J. Physiol. (1981), 313, pp. 369-384 369With I plate and 11 text-figuresPrinted in Great Britain

THE MATURE ELECTRICAL PROPERTIES OF IDENTIFIED NEURONESIN GRASSHOPPER EMBRYOS

BY COREY S. GOODMAN*t AND NICHOLAS C. SPITZER*From the *Department of Biology, B-022, University of California, San Diego,

La Jolla, California 92093, and the tDepartment of Biological Sciences,Stanford University, Stanford, California 94305, U.S.A.

(Received 21 May 1980)

SUMMARY

1. We have examined the mature electrical properties of five identified neuronesin embryos of the grasshopper Schistocerca nitens. The five cells arise from twodifferent precursor cells: the median neuroblast, whose first three progency are calledDUM 3,4,5; DUM 4,5; and DUM 5; and mid-line precursor 3, which divides once toproduce the H cell and the H cell sibling.

2. Electrical coupling was investigated by dual intracellular penetrations. Actionpotentials were elicited by intracellular stimulation of cell bodies and by extracellularstimulation of axons. The ionic basis of action potentials was investigated bychanging the ionic environment and by applying various blocking agents.

3. Most of the mature electrical properties of all five cells appear by day 13 ofembryonic development. They change little through hatching on day 20.

4. The recorded resting potential for all five cells varies from -55 to -60 mV andthe recorded input resistance varies from 200 to 450 MQ. All five cells show delayedrectification, much of which is blocked by tetraethylammonium (TEA). Theirresistance increases as they are hyperpolarized.

5. The five cells do not appear to be electrically coupled between days 13 and 20.6. All five neurones generate mature action potentials and in several cases show

cell-specific electrical properties by day 13. The ionic dependence and depolarizingphase of the action potential change little between days 13 and 20; some changesoccur in the after-hyperpolarization.

7. One cell-specific difference is observed in normal saline. Four of the cells haveaxons, median neurites, and somata which generate action potentials in normal saline;but one of the cells (the H cell sibling) has an inexcitable soma and generates actionpotentials only in its axon.

8. Another cell-specific difference in the soma action potentials of DUM 3,4,5,;DUM 4,5; DUM 5; and the H cell is observed when outward current is blocked byTEA. In three of the cells TEA causes the short-duration action potential (2-4 msec)to be converted into a long-duration action potential (100-1000 msec) in which thereis an initial spike (Na+-dependent) followed by a long plateau (Ca2+-dependent). Inthe other cell, DUM 5, at resting potential the addition ofTEA only causes a shoulder

t To whom correspondence should be sent.

0022-3751/81/3620-0987 $07.50 ©) 1981 The Physiological Society

370 C. S. GOODMAN AND N. C. SPITZER

(Ca2+-dependent) on the falling phase of the action potential. DUM 5 and DUM 4,5thus have different electrical properties, even though they differ only by a single celldivision from the precursor cell.

9. In all four neurones which normally generate soma action potentials (DUM3,4,5; DUM 4,5; DUM 5; and the H cell), the inward current is carried by both Na+and Ca2+. On day 13, either inward current alone can generate the overshooting actionpotential; in contrast, by day 18, neither inward current alone can generate anovershooting response. The inward current of the axon action potential in all five cellsis carried predominantly by Na+. I

10. Thus, the progeny oftwo differentembryonic precursor cells (median neuroblastand mid-line precursor 3) show a broad spectrum of electrical properties. The maturephenotype of electrical excitability is not a property shared in common by all theprogeny of a single embryonic precursor cell in the grasshopper. Conversely, progenyfrom different precursor cells can share the same mature phenotype of electricalexcitability.

INTRODUCTION

Early in embryonic development, cells are often electrically inexcitable and highlyelectrically coupled (e.g. Potter, Furshpan & Lennox, 1966; Warner, 1975; Blackshaw& Warner, 1976; Goodman & Spitzer, 1979a). During differentiation, many nervecells become excitable and also become electrically uncoupled from one another. Wehave studied the temporal sequence of appearance of these two phenotypes duringthe embryonic development ofidentified neurones in grasshoppers. We have examinedfive identified neurones that arise from two different embryonic precursor cells. Theprogeny of these two precursor cells can be visualized with a compound microscopeand impaled with micro-electrodes from the time of their birth to their maturation(Goodman & Spitzer, 1979, 1980a; Goodman, O'Shea, McCaman & Spitzer, 1979;Goodman, Bate & Spitzer, 1979; Spitzer, Bate & Goodman, 1979; Goodman, Pearson& Spitzer, 1980; Goodman, Bate & Spitzer, 1981; Bate, Goodman & Spitzer, 1981).

