lond. B. 234:199-218. POO, M.-M. (1985). Mobility and localization of proteins in excitable membranes. Ann. Rev. Neurosci. 8~369-406. PRZYWARA, D. A., BHAVE,S. V., BHAVE,A,, WAKADE, T. D., and WAKADE,A. R. ( 199 1 ) . Stimulated rise in neuronal calcium is faster and greater in the nucleus than in the cytosol. FASEB J. 5:217-222. RAYNAUD,B., FAUCON-BIGUET, N., VIDAL,S., MALLET, J., and WEBER,M. J. ( 1987). The use of a tyrosine-hydroxylase cDNA probe to study the neurotransmitter plasticity of rat sympathetic neurons in culture. Dev. Biol. 119:305-312. RENAUD,J. F., BARHANIN, J., CAVEY,D., FOSSET,M., and LAZDUNSKI, M. ( 1980). Comparative properties of the in ovo and in vitro differentiation of the muscarinic cholinergic receptor in embryonic heart cells. Dev. Biol. 78: 184-200. REPRESA,A., TREMBLAY: E., and BEN-ARI,Y. (1989). Transient increase of NMDA binding sites in human hippocampus during development. Neurosci. Lett. 9 9 ~ 1-66. 6 RIBERA,A. B. ( 1990). A potassium channel gene is expressed at neural induction. Neuron 5 6 9 1-70 1 . RIBERA,A. B. and SPITZER,N. C. ( 1989). A critical period of transcription required for differentiation of the action potential of spinal neurons. Neuron 2:10551062. RIBERA,A. B. and SPITZER,N. C. ( 1990). Differentia-

tion of I,, in amphibian spinal neurons. J. Neurosci. 10: 1886-1991. RIBERA,A. B. and SPITZER,N.C. ( 199 1 ). The differentiation of potassium current in embryonic amphibian myocytes. Dev. Biol. 144:119-128. RITCHIE,A. (1979). Catecholamine secretion in a rat pheochromocytoma cell line: Two pathways for calcium entry. J. Physiol. 28654 1-56 l . RITCHIE,J. M. ( 1982). Sodium and potassium channels in regenerating and developing mammalian myelinated nerves. Proc. R. Soc. Lond. B. 215:273-287. ROACH,A., ADLER,J. E., and BLACK,I. B. (1987). Depolarizing influences regulate preprotachykinin mRNA in sympathetic neurons. Proc. Nut. Acad. Sci. 845078-508 1. ROBERTS,W. M., JACOBS, R. A., and HUDSPETH,A. J. ( 1990). Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. J. Neurosci. 10:3664-3684. ROJAS,E., HIDALGO,J., CARROLL, P. B., LI, M.-X., and ATWATER, L. ( 1990). A new class ofcalcium channels activated by glucose in human pancreatic beta cells. FEBS Lett. 261:265-270. SALKOFF,L. (1985). Development of ion channels in the flight muscle of Drosophila. J. Physiol. (Paris) 80~275-282. SHATZ,C. J. ( 1990). Impulse activity and the patterning of connections during CNS development. Neuron 5745-756. SHEN,S. S. and STEINHARDT, R. A. ( 1984). Time and voltage windows for reversing the electrical block to fertilization. Proc. Nat. Acad. Sci. 81: 1436-1439. SHENG,M., MCFADDEN,G., and GREENBERG, M. E. (1990). Membrane depolarization and calcium induce c-,fos transcription via phosphorylation of transcription factor CREB. Neuron 457 1-582. SHENG,M., THOMPSON, M. A., and GREENBERG, M. E. ( I99 1 ). CREB: a Ca2+-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252:1427-1430. SHERMAN, S. J. and CATTERALL, W. A. ( 1984). Electrical activity and cytosolic calcium regulate levels of tetrodotoxin-sensitive sodium channels in cultured rat muscle cells. Pruc. Nut. Acad. Sci. 81:262-266. SIEGEL,R. E. and FISCHBACH, G. D. ( 1984). Muscarinic receptors and responses in intact embryonic chick atrial and ventricular heart cells. Dev. Biol. 101:346356. SMART,T. G. and CONSTANTI, A. ( 1990). Differential effect of zinc on the vertebrate GABA,-receptor complex. Br. J. Pharm. 99:643-654. SMITH,P. A., RORSMAN,P., and ASHCROFT,F. M . ( 1989). Modulation of dihydropyridine-sensitive Caz+channels by glucose metabolism in mouse pancreatic beta-cells. Nature Lond. 342550-553. SOMMER,B., K E I N ~ E N K.,, VERDOORN,T. A., WISDEN, W., BURNASHEV, N., HERB,A., KOHLER,M.,

