Electrophysiologic and morphologic properties of neurons in dissociated chick spinal cord cell cultures

DEVELOPMENTAL

BIOLOGY

37, 10%116 (1974)

Electrophysiologic Dissociated

and Morphologic

Properties

of Neurons

in

Chick Spinal Cord Cell Cultures

GERALD D. FISCHBACH’ AND MARC A. DICHTER~ Behavioral

Biology Branch, National

Institute

of Child Health

Accepted

and Development,

Bethesda, Maryland

20014

October 25, 1973

Neurons dissociated from embryonic chick spinal cords mature in relatively sparse cell culture and survive in vitro for several weeks. They generate action potentials and form both excitatory and inhibitory chemical synapses with one another. By electrophysiologic and morphologic criteria, it appears that the neuronal population (after 2-3 weeks) is made up of a variety of different cell types; few, if any, are motoneurons. Neuron cell bodies are not covered by glia or satellite cells and nerve processes are not myelinated. Thus, the cultures should permit more direct microelectrode and pharmacologic analysis of differentiation of cell specific properties and of synapse formation than is possible in the intact central nervous system. INTRODUCTION

Cells can be dissociated from embryonic chick spinal cord and maintained for several weeks in vitro (Cavanaugh, 1955; Shimada et al., 1969; Fischbach, 1972). When plated at low density, cell cultures, in some regards, represent a significant simplification over the intact nervous system or cultured explants of neural tissue (see Crain, 1966). The growth of individual cells can be directly observed over long periods under well controlled conditions and structures that are likely synaptic boutons can be visualized. Only one other interneuronal synapse has been visualized in unfixed tissue (McMahan and Kuffler, 1971). Isolated young spinal cord neurons or neuroblasts do not dedifferentiate in’culture; they generate action potentials, form synapses with one another, and, a few form functional synapses on previously plated muscle cells (Fischbach, 1970, 1972). This degree of differentiation is not unique to avian neurons. Similar findings in dis1Present address: Department of Pharmacology, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115. *Present address: Department of Medicine, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215. 100 Copylyht All rights

0 1974 I,y Academic Press. Inc. 01 rrproductwn in an\ form reserved.

sociated mouse spinal cord cultures have recently been reported (Crain and Bornstein, 1972; Peacock et al., 1973). This paper presents a more detailed description of the morphology and electrophysiological properties of chick spinal cord neurons grown in the absence of muscle cells or cells from other regions of the nervous system. Our initial experiments, performed on 2- to 8-week cultures were designed simply to explore the system, are clearly only a beginning. They indicate, however, that cell cultures might allow a more direct analysis of the formation, function and long-term alteration of interneuronal synapses than has been possible in the past. METHODS

The method of preparing the cultures has been described (Fischbach, 1972). Spinal cords were removed from 7-day embryos. At this age, the sensory ganglia are still located within the spinal canal and usually remained attached to the excised cord (Fig. 1A). Ganglia and meninges were carefully stripped from the cords prior to the dissociation procedure (Fig. 18) so the cultures contained no primary sensory neurons. Spinal cord cells were dissociated by

FISCHBACH AND DICHTER

mechanical disruption (repeated suction through a fire-polished Pasteur pipette) of trypsinized tissue fragments (0.25% trypsin Ca2+-, Mg2+-free Earle’s Salt Solution) and plated in 60-mm, collagen-coated plastic tissue culture dishes. The cells were counted in a hemacytometer and, usually, 2.0 x lo5 cells were added to each dish in 3 ml of media. Large tissue fragments and reaggregated clumps of cells were removed from the suspension by filtration through a double layer of lens paper in a Swinnex filter. The plating medium was composed of Eagles’ Minimum Essential Medium (80% by volume), heat inactivated horse serum (lo%), a saline chick embryo extract (lo%), glutamine (2 mM), penicillin (50 u/ml) and streptomycin (50 pg/ml). The cells were maintained, after 2-3 days, in an identical medium except that the concentration of embryo extract was reduced to 2%. The medium was changed every 2-3 days. In a few experiments, 2-3 day cultures were exposed to cytosine arabinoside (10 -’ M) for 48 hr. Intracellular microelectrode techniques have also been described (Fischbach, 1972). In studies of the incidence and polarity of synaptic potentials, microelectrodes were filled with 2 M K citrate rather than 3 M KCl. As before, simultaneous intracellular stimulation and recording was usually achieved through a single electrode by the use of a resistance bridge circuit. With selection of electrodes and careful checks of bridge balance, this technique was free of artifact over the range of currents employed (less than 5 nanoamps). Identical results were obtained when cells were penetrated with two electrodes; one for passing current and the other for recording. Extracellular stimulation of neurites was achieved through glass microelectrodes filled with 1.0 M NaCl or insulated tungsten microelectrodes (2 pm tip). The cultures were studied on the stage of an inverted microscope and during most

