Synaptic potentials and effects of amino acid antagonists in the auditory cortex

Rrarn Rermn~l~ Bu//c/in. Vol. 28. pp. 401-410.

1992 CopyrIght

Printed in the USA.

03hl-1)X0/9? $5.00 t 00 c. 1491 Pergamon Press 1 td.

Synaptic Potentials and Effects of Amino Acid Antagonists in the Auditory Cortex CHARLES

L. COX,*

RAJU

METHERATE,*t

NORMAN

Received

13 June

M.

WEINBERGER”r

AND

JOHN

H. ASHE*t’

199 I

COX. C. L.. R. METHERATE. N. M. WEINBERGER and J. H. ASHE. ~~~rluplicpo/c~rr/iu/,tmdc#wts of mww ad rrr~~u,q~~rcv~~ in //w uuditovy COTI~.Y BRAIN RES BULL 28(3) 401-410, 1992.-Neurons of in vitro guinea pig and rat auditory cortex receive a complex synaptic pattern of afferent information. As many as four synaptic responses to a single-stimulus pulse to the gray or white matter can occur: an early-EPSP followed. sequentially. by an early-IPSP. late-EPSP. and late-IPSP. Paired pulse stimulation and pharmacological studies show that the early-IPSP can modify information transmission that occurs by way ofthe early-EPSP. Each of these four synaptic responses differed in estimated reversal potential. and each was differentiallq sensitive to antagonism by pharmacological agents. DNQX (6.7-dinitroquinoxaline-2.3-dione). a quisqualate/kainate receptor antagonist, blocked the early-EPSP, and the late-EPSP was blocked by the NMDA receptor antagonist APV (D-2-amino-5-phosphonovalerate). The carlyIPSP was blocked by the GABA-a receptor antagonist bicuculline. and the late-IPSP by the GABA-b receptor antagonists ?-OH saclofen or phaclofen. Presentation ofstimulus trains. even at relatively low intensities, could produce a long-lasting APV-sensitive membrane depolarization. Also discussed is the possible role of these synaptic potentials in auditory cortical function and plasticity. Synaptic potentials Excitatory amino acid receptors Quisqualateikainate receptors Auditory cortex 2-hvdroxv-saclofen NMDA receptors GABA receptors GABA-a receotors GABA-b receutors Bicuculline DNQX (6.7-dinit;oquinoxaline-2,3-dionej ,\PV (D-2.aminoiS-phosphonoCalera;e) Phaclofcn -

ceptive field characteristics of neurons in a manner similar to those obtained during classical conditioning (2.4 I .42). Relevant

PLASTICITY ofsensory cortical neuron function accompanies nervous system development and recovery from peripheral neural injury [for reviews see ( I2,20)]. Modification of functional properties of sensory cortical neurons can also be produced by training procedures that result in learned changes in behavior. In the auditory cortex, the use of classical conditioning procedures that use an acoustic conditioned stimulus (CS) may produce changes in the rate of neuronal discharge and also modification of frequency-receptive fields (6.18.64.67). For the most part. modification of frequency-receptive fields consists of an enhancement of responses to a frequency selected as a conditioned stimulus. In contrast, responses to frequencies adjacent to the CS remain unchanged or are reduced (6). These alterations in the receptive held can result in a shift of the neuron’s “best” frequency to correspond to the frequency of the conditioned stimulus. These changes occur rapidly (i.e.. within only a few conditioning trials), and this rapid time course suggests that physiological modification of synaptic function may underlie the effect of classical conditioning on the auditory cortex. It is likelv that sensory cortex plasticity involves the harmonious action of several neurotransmitters that are intrinsic to local cortical circuitry. For example, the involvement of acetylcholine (ACh) and norepinephrine (NE) in cortical plasticity is well documented (2,7,24.32,4 I .42,49,58,62). In the auditory cortex. microapplication of cholinergic agonists can modify re-

issues concerning the functional significance of muscarinic actions of ACh in the sensory cortex and its relation to learning have been reviewed recently (3.64). The identity of neurotransmitters involved in rapid transmission between thalamic sensory relay nuclei and the sensory cortex is unknown. However. biochemical assays for glutamate in combination with selective lesion techniques suggest that dicarboxylic amino acids are important transmitters in afferent and efferent cortical pathways (19,35). High-affinity. saturable glutamate uptake into cortical synaptosomes has been shown (68). as has stimulated release from cortical slices (33). Electrophysiological and anatomical evidence also suggests that an cxcitatory amino acid functions in rapid thalamocortical transmission (26,3 1.46.60). lntracortical transmission also may involve excitatory amino acids ( I3,25.27). As the nature and interactions of synaptic inputs to the auditory cortex must be resolved to understand fully the relationship between learning-induced and transmitter-induced cortical plasticity. the present study was initiated I) to determine the types of synaptic responses elicited by afferent stimulation and 2) to characterize the identity of the neurotransmitter(s) involved in excitation and inhibition of auditory cortical neurons. Our long-range goal is to elucidate the physiological mechanisms that

I Requests for reprints should be addressed to Dr. John H. Ashe, PhD. Departments Riverside.

CA 9252 1.

401

of Neuroscience

and Psycholog>-075,

Ilniversity

of California,

40’

C-ON t. t .-\!

underlie learning-induced cortical plasticity and to clarify the regulation of these mechanisms by the nl~ulatory actions of neurotransmitters. Portions of these data appear in abstract form (14).

