A slow NMDA-mediated synaptic potential underlies seizures originating from midbrain

Brain Research, 486 (1989) 381-386

381

Elsevier BRE 23489

A slow NMDA-mediated synaptic potential underlies seizures originating from midbrain Martha G. Pierson, Karen L. Smith and John W. Swann Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201-0509 (U.S.A.)

(Accepted 24 January 1989) Key words: Inferior colliculus; Brain slice; Epilepsy, N-Methyl-D-aspartate; Audiogenic seizure; Rat

After bath-perfusion with y-aminobutyric acid antagonists, slices of the rat's inferior colliculus were studied electrophysiologically. Synchronized epileptiform events were found to occur. The most prominent intracellular event was a sustained 30 mV depolarization which was pharmacologically and electrophysiologicallycharacterized as an N-methyl-t)-aspartate-mediated event. We propose that elicitation of this slow synaptic potential is the a priori basis of seizures arising in this midbrain nucleus.

In the mammalian brain the inferior colliculus (IC) is the most peripheral site at which the integrative processing of binaural auditory information occurs 1. However, the IC also appears to be a key element in the genesis of a unique type of subcortical seizures, aptly called wild running seizures (WRS). Evidence of this involvement includes that: (1) sound-triggered WRS are prevented in previously susceptible rodents by ablation of the IC but not by more central lesions2"9'12"27; (2) WRS can be elicited in normal rats by direct electrical stimulation of the IC using exceptionally low current strength13"16"19;.and (3) WRS occur in normal rats after bilateral stereotaxic microinjections of convulsants including N-methyl-o-aspartate (NMDA) 19, picrotoxin (PTX) 1° or bicuculline (BIC) 9'16'17. Despite implicit involvement in this experimental seizure disorder, no evidence has existed of intrinsic epileptogenicity within the IC. Nonetheless, the techniques exist for studying such a question. In vitro slice procedures have been used recently for studying local circuit interactions in isolated brain regions 8'25'29. Results from brain slice studies have led to the appreciation that epileptogenic circuits

exist in both hippocampus 23'24'26'28 and neocortex 3'4. A similar application of brain-slice techniques to the study of the IC is reported here. Portions of these data have been presented elsewhere in abstract fOnT121,22. Brain slices were obtained from rats between 13 and 20 days old. Rats were etherized and decapitated prior to removing the inferior colliculus and underlying brainstem en bloc. The IC was sliced into 375- or 500-/~m thick sections, generally in a horizontal plane. The slices were immediately transferred to an experimental chamber where they rested on a net at the interface between circulating artificial cerebrospinal fluid (ACSF) and humidified air (95% 02/5% CO2). Slices were kept at 36 °C and were maintained by procedures described elsewhere 25. The millimolar composition of the ACSF was: 122.8 NaCI; 5.0 KC1; 2.0 CaCI2; 2.0 MgSO4; 1.3 NaH2PO4; 26.0 NaHCO3, 10.0 glucose. Field stimulated responses were studied in more than 100 slices of IC tissue. Field stimulation employed monopolar tungsten electrodes which were shielded with silver conducting paint. Extracellular recordings were made with 2 M NaCI electrodes (R = 10 MSg).

Correspondence: M.G. Pierson, Wadsworth Center for Laboratories and Research, New York State Department of Health, Box 509, Albany NY 12201-0509, U.S.A.

0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

382 Intracellular recordings were obtained with either 4

of which were in the dorsal cortex and which met the arbitrary criteria of having resting potentials equal to

M potassium acetate (KAc) (R = 100-200 MQ) or 1 M cesium acetate (CsAc) (R = 60-150 M~2)

or more negative than - 5 0 mV and having action potentials with amplitudes greater than +50 inV. In recordings made from IC slices maintained in

electrodes. Recordings were made by standard electronic means. Responses were tape-recorded (Hewlett Packard, model 3968A). Response averaging,

normal ACSF, local stimulation, at low current strengths caused virtually all neurons to respond

integration, and electronic subtractions could be subsequently performed on tape-recorded signals with a signal averager (Princeton Applied Research, model 4203). Recordings reported here were representative of those made from more than 150 cells, all

with inhibitory postsynaptic potentials (IPSPs). These IPSPs were reduced or eliminated by the local application of PTX or BIC (ejected as microdroplets in the vicinity of the intracellular recording elec-

