JOURNALOFNEUROPHYSIOLOGY Vol. 64, No. 5, November 1990. Printed

in U.S.A.

Epileptiform Activity in Microcultures Containing Small Numbers of Hippocampal Neurons MICHAEL

M. SEGAL

Department Department

of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston 02114; and

AND

of Neurobiology,

EDWIN

J. FURSHPAN

Harvard Medical

School, Boston, Massachusetts 0211.5

and Potter (1989). In that model mass cultures containing several thousand neurons are grown chronically in the presence of agents that suppress synaptic activity (kynurenate and elevated Mg*+); intense epileptiform activity arises spontaneously when the blocking agents are removed. This activity displays many features of seizures experimentally induced in intact cortex: large paroxysmal depolarization shifts (PDSs), characteristic of both interictal and ictal activity (Kandel and Spencer 196 1; Matsumoto and Ajmone Marsan 1964a,b); sudden transitions to ictuslike activity; sustained depolarizations, characteristic of ictal activity (Hablitz 1987; Haglund and Schwartzkroin 1984; Kandel and Spencer 196 1; Matsumoto and Ajmone Marsan 1964b); activation of inhibitory as well as excitatory mechanisms (cf. Kandel and Spencer 196 1; Rutecki et al. 1985); “postictal” hyperpolarizations when the episodes are terminated (cf. Kandel and Spencer 196 1), and extensive neuronal death when episodes are not terminated (cf. Siesjo and Wieloch 1986). There are obvious differences between intact-tissue and dissociated-cell culturesINTRODUCTION the connections of the hippocampal neurons to other brain Our understanding of the mechanisms underlying epi- regions are lacking; the specificity of connections among leptiform activity has been obtained from work with intact the neurons is presumably altered, as are the relationships brain, cortical slices, and mathematical models (see review between the neurons and glia cells; there might be selective by Dichter and Ayala 1987). Each model has advantages as loss of certain types of cells, etc. Nevertheless, it appears well as drawbacks, and each has been useful in addressing a that the seizurelike activity must arise from basic cellular particular range of issues. In intact brain, the architecture properties that are preserved and interactions that are reesand normal function of the tissue is best preserved, but tablished in the simplified environment of cell culture. A more reduced system, based on microcultures conbiophysical experiments are difficult to perform, as is the rapid application and withdrawal of drugs of known con- taining only one or a few neurons, was used to study the centration. Slice preparations are particularly useful for synaptic functions of sympathetic neurons (Furshpan et al. biophysical and pharmacological experiments, but even in 1976, 1986). The synaptic interactions in such microculsuch a reduced system it is difficult to study circuitry. tures were invariably intense, presumably because the Mathematical models (see Traub et al. 1987) have been growing, branching axons were confined to a limited area useful in testing the plausibility of explicit hypotheses, but and made many synapses on each target cell; in addition, they must incorporate data from biological experiments. with only one or two neurons present, monosynaptic, including autaptic, interactions could be identified unambigA potentially useful approach is to examine which aspects of epileptiform activity persist as the neuronal cir- uously. cuitry is made simpler. Such studies have been carried out We describe here a similar microculture approach to the on reduced fragments of cortical tissue (Crain and Born- study of synaptic interactions underlying epileptiform acstein 1964; Miles et al. 1984). Picrotoxin-induced epileptitivity in hippocampal neurons. The microculture techform activity occurred only in isolated pieces of the CA3 niques used with sympathetic neurons were not directly region of the hippocampal formation that contained at applicable to hippocampal neurons because the CNS glial least - 1,000 pyramidal neurons (Miles et al. 1984). To cells migrated on the putatively nonadhesive substrates and study epileptiform activity in a preparation containing formed bridges between microcultures. Instead, we 10 wk. One can select microcultures containing any desired number of neurons for study. We report here that in microcultures containing only a small number of neurons, the neurons generate elements of epileptiform activity (e.g., PDSs) after chronic exposure to synaptic blocking agents. In addition, in microcultures, synaptic actions can be observed uncontaminated by polysynaptic interactions.