Grasshoppers are segmented animals whose neuronal precursor cells are arrangedin a reiterated segmental pattern (Wheeler, 1893). Two distinctly different classes ofembryonic precursor cells give rise to the - 3000 neurones in each thoracic ganglion.The first class of precursors is the neuroblasts (Bate, 1976). There is a left and rightventral plate of thirty neuroblasts each, with a single median neuroblast (MNB)located dorsally at the posterior mid line (also called the dorsal unpaired median orDUM neuroblast). Each neuroblast undergoes repeated asymmetric divisions andgives rise to 10-100 progeny. The second class of precursors is the mid line precursors(Bate & Grunewald, 1980). There are seven ofthese (MP1, MP2L, MP2R, MP3-6) alongthe dorsal mid line, just anterior to the median neuroblast. Each mid line precursordivides only once and gives rise to two progeny.We have studied the first three progeny of the median neuroblast and the two

progeny of MP3. In the metathoracic ganglion (T3), the median neuroblast gives riseto about one hundred progeny; the first two cell divisions give rise to the identifiedneurones DUM 3,4,5; DUM 4,5; and DUM 5 (also known as DUMETi) (Goodman& Spitzer, 1979). In the T3 ganglion, the MP3 gives rise to the 'H' cell and the Hcell sibling (Goodman, Bate & Spitzer, 1979). The somata of these five identified

MATURE ELECTRICAL PROPERTIESneurones are close to each other along the mid line on the dorsal surface of theganglion. The soma location and schematic axonal morphology of each of the fiveidentified neurones are shown in a 14 day embryo in Fig. 1.

In this paper we describe the mature (fully developed) electrical properties of thesefive identified neurones, in embryos between days 13 and 20, and describe cell-specificdifferences in their electrical properties. There are no signs of electrical couplingamong the mature neurones. We show that by day 13, most of these phenotypes haveappeared, including the cell-specific differences, although some changes occur afterday 13.

In the following paper (Goodman & Spitzer, 1981), we describe the developmentof electrical properties in these five neurones between days 10-13, including the onsetof electrical excitability and the cessation of electrical coupling. Brief accounts ofsome of these results have already appeared (Spitzer and Goodman, 1978; Goodman& Spitzer, 1979; Spitzer et al. 1979).

METHODS

Embryos of the grasshopper Schistocerca niten8 are generated by a crowded laboratory colony.Embryonic development takes 20 days at 35 0C (Bentley, Keshishian, Shankland & Raymond,1979). In the developing metathoracic ganglion, the progeny of the precursor cells the medianneuroblast and MP3 are located on the dorsal surface (fig. 1), quite separate from the neuronalprogeny of the ventral neuroblasts. The somata of the two progeny of MP3 (the H cell and theH cell sib) are separate from other developing neurones, and before day 10 the soma of the H cellenlarges and becomes the larger of the two. The somata of the progeny of the median neuroblastare in a packet which appears to be surrounded by a glial sheath. The oldest three progeny andlargest of the somata are on the left or right anterior lateral margin of the packet and are, fromanterior to posterior (1st to 3rd born): DUM 3,4,5; DUM 4,5; and DUM 5. The five identifiedneurones examined are individually identified by the position and size of their somata on thedesheathed dorsal surface of the metathoracic ganglion (Fig. 1, P1. 1). The identities of all five areconfirmed in representative preparations from days 10-20 by intracellular injection of thefluorescent dye Lucifer Yellow (Stewart, 1978). The morphology of these five cells is describedelsewhere (Goodman & Spitzer, 1979; Goodman, Bate & Spitzer, 1981).

Di~sections. Somata are viewed after removing embryos from egg cases at different stages ofdevelopment. A chain of segmental ganglia is removed from an embryo and the dorsal surface ofa ganglion is desheathed and viewed through a 40 x water immersion lens of a compound microscopewith Zeiss-Nomarski interference contrast optics.

Intracellular stimulation and recording. Cells are impaled with a single micro-electrode withrecording and current passing cability (80-120 MCI when filled with 3 M-K acetate). Voltage groundis indicated in most records. The most negative steady value reached after impalement of a cellis taken as its resting potential. The resting potentials for all five neurones usually range from -55to -60 mV between days 10-20; impalements yielding smaller values are rejected. The membraneinput resistance (Rin) is estimated by passing hyperpolarizing current pulses (10-10 A or less) oflong duration (100 msec or more) while monitoring the transmembrane voltage. The bridge isbalanced for 10-10 A pulses of both polarities before each impalement, and records are acceptedonly if the balance is the same upon withdrawal. In some experiments the membrane potential isshifted from rest by constant current passed through the recording electrodes. Action potentialsare elicited by depolarizing current pulses (usually 10-10 or less), also passed through the recordingelectrode.

