Acetylcholine and norepinephrine mediate slow synaptic potentials in normal and epileptic neocortex

Neuroscience Letters, 126 (1991) 137-140

137

0 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/%03.50 ADONIS030439409100234T NSL 07753

Acetylcholine and norepinephrine mediate slow synaptic potentials in normal and epileptic neocortex Larry S. Benardo Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (U.S.A.)

and Departments of

Pharmacology and Neurology, State University of New York, Health Science Center at Brooklyn, Brooklyn, NY 11203 (U.S.A.)

(Received 11 December 1990; Revised version received 1 February 1991; Accepted 11 February 1991) Key words:

Neocortex; Acetylcholine; Norepinephrine; blocker

Muscarinic; p-Adrenergic;

Epilepsy; Cholinesterase inhibitor; Catecholamine

reuptake

Slow excitatory postsynaptic potentials (EPSPs) were identified in rat neocortical slices. Such potentials, resistant to blockade of glutamate and y-aminobutyric acid-A (GABA*) receptors, were partially antagonized by muscarinic or B-adrenergic antagonists separately, and completely blocked when these agents were added in combination. Slow EPSPs were enhanced by a cholinesterase inhibitor or catecholamine reuptake blockers. Spontaneous epileptic discharges induced by picrotoxin also triggered slow EPSPs. Such potentials were pharmacologically identical to those induced by electrical stimulation under normal conditions. A non-conventional mechanism for synaptic transmission is postulated to account for triggering of slow EPSPs by epileptic discharges.

Acetylcholine (Ach) and norepinephrine (NE) applied to neocortical neurons cause depolarization [4, 7,9], but the physiological action of these neurotransmitters remains unclear. Stimulating cholinergic pathways in other CNS structures [2, 141induces slow excitatory postsynaptic potentials (EPSPs). Technical difficulties of stimulating diffuse afferents comprising these two transmitter systems had impeded efforts to effect synaptic release in physiological experiments in neocortical slices. I now report both Ach and NE independently generate slow EPSPs in rat somatosensory neocortical neurons following electrical stimulation of viable afferents in in vitro slices. Moreover, slow EPSPs mediated by Ach and NE may be triggered by epileptic discharges in disinhibited slices. Since most cells of origin are not present in the preparation, a novel mode of synaptic transmission must be involved in the generation of these potentials by epileptic discharge. Coronal slices (400 pm) of rat somatosensory cortex (>25 days old) were prepared, including portions of both hemispheres connected by an intact corpus callosum [17]. General techniques for preparing and main-

Correspondence: L.S. Benardo, Department of Pharmacology, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Ave., Box 29, Brooklyn, NY 11203, U.S.A.

taining neocortical slices have been described [3]. Slices were maintained at 355°C in an interface type chamber. Physiological saline composition (pH 7.4) was in mM: NaCl 124, KC1 5, MgC12 1.6, CaC12 2, NaHCOs 26, Dglucose 10. Intracellular recordings from layer V cells were obtained with potassium acetate (2 M)-filled microelectrodes (3&70 MG). Drugs applied to the bathing medium were (final bath concentration in PM): 3-(2-carboxypiperazin-4-yl)propyl- 1-phosphonic acid (CPP; 515) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 515) picrotoxin (33-50), atropine (lO--20), eserine (lo), atenolol (l&50), propranolol (2&100), imipramine (lo), cocaine (lO--20), and tetrodotoxin (lO,~g/ml). Standard recording techniques were employed using an Axoclamp 2A amplifier. Orthodromic stimuli were delivered using uni- or bipolar electrodes. The usual stimulation paradigm consisted of 20 Hz stimulation for 50-400 ms (l-8 stimuli). Recordings from over 125 neurons were made from layer V pyramidal cells in somatosensory cortex (SmI), identified by their dorsal-ventral position in the slice, their physiology [8], and in some cases by intracellular staining using Lucifer Yellow CH (551:in 1M LiCI; n = 7). In a representative, randomly selected group of cells (n = 12), resting membrane potential and input resistance averaged72.9&6.3(S.D.)mVand25.9f4.6(S.D.)MS2, respectively. The optimal stimulation site to elicit slow