10 U/ml papain latex and cysteine (3.7 mM) (Huettner and Baughman 1988) in dissecting medium. The hippocampi were then rinsed with dissecting medium; the remnants of enzyme were neutralized by three, 5-min incubations with 10 mg/ml trypsin inhibitor (Sigma type II-O) in dissecting medium. The hippocampi were placed in a growth medium and triturated with 50 passes through the tip of a 2-ml plastic pipette; the supernatant was collected, and the sediment was triturated another 50 times. The growth medium was a modification of L-15 CO2 medium (Hawrot and Patterson 1979), with the omission of nerve growth factor, fresh vitamin mix, aspartate, glutamate and P-alanine, the use of only one-half of the usual concentrations of glucose, glutamine, penicillin, and streptomycin, and the addition of the Nl additives of Bottenstein and Sato (1979) at the following concenMETHODS trations: 2.5 pg/ml insulin, 25 pg/ml transferrin, 10 nM progesterone, 90 PM putrescine, and 15 nM selenium. Cells were plated Culture dishes into new culture dishes at densities of 0.4 hippocampi/cm2, and after 2.5 h the unattached cells and debris were washed away by The agarose layer was formed from a drop of 0.15% agarose dropping 1 ml of fresh growth medium directly onto the culture solution (Sigma type II-A) placed on one surface of a 22-mm, wells and then changing to new growth medium. The initial platglass cover slip (Gold Seal). The height of each drop was diminished as much as possible by removing excess solution with a ing resulted in attachment to collagen or palladium substrates of mostly glial cells and very few neurons; virtually no cells attached pipette before the agarose gelled. The gel was allowed to dry overnight at room temperature to form a thin film. Culture dishes to the agarose. After 3 days, when the glial cells formed a near areas, the cultures were prepared by drilling an &mm hole in the bottom of a monolayer on the collagen or palladium-coated were irradiated with 1,200 rads from a 6oCo source at a dose rate 35mm, plastic tissue-culture dish and attaching the agaroseof ~27 rads/s. One to 3 wk later, cells from another dissociation coated cover slip with silicone elastomer (Sylgard, Dow Corning) to form a 0.7-mm-deep central culture well (see Furshpan et al. were plated onto the glial layer at a density of 0.2 hippocampi/ 1986). Most dishes with microcultures were made with dots of the cm2, with the use of a lo-mm glass ring to contain the cells over substrate collagen sprayed onto the agarose background. A the 8-mm culture well; unattached material was washed off after 0.075% solution of human placental collagen (Sigma type VI) in 2l/2 h. After 1 day the cultures were again irradiated with 1,200 rads, and (in cultures in which these blockers were used) the 0.125% acetic acid (by volume) was sprayed forcefully from a growth medium was changed to one containing 1 mM kynurenmicro-atomizer (Thomas Scientific) held 25 cm above and 25 cm to the side of the culture dishes, leaving drops 50-300 pm in ate and 11.3 mM MgC12, and the culture well was covered with a dialysis membrane (Spectra/Par; 12- 14 kD molecular weight cutdiameter on the agarose in the culture well. Afterwards, one-half off) held in place with an assembly of rubber and glass rings of the 8-mm-diam culture well area was covered with large drops (Furshpan and Potter 1989). Cultures were grown at 37OC in 4.9% of the collagen solution to allow a mass culture to grow there; the CO2 (balance air) and fed once every 2 wk by replacing the meliquid in the collagen solution was allowed to evaporate at room dium above the dialysis membrane. temperature overnight. For some microculture dishes palladium Adhesive substrates other than collagen and palladium formed was used as a substrate (Carter 1965, 1967; Ponten and Stolt microcultures but were less useful for our purposes. Microcul1980; Westermark 1978). A thin layer of palladium was vacuum tures made by spraying high concentrations of polylysine and deposited with the use of a Denton Vacuum model 502 vacuum laminin (1 mg/ml and 60 pg/ml, respectively) resulted in the evaporator, forming a layer of sufficient thickness to appear light density of all the adhesive substrates, but gray when viewed against a white background. By the use of highest neuronal neurons were too densely packed. Lower concentrations of polyelectron-microscopy grids as masks, islands of palladium were formed on a sea of agarose. In most cases we further decreased the lysine and laminin (0.25 mg/ml and 15 pg/ml, respectively) gave highly variable neuronal attachment. In microcultures made with open area of the grids by filling in some of the grid squares with dots of a mixture of polyphenoic proteins of the marine mussel Sylgard. Typically, about one-half of the 8-mm-diam culture area Mytilis e&/is (Cell-Tak, 1 mg/ml) (Munoz-Blay et al. 1987) apwas covered with grids, and the rest was left open to allow pallaplied with a broken glass micropipette, glial cells initially adhered dium deposition to support a mass culture. The internal surfaces well but spread poorly within the applied dot. of dishes in both methods were sterilized using ultraviolet light The lack of adhesion of cells to agarose was observed for several before use. Collagen microcultures were used for most electrotypes of agarose that differ in their electroendosmosis and sulfate physiological recordings because of the excellent and reproducible spreading of glial cells on collagen, providing a clear optical path groups (Sigma type IIA, used in the experiments described here, and Sigma types IV, TVA, VIII, and SeaKem ME). Agarose drops that enabled accurate counts of the number of neurons. more dilute than 0.15% beaded rather than forming a continuous layer. More concentrated agarose solutions rehydrated to thicker Dissociation methods layers that increased the optical path length and were more likely to buckle and to detach from the underlying glass cover slip. The method of dissociating cells is based on that of Furshpan Attempts to use GelBond plastic (to which agarose has been and Potter (1989). Cerebral hemispheres were removed from chemically bonded) were associated with widespread death of newborn Long-Evans rats anesthetized with 0.25 ml of chloral cells within days. hydrate (20% wt/vol). Intact hippocampal formations were reUnsuccessful attempts were made to separate microcultures moved and placed in dissecting medium. The dissecting medium with the use of the nonwetting substrates Hydron NCC [poly contained (in mM) 8 1 Na2S04, 27 K2S04, 15.7 MgCIZ, 0.23 (2-hydroxyethyl methacrylate)] (Folkman and Moscona 1978), CaC12, and 1 kynurenate; it was buffered with 1 mM HEPES, and Petriperm gas permeable membrane, Sylgard (Huettner and the pH was adjusted to 7.4. The hippocampi were incubated at Baughman 1988), and the hydrophobic polysiloxane resin DC648 37°C for two, 20-min periods in dissociation medium containing (Lucas et al. 1986). Glial cells (present with the dissociated