Neocortical slices were obtained from the auditory cortex of male Hartley guinea pigs (350-450 g) and Sprague-Dawley rats (150-250 g). Animals were lightly anesthetized with sodium pentobarbital (Nembutal, 35 mg/kg intraperitoneally, IP), decapitated, and the brains were removed rapidly and placed in a physiological solution (0-3°C). The physiological solution consisted of a modified Krebs medium that contained (in mM): NaCl 124.0, KC1 2.5, KHzPO.+ 1.0, NaHCOz 25.0, MgSO, 1.2. CaC& 3.0, glucose 10.0. The medium was saturated with 95% Or. 5% CO* to give a final pH of 7.4. Brain slices (400-600 pm) were prepared and placed in cold physiological solution. Auditory cortex slices were obtained from guinea pigs at an angle perpendicular to the sylvian sulcus and from rats at an angle horizontal to the rhinal fissure (Fig. IA). The slices were then immediately transferred onto a nylon net platform in an interphase-type recording chamber that contained oxygenated Krebs solution at 32*C. At least 2 h were allowed for equilibration prior to recording. Introduction of physiological medium into the chamber was via a peristaltic pump at a rate of about I.0 ml/min. Warmed moistened gas (95% Oz. 5% CO*) passed over the upper surface of the slices. Slices were transilluminated and observed with a dissecting microscope to permit positioning of electrodes. Monophasic stimulus pulses (0.1-0.2 ms duration) were delivered to the underlying white matter or to the gray matter with insulated stainless-steel bipolar electrodes ( 100 pm tip diameter). Stimulation electrodes were positioned at a distance of 0.2- 1.7 mm from the recording electrode. Recording micr~1ectrodes were made from glass capillary tubes ( 1 mm OD) and contained 4 M K+-acetate (DC resistance 60- 120 MB). Recordings were obtained using a WPI-M701 or Axoclamp 2A amplifier. Changes in ohmic input resistance of the membrane were inferred from the change in the size of electrotonic potentials produced in response to rectangular hyperpolarizing current pulses across the membrane (0.2-0.4 nA, 200 ms duration). Prior to experimental manipulation, the resting membrane potential was allowed a period of IO-15 min for stabilization. Membrane responses were recorded on a multichannel videotape recorder (DC-6 KHz) and continually monitored on a chart recorder. Data were digitized and analyzed using a computer, or were replayed from tape and photographed on S-mm film from an oscilloscope display. Phrmacological

Agents und Applications

Pharmacological agents used in these experiments were APV (D-2-amino-5-phosphonovalerate) and bicuculline methiodide obtained from Sigma Chemicals, and DNQX (6,7_dinitroquinoxaIine-2,3~ione), 2-hydroxy-~clofen [(3-amino-2-(4-chlorphenyl)-2-hydroxy-propylsulphonic acid], and phaclofen [(3amino-2-(4-chlorophenyl)-propyl phosphonic acid] obtained from Tocris Neuramin Pharmaceuticals. Concentrated solutions were prepared with distilled water on the day of experiment and diluted to final concentration in modified Krebs solution just prior to use. Antagonists were delivered either by bath application or by a modified microliter syringe (2 1f for application of very small volumes (~0.5 ~1) to the surface of the cortical slice near the recording electrode. Local drug application was primarily

FIG. I. Schematic representation of guinea pig and rat brain (A) and the dist~but~on of imnaled cells (B). CA)Auditorv cortex is located in the temporal region in both species and is represented by the stippled area. The solid lines drawn through these regions represent the orientation in which cortical slices were prepared. (B) Distribution of ceils, within the gray matter, from which recordings of guinea pig (dark bars) and rat (open bars) neurons were obtained. The position ofeach cell is indicated as a percentage of the width of the gray matter of the corresponding cortical slice; 0 and 100% correspond to the location of the pia and underlying white matter, respectively.

made to the apical side of the impaled neuron, but occasionally agents were applied to the basal side.

Averages in the text are presented as the mean ? standard error of the mean (X it SE). Differences between means were evaluated for significance with the t test for independent samples or, where appropriate, the t test for paired samples (23). The relationship between membrane potential and input resistance was evaluated for significance by the methods of least squares (9). Differences were considered statistically si~i~cant if the probability of occurrence by chance was 0.05 or less. RESULTS

The location of the auditory region of the guinea pig and rat neocortex is illustrated in Fig. IA. The auditory cortex occupies an area of approximately 1S-20 mm2 on the dorsolateral surface of the temporal lobe, ventrolateral to the syivian s&us in the guinea pig (47,69), and approximately 1 m& dorsal to the rhinai fissure in the rat (37.52). Within these areas. neurons were impaled between the boundaries of the pia and white matter (Fig. 1B). Most cells were located in the deeper half of the gray matter that corresponds to architectonic&y identified layers V and VI (50); however, no attempt was made to sample systematically al1 cortical layers. For either the guinea pig or rat, division of impaled neurons into three groups relative to the width of the cortex, and designated upper (pia, O-30%), middle (31-69%), and lower (70- 1OO%,white matter) revealed no statistically significant differences between cells of these groups in resting membrane potential (V,) or apparent input resistance of the membrane (Ri) (p > 0.1). Intracellular recordings were obtained from a total of 188 neurons from the guinea pig (n = 89) and rat (n = 99) auditory