Extracellular stimulation. The identified neurons DUM 3,4,5; 4,5; and 5 have axons in peripheralnerve 5. The H cell and its sib have axons in the ventral nerve cord. Axons are stimulated byextracellular electrodes pressed against the appropriate nerve bundle, eliciting action potentialsthat are propagated antidromically toward the cell body. The extracellular electrodes (polished glasspipettes filled with saline) are of smaller diameter than the nerves.

371

C. S. GOODMAN AND N. C. SPITZERSolutions. The preparations are perfused continuously (flow rate 8 ml./min; chamber volume 0 5

ml.) with a saline consisting of 125 mM-NaCI, 3 mm-KCI, 10 mM-CaCl2, and 5 mM-HEPES buffer(adjusted to pH 7.4). Some solutions are made by the addition of 10 mM-CoCl2; 10, 20, or 30mM-tetraethylammonium chloride (TEA); or 10-9 tetrodotoxin (TTX)/ml. In other solutions,sodium ions are replaced by eqimolar concentrations of Tris. Replacement by choline yields similarresults. Experiments are performed at 21-23 'C.

14 day Q DUM3,4,5nV45

5~~~~~~~~

2mm~~~~~~

2 mm 200 um .50 PaM MNB

vncD

nv DUM3N 3,4,5 4,5 5 H H sib

4 H

Fig. 1. Grasshopper embryo (Schistocerca nitens) at day 14; diagrams and camera lucidadrawings of living specimens viewed with Nomarski interference contrast optics (hatchingoccurs on day 20 at 35 'C). A, embryo on day 14 viewed within the egg case. B, fusedganglion on day 14 contains the metathoracic (T3) and first three abdominal ganglia(A1-A3). Each segmental ganglion has a single median neuroblast (MNB) shown as a filledcircle. The stippled area is the packet of progeny of the median neuroblast in the T3ganglion. The ventral nerve cord (vnc) runs anteriorly and posteriorly; the peripheralnerves (nv) extend laterally. C, packet of - 100 progeny of the median neuroblast in theT3 ganglion, showing the somata of the neurones arising from the first two cell divisions:DUM 3,4,5; DUM 4,5; and DUM 5. Just anterior to the packet of progeny of the medianneuroblast are the two progeny of mid line precursor 3 (MP3): the H cell and the H cellsibling. D, schematic diagram of the morphology of the five identified neurones: DUM3,4,5; DUM 4,5; DUM 5; the H cell; and the H cell sib. The morphology is revealed byintracellular injection of the fluorescent dye Lucifer Yellow.

RESULTS

Resting potential and input resistanceThe resting potentials recorded from the cell bodies of the five identified neurones

from days 10-20 ofembryonic development usually range from -55 to -60 mV, andthe recorded input resistances (Rin) at resting potential range from 200 to 450 MQ

372

MATURE ELECTRICAL PROPERTIES 373

between days 11-20 (the Rin appeared lower on day 10). The resting potentials donot vary appreciably among neurones between days 10 and 20, while their somataenlarge from 15 to 50 ,cm in diameter. These resting potentials are similar to thoserecorded in the somata of these same neurones in adult grasshoppers, although inadults the Rin is much lower (Heitler & Goodman, 1978; Goodman & Heitler, 1979).

A DUM 5 Day 16 -40-0-15 -0 10 -0-05 50. .

60 0.05 nA

80 E

10000.a)

* C

120 E

140

B 1 B 2

V~~~~~~~~~Om

25 msec

Fig. 2. Mature membrane properties of DUM 5 in a 16 day embryo. A, current-voltagerelationship (800 msec after onset of constant current), showing delayed rectification inthe depolarizing direction, and a resistance increase in the hyperpolarizing direction. B,overshooting soma action potential elicited by antidromic stimulation of the axon in theperipheral nerve, at the resting potential (-55 mV, BR1), and with the soma hyperpolarized(-90 mV, B2); note reversal of the after-hyperpolarization. All records from one cell.