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neurons) were able to attach to each of these putatively nonadhesive substrates even in the absence of other added substrates.

Recording methods Recordings from neurons were made 18-90 days after plating of neurons. Cultures were perfused continuously during recording, with the dish temperature maintained at 3 1“C. The perfusion solution was a 9:l mixture of Ca’+-Mg2+-free Hank’s balanced salt solution and MEM medium, to which was added (in mM) 25 glucose, 5 HEPES, 1.6 glutamine, and CaC12 and MgC12 as desired (usually 1.5 and 1.O mM, respectively). Intracellular microelectrodes were used for all recordings, with resistances of 120-250 MQ when filled with 2 M potassium citrate [3 M potassium methylsulfate (Pfaltz 8~ Bauer, Waterbury CT) was used in early experiments]. Drugs were applied by way of the perfusion system, with a delay of 2-3 min before a new solution reached the bath. Solutions of acidic drugs were neutralized with 0.3 M NaOH. Murine Laminin, Cell-Tak, and high MW POly-D-lySine hydrobromide (ca. 450 kD) were obtained from Collaborative Research, Bedford MA; Papain from Cooper Biomedical; DC648 from Dow Corning, Midland MI; Hydron NCC from Interferon Sciences, New Brunswick NJ; Petriperm from Heraeus, FRG: SeaKem ME agarose and GelBond-NF from FMC Bioproducts, Rockland ME: growth media and balanced salt solution from Flow Laboratories; and phaclofen from Research Biochemicals, Natick MA. All other chemicals were obtained from Sigma. RESULTS

Creuting microcultures As described in METHODS, microcultures were prepared by depositing small areas of an adhesive substrate such as collagen or palladium, which support attachment of neurons and glial cells, atop a nonadhesive layer of agarose, which prevents cell migration. Figure 1 shows one microculture made with palladium on agarose and one with collagen on agarose. In both cases hippocampal cells attached selectively to the small island of substrate. In the first 3 wk after plating of neurons, their processes were clearly visible, but afterwards only the cell bodies of the neurons remained visible with the use of phase-contrast microscopy. Electron micrographs of older microcultures showed that large numbers of processes were present, embedded in several layers of glial cells (Dr. Ann Yee, personal communication). After an initial period of neuronal loss during the first 3 wk, relatively stable survival of neurons in microcultures was observed for > 10 wk, including many microcultures that contained three or fewer neurons. Epileptifwm

activity in microcultures

In recordings from neurons in microcultures we observed epileptiform activity that shared several characteristics with that found in mass cultures (Furshpan and Potter 1989). For comparison with the microculture observations, Fig. 2 shows simultaneous recordings from two neurons in a mass culture that contained several hundred neurons, grown in the presence of synaptic blocking agents (1 mM kynurenate and 11.3 mM Mg*+). As the perfusion solution was changed to standard perfusion medium (1 mM Mg*+ and no kynurenate, beginning 2.7 min before the start of the displayed record, with a dead space time of -2 min), the traces, initially quiet, displayed synchronous synaptic

FIG. 1. Microcultures made with collagen-on-agarose and palladiumon-agarose techniques. Top: 13 neurons growing on glial cells on a square of palladium deposited on an agarose background. Square was 300 pm across. Neurons had been in culture 13 days. Bottom: 2 neurons growing on glial cells on a dot of collagen sprayed onto an agarose background. Island was 175 pm across. Neurons had been in culture 19 days.

complexes, most of which had excitatory and inhibitory components (seen most clearly in the top trace). After a gradual negative shift in the baseline, the inhibitory components were no longer visible (t); only large synchronous depolarizing events remained. These depolarizing events in mass cultures (Furshpan and Potter 1989) were identified on the basis of their size, shape, and synchrony, as PDSs (Dichter and Ayala 1987; Johnston and Brown 198 1; Kande1 and Spencer 196 1; Matsumoto and Ajmone Marsan 1964a). Midway through the figure (*) a slow, sustained depolarization began, and the PDSs increased in frequency.