SYNAPTIC

POTENTIALS

IN AUDITORY

403

CORTEX

cortex. There were no significant ditferences (p > 0. I) between neurons. recorded from either species. in resting V, (-70.6 + 0.7 mV. n = 188). or R, (slope of current-voltage function. 30.5 t 1.9 M12, n 7 29). Spike height averaged 90.3 ? I.9 mV (n = 3 I). Also for either species. there was no significant correlation between values recorded for V,, and R, within a cell (p > 0. I L

Synaptic responses were elicited by stimulation of afferents withm the gray matter adjacent to the recording electrode or the bordering white matter. No qualitative ditferences in response configuration were observed following stimulation of different cortical loci (e.g.. gray vs. white matter). The lowest intensity tested for any given cell varied between 5 PA and I5 PA. At these low intensities. the membrane response consisted of either a single, fast-onset. short-duration early-EPSP or a response that consisted of as many as four synaptic components (Fig. 2A, top trace). The probability of eliciting additional synaptic response components was increased by elevating stimulus intensity. In 22 of 27 (Xl”; ) guinea pig neurons. in which the synaptic responses could be clearly separated according to stimulus intensity. low intensities (IO-20 @A) elicited the early-EPSP alone. Single late-EPSPs were not elicited by low-intensity stimulation. but increasing stimulus intensity to 35-75 PA resulted in the appearance of both early- and late-EPSPs in 24 of 27 (89’8 ) cells. The late-IPSP was elicited in eight of these cells by the higherintensity stimuli. Overlap of hypcrpolarizing and depolarizing synaptic potentials. especially at higher intensities. prevented determination of the intensity dependence of early-IPSPs (see Fig. 2A). The amplitude of the synaptic components was also dependent on stimulus intensity. Furthermore, the position of the early-IPSP and the late-EPSP. relative to the baseline (resting potential). also varied with stimulus intensity (Fig. 2A). These data indicate that low stimulus intensities can elicit an earlyEPSP without additional synaptic responses that are dependent. for the most part. on more intense stimuli. The latencies to peak and the maximum amplitudes of these synaptic responses arc given in Table I. These four components differed signiticantly from each other in their latency to peak amplitude (/I < 0.00 I, Table I ). The early-EPSP had a latency to peak of approximately 8 ms (Table I: Fig. 2A). Stimulation of suthcient intensity resulted in an early-EPSP that elicited a single action potential. Multiple action potentials elicited by the early-EPSP never occurred. Often the early-EPSP was followed by an early-IPSP that reached peak amplitude in about 25 ms. a late-EPSP uith a latency to peak of 50-60 ms, and a late-IPSP with a peak latency of I60- I70 ms. The likelihood ofeliciting responses with more than one synaptic component was about equal for the guinea pig and rat (79”; and 800~. rcspectivcly). However. the frequency of eliciting a given combination of synaptic components (by maximal intensity stimulation) was nonrandom (Table 2). Unaccompanied early-EPSPs occurred mom often than any other single component. Ilnaccompanicd early-IPSPs were rare. and unaccompanied late-EPSPs and late-IPSPs wcrc not observed. Frequency analyses (chi-square tests) were performed to see if the presence or absence of synaptic components was different at different resting potentials. The frequency of depolarizing responses and the resting V,,, were not co-dependent (/I > 0. I). Also, throughout the range of resting V,. late-EPSPs were never seen without a preceding early-EPSP. In contrast to the EPSPs. the frequency ofeliciting an early-IPSP did differ significantly at different resting potentials. Thus. the early-IPSP occurred more frequently in cells with resting V, that were less than -70 mV than in those cells with resting V, more negative than -70 mV (p < 0.01).

A.

75 VA

B. i.

FIG. 2. Membrane responses &cited hy single (A) or trams(B) ofstimuli. (A) Guinea pig neuron. V, = -64 mV. Single-stimulus pulses of vary+ng intensities elicited several synaptic responses in which the individual synaptic components vary in amplitude and in their relationship to the tmlial membrane potential. Low-intensity sttmulalion (I2 PA) to the underlying white matter elicited a fast onset latency early-EPSP followed by an early-IPSP. late-EPSP. and late-IPSP. An additional increase in stimulus intensity ( I5 PA) elicits the same general configuration of synaptic responses. hut with an increase In amplitude of the EPSPs such that the complete response is clearly separated into (bur components. The early-EPSP. early-IPSP. late-EPSP and late-IPSP arc designated A. B. C, D. respectively. Incremental increases in stimulus intensity (27 PA and 35 PA) resulted in an increased amplitude of the EPSPs (A and C) with apparent reduction of the IPSPs (B and D) until the EPSPs completely dominated the response. Calibration: 5 mV. 25 ms. (B) Gurnea pig neuron. V, y ~70 mV. Responses elicited by (i) a single-stimulus pulse, and (ii) a train ofsttmulus pulses. (i) .A single-pulse stimulus ( I5 PA. at arrow) elicited an early-EPSP. (ii) A tram of stimuli ( I5 PA. I!()/ s for 400 ms. between arrows) at the same stimulus intensity elicited a large membrane depolarbation that outlasted the train of stimulr by over 2 s. Note the spike discharge during the prolonged dcpolariration. V, = -70 mV. Calihration: IO mV (i) I5 ms: (ii) 500 ms.