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101

experiments, the cells were bathed in Earle’s BSS which is buffered with NaHCO, (at pH = 7.4) and maintained at 37°C. No difference in survival or results was noted when the NaHCO, was omitted (replaced with NaCl) and the solution buffered with Hepes (15 mhlp or Tris .HCl (10 mM). Tetrodotoxin (TTX) used in studies of action potential generation was obtained from Sankyo Chemicals. For electron microscopy, the cells were fixed for 1 hr at 4°C with either 2% 0~0, or 4% glutaraldehyde in 30 mM barbital buffer that contained 50 mA4 NaCl and 20 mM CaCl,; washed in buffer for 2 hr; blocked-stained with 1% uranyl acetate in 50 mM acetate buffer (pH = 5) for 16 hr at 4°C; dehydrated in ethanol and embedded directly in Epon. Thin sections were stained with uranyl and lead. Neurons were impregnated with silver according to Richardson’s modification of the Bielschowski technique (Sevier and Munger, 1965). RESULTS

The Cultures Between 1.0 x lo6 and 3.0 x 106 cells were liberated from one 7-day spinal cord; more than 90% were viable as judged by their ability to exclude basic dyes (nigrosin or trypan blue) and by the extension of motile microspikes immediately after s&tling on the counting chamber surface. Most were spheres 5-40 pm in diameter, but a few retained a large stump of a former process (Fig. 1C). At the plating density usually employed, collagen was necessary for the attachment and growth of both “supporting” cells and neurons. The rate of attachment of the cells was highly variable: many settled on the collagen substrate within 30 min whereas others remained in suspension after 24 hr. Some of the late floaters were viable neuroblasts because obvious neurons developed in cultures seeded with media withdrawn from 24-hr cultures.

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DEVELOPMENTALBIOLOGY

Immediately after attachment many cells extend processes that reach 100-200 pm in length after 24 hours (see Fischbach, 1972, Fig. 2A), but it is not clear at this stage how many of these cells are neurons. We have never seen a cell with processes longer than 200 pm divide and have never found evidence by autoradiography, that they incorporate 3H-thymidine. Over the next several days, many of the presumptive neurons were located over islands of flat epithelioid cells. This may, in part, be due to the fact (documented with time-lapse cinematography) that the flat cells migrate rapidly and often undermine and drag less firmly attached, round, cell bodies and fine processes. After 2-3 weeks, the cultures were confluent and the neurons, located on top of the background cells, had achieved their final size. Although a few aggregates or clumps of three to ten cells could be found at this time, most of the neurons were either isolated from one another or arranged in loose clusters. The three cells

VOLUME 37,1974

shown in Fig. 2 illustrate the range of morphology of relatively large, multiprocessed cells. Each generated an all-or-none action potential when adequately stimulated (see below) and, therefore, can be unambiguously identified as a neuron. Each of the different types of spinal cord neurons stained with silver and each could be easily distinguished from round sensory ganglion cells grown under the same conditions (Fig. 3). As previously described (Fischbach, 1972) the density of background cells could be reduced by lowering the concentration of embryo extract in the media and virtually eliminated with cytosine arabinoside. Electron microscopy, performed by John Heuser, confirmed our impression that neuron cell bodies were not covered by glia or satellite cells (Fig. 4A). Typical Nissl bodies-stacks of narrow lumened endoplasmic reticulum cisternae covered with ribosomes-were identified in many cells (Fig. 4A). Nerve processes were not myelinated or invested in Schwann cells. Most

FIG. 1. Preparation of isolated spinal cord cells. A and B: Low power bright field views of the same segment of a ‘I-day cord before (A) and after (B) “peeling” off meninges and sensory ganglia. C: Phase contrast view of freshly dissociated cells. Note the variation in size and one cell with a large stunip of a former process. A and B, x 27; C, x 325.