The current-voltage (I-V) relationship of all five cells between days 13 and 20 isnon-linear, as illustrated in Fig. 2A. All five cells show delayed rectification whendepolarized; much of the delayed rectification is blocked by 30 mM-TEA. In DUM3,4,5; 4,5; 5; and the H cell the membrane resistance increases as the cell ishyperpolarized. In a typical example shown in Fig. 2A, the Rin at resting potential(-55 mV) is - 225 MQ. A depolarizing current pulse of the same amplitude causesa smaller depolarization; delayed rectification reduces the Rin to - 110 MQ.The somata of the progeny of the median neuroblast appear to be encased in a glial

sheath. Intracellular recordings demonstrate that, compared to the neurones, this'glial' sheath has a higher resting potential (-80 mV), a lower Rin (10 Mf2), and alsothat it is electrically inexcitable. (We remove this embryonic glial sheath beforepenetrating the somata of the progeny of MNB with micro-electrodes.) The low Rin

374 C. S. GOODMAN AND N. C. SPITZERfor the glial cells may indicate an intrinsic membrane property of the individual cellsor, alternatively, the presence of an electrically coupled network of glial cells. Theneuronal precursor cells in early embryos, just as the glia in later embryos, haveresting potentials of -80 mV, show no signs of electrical excitability, and nonon-linearities in their I-V relationships (Goodman & Spitzer, 1979).

Day 13A _ _ B C

V 2

VI 1OmVV2 25 mV

0.5 nA500 msec

Fig. 3. Absence of electrical coupling in day 13 embryos. A, VI is DUM 3,4,5; V2 is DUM4,5. B, V1 is DUM 3,4,5; V2 is DUM 5. C, V1 is the H cell; V2 is the H cell sib. The zeropotential is indicated for V1; similar values were recorded for V2. Records from threepreparations.

The soma action potential is followed by a hyperpolarization (that by day 16 lastsfor over 200 msec), part of which is blocked by TEA. This afterhyperpolarization hasa reversal potential of -80 mV (Fig. 2B), suggesting that the K+ equilibriumpotential in saline containing 3 mM-KCl is -80 mV. We previously showed thatspontaneous i.p.s.p.s and the response to GABA iontophoresed onto the soma bothhave reversal potentials of -70 mV, are blocked by picrotoxin, are abolished byreplacement of Cl- with isethionate, and that the response of the soma to GABAbecomes depolarizing after injection of Cl- into the soma (Goodman & Spitzer, 1980).These data suggest that the Cl- equilibrium potential is -70 mV. That the restingpotential (-60 mV) is above the Cl- and K+ equilibrium potentials is due at leastin part to a resting conductance of Na+. The neurones hyperpolarize by over 10 mVwhen Na+ is replaced by Tris or choline. The cells are held near -60 mV by constantdepolarizing current during the perfusion with Na+-free solutions.

Mature absence of electrical couplingMost of the possible pairs of the five identified neurones were assayed for electrical

coupling between days 13-20 ofembryonic development. Electrical coupling was notobserved for large hyperpolarizations of up to 80 mV, nor for depolarizations whichgenerated repetitive action potentials in normal saline or long-duration actionpotentials in 30 mM-TEA. Examples of dual penetration in normal saline are shownin Fig. 3. In contrast, the neuronal precursor cells in early embryos are highlyelectrically coupled to one another and to their immature progeny (Goodman &Spitzer, 1979).

Mature action potentials in normal salineOn day 18 of embryonic development, action potentials can be recorded from each

ofthe five neurons examined (Fig. 4B). In normal saline, four of the cells (DUM 3,4,5;

MATURE ELECTRICAL PROPERTIES 3754,5; 5; and the H cell) appear to generate overshooting action potentials in theirsomata. In contrast, only smaller nonovershooting action potentials are recorded inthe soma of the H cell sib; these are likely to be axon spikes that spread passivelyinto the soma. We can elicit the soma spikes by intracellular stimulation (Fig. 4B),and also by extracellular stimulation ofthe nerve bundles that contain the axons (Fig.

A 1 DUM 3,4, 5 DUM 4,5 DUM51

H cell H cell sibA U ,, A 2 A 3D 5 A 4 -A 5

Day 18B 1 DUM 3,4,5 B 2 DUM 4,5 DUM 5 H cell H cell sib

A 83 8 47........ 85____0,/

100 mVA 5,8 2-5nAA 4 05nA 25 msec

Fig. 4. A, action potentials of five identified neurones in day 13 embryos, elicited byextracellular stimulation of the nerves containing their axons (1-3), or by intercellularinjection of current (4, 5). B, action potentials of five identified neurones in day 18embryos, elicited by injection of current into the somata. The three oldest progeny of themedian neuroblast all generate overshooting action potentials in their somata (1-3). Ofthe two progeny of the MP3, the H cell generates an overshooting soma action potential,while the H cell sib produces an action potential only in its axon (4, 5). The depolarizingphase of all five action potentials appears similar on days 13 and 18.

