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The transient potassium current, the A-current, is involved in spike frequency adaptation in rat amygdala neurons
160
Brain Research, 480 (1989) 160-169 Elsevier
BRE 14249
The transient potassium current, the A-current, is involved in spike frequency adaptation in rat amygdala neurons Po-Wu Gean and Patricia Shinnick-Gallagher Department of Pharmacology and Toxicology, The Universityof Texas Medical Branch, Galveston, TX 77550 (U. S.A. ) (Accepted 26 July 1988)
Key words: A-current; 4-Aminopyridine(4-AP); Action potential accommodation; Basolateral amygdala
The possible functional roles of the transient K + current, 1A, in basolateral amygdala (BLA) neurons were studied using a rat brain slice preparation and conventional intracellular recording techniques. Conditioning depolarization, which inactivates 1A, slowed the action potential repolarization while conditioning hyperpolarizationaccelerated the action potential repolarization. 4-Aminopyridine (4-AP, 100/JM), a specific I A antagonist, also caused a clear delay in spike repolarization similar to the effect of conditioning depolarization suggesting that I Ais involved in the action potential repolarization. When BLA neurons were excited by injecting long depolarizing ctn'rent pulses (500 ms), they responded with an initial rapid discharge of action potentials which slowed or ac~mmodated; an afterhyperpolarization (AHP) followed the depolarizing current pulses. Superfusion of 4-AP (100/~M) blocked accommodation restdting in an increase in action potential discharge in 74% (32 out of 43) neurons tested. The remaining l l cells responded with an increased frequency of discharge of the first few action potentials. Unlike the effect of cadmium (Cd2+, 100/~M), a calcium channel blocker, 4-AP did not reduce the AHP. In the presence of norepinephfine (NE, 10#M), a neurotransminer which has been shown to block calcium-activated potassium conductance, 4-AP caused a further increase in the number and frequency of action potential discharge. In addition, in BLA neurons, spontaneous interictal and ictal-like events were observed at low and high concentrations of 4-AP, respectively. We conclude that I Ais involved in the action potential repolarization as well as spike frequency adaptation in BLA neurons and that these actions may contribute to the convulsant effect of 4AP.
INTRODUCTION The A-current (IA) is a fast, transient outward K +current that is activated at subthreshold m e m b r a n e potentials by depolarizing voltage steps from potentials more negative than the resting potential 6. The A-current was first discovered in molluscan neurons ~2and has now been found in a variety of cells iaeluding m a m m a l i a n sympathetic ganglia and hippocampal neurons (for review, see ref. 28). The A-current, like other potassium currents, has a stabilizing effect on the membrane potential. Functionally, it has been shown to play an important role in determining the repetitive firing pattern of the cells6. Activation of the A-current in the subthreshold region of the m e m b r a n e potential lengthens the
interspike interval and slows the spike frequency 7"8-30. Similarly, I A can modulate the efficacy of synaptic transmission by its short-circuiting effect on depolarizing current in the subthreshold range 31. M o r e o v e r , recent evidence from mammalian neurons show that I A is significantly de-inactivated at the resting m e m b r a n e potential and is activated by the spike upstroke 3. Thus, I A appears to be involved in and contribute to the repolarizing phase of the action potential s,33 In the present study, we examine the functional role of I a in the rat basolateral amygdaloid neurons by using a specific antagonist, 4-aminopyridine (4AP). W e found that IA is involved in the regulation ot spike frequency adaptation as well as the action potential repolarization and suggest that these action~,
Correspondence: P. Shinnick-Gallagher, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77550, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
161 may in turn contribute to the convulsant effect of 4AP.