This observation may likely be related to the proximity of the resting potential to the reversal potential of the early-IPSP (see below). The late-IPSP. similar to the earlv-EPSP and late-EPSP. could be elicited throughout the range of resting membrane potentials. The only species differences in synaptic responsiveness were in the probability of obtaining a given synaptic pattern, rather than the nature of the synaptic response per se. The probability of obtaining an early-EPSP/early-IPSP sequence. in response to maximum stimulation. was greater in rat neurons. whereas the

MEAN (&SE) LATENCY-TO-PEAK AND MAXIMUM AMPLITUDE OF DEPOLARIZING; AND HYPERPOLARIZING SYNAPTIC COMPONENTS OF THE RESPONSE TO MAXIMAL AFFERENT STIMULATION

tiumcaPig Component

latency

Fast-EPSP Fast-IPSP Slow-EPSP Slow-IPSP

7.9 24.7 62.0 172.8

-_

Amplitude (mVI

(ms)

f 0.3 + 1.4 + 4.5 _c 7.4

16.0 7.9 8.7 4.9

+- 1.2 k 0.7 t 0.9 _t 0.5

full complement of synaptic responses (i.e., early-EPSP/earlyIPSP/late-EPSP/late-IPSP) was more likely to be elicited from guinea pig neurons (Table 2). The apparent difference between neurons from the rat and guinea pig in the observed frequency of early-EPSP/early-IPSP can likely be accounted for by the difference in resting potential between the two groups of neurons. That is, the average resting potential of rat neurons that had only the early-EPSP/early-IPSP combination was significantly lower (p < 0.05) than those neurons in the guinea pig. The increased probability of early-EPSP/early-IPSP/late-EPSP/lateIPSP in guinea pig compared to rat neurons may be due to the increased probability of obtaining a late-IPSP in guinea pig neurons. There was no statistically significant difference, between species, in the resting membrane potential of neurons that elicited the early-EPSP/early-IPSP/late-EPSP/late-IPSP pattern. Membrane responses to trains of stimuli were investigated in 28 neurons from the guinea pig cortex. In general, the early-

Rat

__)I

87 46 43 44

-_--

Latency (ms)

Amphtude (mV)

7.5 24.1 49.7 158.7

14.s 5.7 7.0 3.7

t + t k

0.2 1.2 4.2 9.0

i + I -t

1.0 0.6 0.9 0.5

)i

92 39 33 20

EPSP was able to follow consistently stimuli up to a frequency of about 60/s (0.5 s). In 16 of 28 cells (57%), repetition rates of 20-60/s (0.25- 1.O s) elicited a membrane depolarization that outlasted the period of stimulation by 1.0-5.0 s (mean duration 3.4 + 0.4 s; Fig. 2B). The mean amplitude of the posttetanic depolarization was 14.9 F 2.2 mV (range 6-35 mV). A portion of the cells ( I 1of 16) in which low-intensity repetitive stimulation resulted in prolonged depolarization did not elicit a late-EPSP in response to a single-stimulus pulse of the same intensity (Fig. 2B).

The amplitude of both the depolarizing and hyperpolarizing synaptic potentials was sensitive to variations of the membrane potential, over the range of about - I20 mV to -20 mV. Passage

TABLE 2 OBSERVED FREQUENCY OF EXCITATORY (A, C) AND INHIBITORY (B, D) SYNAPTIC POTENTIALS ELICITED BY ELECTRICAL STIMULATION IN GUINEA PIG AND RAT CORTICAL NEURONS Guinea Pig Synaptic Potential’ A

B C D AB AC AD BC BD CD ABC ABD ACD BCD ABCD

Mean ”

Rat

Frequency

Frequency

17 2 0 0 9 19 0 0 I 0 7 2 7 0 25

-73.7 -70.0

+ 2.4 f 10.0

-69.3 -71.5

rt It

2.4 2.0

-60.0

i

0.0

~64.3 -+ 1.6 -56.5 + 0.5 -74.3 rt 2.9 -71.8 -70.9

xk I.6 f 0.9 89

20

VIII .YkSE

p**

-73.0

k 2.2

0.03 2.21

NS NS

-61.4 -73.4

t 2.3 ” 2.2

5.95 0.03


1.13

NS

I.65 2.21 0.22

NS NS NS

4.37

<0.05

0

0 0 25 20 0 0 0 0 I4 0 6 0 I4

Chl Square

-66.4

f 1.5

-71.7

t 3.4

~73.3 -69.2

f 2.5 + I.1 99

NS

* A, B, C, D correspond to the early-EPSP,early-IPSP, late-EPSP, and late-IPSP, respectively. ** Chi-square probability of difference in frequency, between rat and guinea pig, occuming by chance.

NS = not significant.