FISCHBACH AND DICHTEH

Spinal Cord Cell Cultures

103

FIG. 2. Representative phase contrast micrographs of 3 cells (26 days in culture) that illustrate the range in morphology of relatively large spinal cord neurons. A: Typical appearance of retractile, round or ellipsoid cells with few relatively unbranched processes. B: Typical appearance of round cells with many processes that extend directly from and branch in the immediate vicinity of the perikaryon. C: Typical appearance of tlat. elongated cells with few broad processes that usually issue from the poles of the cell body and then branch repeatedly. Bar 100 Frn applies to A, B. and C.

FIG. 3. Silver impregnated large (A) and small IB) spinal cord cells compared with a sensory ganglion cell ((‘I grown under the same conditions in another culture. Bar = 50 pm.

neurites contained numerous microtubules (Fig. 4B). It should be emphasized that the cultures were not invariably successful. In a few cases, for still unexplained reasons, either the cells did not attach to the substrate or, after about 1 week, they

became vacuolated and rapidly degenerated. All of the plates from a given dissection shared the same fate. Membrane

Properties

Resting membrane presumptive neurons

potentials, V,. ot ranged widely be-

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DEVELOPMENTALBIOLOGY

tween ca. 30-75 mV. We did not always attempt precise measurement of V, and believe that lower values, i.e., less than 45 mV, reflect membrane damage on penetration. Steady membrane potentials could be “held” in adequately penetrated cells for

VOLUME 37, 1974

longer than 30 min and, it was often possible to repenetrate the same cell severa1 times during the course of an experiment. Nearly every presumptive neuron (see Figs. 2 and 3) generated an action potential

FIG. 4. Electron micrographs of a neuron cell body (A) and nerve fiber (B). Note the bare surface of the neuron (except for a few, contacting neurites) and the organized stacks of rough endoplasmic reticulum. This culture was fixed in 0~0, (see Methods). B: Area from another culture fixed in glutaraldehyde for better preservation of microtubules. A: ca. x 10,000; B: ca. x 30,000. Micrographs kindly provided by J. Heuser.

FISCHBACH AND DICHTER

30 2 _s + g 2 E w -Iz

.

c

20

.

.

. IO

.

.*

**a .

.

0

.

l

i

.

40

50 RESTING

60 POTENTIAL

80

70 (mV)

FIG. 5. Action potentials. A and B: Two spikes evoked by brief, just-threshold current pulses (duration indicated by lines below each trace) illustrate the range in action potential size, rate of rise and duration. The resting potentials of the two cells were comparable (55 mV in A; 52 mV in B). Note the positive afterpotential following the spike in A and the lack of one in B. Bars = 50 mV and 5 msec. C: The relation between resting membrane potential and action potential overshoot in a sample of 31 cells, all with resting potentials greater than 43 mV. The open triangle and square refer to the spikes shown in A and B. respectively.

when stimulated through a microelectrode located in the cell body (Fig. 5A,B). Failures were probably due to membrane damage caused by the microelectrode: V,,,, in these cases, was invariably less than 40 mV. The resting potential of the cell shown in Fig. 5A was 54 mV; a brief, outward current pulse depolarized the membrane to threshold and triggered a 72-mV sharp spike. In this example, the action potential overshot the zero membrane level by 18 mV (V,) and was followed by a prolonged after-hyperpolarization. The maximum rate of rise of the spike was 230 V/set (u,,,,,). and the duration was measured at 0.5 peak amplitude (half width) was 0.5 msec. These parameters varied widely between different cells (Table 1). Some of this variation may be due to