138

EPSPs was in the gray matter ventral to the recording site. Deep white matter stimulation elicited fast synaptic events, but was usually ineffective for triggering slow EPSPs. Significantly displacing the stimulating electrode laterally within the gray matter did not induce slow synaptic excitation. When stimuli suprathreshold for fast synaptic driving were delivered, a slow EPSP occurred in > 95% of cells tested, often preceded by a slow inhibitory postsynaptic potential (IPSP; Fig. 1At). The slow hyperpolarization began with the first stimulus of the train, lasted seconds, and was associated with a decrease in neuronal input resistance (43.3% f 14.1% (S.D.), n = 10). The slow depolarization lasted up to tens of seconds, was capable of reaching firing threshold, but spike threshold

was unchanged. Slow EPSPs were associated with increased input resistance (26.1 f 11.4% (S.D.), n = 11, measured at the same membrane potential), often cumulative, outlasting the depolarization. Under control conditions cells were prone to repetitive spiking at frequencies of 7-12 Hz when depolarized to voltages just above firing threshold. Rhythmic firing appeared to be supported by underlying intrinsic rhythmic oscillations of membrane potential, similar to that described in this [ 151 and other CNS structures [I]. Slow EPSPs appeared to accentuate this underlying oscillatory behavior, releasing rhythmic activity. Slow EPSPs were voltage-dependent, i.e. D.C. depolarization below spike threshold resulted in larger slow EPSPs [2]. Slow potentials were unrelated to

As.

.

i CONTROL

ATENOLOL

CONTROL

-I

5mV

5%

C

.

ATROPINE

l

WASH

ATEiOLOL 2

1

.

i

j Fig. 1. Effects of muscarinic

and Badrenergic

response

train delivered

to a 20 Hz stimulus

slow depolarization,

before (trace

-62

mV. B: response

-75

mV, held at -65

(50 PM) and atropine (50 PM). Resting

I) and following

to a 20 Hz stimulus mV. C: response potential

on slow potentials

(trace 2) bath application

train delivered

in neocortical

washout

neurons.

Chart

by the filled circle, showing of atropine

(10 FM). Resting

records

train delivered

for 400 ms before (trace

of these drugs (trace 3). Bath also contained

- 70 mV, held at - 65 mV. Muscarinic

and /3-adrenergic

of 3 different

slow hyperpolarization membrane

for 100 ms before (trace 1) and after (trace 2) atenolol(50

to a 20 Hz stimulus

(20 PM), and following

membrane

antagonists

for 100 ms at the ‘S’ and indicated

potential

cells (A-C). -70

CNQX

mV, held at

PM). Resting membrane

1) and after (trace 2) application

antagonists

A:

and subsequent potential of atenolol

and CPP (both at 10 PM) and picrotoxin attenuated

the slow hyperpolarizations

to some extent. Whether this is due to antagonism of a pre- or a postsynaptic phenomenon is unclear at this time, and is the subject of current study. Voltage calibration applies to all traces. Time calibration in (B2) applies to A and B. Time calibration in Cs applies to C.

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a muscarinic antagonist and a beta-adrenergic blocker were added in concert was the slow EPSP completely blocked (Fig. 1C; n= lo), which was reversible (Fig.

postsynaptic firing, persisting after spikes were eliminated with intracellular QX-314. Rather, they resulted from evoked transmitter release, since bath TTX (10 pg/ ml) blocked both slow and fast synaptic events. Identifying transmitter agents mediating slow EPSPs first required eliminating contributions of fast EPSPs (glutamatergic) and IPSPs (GABA,; which may be depolarizing at the high resting potentials of neocortical neurons). Both slow potentials persisted after exposure to specific antagonists of excitatory amino acid (CNQX and CPP; [6, 181 and y-aminobutyric acid-A (GABA*) (picrotoxin) receptors (e.g. Fig. lCt), without preceding spike activity. Since previous studies showed exogenous Ach or NE cause slow depolarizations [4, 7, 91, their role in producing slow EPSPs was assessed. Accordingly, slices were exposed to drugs which block muscarinic (atropine) or beta-adrenergic (propranolol or atenolol) receptors. Spike firing and resting membrane resistance were unaffected. Drugs of either class reduced (by 30 to 50%) but did not eliminate the slow EPSP (Fig. lA, B). Only when

A

lC3).