I%~EURONIC CONTROL OF [ONIC CURRENTS

TAKAGI, T., SAKMANN, B., and SEEBURG,P. H. ( 1990). Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science 249: 1580- I 5 8 5 . SPITZER,N. C. ( 1985). The control of development of neuronal excitability. In: Molecular Bases of Neurul Development, G. M. Edelman, W. E. Gall, and W. M. Cowan, Eds., Rockefeller University Press, New York, pp. 67-88. SPITZER. N. c. and LAMBORGHINL J. E. (1976). The development ofthe action potential mechanism ofamphibian neurons isolated in cell culture. Proc. Nu[. Acad. Sci. 73: 164 1 - 1645. STEINBACH, J. H . ( 1975). Acetylcholine responses in clonal myogenic cells in vilro. .I. Phjwiol. 247:393405. STRE~T, J. and Lux, H. D. (1989). Distribution of calcium currents in sprouting PC12 cells. J. Neurosci. 9:4 190-4 199. THOMPSON, S. and COOMBS,J . ( 1988). Spatial distribution of Ca currents in molluscan neuron cell bodies and regional differences in the strength of inactivation. .J. Neurosci. 8:1929-1939. THOMPSON, W., KUFFLER:D. P., and JANSEN. J . K. S. (1979). The effect of prolonged reversible block of nerve impulses on the elimination of polyneuronal innervation of newborn rat skeletal muscle fibers. Areel*rosci. 4:27 1-28 1. TREACY,M. N., HE, X., and ROSENFELD. M. G. ( 1991 ). I-POU: a POU-domain protein that inhibits neuronspecific gene activation. Nature 350:577-584. TREISMAN,R. (1986). Identification of a protein-binding site that mediates transcriptional rcsponse of the c-fos gene to serum factors. Cell 46567-574. TRBMBLAY, E., ROISIN, M. P., REPRESA. A., CHARRIAUT-MARLANGUE, C., and BEN-ARI. Y. ( 1988). Transient increased density of NMDA binding sites in the developing rat hippocampus. Brain Rex 461:393-396. TREMBLAY,E., ROISIN-LALLEMAND. M. P., and BENARI, Y. (1990). Developmental study of [ 3H]TCP and [ 3H]glycine binding sites in the rat hippocampus. Dev. Brain Res. 57:21-28. TSUMOTO,T., HAGIWARA, K., SATO,H., and HATA,Y. (1987). NMDA receptors in the visual cortex of young kittens are more effective than those of adult cats. Nature 3275 13-5 14. VANESSEN,D. C. ( 1982). Neuromuscular synapse elimination. In: Neuronal Developmenl, N. C. SpitLer, Ed., Plenum Press, pp. 333-376.

673

VELASCO,J. M., PETERSEN,J. U. H., and PETERSEN, 0. H. ( 1988). Single-channel Ba2+currents in insulinsecreting cells are activated by glyceraldehyde stimulation. FEBS Lett. 231:366-370. VENTURA, A. L. M., KLEIN,W. L., and DEMELLO,F. G. ( 1984). Differential ontogenesis of D, and D, dopaminergic receptors in the chick embryo retina. Dcv. Bruin Res. 12:2 17-223. VIDAL,S., RAYNAUD,B., and WEBER.M. J. ( 1989). The role of Ca2+channels of the L-type in neurotransmitter plasticity of cultured Sympathetic neurons. Mol. Br. Rex 6:187-196. WALICKE,P. A. and PATTERSON, P. H. ( I98 I ). On the role of Ca++in the transmitter choice made by cultured sympathetic neurons. J. Neurosci. 1543-350. WAXMAN.S. G . and FOSTER,R. E. (1980). Development of the axon membrane during differentiation of myelinatcd fibres in spinal nerve roots. Proc. R. SOC. I,ond. B. 209:44 1-446. WESTBROOK, G. L. and BRENNEMAN, D. E. ( 1984). The development of spontaneous electrical activity in spinal cord cultures. In: Developmental Neuroscience: l'h,ysiological, Pharmacological and Clinicul Aspects, F. Caciagli: E. Giacobini, R. Paoletti, Eds., Elsevier, pp. 11-17. WESTENBROEK, R. E., AHLUANIAN. M. K., and CATTERALL, W. A. (1990). Clustering of L-type CaZt channels at the base of major dendrites in hippocampal pyramidal neurons. AAJuliire347:28 1-284. WILLARD,A. L. (1980). Electrical excitability of outgrowing neurites of embryonic neurones in cultures of dissociated neural plate of Xenopus luevis. J. Physiol. 301:115-128.

YAARI,Y., HAMON,B., and Lux, H. D. ( 1987). Development of two types of calcium channels in cultured mammalian hippocampal neurons. Science 235:680682. YOOL,A. J., DIONNE, V. E., and GRUOL.D. L. ( 1988). Developmental changes in K'-selective channel activity during differentiation of the Purkinje neuron in culture. J. Neurosci. 8: 197 1- 1980. YOUNG,S. H. and Poo, M.-M. (1983). Spontaneous release of transmitter from growth cones of embryonic neurones. Nature 305:634-637. YUSTE,R. and KATZ,L. C. (1991). Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters. Neuron 6:333-344. ZISKIND-CONHAIM, L. ( 1988). Electrical properties of motoneurons in the spinal cord of rat embryos. Dev. Hiol. I28:2 1-29.