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FIG. 2. Epileptiform activity in 2 neurons in a mass culture. Simultaneous records are shown from 2 neurons in this mass culture that was estimated to contain several hundred neurons. Perfusion solution was changed 2.7 min before the beginning of the figure from one containing 1 mM kynurenate and 11.3 mM Mg2+ to one containing no kynurenate and 1mMMg. 2+ Neurons had been in culture 47 days.

Figure 3 shows a similar transition to epileptiform activity in simultaneous recordings from three neurons in a 12-neuron microculture. During the washout of kynurenate ( 1 mM) and excess Mg2+ (reduced from 11.3 to 1 mM), synchronous inhibitory postsynaptic potentials (IPSPs) occurred in each of the neurons; these were then replaced by synchronous excitatory events. In the expanded record (B), it can be seen that all of the IPSPs had relatively fast components (duration - 100 ms); some also had slower components lasting OS- 1 s (seen most clearly in neurons 1 and 3). Midway through B there was a transition to synchronous excitatory potentials that became (with each successive burst) more similar from neuron to neuron, as well as Neuron

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larger in amplitude. This transition to epileptiform activity in a 12-neuron microculture was similar in many respects to the typical mass-culture activity seen in Fig. 2. One obvious difference was the absence of a sustained depolarization in this microculture. Sustained depolarizations were seen only rarely in microcultures (Segal 1990; Segal, unpublished observations). The transition to epileptiform activity also occurred in microcultures with < 12 neurons. In three, two-neuron microcultures in which both neurons were excitatory, epileptiform activity consisting of PDSs was observed in both neurons. Figure 4 is a recording from such a microculture. Stimulation of either neuron in standard perfusion medium (1 mM Mg2+ and no kynurenate) led to bursts of PDSs in both neurons; these postsynaptic responses were reduced to excitatory postsynaptic potentials (EPSPs) triggering action potentials when 1 mM kynurenic acid was applied and the postsynaptic neuron was hyperpolarized to prevent firing of PDSs (not shown). A change from perfusion with 11.3 mM Mg2+ and 1 mM kynurenate to perfusion with 1 mM Mg2+ and 1 mM kynurenate gave rise to the spontaneous events shown in Fig. 4A (expanded in Fig. 4B). Each cluster of activity began with a PDS that evoked many spikelike depolarizations, followed by a phase of recurring, briefer PDSs. Neuron 1 appeared to initiate the bursts, because each PDS in neuron 1 was preceded by a gradual depolarization, whereas those in neuron 2 began abruptly, and because hyperpolarization of neuron 1

1

OmV

I

FIG. 3. Epileptiform activity in 3 neurons neurons. Records in A are shown expanded at the beginning of the figure from one containing 1 mM Mg’+. Neurons had been in culture 20

in a microculture with 12 neurons. Simultaneous records are shown from 3 higher chart speed in B and C. Perfusion solution was changed 18 min before 1 mM kynurenate and 11.3 mM Mg2+ to one containing no kynurenate and days.

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A Neuron 1 Neuron 2

lO mV

40mV FIG. 4. Epileptiform activity in a 2-neuron microculture (both neurons excitatory). Indicated segments of A and C were expanded in B and D. Perfusion solution was changed 5 min before the beginning of A from one containing 1 mM kynurenate and 11.3 mM Mg2+ to one containing 1 mM kynurenate and 1 mM Mg 2+ . Records in A and C are continuous. Immediately after the last cluster of activity in A, the perfusion solution was changed from one containing 1 mM kynurenate to one containing no kynurenate (1 mM Mg2+ in both solutions). Neurons had been in culture 27 days.