This result suggests simultaneous release of both Ach and NE activates slow EPSPs. Given this, agents prolonging the action of either transmitter should augment the slow EPSP. Exposure to the cholinesterase inhibitor eserine increased the size (154 f 94% (S.D.), n =4) and duration (253 f 157% (S.D.), n =4) of the slow EPSP. Similarly, bath application of the catecholamine reuptake blockers imipramine or cocaine enhanced the slow EPSP (i.e., for imipramine amplitude increased 60+ 19% (S.D.), n = 3, duration increased 69f 37% (S.D.), n = 3; for cocaine amplitude increased 79 &-37% (S.D.), n = 4, duration increased 253 &-157% (S.D.), n = 4). These drugs did not independently affect resting membrane properties. These data corroborate those using pharmacologic antagonists, and support the conclusion that extracellular stimulation releases Ach and NE to mediate slow EPSPs.

Pps

I

i

1CONTROL

i ATROPINE +

5mV 5wc

CONTROL

ESERINE

i WASH

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CONTROL

COCAINE

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7:

Fig. 2. Epileptic discharges induced after bath application of picrotoxin (50 PM) trigger slow synaptic potentials. Records from 3 different cells (AC). A: a spontaneous paroxysmal depolarization shift (PDS [lo]) or epileptic discharge occurs as indicated at the filled circle, triggering a slow hyperpolarization and slow depolarization (trace 1). Atropine (10 PM) and atenolol(50 PM) added to the bath blocks the slow depolarization (trace 2). Resting membrane potential -58 mV. B: responses following spontaneous epileptic discharges before (trace 1) and after eserine (10 PM) exposure, and following washout of the drug (trace 3). Resting membrane potential - 68 mV, held - 57 mV. C: Responses following spontaneous epileptic discharges before (trace 1) and after cocaine (10 PM), and following washout of the drug (trace 3). Resting membrane potential -65 mV. Held -61 mV. Calibrations in A apply to all traces.

140

The role of such events in epilepsy is unknown. Blockade of GABA-mediated chloride-dependent IPSPs leads to epileptic discharge presumably by releasing excitability sustained by local recurrent excitatory synapses [16]. Whether similar release phenomena occur with slow potentials was tested by exposing slices to picrotoxin (50 PM), to block fast IPSPs and induce epileptic discharge. Interestingly, this maneuver allowed triggering of slow EPSPs at lower stimulus intensities (half of control, or less), with fewer stimuli. Epileptic discharge occurred spontaneously. Remarkably, this discharge was capable of triggering slow IPSP-EPSP sequences in 21 of 26 cells (most prominent at depolarized potentials; Fig. 2) reminiscent of observations described above. As above, atropine and atenolol exposure in combination, blocked slow EPSPs triggered by epileptic discharge (Fig. 2A; n =4). Moreover, adding eserine (Fig. 2B) or cocaine (Fig. 2C) enhanced slow EPSPs generated by epileptic events (i.e., duration increased 167 +42% (SD.), n=4 and 85 f41% (S.D.), n =4, respectively). This supported the notion that these slow EPSPs were also subserved by cholinergic and noradrenergic transmission. Slow EPSP generation by spontaneous epileptic events seems to implicate a novel mode of transmission. Namely, one involving interactions with terminals of cholinergic and noradrenergic pathways. The present study does not provide evidence on this issue. However, one possibility is that potassium rises, known to accompany epileptic discharge [ 131,might serve as the intermediary in this process, causing afferent depolarization [ 111, leading to release of transmitters mediating slow potentials [lo]. Whether this involves repetitive terminal discharge, as occurs in some cortical epileptic foci [5], is equally speculative. Regardless, under epileptic conditions slow EPSPs may play a role in seizure initiation and spread, and may subserve interictal to ictal transitions [ 121.These results have significant implications for normal neocortical physiology. How the discrete, small subcortical nuclei giving rise to the major cholinergic and noradrenergic pathways in brain exert their effects, and maintain any regional specificity is unclear. The present results suggest activity in these pathways are to an extent, under local control. Thus, very active areas of cortex could trigger slow EPSPs locally, further increasing regional excitability. Under normal conditions slow EPSPs, like fast EPSPs are held in check by GABA*mediated fast IPSPs, and by slow IPSPs, preventing excessive possibly abnormal firing. Initially it seemed Ach and NE co-release occurred as a consequence of stimulation technique. The findings obtained under epileptic conditions suggest their concurrent action may be physiological.