superfused with ACSF of the following composition (in raM): NaCI 117, KCi 4.7, CaCI2 2.5, MgCI2 1.2, NaHCO 3 25, NaH2PO 4 1.2 and glucose 11 saturated with 95% 0 2 - 5 % CO2. Intracellular recordings were obtained from neurons of basolateral amygdala nucleus using conventional intracellular recording techniques. Microelectrodes were pulled from microfiber-filled 1.0 mm capillary tubing (Frederick Haer) on a Brown-Flaming electrode pulier (SuRer). The electrodes were filled with 4 M potassium acetate with resistances ranging from 80 to 180 MfL Electrical signals were amplified using an Axoclamp II amplifier and recorded on a Gould 2400s chart recorder. Fast transient potentials which could not be adequately resolved with the chart recorder were stored on tapes using a Video Cassette Format Instrumentation Recorder (Model 420 B, A.R. Vetter) and digitized using a Data-6000 digital osciiloscope (Data Precision). All data are expressed as
MATERIALS AND METHODS In this study intracellular recordings were made from brain slices of the amygdala complex of male albino rats (125-200 g). The preparation and the maintenance of the brain slices was similar to that previously reported s°. Briefly, rats were decapitated using a guillotine and the brain rapidly removed from the skull and placed in cold oxygenated artificial cerebrospinal fluid (ACSF). Transverse slices of 500 p m thickness were cut and the appropriate slices placed in a beaker of ACSF gassed with 95% 0 2 - 5 % CO 2 at room temperature for at least 1 h before recording. A single slice was then transferred to a recording chamber where it was held submerged between two nylon nets. The slice was maintained at 32 _+ 1 °C and
A -55 mV
-62 mV
__3-'-1.
-74mV
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-74 mV Fig. 1. Effect of conditioning depolarization and hyperpolarizationon action potential repolarization. A: action potentials were elicited at different holding potentials by injection of cathodal or anodal DC current. B: records in A were superimposed at a fast time base to show that a smallbut distinct accelerationof the spike repolarizationfollowingthe conditioning hyperpolarization.Dash line is the action potential elicited at the resting membrane potential of -62 inV. Current monitor is positioned belowthe voltage trace.
162 mean +S.E.M. Statistical analysis was performed using the paired Student's T-test. Membrane resistance was measured by passing currents of 100 ms duration and various intensities across the membrane and recording the resultant electrotonic potentials. The input resistance of the nemon was determined from the slope of the linear (-60 to -90 mV) portion of the current-voltage Curve.
RESULTS The results presented here were obtained from 53 stable intracellular recordings from basolaterai amygdala (BLA) neurons having resting membrane potentials more negative than-55 mV and action potential amplitude greater than 70 mV. The resting membrane potential and neuronal input resistance were-67 _+4 mV (n = 53) and 46 _ 14 MO (n = 48), respectively.
1A and spike repolarization One of the distinct characteristics of 1A is its voltage-dependency of activation and inactivation. For example, in cultured hippocampal cells, IA is activated upon stepping from hyperpolarized levels to - 5 0 to -40 mV and is inactivated at potentials mo:e positive than - 5 0 mV 3°. Thus, the participation of I A in spike repolarization can be tested by altering membrane potential with constant current injection, since IA is enhanced by conditioning hyperpolarization. The effect of varied membrane potentials on the spike repclarization was tested in 10 BLA neurons. The main observation was thai the rate of spike repolarization progressively increased as the holding level was shifted toward hyperpolarized potentials. This behavior is illustrated in Fig. 1, where the time course of the action potential elicited from different holding levels is shown. The spikes shown in Fig. 1A are superimposed at a faster sweep speed in Fig. 1B to demonstrate more clearly the voltage-dependency of the falling phase. In 10 neurons, the duration of action potential (measured from one-third of the peak amplitude) is 0.93 _+ 0.12 ms when membrane potential was held at - 7 4 + 2 mV and 1.08 _+ 0.15 ms when held a t - 5 5 + 2 mV (P < 0.001). To test further the hypothesis that i A contributes to spike repolarization, the effect of 4-AP on the action
potential o~onfiguration was analyzed in 40 BLA neurons. Action potentials, occurring spontaneously or elicited by intraceilular depolarizing current injection, rapidly repolarized to a particular membrane potential termed the point of rapid repolarization 27 (Fig. 2A) and were followed by a slow depolarizing afterpotential (DAP) in BLA neurons. In 40 neurons, the point of rapid repolarization ranged from - 4 2 mV to - 6 4 mV (mean _+ S.E.M.: -53 _+ 5 mV). Superfusion of 4-AP (100 ~M) invariably caused a clear de!ay in the spike repolarization and shifted the point of rapid repolarization an average of 4 mV (-49
A
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Fig. 2. Effect of 4-AP on the spike repolarization. A: action potentials were evoked by current injection (Ai) or occurred spontaneously (A2) in the same BLA neuron. Current monitor is positioned below the voltage recording. Superfusionof 4-AP (100 /~M, 7 min) shifted the point of rapid repolarization (marked by stars) to a less negative potential and resulted in the initiation of second spike riding on the DAP. Resting membrane potential (RMP) was -63 mV. B: action potentials obtained in A2 were superimposed to show the spike-broadening effect of 4-AP. Note the uniform broadening of the spike from the peak to the base.