SYNAPTIC

POfENTlALS

IN AUDITORY

405

CORTEX

of a steady depolarizing current reduced the amplitude of the early- and late-EPSPs, whereas steady hyperpolarizing current increased their amplitude (Fig. 3). Estimated values for the reversal potentials for the early- and late-EPSPs are 32.8 mV 2 6.4 mV (n = 26) and -22.2 mV ? 8.4 mV (n = 13) respectively. But it should be keep in mind that electrical overlap of the lateEPSP with the early- and late-IPSPs (Figs. 2A and 3) has likely confounded measurement of the reversal potential for this response. There were no significant dilferences between species in the e&t of current injection on the early- and late-EPSPs (p > 0.1). The amplitudes of the early- and late-IPSPs were also altered. and frequently reversed, by varying the membrane potential (Fig. 3). The estimated reversal potentials for the early- and late-IPSPs are -70.9 mV + 3.7 mV (n = 16) and -104.4 mV f 3.2 mV (n = 15). respectively. Due to the long latency to peak amplitude of the late-IPSP. electrical overlap with other components did not hamper the ability to determine the maximum amplitude. In contrast. the reversal potential for the early-IPSP was more difficult to determine because of electrical ovjerlap with the earlyand late-EPSPs (cf. Figs. 2A and 3). Therefore, relatively low stimulus intensities were used to minimize the overlap of the early-IPSP with the early- and late-EPSPs. and reversal potential ofthe early-IPSP occurred within 10 mV ofthe resting membrane potential. The proximity of the reversal potential for the earlyIPSP to the resting V,,, likely explains the higher frequency 01 early-IPSPs observed at membrane potentials positive to -70 mV (see above). As just noted. for depolarizing synaptic potentials, there were no significant differences between species in the effect of current injection on the early- and late-IPSPs (p > 0. I). Overall. these data suggest that Nat or Cat+ currents dominate synaptic depolarizations. With regard to synaptic hyperpolarizations, the clear difference in the value of the reversal potentials suggests the involvement ofdifferent ionic currents for the earlyand late-IPSPs.

E.wi[trror~~ .s)nuptic~ po[cwrialv. The dicarboxylic amino acid antagonists DNQX and APV were used to examine the involvement of excitatory amino acid receptors in synaptic responses of the auditory cortex. DNQX is a selective antagonist at kainate/ quisquilate receptors (28) and APV selectively blocks NMDA receptors (63). After recording several control synaptic responses (e.g.. 510 responses). either DNQX or APV was applied to the slice by either microdrop or bath superfusion. There were no differences in results associated with the method of drug application. and these data have been combined. DNQX tested at concentrations of 5, 7.5, and 12.5 FM was without effect on the resting membrane potential or input resistance. DNQX (7.5-12.5 PM) reduced the amplitude of the early-EPSP in 11 of 12 guinea pig neurons by an average of 70.9% -t 5.6%’ (Fig. 4A). DNQX at 5 FM resulted in a smaller reduction in the amplitude of the earlyEPSP (58.9% -t 5.2%. n = 4). Suppression of the early-EPSP by DNQX occurred with only minimal reduction in the amplitude of other synaptic potentials when they were present in the evoked response. In 6 of 7 cells that had an early-EPSP and late-EPSP. DNQX suppressed the early-EPSP by an average of 64% ? 6.2%’ but reduced the amplitude of the late-EPSP by only 6% + 4.8% (Fig. 5A). The differential effect of DNQX on the early versus the late EPSP attained statistical significance (p < 0.002). For the most part. reduction of the early-EPSP by DNQX persisted for the duration that the cell was impaled (40-60 min). although partial recovery did occur occasionally within this period (approximately 60%) recovery. n = 2).

FIG. 3. Effects of varyingmembrane potential on cortical responses clicited by electrical stimulation in both guinea pig and rat. (A) Guinea pig: At the resting potential (-75 mV). stimulation (70 PA) applied to the underlying white matter elicited an early-EPSP followed by a small-amplitude late-EPSP and late-IPSP. Membrane depolarization to -62 mV decreased the amplitude of the early-EPSP and revealed an early-IPSP at the inflection point of apparent separation of the early and late depolarizations. On the other hand. hyperpolarization of the membrane potential from the resting potential increased the amplitude of earlyand late-EPSPs. reversed the early-IPSP. and eventually eliminated the late-IPSP. (B) Rat: Similar to theguinea pig. at resting membrane potential (-70 mV) electrical stimulation (40 PA) of underlying gray matter evoked a typical four-component response. Depolarization of the membrane potential decreased the amplitude of the EPSPs and increased the amplitudes of the IPSPs. Hyperpolarization of the membrane potential resulted in an increase in the amplitude of the EPSPs. a reversal of the early-IPSP. and elimination of the late-IPSP.

In neurons

from the rat. DNQX (2.5-5.0 PM) suppressed by an average of 59% ? 8. I% (n = 6). In two of these cells. the early-IPSP was suppressed an average of 30%,. but the late-EPSP was not reduced appreciably in any of the cells tested. Similar to the data from guinea pig neurons, the effects of DNQX persisted for greater than 40 min and only 2 of 6 cells had partial recovery of the response. In I 1 of I I guinea pig cells, APV (50-100 PM) reduced the amplitude of the late-EPSP by an average of 82% t 5.9% (Fig. 4B), without modification of the resting membrane potential or input resistance. Stimulation elicited both the early- and lateEPSPs in 10 of these cells. The action of APV was selective for the late-EPSP. reducing it by an average of 83% t 6.6%’ while attenuating the early-EPSP by only 3% t 4.3% (Fig. 5B). resulting in a statistically significant differential effect of APV on the lateEPSP (p < 0.00 I). Near complete recovery from the effects of APV occurred within I5 to 25 min in all cells tested. In neurons in which stimulus trains produced a prolonged depolarization. APV (100 FM) was effective in reducing the magnitude of this depolarization by an average of 37.7% f 4.5% (n = 5). Similar findings of reduction and recovery were observed in neurons from the rat (n = 5). APV (25-50 PM) reduced the amplitude of the late-EPSP by an average of 7 I .9% ? 9.9%. with only a small reduction of the early-EPSP (1 I .4% t 4.0%,). Sequential administration of DNQX and APV to the same cell also resulted in differential antagonism of the early and late EPSPs (n = 4). The differential effects of the selective antagonists DNQX and APV suggest that, in both the guinea pig and the the early-EPSP