105

Spinal Cord Cell Cultures

membrane depolarization and partial inactivation of the spike generating mechanism caused by the recording electrode. There was a small trend, albeit statistically insignificant, between valves of V, and V, (Fig. 5C, r = 0.307; p. > 0.05) and hyperpolarization of cells with relatively low V, (less than 55 mV) by steady inward currents often increased V, and vm,, It seems unlikely, however. that injury can explain all the variation in action potentials. The scatter in Fig. 5C is great, and large differences in V,, Vm,, and spike duration were evident when cells with the same V,, were compared (Fig. 5A,B). Variation between cells with comparable membrane potentials (within 5 mV) was also observed in post-spike afterpotentials-and in degree of adaptation. Some spikes were followed by a prolonged after-hyperdepolarization (Fig. 5A), others by an after-depolarization (Fig. 11A) and still others by no afterpotential at all (Fig. ,5B). Many cells fired repetitively during a prolonged depolarizing current pulse (Fig. 6A) whereas others fired only one or two spikes (Fig. 6B). Tetrodotoxin (TTX), a drug that blocks active Na+ channels in other tissues (Kao, 1966), rapidly abolished spontaneous and stimulus evoked action potentials in every case tested. In the presence of low doses of TTX (1.0 to 2.0 7 10 7 gm/ml) large depolarizing pulses merely produced the expected sag (delayed rectification) in the voltage record (Fig. 6C). Many of the fine processes conducted action potentials. In the example shown in Fig. 7, spikes were recorded by a microelecTABLE ELECTRICAL

1

PROPERTIES OF SPINAL

CORD NEVRONS

106

DEVELOPMENTALBIOLOGY

trode located in the soma following focal extracellular stimulation of a neurite at points 135 pm (Fig. 7B,) and 300 pm (Fig. 7B,) from the cell body. The amplitude and shape of the “conducted” spikes are identical to one evoked through the intracellular electrode (Fig. 7B,). The spikes were probably initiated at the point of

A

‘11

1

I

6

d

b----

i

FIG. 6. Differences in degree of repetitive firing in response to a prolonged pulse of outward current (duration marked by arrows). The cell in B adapted after only one spike. C: Abolition of spikes in another cell after addition of TTX (lo-’ gm/ml). Two superimposed traces show the effect of equal but opposite current pulses (lower traces). The initial hump in the depolarizing potential simply reflects the onset of delayed rectification. Bars = 50 mV, 50 msec for A and B. Calibration pulse in C = 10 mV, 5 msec.

VOLUME 37, 1974

stimulation rather than at a more proximal site because: (1) the location of the stimulating electrode was critical at just-threshold stimulus intensities (the response was lost following movement of 2-3 pm away from the neurite), (2) the spikes arose from a flat baseline, (3) the latency varied as expected with change in stimulus site (cf. Fig. 7B, and B,). The slow conduction velocity, 0.15 mlsec, is consistent with the small diameter of the process and its lack of myelin. In many cases, it was possible to evoke spikes that were conducted over several hundred microns by stimulation of several distinct neurites of the same cell. As in most neurons recorded in uiuo, the rising phase of the antidromic spike was marked by an inflection or “notch” (Fig. 7Bz, B,). The notch could be accentuated and the second component ultimately blocked by hyperpolarization of the soma membrane (Fig. 7BJ. Thus, by analogy, it is likely that neurons in culture possess a low threshold trigger zone that fires before the remainder of the soma-dendrite membrane. Membrane current-voltage characteristics were measured by injecting prolonged square pulses of current through the intracellular electrode. Superimposed tracings of the resulting electrotonic potentials in one cell are shown in Fig. 8A and the steady state (measured at the end of the current pulse) values are plotted in Fig. 8B. In most cells, the relation was linear in the hyperpolarizing direction; a few, like that shown in Fig. 8, showed a delayed sag or rectification (see Ito and Oshima, 1965; Nelson and Frank, 1967) in response to relatively large inward currents. Most cells showed a marked, delayed rectification in response to outward (depolarizing) currents. Input resistances, Ri,, taken as the initial slope of the relation in the hyperpolarizing quadrant, varied in different cells between 3.0-60.0 m0 (Table 1). This range is consistent with the observed spectrum of cell size and shape and does not

FISCHBACH

AND

DICHTER

Spinal Cord Cell Cultures

FIG. ‘i. Electrical excitability of nerve processes. All traces were recorded from an intracellular electrode placed in the soma of the neuron shown in A. Action potentials were evoked by intracellular stimulation through the recording electrode, B, (current pulse on lower trace). and by extracellular stimulation B,, H, at the indicated points. The break in the records marks the stimulus artifact. In B,. superimposed traces show the effect of hyperpolarization of the soma membrane (inward current pulse not shown) on the extraceliularly .iO urn. evoked spike (see text I. Calibration pulse = 20 mV, 1 msec. Bar in A

A -I I

B T -‘? A;/ .a

Ii

i.0 1:

o-5

i/,.