aborted the spontaneous PDSs in both neurons (not shown). In the absence of kynurenate (Fig. 4C) there were fewer PDSs in each cluster, and individual PDSs became longer and more wedge-shaped (records in Fig. 4A and C are continuous; records in C were made as the 1 mM kynurenate was washed out with [Mg2+] still at 1 mM; the solution change began immediately after the last cluster of activity in A). Action potentials were often small or absent during the PDSs, presumably because of inactivation of voltage-gated Na+ channels. The clusters of PDSs illustrated in Fig. 4 all terminated spontaneously, and despite the absence of any inhibitory neurons in this microculture, each cluster was followed by an afterhyperpolarization. [Throughout this paper, the use of the terms excitatory and inhibitory is intended only to characterize the most obvious recorded effect of the neuron. It is not meant to imply that a transmitter always has the same effects on different postsynaptic neurons or that a neuron can release only one transmitter (Potter et al. 198 1; Segal 1983.1 Epileptiform activity was also observed in five, twoneuron microcultures that each contained one inhibitory and one excitatory neuron. Records from one of these microcultures are shown in Fig. 5. In Fig. 5A, the spontaneously occurring action potentials in the neuron identified as inhibitory (bottow2 traces) produced IPSPs in the other neuron (+ W, top trace). Stimulation of the neuron identi-

fied as excitatory produced EPSPs and PDSs in the inhibitory neuron (not shown). At the beginning of Fig. 5A, the perfusion fluid was changed from one with 1 mM kynurenate and 1 mM Mg2+ to standard perfusion medium (no kynurenate and 1 mM Mg2+), and PDSs appeared in both neurons (Fig. 54; at the slow chart speed used for Fig. 5, A and &, the PDSs appear as spikelike events; compare with expanded records in Fig. 5, B2 and B3). After PDSs were recorded for 20 min, the perfusion medium was changed to one containing bicuculline (during the course of Fig. 5BI). Individual PDSs in both neurons became longer and more wedge-shaped, and action potentials were usually indiscernible during the PDSs (compare Fig. 5B2, before bicuculline, with Fig. 5B3, with bicuculline). Bicuculline attenuated not only the IPSPs produced by the spontaneous firing of the inhibitory cell (t, Fig. 5BI) but also the afterhyperpolarization in the inhibitory neuron. This suggests that the inhibitory neuron produced IPSPs in the excitatory neuron, as well as autaptic IPSPs in itself, and these inhibitory currents were components of the PDSs. It also appears that there were autaptic EPSPs; the PDSs in the excitatory neuron were presumably the result of self-excitatory synapses. In seven, two-neuron microcultures in which both neurons were inhibitory, no epileptiform activity was seen. In these cultures there was no spontaneous activity in stan-

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’ 1 min

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FIG. 5. Epileptiform activity in a 2-neuron microculture (1 neuron excitatory and 1 inhibitory). Perfusion solution was changed 2 min before the beginning of A from one containing 1 mM kynurenate to one containing no kynurenate. Twenty minutes elapsed between the end of A and the beginning of BI; same calibrations apply to A and Bl . In the middle of BI, the perfusion solution was changed from normal perfusion medium to one containing 10 PM bicuculline methiodide. Segments of B, were expanded in B2and B3.Neurons had been in culture 34 days. All solutions contained 1 mM Mg2+.

dard perfusion medium (1 mM Mg*+ and no kynurenate), and stimulation of either neuron produced IPSPs in the other neuron and autaptic IPSPs in the stimulated neuron (not shown). Synaptic activity in microcultures A majority of the IPSPs recorded in microcultures were fast IPSPs, lasting - 100-200 ms, and fully blocked by bicuculline (10-50 PM). Such IPSPs were fully abolished and then reversed in polarity as the membrane potential of Phaclofen II the postsynaptic neuron was made more negative (not shown). The amplitudes of the IPSPs in these cultures were usually 5-20 mV, even for neurons with resting potentials more negative than -60 mV. We have not observed pure slow IPSPs in microcultures. However, in simultaneous recordings from pairs of neurons, IPSPs with both fast and slow components were occasionally evoked by stimulation of one of the neurons. The pharmacology of six such connections was studied-in two cases the slow component of the IPSP was blocked by phaclofen (1 mM); in four cases it was not blocked. Figure 6 shows simultaneous recordings from two neurons in a four-neuron microculture. An action potential evoked di20mV rectly in one neuron (bottom trace) produced a two-com’ 1 set ponent IPSP in the other neuron (top trace). The slow comFIG. 6. A conjoint fast IPSP-slow IPSP. Microculture contained 4 ponent was reversibly blocked by phaclofen; the fast com- neurons; recordings were made from 2 of them. A: IPSP with fast and slow ponent was reversibly blocked by bicuculline, and both components evoked in the neuron in the top trace by single action potencomponents were blocked by the combination of the two tials (bracketed by symmetrical stimulus artifacts) in the neuron in the drugs (Fig. 6, B-D). Both IPSP components persisted in the bottom trace (in regular perfusion medium). Resting potential of the presence of 5 mM kynurenate, making it unlikely that the neuron in the top trace was -60 mV, and the amplitude of PDSs was 30-38 mV (not shown). B: phaclofen (1 mM) blocked the slow compotwo-component IPSP was due to polysynaptic activation of nent. C: bicuculline methiodide (50 PM) blocked the fast component. D: separate “fast-inhibitory” and “slow-inhibitory” neurons. both drugs blocked both components. Neurons had been in culture 39 These results indicate that both GABA* and GABAB re- days. Neuron represented in the top trace was found to be excitatory, ceptors (Dutar and Nicoll 1988) were activated bv a single evoking EPSPs in the neuron represented in the bottom trace. Downloaded from www.physiology.org/journal/jn by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 9, 2018. Copyright © 1990 American Physiological Society. All rights reserved.