4A for DUM 3,4,5; 4,5; 5; not illustrated for the H cell). The soma action potentialis often not elicited during repetitive antidromic stimulation. Under such conditions,we observe what appear to be axon spikes passively spreading into the soma, as shownin Fig. 5A3. These axon spikes are often double-peaked; each peak probablycorresponds to a separate site of spike initiation in each of the two lateral axons aspreviously described for DUM 5 (DUMETi) in adult grasshoppers (Heitler &Goodman, 1978).During repetitive stimulation ofDUM 5 in adult grasshoppers, Heitler & Goodman

(1978) observed an additional action potential, intermediate in amplitude betweensoma and axon spikes (when recorded in the soma), which they termed a neurite spike.Such action potentials probably arise in the median neurite between the soma andthe T-junction leading to the two symmetrical axons. We rarely observed neuritespikes in mature embryonic neurones. As discussed in the following paper, we didobserve neurite spikes as an intermediate stage in the development of electricalexcitability, before the appearance of the overshooting soma action potentials. Inmature embryonic neurones, the electrotonic distance from axon to soma is muchshorter than in adult neurones, as indicated by the several-fold larger amplitude axonspikes passively propagated into the soma. It is likely that the median neurite ofmature embryonic neurones is capable of generating action potentials, but thatbecause of the high Rin and short electrotonic distance from neurite to soma, the

376 C. S. GOODMAN AND N. C. SPITZERneurite action potential is never observed alone but rather always elicits soma actionpotentials.As early as day 13 of the embryonic development, all five identified neurones

generate action potentials whose depolarizing phase is similar in amplitude, duration,

DUM 5 Day 16

0, -'

-Na'

8 1 Normal B 2 +Co2+ B 3

+TEA C2 +TEA C 3 +TEAC 1

-60 mV -50 mV -40 mV

100 mV0-5 nA

25 msec

Fig. 5. Action potentials (AP) in the soma and axons of DUM 5 in a day 16 embryo; allrecords from one cell. Somatic stimulation (Al) and antidromic invasion (A2) elicit theAP in the soma. Repetitive stimulation often fails to elicit the overshooting soma spikeand reveals the double peaked response (A3), due to separate action potentials in the twoperipheral axons. B, ionic basis of action potentials in the soma and axons. Co2+ reducesthe amplitude and increases the duration of the soma action potential but has little effecton the axon action potential. Removal of Na+ abolishes the axon action potential (notshown); the remaining component of the soma action potential is elicited only byintracellular injection of current (B3). C, effect ofTEA on soma and axon action potentials.TEA has little effect on the axon action potential at all membrane potentials tested. Incontrast TEA causes the appearance of a shoulder on the soma AP at -60 mV, thatbroadens at -50 mV, and appears as long overshooting plateau when the soma isdepolarized to -40 mV.

and shape to day 18 action potentials, as shown in Fig. 4. DUM 3,4,5; 4,5; and 5generate double-peaked axon spikes as well as soma spikes on day 13 (like those onday 16, Fig. 5A3). However, there is a change after day 13 in the shape and durationof the afterhyperpolarization (Fig. 6A, B; see also Fig. 4).

Ionic dependence of action potentials in normal salineIn the four identified neurones whose somata normally generate action potentials

(DUM 3,4,5; 4,5; 5; and the H cell), the inward current is carried by Na+ and Ca2 .On day 16, the addition of 10 mM-Co2+ to block Ca2+ channels reduces the overshootbut does not abolish the action potential (Fig. 7). Removal of Na+ or addition of

MATURE ELECTRICAL PROPERTIES 377

DUM 4,5A 8 C +Co,+Day 13 Day 16 Day 16

50 mV0-5 nA

50 msec

Fig. 6. Development of after-hyperpolarization. A, B, increase in the duration andamplitude between days 13 (- 50 msec) and 16 (- 500 msec or more). Records from DUM4,5. C, addition of 10 mM-Co2+ on day 16 reduces the after-hyperpolarization to its formon day 13. Records from two cells-.

DUM4,5 Day 16A 1 A2 A3

V- --Na+

Normal +002+ B 3DU.M 58 1 82 - __

100 mV

0.5 nA

50 msec

Fig. 7. Ionic dependence of action potentials in the somata of DUM 4,5 (A) and DUM5 (B) in day 16 embryos. Records from two cells. Addition of 10 mM-Co2+ or removal ofNa+ reduces but does not abolish the action potential.

10-9 gTTX/ml. causes a greater reduction in the soma action potential, but does notabolish it. Simultaneous blockade ofNa+ and Ca2+ channels (not illustrated) abolishesthe soma action potential. One day 13, either treatment alone has little effect, whilesimultaneous application of both treatments abolishes the soma action potential. Byday 18, blockage of either inward current causes a greater reduction in the amplitudeof the action potential than on day 16. For DUM 5 in the adult, the blockage of eitherinward current will abolish the action potential (Goodman & Heitler, 1979). Thischange may be due to the progressive decrease in the specific membrane resistancefrom embryonic day 13 to day 20 to adulthood. Alternatively, it may be due to anactual change in the density of inward vs. outward current channels, or in theproperties of the channels. The Rin decreases from ' 300 MCI on day 13 in the embryoto 10 MQ in the adult, but this may reflect an increase in membrane surface area,a decrease in specific membrane resistance, or a combination of both factors.