I::

.

l

05 ,

I(nA) 7’; 4

2c

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40

AV(mV) 60

FIG. 8. Current-voltage characteristic. A: Superimposed, selected tracings of current pulses (upper records) and resulting electrotonic potentials (lower records). The largest outward current pulse triggered a spike (break in voltage trace). Bar : 1 nanoamp; calibration pulse = 10 mV, 5 msec. The complete series is plotted in B. Electrotonic potentials (AV, were measured at the end of the current pulse. Inward currents are plotted to the left of the origin and hyperpolarizing potentials are below the abscissa. Resting potential (origin) 64 mV. The dashed line was drawn by eye through the first 8 points in the hyperpolarizing quadrant.

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DEVELOPMENTAL BIOLOGY

necessarily imply differences in membrane conductance per unit area.

Synapses-Spontaneous

Potentials

Spontaneously occurring synaptic potentials were detected in the majority of neurons tested 2-3 weeks after plating. A given cell exhibited depolarizing, excitatory postsynaptic potentials (epsps) (Fig. 9A) or hyperpolarizing inhibitory potentials (ipsps) or both (Fig. 9B). The largest epsps triggered action potentials (Fig. 9A,C). Synaptic potential amplitude varied with change in resting membrane potential as expected for chemically mediated synapses. As shown in Fig. lOA, rapidly rising epsps decreased in size and ultimately reversed in polarity as the membrane was depolarized by current injected into the cell body. We have not accurately determined epsp reversal or true equilibrium

A

VOLUME37, 1974

potentials. Further analysis is complicated by the fact that, in most cases, the membrane resistance decreased drastically as the cells were depolarized (Fig. 10, Fig. 8). Moreover, the exact sites of transmitter action have not yet been identified, and they may be located at relatively great electrotonic distances from the polarizing electrode. In fact, slowly rising epsps, which presumably originate in distal branches of the dendritic tree (Rall, 1967), varied directly with membrane potential but could not be inverted. Ipsps recorded with K citrate filled electrodes varied inversely with changes in membrane potential and, in every case, inverted to depolarizing potentials when the membrane was hyperpolarized by lo-20 mV (Fig. 10B). As in adult motoneurons, where inhibition is due in large part to an increase in Cl- conductance (Coombs

I B

FIG. 9. Spontaneous synaptic potentials. A and B: Superimposed traces showing depolarizing potentials (epsps) in one cell (A), hyperpolarizing potentials (ipsps) in another (B-upper record), and both in a third cell (B-lower record). Vertical bar = 10 mV for A, 12 mV for B. Horizontal bar = 5 msec for A, 10 msec for B-upper, 20 msec for B-lower. C: A continuous record showing regularly occurring, large epsps that often trigger action potentials. Note the few small psps. D: Small psps recorded in the presence of TTX (lo-‘gm/ml). Vertical bar = 10 mV for C, 5 mV for D. Horizontal bar = 1 set for C, 5 set for D.

FISCHBACH AND DICHTER

+4

I

6 ~~-28

FIG. 10. Inversion of synaptic potentials. A: Superimposed traces showing decrease in epsp size and change in polarity as the membrane potential was decreased to the values indicated to the right of each trace. A constant pulse of inward current was injected into the cell in the center of each sweep. Note the marked decrease in size of the electrotonic potential as the cell was depolarized (see text). B: Inversion of ipsps as the soma membrane was hyperpolarized by only 12 mV. Vertical bar = 10 mV in A, 20 mV in B. Horizontal bar = 40 msec in A, 30 msec in B.