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presynaptic neuron. The neuron in which this two-component IPSP was recorded was found to be excitatory; when stimulated it evoked EPSPs in the inhibitory neuron. Including this neuron, there were six cases in which the synaptic action of a neuron receiving a two-component IPSP was tested (including the 2 neurons with phaclofenblocked, slow IPSPs and 2 of the 4 neurons with phaclofen-insensitive IPSPs); in each of these cases the neuron receiving the slow inhibition was excitatory. The properties of EPSPs were more difficult to study than those of the IPSPs. In microcultures grown in blockers, the EPSPs in all but one case led to firing of the postsynaptic neuron unless the postsynaptic neuron was substantially hyperpolarized or the EPSPs were substantially blocked (with kynurenate), making it difficult to measure EPSP amplitudes. It will be instructive to do voltage-clamp studies of the amplitude of the excitatory synaptic currents in blocked and nonblocked cultures. In this preliminary series of 36 microcultures grown in glutamate blockers, epileptiform activity was observed in 25 of 26 microcultures that were known to contain an excitatory neuron (as judged by kynurenate sensitivity of EPSPs). In the 10 microcultures in which all tested neurons were inhibitory, no epileptiform activity was observed (7 of these microcultures were 2-neuron microcultures in which simultaneous recordings were made from both neurons). We also carried out experiments to exclude the possibility that there were unseen neuronal processes traversing the agarose between microcultures. With the sprayed collagenon-agarose technique, processes crossing the agarose were occasionally present between nearby collagen dots, and these were readily seen on the optically clear agarose background. Only microcultures with no visible connections were used in this paper. To test for unseen processes outside microcultures, two types of experiments were conducted. One control was to detach the agarose underlying a single microculture from surrounding agarose by the use of a microelectrode to tear the agarose. An additional five, two-neuron microcultures were studied in this manner. Three microcultures had one inhibitory neuron and one excitatory neuron; perfusion with medium without kynurenate resulted in synchronous PDSs in both neurons, similar to the records shown in Fig. 5. The other two microcultures studied had two inhibitory neurons each; no epileptiform activity was seen. These results are similar to those from the nondetached microcultures. A second control for unseen processes outside microcultures was injection of neurons with Lucifer yellow and observation with fluorescence microscopy. An additional 14 such experiments were carried out and demonstrated that very fine processes were present within the microculture, including processes not visible against the glial cells of the microculture, but no processes were revealed on the agarose (Segal, unpublished observations). Additional assurance of the lack of unseen processes outside the microcultures is provided by the fact that in microcultures in which simultaneous recordings were made from all neurons, there were no synaptic inputs unaccounted for by the neurons in the microculture. Observations have also been made on 38 microcultures grown in control medium (without kynurenate or elevated

E. J. FURSHPAN

Mg*+). Such microcultures rarely showed any spontaneous activity. When excitatory neurons were stimulated (in 13 cases in 12 microcultures), the resulting activity in the recorded neuron consisted of brief EPSPs (3-12 mV) that evoked no action potentials (in 6 cases), single action potentials or short bursts of action potentials (in 3 cases), or depolarizing plateaus of lo- to 20-mV amplitude without inactivation of action-potential generation (in 4 cases). Wedge-shaped PDSs were never observed. In contrast, IPSPs appeared to be similar in control and blocked microcultures (Peters and Segal, unpublished results). DISCUSSION