rat, mediation of the early- and late-EPSPs is by activation of non-NMDA and NMDA receptors, respectively. Inhibitorj~ svnuptic potenfkds. Gamma aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the brain. and experiments were designed to explore its function in inhibitory synaptic transmission of the auditory cortex. GABA receptors can be divided into two major subtypes, GABA-a and GABAb subtypes, based on differential sensitivity to pharmacological agents (8). The GABA-a receptor subtype involves an increased chloride conductance (I 5). Responses mediated by the GABAb receptor subtype involve an increase in K ’ current (45). Voltage changes that result from activation of the GABA-b subtype are longer in onset latency and duration in comparison to voltage changes that follow binding to GABA-a subtype ( 1 I. 16.29). The GABA-a antagonist, bicuculline. and the selective GABA-b antagonists. 2-hydroxy-saclofen and phaclofen ( 16,34.38), were used to test the role of GABA receptors in the synaptic potentials of the auditory cortex. Bath application of the GABA-a antagonist bicuculline (2.5 or 10 PM) selectively reduced the amplitude of the early-IPSP recorded from either guinea pig or rat neurons (n = 15) (Fig. 6). Usually, reduction of the early-IPSP amplitude was accompanied by an increase in the amplitude of the early-EPSP of sufficient magnitude to discharge the cell (Fig. 6A). The duration of the early-EPSP was also increased in the higher concentration of bicuculline (10 PM). The overlap of potentials prevented meaningful quantification of the degree of reduction of the early-IPSP by bicuculline. Complete recovery from the effects of bicuculline occurred within 20-30 min after switching to drug-free physiological solution. These findings suggest that the early-EPSP and early-IPSP overlap electrically. and that the early-IPSP can influence the magnitude and duration of the fast excitatory response. To study further the influence of the early-IPSP on cellular excitability, we used a paired stimulus pulse design (n = 6). Presentation of pairs of identical stimulus pulses. with varying interpulse intervals (IPI), is a procedure that can yield considerable information concerning temporal aspects of synaptically induced changes in cellular excitability. In control conditions, at somewhat short IPIs (IO-30 ms), the amplitude of the earlyEPSP in response to the second (test) of two pulses was smaller than the response to the first (conditioning) pulse. Following suppression of the early-IPSP by bicuculline (2.5 or IO FM), the early-EPSP in response to the test pulse, over the same IPIs. was larger in amplitude than the early-EPSP in response to the conditioning response. These data clearly suggest that I ) the earlyIPSP reduces the excitability of these neurons and 2) mediation of the early-IPSP is by GABA-a receptors. The GABA-b antagonist, 2-hydroxy-saclofen (100 PM). reduced the amplitude of the late-IPSP (90.9% + 5.8%; n = 5) while having little affect on the amplitude of the early-IPSP (Fig. 68). In two additional cells, phaclofen (40 PM) selectively reduced the late-IPSP by an average 69.5% (t4.5%). Reduction of the amplitude of the late-IPSP was also accompanied by an increase in the amplitude of the late-EPSP, thus suggesting an overlap of these two components. These data show that GABA initiates the early and late IPSPs; however, different GABA receptor subtypes are involved: the GABA-a subtype for the earlyIPSP and the GABA-b subtype for the late-IPSP. DISCUSSIOK

The auditory cortex was studied because of its ability to rapidly develop plasticity of neuronal discharge under controlled conditions that also result in behavioral learning (3.6,64,66).

A. PRE-DRUG

DNQX

PRE-DRUG

APV

RECOVERY

d

FIG. 4. Differential effects of excitatory amino acid antagonists on the early- and late-EPSPs of guinea pig neurons. (A) Recordings from two different cells that responded to stimulation ofadjacent gray matter with early- and late-EPSPs (predrug). In (i). the synaptic response consisted of an early-EPSP and a late-EPSP. Depolarization of the membrane potential (V, range from -80 mV to -37 mV) did not reveal either the earlv-IPSP or late-IPSP. Bath aoolication of 10 I.LMDNOX resulted in a 66% suppression of the early%PSP while the’amplitude of the lateEPSP was essentially unaffected. In (ii), the microdrop application of DNQX (0.2 ~1, I2 PM) reduced the amplitude of the early-EPSP resulting in the apparent unmasking (or possibly facilitation) of the early-IPSP. (The arrow indicates an action potential elicited by the late-EPSP). V, = (i) -80 mV. (ii) -60 mV. Calibrations: (i) and (ii) 5 mV, IO ms. (B) In this cell. microdroo. aoolication of APV (0.2 ul. 100 rrM) resulted in .. a strong reduction of the late-EPSP amplitude ‘with vi&ahy no effect on the amplitude of the early-EPSP. APV suppression of the late-EPSP was partially reversed by 40 min of wash in drug-free medium. V, = -52 mV. Calibration IO mV. 20 ms.

This work is directed toward the goal of identifying cellular and synaptic mechanisms that govern plasticity in the auditory cortex. The major results can be summarized as follows: I) Neurons of the guinea pig and rat auditory cortex receive a complex synaptic pattern of afferent information that consists of an early-EPSP followed, sequentially, by an early-IPSP, late-EPSP, and lateIPSP. 2) The mechanisms for production of the early-EPSP and late-EPSP differ in terms of involvement ofthe excitatory amino acid receptor subtype. Production of the early-EPSP requires non-NMDA type receptors, whereas production of the late-EPSP requires the NMDA subtype of excitatory amino acid receptor. 3) The early-IPSP can modify information transmission that occurs by way of the early-EPSP. 4) The mechanisms for production of the early-IPSP and late-IPSP differ in terms of involvement of GABA receptor subtypes and ionic conductances. Production of the early-IPSP requires the GABA-a receptor subtype that apparently gates chloride conductance, and the late-

SYNAPTIC

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A. DNQX

B.