C. S. GOODMAN AND N. C. SPITZERIn contrast to the constancy ofthe depolarizing phase ofthe soma action potential,

the after-hyperpolarization increases from - 50 msec on day 13 to more than500 msec by day 16. Paralleling the increase in duration is a less dramatic increasein amplitude. This increase in both amplitude and duration of the after-polarizationcan be eliminated on day 16 by Co2+ (Fig. 6C). This finding suggests an increase inCa2+ dependent outward K+ current (Meech & Standen, 1975).

+TEA Day 16A DUM 3,4,5 8 DUM4 5 C DUM 5 H cell E H cell sib

0,o

100 mVI0-5 nA

A,B,D 100 msecC, E, 25 msec

Fig. 8. Action potentials of five identified neurones in day 16 embryos, in the presenceof TEA. At the resting potential (-55 to -66 mV), the soma action potential of DUM5 is not converted to an overshooting plateau of long duration, as observed in DUM 3,4,5;DUM 4,5; and the H cell. The action potential in the H cell sib fails to overshoot evenin the presence of TEA.

In all five neurones examined, the inward current of the axon action potential iscarried predominantly by Na+ as illustrated for DUM 5 in Fig. 5B. When recordedas it antidromically invades the soma, the axonal action potential is unaffected byaddition of 10 mM-Co2+ but is abolished by removal of Na+ or addition of TTX.

Mature action potentials in TEACell-specific differences in the soma spikes ofDUM 3,4,5; DUM 4,5; DUM 5; and

the H cell are revealed when outward current is blocked by 10-30 mM-TEA (Fig. 8).At the normal resting potential (-60 mV), TEA causes the brief action potentials(2-4 msec) in the somata of DUM 3,4,5; DUM 4,5; and the H cell to become muchlonger (100-1000 msec), consisting of an initial spike followed by a long plateau (Fig.8). Similar results are obtained at membrane potentials from -90 to -50 mV, asshown for DUM 4,5 in Fig. 9.

In contrast, 30 mM-TEA causes the action potential of DUM 5 to lengthen muchless dramatically (from 2-4 to 5-10 msec) at resting potential (-60 mV); the initialspike is followed by a shoulder on the falling phase. The action potential of DUM5 lasts less than 20 msec at membrane potentials between -80 and -50 mV. At -40mV, however, DUM 5 generates a long-duration action potential (100-1000 msec) in30 mM-TEA (Figs. 503, 9). This characteristic difference in the effects of TEA onDUM 5 has been observed in over twenty preparations between days 13-20. Thiscell-specific property of DUM 5 is observed as early as day 13 of embryonicdevelopment. This finding is consistent with a previous report that at the restingpotential (-60 mV), 50 mM-TEA causes only a hump on the falling phase of theaction potential of DUM 5 in adult grasshoppers (Goodman & Heitler, 1979). In allfour embryonic neurones that normally generate soma action potentials, the after-

378

MATURE ELECTRICAL PROPERTIES 379

1000

500 DUM 4,5

TIA7

a200 < ()E ij(13)(11)6/

<100 (5) (6) ~ (20) ~ (9)W _()(4)

C

C0

50 DM

0)20

0

(12)5 ~~~~~~~~(14)

2

1 _ I

-90 -80 -70 -60 -50 -40

Membrane potential (mV)

Fig. 9. Action potential duration in TEA as a function of membrane potential for twoidentified neurones in day 16 embryos. Means + standard deviations from recordings fromthree specimens each ofDUM 5 and DUM 4,5. Values in parentheses indicate the numberof action potentials used in determinations of the mean.

hyperpolarization persists in 30 mM-TEA, suggesting an outward K+ current notblocked by this concentration of TEA. The soma of the H cell sib, which does notgenerate overshooting action potentials in normal saline, does not generate them in30 mM-TEA either (Fig. 8).

Ionic dependence of action potentials in TEATEA causes long-duration action potentials in DUM 3,4,5; DUM 4,5; and the H

cell at the normal resting potential (-60 mV), and in DUM 5 when the membranepotential is depolarized to -40 mV. The inward current of the action potentials isbased both on Na+ and Ca2+ (Fig. 10). The plateau in all four cells is abolished by10 mM-Co2+ and is thus based primarily on Ca2+ inward current; the initial spike islargely eliminated by the removal of Na+ (or addition of TTX) and is thus based onlarge part on Na+ inward current.