et al. 1955b), ipsps decreased in size, inverted and eventually triggered spikes when the cells were penetrated with KC1 filled microelectrodes. This sequence was extremely rapid (less than 1 minute) even with high resistance (50-100 MQ) electrodes. Spontaneous synaptic potentials recorded in adult vertebrate motoneurons can be divided into two categories (Katz and Miledi, 1963; see Kuno, 19’il for review). One type reflects the random discharge of individual quanta of transmitter from presynaptic terminals and, by analogy with the neuromuscular junction, have been termed miniature synaptic potentials (mepsps or mipsps). The other results from the synchronous discharge of

109

Spinal Cord Cell Cultures

many quanta during spontaneously arising action potentials in presynaptic nerve terminals. Large epsps and ipsps in our cultured neurons were undoubtedly due to presynaptic impulses because they usually recurred in a regular pattern (Fig. 9C) and were invariably abolished by TTX (10 7 gm/ml). Small psps less than 1 mV that persisted in the presence of TTX, i.e.. true mpsps, were detected in many but not all cells (Fig. 9D). The regular pattern and simple wave form of large synaptic potentials (Fig. 9A,C) strongly suggests that they were generated by the rhythmic discharge of a single presynaptic neuron, e.g.. were “unitary” psps (Burke and Nelson, 1966; Mendell and Henneman, 1968). Interestingly, the degree of spontaneous synaptic activity was markedly decreased when the cells were tested in complete media (salts plus amino acids. vitamins, embryo extract, and horse serum). For example, in one series synaptic potentials were detected in 33 of 72 cells in four different cultures bathed in Earle‘s BSS; and only 1 of 24 cells bathed in complete media. Potentials less than 0.5 mV would not have been detected in these experiments. Large synaptic potentials reappeared within a few minutes after the media was replaced with balanced salt solution. It will be of interest to determine whether this suppression is a pre- or post synaptic effect and to identify the factor(s) responsible.

Synapses-Euoked

Potentials

It was often possible to identify “input” or presynaptic neurons by recording from one cell and stimulating another, within the field of view through a second intracellular electrode (Fig. 11A). In most cases, the two cells appeared to be connected by at least one fine neurite and the simple. monophasic psp was consistent with a direct monosynaptic connection. Such psps were often larger than 15-20 mV; and, considering the size of individual mepsps

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DEVELOPMENTALBIOLOGY

VOLUME 37. 1974

(Fig. 9D), they must have been composed of more than 30 quanta. The quanta1 efficacy of synapses between excitatory interneurons in the mature spinal cord is not known. In comparison, epsps generated in adult motoneurons by single I” afferent fibers are usually less than 1.0 mV (Kuno, 1964; Burke, 1967; Mendell and Henne-

man, 1971) and the largest unitary epsps recorded to date (in Clarke’s column neurons) are less than 4.5 mV (Eide et al., 1969; Kuno et al., 1973). More complex evoked responses suggesting polysynaptic interaction and recurrent synaptic potentials were commonly observed in cells located in small clusters of neurons (ca. 3-6 cells within a 500 pm circle). In the example shown in Fig. llB, a prolonged, recurrent polyphasic potential followed an action potential that was directly evoked by a brief suprathreshold stimulus. We have never observed, during simultaneous intracellular stimulation and recording, direct electrotonic spread of current between two neurons even when the two cell bodies appeared contiguous. Electron micrographs of such “touching” neurons show that their membranes are not separated by intervening connective tissue cells I or glia. It was also possible to evoke psps by focal extracellular stimulation of a contacting neurite. Suprathreshold stimulation of most input processes usually elicited a simple psp in the follower cell over a wide range of stimulus strengths. In a few cases, however, a second, clearly different, psp appeared as the stimulus was increased beyond threshold for the first. In Fig. llC, first a small epsp and then another larger epsp were elicited as the stimulus strength was gradually increased. Thus, some neurites that appear single by light microscopy must be composed of more than one fiber. FIG. 11. Stimulus evoked synaptic potentials. A: Synaptic connections have been identiIntracellular stimulation of a neuron (lower trace; bar = pulse duration) evoked a large epsp (upper trace) in fied between several different types of a nearby cell. Vertical bar = 25 mV for spike and 15 spinal cord neurons and, in mixed cultures, mV for epsp. Horizontal bar-10 msec for both. The between sensory ganglion and spinal cord long latency between spike and epsp reflects the slow cells.3 By simultaneous recording from two conduction velocity of the process. B: A complex neurons while stimulating several others in barrage of potentials following a directly evoked spike

,

I -m

B

,

(bar). Bars-50 mV and 20 msec. C: Synaptic potentials (superimposed traces) evoked by extracellular stimulation of an incoming “process” showing recruitment of fibers (largedepsp) within the “process” with increasing stimulus strength. Calibration pulse = 5 mV and 5 msec.