FIG. 5. Dilierential attenuation of the early- and late-EPSP amplitude by excitatory amino acid antagonists, observed in cells that responded to stimulation with both an early- and late-EPSP. (A) Exposure to DNQX (7.5-12.5 PM) resulted in a significant reduction ofthe amplitude ofthe early-EPSP to an average of 36 ? 6.2% (n = 6. p i 0.001) ofthe control amplitude. In contrast, the late-EPSP was. on average, not significantly affected by DNQX (6 t 4.W reduction from control; n = 6: 17 > 0. I). The differential effect of DNQX on the early-EPSP and late-EPSP attained statistical significance (paired / test. p < 0.0 I). (B) Exposure to APV ( IO0 PM) significantly suppressed the amplitude of the late-EPSP to an average of 17 + 6.6% ot control (n = IO. p i 0.001). In contrast. exposure to APV did not signilicantly suppress the amplitude ofthe early-EPSP (97’~ i 4.3% of control: n = 8. /I> 0. I ). The differential effect of APV on the late-EPSP and early-EPSP also attained statistical significance (paired I test. p i 0.0 I).

IPSP requires the GABAh receptor subtype that apparently potassium conductance. DNQX, which is selective for glutamate receptors

gates

of the quisquaIate/kainate type (28). antagonized the early-EPSP. whereas APV, an effective NMDA receptor antagonist (63). differentially attenuated the late-EPSP. Thus, it appears that the early-EPSP is mediated primarily by activation of kainate/quisqualate receptors, whereas the late-EPSP is mediated primarily by activation of NMDA receptors. Responses similar to the short and long latency EPSPs recorded in the present work from the auditory cortex have also been reported for other cortical areas (5,22,30.56,57,59,61). Generation of the late-EPSP might be thought to require depolarization by the early-EPSP to an activation threshold. especially considering the voltage dependence of NMDA receptor activation (40). But the interaction of the late-EPSP with the two IPSP responses in the present study has made it difficult to assess its voltage dependence for comparison to the voltage de-

pendence of the NMDA receptor. It is noteworthy, however. that APV-sensitive EPSPs have been observed to follow either conventional [linear (57)] or NMDA receptor-like (30.59) voltage dependence. Conventional voltage dependence may be observed if the NMDA receptors are actually located on an excitatory interneuron projecting to the impaled neuron (56). Interestingly. in the present study. some cells in which single-pulse stimulation did not elicit the late-EPSP responded to stimulus trains with a prolonged (c.g.. l-5 s). APV-sensitive depolarization. This suggests that for these cells, the additional synaptic drive produced by a stimulus train may be required for activation of an APVsensitive conductance. Indeed, the presence of the late-EPSP during antagonism of the early-EPSP by DNQX suggests that production of the late-EPSP does not. in fact. strictly depend on the presence of the early one. This may indicate that NMDA receptors can be activated at the resting membrane potential. as has been shown for pyramidal cells in the hippocampus (5 I ). The data from the present study indicate that the early- and late-IPSPs are differentially mediated b! GABA-a and C;AHAb receptors. rcspectivcly. Examination of the early- and latcIO0 IPSPs revealed mean reversal potentials near 70 mV and mV. respectively. suggesting that diherent ion conductanccs underlie these IPSPs. The reversal potential of the early-IPSP lies within the range ofthe average resting mcmbmnc potential. Thus it is likely that the proximity of the reversal potential for the early-IPSP to the resting V,, governs the prohdbiiity of eliciting 70 mV were an early-IPSP. Neurons uith resting V,,, less than more likely to elicit an early-IPSP than neurons with a resting V, greater than ~70 mV. In contrast to the oarly-IPSP, the probability. of eliciting the early- and late-EPSPs was not related to the resttng membrane potential. This is probably the result of the extrapolated reversal potentials for the early- and latcEPSPs being positive to the range of the resting mcmbranc potentials for all neurons tested. In the rat and guinea pig sensorimotor cortex. evoked membrane responses include a hyperpolariration with carly and Iatc

A.

xn_

.-A “’

FIG. 6. Dillerential etfects of selective GAB.A antagonists on the earlyand late-IPSPs recorded from guinea pig auditory cortical slices. (A) V, = -64 mV. Electrical stimulation (30 PA) of underlying white matter evoked an early-EPSP followed by both an early- and late-IPSP (predrug). Following a I5-min bath application of 2.5 JIM bicuculline, the amphtudc ofthe early-IPSP was suppressed. whereas the amplitude of the late-IPSP was unattected. Note that during the bicuculhne effect there is an action potential (chopped) that occurs during the early-EPSP. The elfect of bicuculline was reversed following a 25.min wash in drug-free krcbs solution (wash). (B) V, = ~60 mV. Synaptic response of a diHcrent neuron evoked by electrical stimulation of layer I (60 @A). that consists of an early-EPSP, a depolarizing early-IPSP. a late-EPSP. and a lateIPSP (predrug). The late-IPSP is signihcantly suppressed following a 20min bath application of 100 JLM 2-hydroxy-saclofen. The suppression occurs with a slight increase in the amplitude of the late-EPSP. but no apparent effect on the early-EPSP or early-IPSP. The ellect of the 2hydroxy-saclofen is partially reversed following a 30-min wash with drugfree physiological solution.