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Fig. 1. Synaptic inhibition and epileptiform activity in IC slices. A: averaged IPSPs (n = 5) before (left trace) and after (right trace) pressure ejection of 25 aM BIC onto slice surface. Slice was a 375-/~mthick horizontal section, removed 1125#m below dorsal surface of 13-day-old rat's IC. Stimulus strength was 30/~A. Resting membrane potential was -67 mV. B: amplitude and polarity of IPSP as function of cell's membrane potential. Slice was 375-/tm thick horizontal section taken 750 jtm below dorsal surface of IC of 16-day-old rat. Stimulus strength was 45 /tA. Cell's resting membrane potential was -63 mV. C: epileptiform responses in a disinhibited IC slice (50/~M PTX). Traces (A) are intracellular and traces (B) are extracellular. Stimulus strength was 50/~A. Slice was 500-am thick sagittal section taken 500/~m lateral to the midline of 15-day-old rat brain. Resting membrane potential was -58 mV. Event, labeled '1', was 'expanded' by using a faster time base in the lower two traces. The abbreviation, ifp, refers to the initial field potential; sfp refers to the slow field potential, the duration of which is indicated by the hatched line. Dots indicate individual cycles of synchronous repetitive oscillations (sro's).

383 trode), as shown in Fig. 1A. Such blockade implies that the IPSPs were 7-aminobutyric acid (GABA)mediated. The mean reversal potential of these IPSPs (Fig. 1B) was -70.8 + 3.9 mV (n = 5), a value which is consistent with GABAergic potentials which are mediated by an increase of membrane permeability to C1-. When PTX or BIC was bathapplied, such GABAergic antagonism could have a further effect. If stimulation strength was sufficiently intense, remarkably large prolonged epileptiform events occurred in the disinhibited slices. The nature of these latter events is described below. Extracellular recordings in disinhibited IC slices were composed of 3 events as shown in Fig. 1CB. Because these events are recorded extracellularly, it is inferred they reflect synchronous and, therefore, potentially epileptiform cellular events. Two of these extraceUular events were named respectively the initial field potential (ifp) and the slow field potential (sfp). The ifp was the first event to occur following stimulation. It ranged in size from about +2 mV to - 4 mV and lasted less than 200 ms. The sfp ranged in size from +2 mV to -10 mV but was often of extreme duration. The sfp sometimes lasted longer than 15 s. Polarities and magnitudes of both potentials were highly dependent on recording location. This dependency suggests that epileptogenesis in the IC, even within single slices, is extremely focal in nature. Riding on the envelope of the sfp was often the third field event, which we are calling synchronous repetitive oscillations (sro's). Such oscillations occurred with a low frequency (2-4 Hz). Examples are indicated by dots in the lower panel of Fig. 1C B. The sro's appear to be similar to events recorded extracellularly in disinhibited neocortex or hippocampus and referred to as afterdischarges 14. It was examined whether any intracellular events correlated one-for-one with the above field potentials. Simultaneous intracellular recordings (Fig. 1CA) were made in regions of slices where the sfp exhibited a large negative amplitude. Correlating with the sfp was a graded sustained depolarization (SD) which sometimes was as large as +30 mV and which, like the sfp, could last upwards of 15 s. Identification of the intracellular correlate of sro's was also straightforward. Each oscillation in the field was matched by a synchronous repetitive depolarization (SRD) in the cell. Nonetheless, a simple