163 -4- .5 mV, n = 40, P < 0.0001) to less negative potential. This effect was usually accompanied by initiation of a second spike riding on the DAP. 4-AP at a concentration of 100/~M has no significant effect on resting membrane potential or neuronal input resistance. The resting membrane potential and input resistance w e r e - 6 7 + 4 mV and 47 +_ 14 Mf~ before a n d - 6 6 _+ 4 mV and 44 _+ 10 MQ after application of 4-AP. These results suggest that 1A contributes to rapid repolarization of the action potential in B L A neurons.
IA and spike frequency adaptation An electrophysioiogical characteristic of many CNS neurons is that the frequency of spike discharge declines during a constant depolarizing pulse. This accommodation or adaptation in B L A neurons, similar to that of hippocampal cells 21, is due largely to a calcium-activated potassium conductance. This can
A
control
B
be demonstrated by the application of cadmium (Cd 2+) or norepinephrine (NE). Fig. 3 shows that, on injecting a long depolarizing current pulse (500 ms), B L A neurons responded with an initial rapid action potential discharge which slowed or accommodated after 100-250 ms (Fig. 3At,3Bt). The end of the catelectrotonic pulse was followed by an afterhyperpolarization (AHP) (Fig. 3Ct). Superfusion of Cd 2+ ( 1 0 0 / z M , n = 8) or NE-containing solution ( 1 0 ~ M , n = 8) reduced accommodation resulting in an increased frequency and duration of action potential discharges (Fig. 3A2,B2). The A H P was also decreased in amplitude (Fig. 3C2). The effect of 4-AP on spike frequency adaptation was tested in 43 B L A neurons. Normally, intracellular injection of long depolarizing current pulses (500 ms) elicited trains of action potentials which rapidly accommodated (Fig. 4Al). 10 min after superfusing C Cont;og
Control
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3 Cadmium and noreoineDhrine block accommodation ~nd the AHP in BLA neurons. At,B~: control responses of BLA neurons
* pulses . (500 ms) . which elicit . . . tram of action . tog" long" depolarizing current accommodatmg potentials. A,,B,: m Cd-'+ (100pM) or NE (l 0 /.tM) solution, the accommodation was reduced such that the frequency of action potential discharge increascd and the duration of this discharge was prolonged. A3,B3: responses after washing preparations with control solution for 20 rain. RMP was -68 mV in A and -67 mV in B. C1: chart records of response of the neuron to injection of a 70 msec depolarizing current pulse. Membrane potential was held at -60 inV. C2:10 min after the superfusion of NE (10pM), the slow AHP was blocked. B and C were recorded from the same neurons.