3 We have also, on rare occasion, recorded depolarizing potentials in one sensory ganglion cell after stimulating another-in cultures to which no spinal cord neurons had been added (Dichter and Fischbacb, unpublished observations.)

FISCHBACH AND DICHTER

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111

FIG. 12. A presumptive synaptic bouton. Komarski interference contrast optics. A: Low power view ot a neuron and initial segments of processes. B: Higher magnification of area outlined in A showing a relatively large terminal swelling on the upper surface of the process. The fine, incoming, preterminal neurite passes out ot’ the plane of focus at the lower edge of the process. Bars 25 wrn in A: 5 p in R.

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FIG. 13. Electron micrographs of synaptic contacts. A: Several boutons contact a single process. Note the accumulations of small, clear vesicles and the increased density of presynaptic membranes. x 20,000. B: Two boutons that contain large dense-core vesicles in addition to smaller, clear vesicles. x 40,000. Fixed in 0~0,. Micrographs kindly provided by J. Heuser.

we found that one neuron can receive synaptic input from more than one cell and that one neuron can innervate more than one cell. Contiguity was not a good criterion for functional contact. In fact, imme-

turn,

diately adjacent cally connected.

cells were rarely synapti-

Synapses-Morphology Bulbous swellings were identified

on the

FISCHBACH AND DICHTER

surface of neuron cell bodies and along their processes with Normarski interference contrast optics (Fig. 12A,B). To resolve these structures, it was necessary to grow the cells on collagen-coated, glass coverslips. The swellings appeared to be terminals of fine (ca. 0.5 pm) neurites that could often be traced for several hundred microns away from the cell. Although McMahan and Kuffler (1971) have clearly demonstrated that similar swellings on parasympathetic neurons in the atrium of the frogs heart are synaptic boutons, direct physiological tests of the spinal cord terminals are required. Synaptic structures were identified by electron microscopy. Figure 13A shows a section through a cluster of neurons in which several terminals contact a single process. Accumulations of clear vesicles (ca. 500 A) and increased membrane density at the points of contact are typical of adult synapses. Other endings contained many larger dense-core vesicles (Fig. 13B). The terminals are not covered by glia. DISCIrSSIOPi

Apparently healthy neurons-cells that extend elaborately branched processes, stain with silver, generate action potentials and form chemical synapses with one another-can be isolated from embryonic chick spinal cord and maintained for several weeks in relatively sparse cell cultures. In recent descriptions of dissociated mouse spinal cord neurons in uitro, the cells were cocultured with brain (Crain and Bornstein, 1972) or sensory ganglion (Peacock et al., 1973) cells. Chick cord neurons, at least, survive without synaptic or “trophic” interaction with these other cell types. The fact that many neurites, most of which must be dendrites, conducted spikes might be taken as a sign of immaturity or pathology. The dendrites of neurons in several regions of the brain are thought to be “passive” and normally generate only graded synaptic responses (see Purpura,