C. S. GOODMAN AND N. C. SPITZER

A 1 Normal

0, /-

Normal8 1

0,/- 1

VI

+TEA

cl 1\

-40 mV

DUM4,5 Day 18A 2 '+TEA

DUM 5 Day 18+TEA

B 2

-60 mV

+TEA +Co2+C2

A 3 +TEA

+TEAB 3

-40 mV

+TEA -Na+C3

100 mV

0.5 nA

A 1,2;B;C2,3 50msecA3,C1 250msec

Fig. 10. A, action potentials elicited from the soma of DUM 4,5 in the presence of TEA;all records from one cell in a day 18 embryo. Soma action hpotential broadens in TEAand the spike(s) is followed by an overshooting plateau of long duration. B, actionpotentials in the soma of DUM 5 in the presence of TEA. Soma action potential broadensslightly in TEA at the resting potential. Only when the membrane potential is depolarizedto -40 mV is the initial spike(s) followed by an overshooting plateau of long duration.C, ionic dependence of action potentials in the soma of DUM 5 in the presence of TEA.The plateau is abolished by addition of Co2+ and the spike(s) is abolished by removal ofNa+. All records in B, C from one cell in a day 18 embryo. Note compressed time basedin A3 and C1.

A +TEA

0,/-DUM4,5 Dayl5BT +TEA -Na+ B 2 - +TEA -Na+

100 mVA 2-5 nA8 0-5 nA

A 100 msec

B 250 msec

Fig. 11. Repetitive stimulation of DUM 4,5 in the presence of TEA elicits long durationCa2+ action potentials that fail to overshoot the zero potential. Records from two cellsin day 15 embryos. Second stimulus in A has small amplitude plateau. B2 is secondstimulus in B.

380

MATURE ELECTRICAL PROPERTIES

TEA permits generation of action potentials in neuropilThe shape and duration of the axon action potential, as recorded while antidrom-

ically invading the soma, changes little in 30 mM-TEA (Fig. 5C). In Na+-free salinewith 30 mM-TEA, we are unable to evoke an actively propagated action potentialby extracellular stimulation of the peripheral nerves containing the axons. We thushave no evidence for Ca2+ channels in the axon membrane in the peripheral nerve.However, when repetitive intracellular stimulation of the soma (in TEA or TEA-Na+)fails to elicit an overshooting Ca2+ action potential, we often observe a smallamplitude and long-duration response (Fig. 11) that is abolished by 10 mM-Co2+.Because of its pharmacology, amplitude, duration, and shape, this response is likelyto be a Ca2+ action potential that is actively generated (and probably overshooting)somewhere in the neuropil processes of the neurone, and then passively propagatedinto the soma. These Ca2+ action potentials do not appear to arise in the axon butrather somewhere else in the neuropil.

DISCUSSION

Electrical properties of the progeny of the median neuroblastThe first three progeny of the median neuroblast in the metathoracic (T3) ganglion

of the grasshopper embryo are the identified neurones DUM 3,4,5; DUM 4,5; andDUM 5 (Goodman & Spitzer, 1979). All three cells have peripheral axons, and thoseof DUM 5 (the youngest of the three) extended over their peripheral targets, theextensor tibiae muscles, by day 13 (hatching occurs on day 20 at 35 0C). By day 13,all three cells have most of their mature electrical properties: they are electricallyuncoupled from one another, their axons generate Na+-dependent action potentials,and their somata generate Na+-Ca2+-dependent action potentials. Cell-specificdifferences in the action potentials, generated by the somata of these three cells, arealso present by day 13. Whereas TEA causes a 50-500 fold increase in the durationof the action potentials of DUM 3,4,5 and DUM 4,5, it causes only a shoulder onthe falling phase of the action potential of DUM 5. If DUM 5 is depolarized fromits resting potential (-60 mV) to -40 mV, it can also produce a long-duration actionpotential in TEA. One possible explanation for the effect of steady depolarizationwould be the inactivation of delayed outward current (not blocked by TEA), as seenin molluscan neurone somata (Aldrich, Getting & Thompson, 1979). We have not yetinvestigated the mechanisms responsible for the difference between DUM 5 and theother two cells. Nevertheless, it clearly represents a biophysical difference in cells thatdiffer only by a single cell division and at most by 6 h in birthdate.We have shown that there is a cell-specific difference among the first three progeny

whose somata generate action potentials. When Goodman et al. (1980) examined therange of electrical properties in the - 100 progeny of the median neuroblast, theyfound a broad spectrum of electrical excitability. Some of the later progeny generateonly neurite and axon action potentials, some generate only axon action potentials,and many (including all of the last born) do not generate action potentials. Thenon-spiking progeny are local, intraganglionic neurones that appear to comprise amajor proportion of the progeny of the median neuroblast. All of the non-spiking