Creating microcultures The use of agarose as a nonadhesive substrate appears to be important in forming microcultures of central neurons because glial cells adhered to and migrated on all other substrates tested. The reason that cells avoid agarose is not clear. It might be due to the negative charge of agarose (Costachel et al. 1969), to specific chemical groups on the agarose, or to the high water content of the rehydrated agarose. Synaptic activity in microcultures One important advantage of microcultures with only a few neurons is that it is possible to record from all neurons in the network simultaneously. Monosynapticity of connections can be determined without using techniques for reducing polysynaptic activity such as high Ca*‘, high Mg*+ solutions (Austin et al. 1967) that distort responses -for example, by blocking N-methyl-D-aspartate (NMDA) receptors (Mayer et al. 1984; Nowack et al. 1984). The ability to establish that synaptic actions are monosynaptic also allows one to determine by use of blocking drugs whether individual neurons are excitatory or inhibitory (or both). A second important advantage of microcultures is that PSPs are large and thus amenable to pharmacological dissection. At least two effects may be operating to increase synaptic strength. One is that the growing axonal arborization is restricted to relatively few target cells (Furshpan et al. 1976, 1986). The IPSPs and EPSPs in both the chronically blocked microcultures shown here and in nonblocked microcultures (Peters and Segal, unpublished observations) were often 5-20 mV; PSPs seen in neurons with similar resting potentials in hippocampal slices were 54 mV (Miles and Wong 1984, 1986). There may also be a specific upregulation of excitatory transmission in cultures grown under chronic blockade of glutamate receptors. It will be important to compare excitatory connections in blocked and nonblocked microcultures that are matched for their composition of excitatory and inhibitory neurons. A third advantage of microcultures is the high degree of synaptic connectivity and the variety of synaptic phenomena that were observed. There were fast EPSPs, fast IPSPs, and conjoint fast IPSPs-slow IPSPs, as well as autaptic EPSPs and autaptic fast IPSPs. The records shown in Figs. 4-6 were typical in that chemical synaptic connections were present in almost all instances between any two

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neurons in a small microculture. In contrast, the connectivity in hippocampal slices is estimated at l-5% for CA3 pyramidal neurons (MacVicar and Dudek 1980; Miles and Wong 1986). Recordings from hippocampal slices have suggested that slow IPSPs may be produced by different neurons from those producing fast IPSPs (Miles and Wong 1984). Experiments of the type illustrated in Fig. 6 (in the presence of kynurenate) suggest that, at least in culture, one neuron can evoke both fast and slow IPSPs in the same postsynaptic cell. A fourth advantage of microcultures is the ability to compare the activity and pharmacology of small networks that differ in their cellular composition. The effects of a particular cell type, receptor type, or voltage-sensitive channel subtype could be studied. For example, it could be presumed that an anticonvulsant with similar effects on microcultures containing or lacking inhibitory neurons has its most significant effect at a locus other than inhibitory transmission. Epileptlyorm

activity in microcultures

PDSs are one of the hallmarks of epileptiform activity (Kandel and Spencer 196 1; Matsumoto and Ajmone Marsan 1964a). PDSs are large, complex events in which the cell rapidly depolarizes, often to a voltage at which actionpotential generation may become inactivated after a few impulses, and more slowly repolarizes. The activation of excitatory receptors during the PDS activates voltage-sensitive channels that contribute to generation and propagation of the PDS (Dichter and Ayala 1987; Johnston and Brown 198 1; Kandel and Spencer 196 1; Matsumoto and Ajmone Marsan 1964a). PDSs usually have amplitudes of 15-50 mV and durations of 40-400 ms (Matsumoto and Ajmone Marsan 1964a). An essential characteristic is synchronous occurrence in a population of neurons (Dichter and Ayala 1987; Johnston and Brown 198 1; Kandel and Spencer 196 1; Matsumoto and Ajmone Marsan 1964a). The synchronous large excitatory events shown in Figs. 2-5 fulfill the criteria for PDSs. The ability of kynurenate to abolish them or reduce their size indicates the central role of EPSPs in their generation. The PDSs in microcultures are very similar to those recorded in mass cultures (Furshpan and Potter 1989). The mechanism by which the chronic blockade of synaptic transmission induced the epileptiform activity is not clear. There has been discussion (e.g., Ayala et al. 1973) as to whether epilepsy is due to changes in individual neurons (the “epileptic neuron” hypothesis), or whether the behavior of an entire population has been altered (the “circuit” hypothesis). The data presented here do not resolve this question because it is not yet clear whether the differences in behavior between blocked and nonblocked cultures are due to induction of different neuronal properties or selection of different neuronal types (see Furshpan and Potter 1989). Although it is possible that epileptic neurons were present in virtually all of our cultures that contained excitatory neurons, it is also possible that epileptiform activity occurs when normal neurons are permitted to make unusual and highly simplified circuits. Detailed comparisons of the behavior of microcultured neurons grown in the