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1967, for review) whereas the dendrites of neonatal cortical cells and chromatolytic motoneurons generate all-or-none action potentials (Purpura, 1967; Eccles et al., 1958; Kuno and Llinas, 1970). This generalization is not always accurate, however (Purpura, 1967). Dendrites of a variety of normal, adult, vertebrate neurons generate partial or full spikes (Spencer and Kandel. 1961; Nelson and Burke, 1967; Kriebel et al., 1969; Llinas and Nicholson, 1969) and processes of neurons in organized cerebellar explant cultures conduct impulses (Hild and Tasaki, 1962). One explanation for the passive behavior of some dendrites is that they are specialized to receive synaptic input and that patches of subsynaptic membrane are not electrically excitable (Grundfest. 1957). It might be possible, in cell cultures, to correlate the electrical excitability of a process with the density of synaptic terminals on the same process. Variation in light microscopic appearance and in spike electrogenesis, i.e.. action potential configuration and degree of repetitive firing, suggest that the neuronal population in cultures derived from ‘i-day chick embryonic spinal cord is comprised of several different cell types. This suggestion is reinforced by the consideration that apparently all the neurons present in older cultures were postmitotic and, hence. probably already “specified” at the time of dissociation. The categories suggested in Fig. 2 are arbitrary. But there is a wide range in cell size in the initial cell suspension and techniques which have recently been used to separate cells according to size from embryonic retina and cerebellum (Lam, 1972; Barkley et al., 1973) might lead to a more refined and useful classification of spinal cord cells. Differences in action potentials have led to an unambiguous identification of different types of leech sensory neurons (Nicholls and Baylor, 1968), and similar differences may prove an adequate assay in vitro. It would obviously be an advantage to identify other.

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BIOLOGY

more qualitative, differences between cells, such as chemosensitivity, transmitter related enzymes, or synaptic specificities. As a limiting statement, it seems clear that most of the excitable cells are probably not motoneurons: less than 10% form functional neuromuscular junctions when added to previously plated muscle cells (Fischbach, 1972). The high input resistances of the neurons examined in this study, the large-amplitude and quantum content of unitary synaptic potentials, and the ease with which epsp amplitude was altered by polarization of the postsynaptic membrane are all distinctly atypical of adult motoneurons (Coombs et al., 1955a; Smith et al., 1967; Kuno, 1971) and support this conclusion. Variation in psp size and in many cases, reversal in polarity with change in postsynaptic membrane potential, the occurrence of true miniature psps, and the ultrastructure of synaptic endings all strongly suggest that transmission at most, if not all, synapses is chemically mediated. The possibility of electrotonic transmission at some of the synapses cannot be excluded, however. Electrical coupling was detected in a few motoneuron-muscle cell pairs in mixed cultures and the possibility that specialized contacts (gap junctions) may represent a stage in formation of a chemical synapse was raised (Fischbach, 1972). It may be that electrical coupling occurs at the earliest interneuronal contacts but is absent in older cultures. Judging from the occurrence of spontaneous synaptic potentials, the great majority of neurons in cultures seeded with ca. 2 x lo5 cells/60 mm dish were innervated when tested 2-3 weeks after plating, and it is possible that a more exhaustive screening method, such as stimulation of all nearby processes and cell bodies, would reveal that every neuron was innervated. In any case, the high incidence of both excitatory and inhibitory contacts makes spinal cord cell cultures a good system in

VOLUME 37,1974

which to assay factors that influence the formation of different types of synapses. The incidence of large spontaneous synaptic potentials was markedly and reversibly reduced when the cells were bathed in the complete medium in which they normally grow, so it seems unlikely that impulse evoked synaptic function is necessary for synapse formation between spinal cord cells. Electrophysiologic and electron microscopic evidence has been presented that synapses form in explants of mouse cerebrum grown in the presence of concentrations of local anesthetics that block all impulse activity (Crain et al., 1968; Model et al., 1971). It will be of great interest to determine the specificity of synapse formation (or reformation) in spinal cord and in mixed spinal cord-sensory ganglion cell cultures. Two observations indicate that the connections are not completely random. First, neurons do not invariably synapse on adjacent cells or on nearby (200-300 pm away) neighbors that they appear to contact. In fact, proximity was not a good criterion for functional connection. Second, most inhibitory endings were probably located on or near the cell body: inhibitory potentials inverted within seconds after injection of chloride ions into the soma. This proximal location of inhibitory input is characteristic of many neurons in uiuo and suggests that a degree of topographic specificity is retained in uitro. If the bulbous swellings observed with interference contrast optics are synaptic boutons, then it may be possible to test this hypothesis directly by selective stimulation of individual terminals. We would like to thank Dr. John Heuser for providing the electron micrographs and for his advice on the silver impregnation and Mrs. Marie Neal for expert technical assistance in preparation and maintenance of the cultures. REFERENCES BARKLEY,

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