164 100pM 4-AP, this behavior changed radically in 74% (32 out of 43) cells, such that the same intensity current injection now produced sustained trains of spike firing at frequencies up to 30 H z (Fig. 4A2). The remaining 11 cells responded with an increased frequency of discharge of the first few action potentials during the pulse (Fig. 5). In all cells studied, 4-AP slowed the action potential throughout the entire repolarization phase beginning at the peak of the spike. Additionally, in the presence of 4-AP, action potentials were triggered at a more hyperpolarized level. In 15 neurons, 4-AP (100/~M) lowered the threshold for triggerine an action potential from -50.7 +_ 5.1 mV to Z,3.5 + 4.1 mV (P < 0.005). Action potentials in the presence of 4-AP are larger in amplitude and
A 1
8
Control
_
longer in duration (Figs. 4B and 5B). Spike amplitude and duration (measured at the midpoint between the threshold and the peak) were 85 _+ 7 m V and 0.73 _ 0.15 ms before and 88 _+ 7 m V (n = 16, P < 0.0005) and 0.87 + 0.24 ms (n ffi 16, P < 0.0005) after superfusion with 100pM 4-AP. The next question to be tested was whether 4-AP exerted its effect on accommodation through blocking a Ca2+-dependent K+-conductance. We therefore examined the effect of 4-AP on the amplitude and duration o f the A H P following a cathodal current injection. In all cells tested, 4-AP blocked the spike frequency adaptation without affecting the A H P (Fig. 4D). In 18 neurons, the amplitude and duration of the A H P were 3.4 _+ 1.3 m V and 1.6 + 0.4 s before
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Fig. 4.4-AP blocks spike frequency adaptation without reducing the amplitude and duration of the AHP. A: responses of a BLA neuron to long depolarizing current injections. The strong adaptatior_~,'~,~.,:sreversibly suppressed resulting in repetitive firing in 100/~M4AP. RMP -- -63 mV. B: superimposed action potentials taken from the beginning of each train of spikes in A~ and A 2 to show that 4AP increased amplitude and duration of the action potential. The initial part of action potentials indicated (star) in B are shown at a higher gain in C to show that 4-AP also lowered the action potential threshold. Dash fine represents the control action potential; the sofid line, the effect of 4-AP. D: traces of AHPs taken from A~ and A 2were superimposed to show that 4-AP blocked spike frequency adaptation without ,'educing the amplitude or the duration of th:~.AHP.
165 A 1
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Fig. 5. Partial blockade of spike frequency adaptation by 4-AP in some BLA neurons. A l: control response of a BLA neuron to a long depoiarizing current injection. A2: in 11 of 43 neurons tested, superfusion of 4-AP (100/¢M, 10 min) only increased the first few action potentials during the pulse. A3: the effect of 4-AP was reversible after 20 min of washing. RMP = -64 InV. B: action potentials taken. from the beginning of each train in A I and A 2 were superimposed to show that, in all cells studied, 4-AP increased amplitude and duration of the action potential. A
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Fig. 6.4-AP caused a further increase in the number of action potentials in NE-containing solution. All records taken from the same ,euron. A: response of a BLA neuron to a depolarizing current stimulus. B: 10 min after the application of NE (10 gM). NE caused a slight hyperpolarization (3 mV) and the membrane potential was returned to control level by a continuous passing cathodal current through the recording electrode. C: as in B, but 7 min after the addition of 4-AP (100gM). D and E: 3 and 20 min after the 4-AP was washed from the recording chamber. F: 10 min after returning to control solution. RMP = -69 inV.
166 and 3.5 _+ 1.3 mV and 1.8 + 1.0 s (n = 18) after the application of 4-AP (100-200/~M). Neurotransmitters such as norepinephrine (NE) have been shown to block the Ca2+-activated K +conductance and reduce spike frequency adaptation in hippocampai pyramidal cells22. To minimize the
i " c
ilOm
CaZ+-activated K+-conductance, a NE (10/~M) containing solution was applied to the preparation. As expected, application of NE caused an increase in repetitive tiring (Fig. 6B). Addition of 4-AP (100/~M) to the medium resulted in a further increase in the number of action potentials and the frequency of action potential discharge (Fig. 6C, n = 4) elicited by the stimulus of the same intenaity. These results together with a lack of an effect on the A I I P suggest that the effect of 4-AP on the spike frequency adaptation is not mediated through a blocking of a Ca z+dependent K+-conductance. This timing is consistent with observations in other neuronal membranes which suggest that 4-AP at concentration of 100 # M did not block Ca2+-dependent K+-conduc tance 3°. In some cases, it has been reported that 4-AP actually enhanced a Ca2+-dependent K+-conduc tance ~4~3°. Thus, it is probable that, in addition to a cae+-activated K+-conductance, IA is involved in the regulation of spike frequency adaptation.