(single) intracellular correlate of the third field event, the ifp, did not exist. Instead, early intracellular responses were found to be mixtures of several potentials. At least 4 synaptic events could, on occasion (depending upon concentration of PTX or BIC, and stimulus strength), contribute to early intracellular responses. Such potentials included: IPSPs (Fig. 1A), sudden intense depolarization shifts (see Fig. 1CA) and two additional types of brief depolarizing potentials (not shown in isolation in this article). Our initial attention has focused on elucidating the cellular mechanisms responsible for the generation of the most prominent of the above potentials, the sfp/SD pair. To isolate these long-lasting potentials, we found it useful to reduce the PTX or BIC concentration below the levels where sro's or SRDs occurred. While this strategy selectively favors the sfp/SD event (Fig. 2A), it also caused a reduction of the sfp/SD event's magnitude and duration (Fig. 2Ba). Nonetheless, the event's salient features seemed not to be altered by this slight modification in technique (see Fig. 2B). We hypothesized that the sfp and the SD were synaptically mediated, despite the fact that the potentials could be extremely protracted. Thus, assuming the SD was a 'giant' excitatory postsynaptic potential, one might anticipate that membrane resistance would decrease during the SD response (due to an increase in membrane permeability). However, in many IC cells it was found that intracellular responses to hyperpolarizing current pulses did not exhibit dramatic changes in amplitude during the SD (see insets Fig. 2B). Although historically such results might have been interpreted as suggesting that an event was not synaptic in nature, it has been reported recently that similar small changes of membrane resistance are characteristic of depolarizing responses mediated by NMDA receptors 15. Moreover, since local in vivo application of NMDA antagonists to the IC blocks WRS in rodents TM, it was a possibility that the sfp and its intracellular correlate, the SD, were NMDAmediated synaptic potentials. Indeed, when the NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid 7 (APV) was applied to slices (10-25 /~M), it caused the complete elimination of the sfp and the SD. In Fig. 2B are shown original responses

384 (traces a), APV-insensitive components (traces b) and, by subtraction, APV-sensitive components (traces c). From their APV-sensitivity it can be inferred that sustained potentials such as those shown in traces c, are NMDA-mediated. By con-

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Fig. 2. Effect of BIC and APV on epileptiform events recorded in IC. Responses in left column obtained in ACSF containing 25 /tM BIC; right-column responses obtained in same cells but ACSF contained 2.5/~M BIC. Panel A shows intracellular (traces a) and extracellular (traces b) responses at two BIC concentrations. Stimuli were 60 p A current pulses. Cell's resting potential was -59 mV. Slice was 375-/~m thick horizontal section from a location 1125 /~m below dorsal surface of 13-day-old rat's IC. Panel B shows an experimental characterization of intracellular responses to 70 HA current stimulation. Cell's resting potential was -69 mV. Slice was 375-pm thick horizontal section derived from a depth 375/~m below the dorsal aspect of the IC of a 13-day-old rat. Traces (a) compare cell's response in high (left) and low (right) BIC concentrations. Insets demonstrate responses during which hyperpolarizing current pulses (5 Hz) were applied intracellularly. Traces (b) demonstrate effect of APV application (i.e. only APV-insensitive portions of responses remain). To generate the left trace, ACSF containing 25/~M APV and 25 /tM BIC was used; to obtain the right trace, a microdroplet containing 100 #M APV was ejected onto slice surface. Traces (c) were obtained by subtracting APV-insensitive potentials (traces b) from corresponding baseline response (traces a). As such, traces (c) represent the APV-sensitive portion of IC responses.

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Fig. 3. Voltage dependence and reversal potential of inferred NMDA-mediated potentials in IC neurons. BIC concentration is 2.5 /tM. Panel A shows intracellular KAc electrode) responses to an 80-/~A stimulation at 3 membrane potentials. Cell's resting potential was -72 mV; slice was 375-/~m thick; tissue was originally located 375/~m below IC surface; rat was 13 days old. Panel B shows relationship between membrane potential and magnitude and polarity of NMDA-mediated as well as non-NMDA-mediated potentials. Membrane potentials were varied by passing current through a 1 M CsAc intracel|ular electrode. Magnitudes of NMDA-mediated potentials were obtained by subtracting APV-insensitive (25/~M APV) responses (e.g. see Fig. 2B, traces b) from baseline responses (e.g. Fig. 2B, traces a). The difference potentials so-derived (e.g. Fig. 2B, traces c), were integrated between 0 and 500 ms in order to generate the curve shown by closed symbols (O). Potentials remaining after APV perfusion were integrated between the same limits and are shown by open symbols (O). Cell's resting potential was -53 mV; 375-/tm thick horizontal slice was obtained at a depth of 750 Hm from a 14-day-old rat IC. All stimuli were 40/~A.