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C. S. GOODMAN AND N. C. SPITZERneurones have Ca2+ inward current channels and can make action potentials whenoutward current channels are blocked by TEA. Thus, all of the different types ofprogeny of the median neuroblast, whether normally spiking or non-spiking, canproduce Ca2+ action potentials that appear to arise in their neuropil processes whenoutward current is block by TEA. One possibility is that these Ca2+ action potentialsare generated at synaptic terminals.

Electrical properties of the progeny of the MP3The two progeny ofthe MP3 in the metahoracic ganglion ofthe grasshopper embryo

are the H cell and the H cell sib (Goodman et al., 1979; Goodman, Bate & Spitzer,1981). Both cells have one or more axons in one of the ventral nerve connectives.By day 13, the mature electrical properties of both cells have appeared; they areelectrically uncoupled from one another (and from the progeny of the medianneuroblast), they both generate Na+-dependent axon action potentials, but only theH cell generates a Na+-Ca2+-dependent soma action potential. In normal saline andin 30 mM-TEA, the soma action potential of the H cell is indistinguishable from thoseof DUM 3,4,5 and 4,5. We could find no evidence, however, for either Na+ or Ca2+inward current channels in the soma of the H cell sib.

Thus, the progeny oftwo different embryonic precursor cells (MNB and MP3) showa broad spectrum ofelectrical properties. Electrical excitability is a mature phenotypenot shared by all the progeny of a single precursor cell. In contrast, progeny fromdifferent precursor cells can share the same mature phenotype ofelectrical excitability(e.g., DUM 3,4,5 and the H cell).

Changes of electrical properties after day 13The amplitude, duration, and shape of the depolarizing phase of the action

potentials in all five neurones remain relatively unchanged from days 13 to 20 ofembryonic development. However, some changes do occur after day 13. On day 13,either Na2+ or Ca2+ inward currents alone are capable of generating responses thatare larger in amplitude than those responses they can generate alone on day 20. Thismay be due to a decreased specific membrane resistance, or to an actual change inthe relative density or properties of the ionic current channels.

After day 13 there are dramatic changes in the duration of the after-hyperpolarization. Delayed rectification that is blocked by TEA appears early in thedevelopment of these cells, before the appearance of Na+ or Ca2+ inward currents(Goodman & Spitzer, 1981). The later increase in the after-polarization is blocked byCo2+, however, suggesting an increase in Ca2+-activated K+ current after the initialappearance of Na+ and Ca2+ inward currents. One possibility is that there is anincrease in Ca2+ dependent K+ channels; however, an increase in Ca2+ influx or adecrease in intracellular Ca2+ buffering could also account for this developmentalchange. The resolution ofthese issues will require close examination ofthe developmentof different types of outward current channels.

Development of electrical properties between days 10-13The neuronal precursor cells and their undifferentiated progeny in early grasshopper

embryos are electrically inexcitable and highly electrically coupled (Goodman &

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MATURE ELECTRICAL PROPERTIESSpitzer, 1979). Here we have shown that by day 13, the first three progeny of themedian neuroblast and both progeny of the MP3 are capable of generating actionpotentials and are electrically uncoupled. In the next paper, we describe the onsetof electrical excitability and the cessation of electrical coupling in these five identifiedneurones between days 10-13 ofembryonic development (Goodman & Spitzer, 1981).

We thank Jonathan Raper and Alan Willard for their helpful criticisms of the manuscript, andJames Coulombe, Amanda Iles and Kim Ridge for technical assistance. Supported by the HelenHay Whitney Foundation and the N.S.F. (C.S.G) and the N.S.F. and the N.I.H. (N.C.S.).

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EXPLANATION OF PLATE

Photomicrograph of the somata of some of the neurones which are the progeny of the medianneuroblast and mid-lines precursor 3 (MP3), in the metathoracic ganglion of a living day 14 embryo.The dorsal surface of the ganglion has been desheathed and the glial covering removed; the cellsare viewed with a 40 x water immersion objective and Nomarski interference contrast optics. Fourlarge somata are identified: the three oldest progeny of the median neuroblast (DUM 3,4,5; DUM4,5; and DUM 5 or DUMETi) and one of the two progeny of MP3 (the 'H' cell). Anterior at top.

The Journal of Physiology, Vol. 313 Plate 1

(Facinrg p. 384)(OREY S. OODI)MAN ANi) NICHOLAS C. SPITZER