Convulsant effect of 4-A7
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C
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120
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Fig. 7. Spontaneous interictal-like and ictal-like activity induced by 4-AP. A: intracellular recording of typical spontaneous interictal-likebursts in a BLA neuron 20 rain after the superfusion of 1001~M4-AP. RMP = -66 inV. B: chart recording of a spontaneous ictal-like event seen after 15 rain in 2 mM 4AP solution. RMP = -72 mV. C: exparided record of the tonical bursting phase of the ictal-likeevent shown in B. D: following the tonic phase, the frequency of bursting decreased, similar to the clonic phase of seizures.
During the superfusion of 4-AP (100/~M-2 mM), we often (66%, 33 out of 50 neurons) observed the appearance of spontaneous epileptiform activity in BLA neurons. In a low concentration of 4-AP (100-200/~M), this spontaneous epileptiform activity resembled spontaneous interictal-like bursting activity consisting of a number of spikes riding on a large depolarizing potential termed a paroxysmal depolarizing shift (PDS) 23 and followed by an AHF (Fig. 7A). At higher concentrations of 4-AP (500 g M - 2 mM, n = 5), in addition to interictal-like activity, spontan e o l i g ;e'tal-l;lre . . . . . . . . . . . .h'~mv~';nr* . . . . . 6 a. v. e. n.t e were s¢en. These ictal-like events consisted of 20-50 recurrent bursL, tiding on long-duration, large depolarizing potentials (Fig. 7B). During ictal-like activity, there was usually an initial increase in burst frequency (Fig. 7C) for a period of 20-30 s which resembled the tonic phase ot seizures. Following the tonic phase, the frequency ot the interictal-like burst decreased (Fig. 7D), these are similar to the clonic Fhase of seizures. DISCUSSION The primary finding of this work is that a transienl outward K-current (IA) is involved in the regulatior
167 of spike frequency adaptation as well as action potential repolarization in BLA neurons. This conclusion is based on the following observations: (1) Conditioning hyperpolarization accelerates the rate of action potential repolarization. (2) 4-AP at concentration of 100/~M broadened the spike and the spike-broadening effect of 4-AP showed the same characteristics as that caused by conditioning depolarization: a uniform broadening from the peak to the base of the spike. (3) 4-AP blocked spike frequency adaptation without affecting the amplitude and duration of the AHP. (4) In the presence of NE (10/zM), a near-maximal concentration needed to reduce the calcium-activated potassium conductance in hippocampal pyramidal cells26, 4-AP was still effective and caused a further increase in the number and frequency of action potential discharge elicited by the same intensity stimulus. Furthermore, in unpublished experiments using the single-electrode voltage-clamp we have found that 4-AP (1 mM) blocks the fast transient but not the sustained TEA-sensitive (10 mM) outward current suggesting that the effect of 4-AP on accommodation and spike repolarization is due to its effect on the Acurrent. Traditionally, the ionic currents reponsible for the repolarization of the action potential in squid axon were thought to be the delayed rectifier K+-current, I K, and Na+-current inactivation 15. However, subsequent studies in vertebrate neurons have revealed that currents other than I x may be important. For example, in frog sympathetic ganglia, spike repolarization is mainly due to Ca2+-activated K+-currents ~'~9. A fast transient ,outward current (/.~.) has been shown to participate in the spike repolarization in rat superior cervical gang!iona. Recent evidence from rat hippocampal Ca-1 cells indicated that a Ca2+-depen dent outward current, probably the fast TEA-sensitive K+-current, I c, contributes substantially to spike repolarization. In addition to I c, a 4-AP sensitive presumably I A also seems to contribute to spike repolarization 33. The present study suggests that I A contribute to spike repolarization in BLA neurons. The effect of the conditioning membrane potential on the action potential falling phase is a clear indication for the presence of this transient outward current (IA). Trains of action potentials in BLA neurons, as in