40x

(‘OX

components that resemble the early- and late-IPSPs reported here (4.10). Data from ion substitution experiments suggest that Cl and K+ ion conductances underlie the early- and late-IPSPs. respectively. Similarly, bicuculline antagonized the early-IPSP, implicating GABA-a receptors in its production (I 1,29), whereas the longer-latency IPSP is relatively insensitive to bicuculline and picrotoxin (1 1). The late-IPSP is differentially suppressed by the GABA-b antagonists, 2-hydroxy-saclofen and phaclofen. These data are in agreement with the late-IPSPs in the sensorimotor cortex that display a characteristic conductance change and reversal potential similar to those of membrane responses to the selective GABA-b agonist baclofen, suggesting the involvement of GABA-b receptors in late IPSP production (1 1). Thus, the early and late hyperpolarizations are likely to be synaptically mediated and not merely activated by membrane depolarization, and may be differentially mediated by GABA-a and GABA-b receptors, respectively.

A few investigators have documented synaptic responses to acoustic stimuli in the in vivo auditory cortex (43,48,53). Acoustic stimuli typically elicit an EPSP/IPSP sequence (43,53). De Ribaupierre et al. (1972) reported that long-duration IPSPs can have an early and late component. With hyperpolarizing current, the early component reversed near the resting potential. whereas further hyperpolarization reversed the late component. Also, the early-IPSP reversed over time during recordings though KCI-filled electrodes, suggesting a dependence on Cl conductance. Finally, a putative late-EPSP occasionally separated the early and late IPSP components, resulting in a “positive bump” in the hyperpolarization (48). Thus, the four synaptic potentials observed in the present study appear to be elicited by sensory stimuli in the intact animal. If sensory stimulation can elicit the synaptic potentials described in the present study, then it seems probable that threshold intensity acoustic stimuli could evoke only the early-EPSP, and that higher-intensity acoustic stimuli could evoke additional potentials (43,48,53). Because the late-EPSP can potentially dominate the evoked response, it is of interest to determine the conditions under which it is elicited. One possibility is that intense acoustic stimuli could evoke the late-EPSP. Another possibility, discussed next, is that very different conditions generate the lateEPSP (i.e., the conditions that result in neuronal plasticity).

Synaptic Potentials and Plasticity in Sen.wy3 C’orter Knowledge of the synaptic potentials intrinsic to the auditory cortex is critical to understanding mechanisms of cortical plasticity. It is likely that each of the early- and late-EPSPs and IPSPs contribute to cortical function in different ways and each may be modified to a different degree by conditions leading to changes in auditory function. Receptive fields in sensory cortex are modified by the GABA-a receptor antagonist bicuculline

El

‘\I.

(1,17,54). These modifications may result from suppresslon of the early-IPSP that indirectly leads to facilitation of the earlyand late-EPSPs. However, the uniform facilitatory effect of btcuculline on receptive fields stands in contrast to the complex facilitatory and suppressive effects of cholinergic agonists on auditory frequency receptive fields (4 1). It is possible that acctylcholine exerts different actions on the IPSPs and EPSPs and its overall effect depends on the combination and magnitude of synaptic potentials elicited by a given input to a neuron. The late-EPSP. recorded in the present study, appears to depend on glutamate receptors ofthe NMDA type that have been implicated in some forms of neural plasticity. For example. in the visual cortex, injury-induced modification of receptive fields is disrupted by blockade of cortical NMDA receptors (36). In any case, detailed information on synaptic interactions in the auditory cortex is needed to understand different kinds of receptive field plasticity. This study is the first to provide such information for the auditory cortex. A recent theory of auditory function posits that learninginduced plasticity of neuronal discharge in the auditoo cortex requires the convergence of inputs from the lemniscal and nonlemniscal divisions of the auditory thalamus (65). The lemniscal auditory thalamus (medial geniculate body, ventral division) rapidly relays acoustic information to the auditory cortex; it does not display plasticity of neuronal discharge during associative learning [for review, see (65)J. In contrast, neurons in the nonlemniscal auditory thalamus (medial geniculate body, magnocellular division), do display discharge plasticity during associative learning. The cortical early-EPSP and the early- and late-IPSPs occur under conditions that are not learning related (43,48.53). Thus, they may mediate inputs from the nonplastic, lemniscal auditory system. The results of our study show that the late-EPSP is considerably more labile than the other components and can dominate the full synaptic response to afferent stimulation. The late-EPSP may thus mediate inputs from the nonlemniscal auditory system and could potentially underlie discharge plasticity in the auditory cortex. Further support of this model comes from the very high density of NMDA receptors in the superficial cortex (layers I-III) (39,44), the site of termination of afferents from the magnocellular division of the medial geniculate (55). Thus. learning-induced discharge plasticity in magnocellular medial geniculate neurons may result in the activation of NMDA receptors and the generation of the late-EPSP in the auditory cortex. The resulting activation of intracellular mechanisms and subsequent changes in functional neuronal characteristics could comprise the cellular basis of learning-induced plasticity of the auditory cortex. ACKNOWLtDGEMENTS

We thank B. Kolls, S. Reidy, and S. Shatkin for technical assistance during various phases of this project. This work was supported by NSF BNS 9008818, NIH BRSG RR07010-17, and the Center for the Neurobiology of Learning and Memory.

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