trast, one infers that early depolarizing potentials, such as those shown in traces b, are not. During the course of these studies, we had observed that the magnitude and duration of the SD could vary with membrane potential. In Fig. 3A, hyperpolarizing and depolarizing currents were passed into a neuron via the recording electrode. As shown, the SD became larger and more protracted when the neuron's membrane potential was -72 mV than it was when the membrane was polarized to -94

385

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a) 10m V b) 15mVl 500m s e c Fig. 4. The effect on the slow potential of reducing Mg 2+ concentration. BIC concentration is 2.5/~M. Panels (a) show intraceUular response. Panels (b) show simultaneously recorded extracellular response. Cell's resting membrane potential was -67 mV, but cell was kept at a membrane polarization o f - 7 0 mV during experiment; tissue was a 375-/~m horizontal section removed 375/~m below the dorsal surface of the IC of a 14-day-old rat. Stimulus strength was 60/~A.

or -53 mV. These observations were also consistent with the idea that the SD was NMDA-mediated since NMDA responses have been characterized as displaying voltage-dependency 15. To further support the notion that the SD is synaptic in nature, and possibly NMDA-mediated, we systematically examined whether variations occurred in its polarity and amplitude as a function of membrane potential. To perform such experiments, we passed polarizing current into a cell through a CsAc recording electrode. We recorded responses both before and after adding APV to the perfusate. As a result of these experiments we found the APV-sensitive portion of responses (e.g. see Fig. 2Be) became reversed when cells were depolarized to membrane potentials of approximately 0 mV (see closed symbols, Fig. 3B). The mean reversal potential of such long-lasting APV-sensitive potentials was +0.5 _ 5.1 mV (n = 21 cells). Such values approximate the usual reversal potential of excitatory amino acid-mediated synaptic potentials 15. Additionally, the SD in Fig. 3B demonstrates a clear voltage-dependency not shown by simultaneously recorded APV-insensitive early potentials (e.g. see Fig. 2Bb). Also shown in Fig. 3B is reversal of the APV-insensitive potential at a different (more hyperpolarized) potential than where reversal of the

SD occurred. However, this reversal of the APVinsensitive potential (at -30 mV) probably reflects its mixed synaptic origin. In keeping with the notion of a mixed synaptic origin, the reversal of APVinsensitive potentials was found to vary according to the concentration of GABAergic antagonist (PTX or BIC) as well as according to stimulus intensity. Thus, in these same 21 cells (for which reversal of the SD was considered in foregoing), reversal of the APVinsensitive early potentials, not unexpectedly, had a wide range of values (from -60 mV to 0 mV). Nonetheless early potentials never exhibited voltage dependency. We examined whether the sustained depolarization was modified by magnesium ion concentration. It is demonstrated in Fig. 4, that by using 1 mM instead of 2 mM Mg 2÷ in the perfusate, the SD (and sfp) became selectively enhanced (amplitude and duration). Since, hypothetically, the NMDA receptor's voltage-dependency is due to Mg 2÷ blockade of a polar (i.e. voltage-sensitive) binding site within its ionophore 15, these data are also consistent with the inference that the SD is an NMDA-mediated synaptic potential. These studies demonstrate for the first time, an intrinsic capacity of isolated IC tissue to sustain seizure-like events. Such an ability establishes the possibility that focal epileptiform activity, within the IC itself, may be the initiating event in the genesis of the type of behavioral seizures known as audiogenic WRS. Furthermore, results described here support the idea that focal onset seizures do not occur by a single mechanism. That is, compared with synaptic events reported in other in vitro models of epilepsy, the NMDA-mediated event described here seems particularly unique. In neither neocortical nor hippocampal slice models do NMDA-mediated potentials appear to play the prominent role they do in the IC. Relevant to this, it seems, are the previous reports that direct bilateral injections of NMDA into the ICs initiate behavioral W R S 19. By juxtaposing such observations with the data presented here, we are led to suggest that the extraordinarily large, sustained, NMDA-mediated depolarization occurring in the IC is the physiologic event which is responsible for seizure generation in the auditory midbrain.

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