I thank R.K.S. Wong for comments on this manuscript. Supported in part by Clinical Investigator Development Award K08 NS01386-02 and the Dana Foundation. I Benardo, LX and Foster, R.E., Oscillatory ive neurons:

Mechanism,

modulation

behavior

and

in inferior

neuronal

ol-

aggregates,

Brain Res. Bull., 17 (1986) 7733784. 2 Cole, A.E. and Nicoll, R.A., Acetylcholine tic potential

in hippocampal

pyramidal

mediates

a slow synap-

cells, Science,

221 (1983)

129991301. 3 Connors,

B.W.,

Benardo,

between neurons

L.S.,

and

of the developing

Prince,

neocortex,

D.A.,

Coupling

J. Neurosci.,

3 (1983)

7733782. 4 Foehring,

R.C.,

Schwindt,

P.C. and Grill, W.E., Norepinephrine

selectively reduces slow Ca2+ and Na+-mediated neocortical

neurons,

5 Gutnick,

J. Neurophysiol.,

M.J. and Prince,

antidromic

invasion

K+ currents

in cat

61 (1989) 245-256.

D.A., Thalamocortical

of spikes form a cortical

relay neurons:

epileptogenic

focus,

Science, 176 (1972) 424426. 6 Honore,

T., Davies,

Lodge,

S.N., Drejer,

D. and Nielsen,

J., Fletcher,

E.J., Jacobsen,

F.E., Quinoxalinediones:

tive non-N-methyl-o-aspartate

glutamate

potent

receptor

P.,

competi-

antagonists,

Sci-

ence, 241 (1988) 701-703. 7 Krjevic,

K., Pumain,

R. and Renaud.

tation by acetylcholine

in the cerebral

L., The mechanism cortex, J. Physiol.,

of exci-

215 (1971)

447465. 8 McCormick, D.A..

D.A.,

Comparative

neurons

Connors,

of the neocortex,

9 McCormick, tylcholine

B.W.,

physiology

Lighthall,

J.W.

of pyramidal

J. Neurophysiol., cerebral

Prince, spiny

54 (1985) 782-806.

D.A. and Prince, D.A., Mechanisms in the guinea-pig

and

and sparsely

of action of ace-

cortex in vitro, J. Physiol.,

375

(1986) 1699194. 10 Mann,

P.J.G.,

metabolism

Tennenbaum,

M. and Quastel,

in central nervous

er cations

on acetylcholine

J.H., Acetylcholine

system; effects of potassium

liberation,

Biochem.

and oth-

J., 33 (1939) 822.

835. 11 Prince, D.A., Cellular and F. Coceani vulsions,

activities in focal epilepsy.

(Eds.), Brain Dysfunction

Raven, New York,

12 Prince,

D.A.,

underlying

Connors.

Brazier

Febrile Con-

1976. pp. 187-211.

B.W.

interictal-ictal

In M.A.B.

in Infantile

and

Benardo,

transitions,

L.S.,

Adv. Neural.

Mechanisms

Status Epilepti-

cus, 34 (1982) 1799189. 13 Prince, D.A., Lux, H.D. and Neher, E., Measurement lar potassium 14 Sastry, central

B.R., Excitatory

postsynaptic

nervous

associated

system

brane resistance, 15 Silva,

L.R.

potentials

in the mem-

Life Sci., 27 (1980) 1403-1407.

and

Connors,

B.W.,

Layer

5 neurons

447 Hz synchronized

Sot. Neurosci.

16 (1990)

Abstr.,

R.D. and Wong,

synchronization

of the corpus

rhythms

can

initiate

in neocortex,

I 134.

R.K.S.,

in epilepsy,

17 Vogt, B.A. and Gorman, stimulation

in the mammalian

with an increase

NMDA-independent, 16 Traub,

of extracellu-

activity in cat cortex, Brain Res., 50 (1973) 4899495.

Cellular

mechanism

of neuronal

Science, 216 (1982) 7455747.

A.L.F.,

Responses

callosum

of cortical

neurons

in vitro. J. Neurophysiol.,

to 48

(1982) 1257-1273. 18 Watkins, excitatory 951.

J.C. and Olverman, amino acid receptors,

H.J., Agonists Trends

and antagonists

Neurosci.,

for

10 (1987) 938-