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presence or absence of blocking agents will be important in this regard. It is not known whether the formation of new autaptic or collateral connections can occur pathologically in cortex in vivo. The question of how many neurons are needed to produce epileptiform activity has been addressed by Miles et al. ( 1984), who recorded from progressively smaller pieces of the CA3 region of the hippocampal formation. The smallest pieces that supported picrotoxin-induced epileptiform activity were estimated to contain about 1,000 pyramidal neurons. The presence of epileptiform activity in microcultures with only two neurons presumably reflects the fact that the probability and strength of synaptic connections are greatly increased in microcultures. It may be useful to think of each neuron in a microculture as representing many neurons in vivo. The presence of epileptiform activity in a microculture with only one excitatory neuron and one inhibitory neuron (Fig. 5) suggests that the excitation was maintained by a single excitatory neuron, by means of autapses. In other studies of single-neuron microcultures, both PDSs and sustained depolarizations were seen in microcultures containing only an excitatory neuron (Segal 1990; Segal, unpublished observations). Also, in Fig. 5&, blocking the action of the inhibitory neuron with bicuculline served only to augment and prolong the PDSs in both the inhibitory and excitatory neurons. Sustained depolarizations can be studied in microcultures, although they occur infrequently (Segal 1990; Segal, unpublished observations). The ability to study both ictaland interictal-type epileptiform activity in physiological solutions not containing blocking drugs is one of the advantages of this system. The termination of activity in the microcultures in which GABAergic neurons were absent (e.g., Fig. 4) or GABA* and GABAB receptors were blocked (e.g., neurons recorded from in Fig. 6, PDSs not shown) suggests the presence of repolarization mechanisms other than GABAergic inhibition. Possibilities would include Ca*‘mediated K+ currents (Alger and Nicoll 1980; Hotson and Prince 1980; Schwartzkroin and Strafstrom 1980) ion pumps (Ayala et al. 1970; Bergmann et al. 1970), inactivation of excitatory currents, or the release of other inhibitory transmitters such as a peptide (Tortella and Long 1985), a classical transmitter (Potter et al. 1986), or adenosine (Dunwiddie and Fredholm 1989). There are a number of obvious differences between synaptic interactions in microcultures and in the intact cortex. These include the smaller numbers of neurons, larger synaptic potentials, altered glial ensheathment, presumably altered connectivity patterns (Schacher et al. 1985), and perhaps selective survival of certain neuronal types (Mattson and Kater 1989; Tecoma and Choi 1989), as well as possible alterations in transmitter repertoires (Potter et al. 1986). Nevertheless, the presence of epileptiform activity in this highly reduced system provides obvious advantages for the study of this activity. In addition, in both the mass culture and microculture models the epileptiform activity occurs without the need to modify the ionic environment (e.g., elevating K+ or decreasing Ca*+) or to add drugs to block GABA-mediated inhibition (reviewed by Dichter

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M. M. SEGAL AND E. J. FURSHPAN

and Ayala 1987; Dichter and Spencer 1969; Matsumoto and Ajmone Marsan 1964a,b; Ogata et al. 1976); therefore the interplay of excitation and inhibition in regulating epileptiform activity can be better studied. Microcultures should also be useful for a variety of other studies of synaptic actions of central neurons. We thank L. Weiss, C. Fischer, S. Ray, R. Bosler, J. Gagliardi, and M. LaFratta for expert technical assistance and W. Koroshetz, E. Liman, S. Lin, B. Peters, and A. Yee for reading an earlier version of this manuscript. This work was supported by a grant from the Dana Fellowship Program in Neuroscience (to M. M. Segal), National Institute of Neurological Disorders and Stroke Grants NS-0 1407-O 1 (to M. M. Segal) and NS-PO l02253 (to E. J. Furshpan), and an Esther and Joseph Klingenstein grant (to E. J. Furshpan). Address for reprint requests: M. M. Segal, Neurobiology Department, Harvard Medical School, 220 Longwood Ave., Boston, MA 02 115. Received 23 January 1990; accepted in final form 14 June 1990. REFERENCES B. AND NICOLL, R. Epileptiform burst afterhyperpolarization: calcium-dependent potassium potentials in hippocampal CA 1 pyramidal cells. Science Wash. DC 2 10: 1122- 1124, 1980. AUSTIN, G., YAI, H., AND SATO, M. Calcium ion effects on Aplysia membrane potentials. In: Invertebrate Nervous Systems, Their SigniJicance for Mammalian Neurophysiology, edited by C. A. G. Wiersma. Chicago, IL: Univ. of Chicago Press, 1967, p. 39-53. AYALA, G. F., DICHTER, M., GUMNIT, R. J., MATSUMOTO, H., AND SPENCER, W. A. Genesis of epileptic interictal spikes. New knowledge of cortical feedback systems suggests a neurophysiological explanation of brief paroxysms. Brain Res. 52: 1-17, 1973. AYALA, G. F., MATSUMOTO, H., AND GUMNIT, R. J. Excitability changes and inhibitory mechanisms in neocortical neurons during seizures. J. Neurophysiol. 33: 73-85, 1970. BERGMANN, F., COSTIN, A., CHAIMOVITZ, M., AND ZERACHIA, A. Seizure activity evoked by implantation of ouabain and related drugs into cortical and subcortical regions of the rabbit brain. Neuropharmacology ALGER,

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