hippocampal pyramidal cells21, are followed by an AHP. This AHP characteristically has two components, one fast and one slow, both of which are due to Ca2+-activated K+-conductance and are blocked by calcium channel blockers 17as. There is general agreement that the slow component (slow AHP, IAHP) plays a role in spike frequency adaptation. Neurotransmitters such as norepinephrine n~°, histamine n and acetylcholine4.5 block the slow AHP, reduce spike frequency adaptation and increase neuronal excitability. In the present study~ we show that a Ca2+-activated K+-conductance, the slow AHP, does not appear to be the only factor that controls accommodation in BLA neurons. After exposure to norepinephrine at a concentration of 10/~M which has been reported to be a near-maximal concentration of norepinephrine necessary to block the slow Ca2+-activate~ K+-conductance, some residual accommodation was still present. Previous studies3° in cultured hippocampal neurons have reported that the presence of 1A would tend to retard action potential generation and thus dampen excitability. Application of 4-AP lowered the threshold for triggering action potential and the frequency of repetitive firing was increased 3°. Furthermore, it has been suggested that the mechanism underlying the acetylcholine block of accommodation is mainly through the inhibition of IA25. We tested the possible involvement of IA in spike frequency adaptation by applying 4-AP in the presence of norepinephrine. 4-AP caused a further increase in the number and frequency of action potential discharge elicited by same stimulus sttggesting that 1A does play a role in accommodation. Accommodation appeared to be due to at least two potassium currents, the calcium-activated potassium current, and the A-current. Thus, the A-current in amygdala neurons exerts a braking action on action potential discharge. In conclusion, we have shown that the transient K +'current (IA) is involved in the action potential repolarization and regulation of spike requency adaptation in BLA neurons. 4-AP slows the spike repolarization and blocks accommodation. These findings are particularly interesting in light of the recent reports that 4-AP is a potent convulsant9"29"34and that dendrotoxin, a neurotoxin whose convulsive action can be attributed to a reduction of I A, binds to synaptic region of the hippocampus 13suggesting that potas-
168 sium channels affected by dendrotoxin and 4 - A P m a y be present presynaptically, A n enhancement o f action potential firing b y reducing accommodation and a prolongation o f individual spikes would exert a powerful excitatory action if these changes t o o k place at presynaptic nerve terminals, where small changes of action potential duration and frequency produce remarkable changes in the amount o f transmitter released t6.32. Thus, blocking of presynaptic I A by 4-AP may predispose the neurons to a situation in which transmitter could be released synchronously. In addition, postsynaptic neurons responding to excitatory stimuli without accommodation would conREFERENCES 1 Adams, P.R., Constanti, A., Brown, D.A. and Clark, R.B., lntraceUular Ca ++ activates a fast voltage-sensitive K + current in vertebrate sympathetic neurons, Nature (Lend.), 296 (1982) 746-749. 2 Ayala, G.F., Matsumoto, H. and Gumnet, R.J., Excitability changes and inhibitory mechanisms in neocortical neurons during seizures, 1. Neurophysiol., 33 (1970) 73-85. 3 Belluzzi, O., Sacehi, O. and Wanke, E., A fast transient outward current in the rat sympathetic neurons studied under voltage-clamp conditions, J. Physiol. (Lond.), 358 (1985) 91-108. 4 Benardo, L.S. and Prince, D.A., Cholinergic excitation of mammalian hippocampal pyramidal cells, Brain Research, 249 (1982) 315-331. 5 Cole, A.E. and Nicoll, R.A., Characterization of a slow cholinergic post-synaptic potential recorded in intro from rat hippocampal pyramidal cells, 1. Physiol. (Lond.), 352 (1984) 173-188. 6 Connor, J.A., and Stevens, C.F., Prediction of repetitive firing behavior from voltage clamp data on an isolated neuron soma, I. PhysioL (Lond.), 213 (1971) 31-53. 7 Connur, J.A., Neural repetitive firing: a comparative study of membrane properties of crustacean walking leg axons, Z Neurophysiol., 38 (1975) 922-932. 8 Connor, J.A.. Slow repetitive activity from fast conductance changes in neurons, Fed. Proc., 37 (1978) 2139-2145. 9 Galvan, M., Gra.fe, P. and Ten Bruggencate, G., Convu!sant actions of 4-aminopyridine on the guinea-pig olfactory cortex slice, Brain Research, 241 (1982) 75-86. 10 Gean, P.W. and Shinnick-Gallagher, P., Picrotoxin induced epileptiform activity in amygdaloid neurons, Neuresci. Lett., 73 (1987) 149-154. 11 Haas, H.L. and Konnerth, A., Histamine and noradzenaline decrease calcium-activated potassium conductance in hippocampai pyramidal cells, Nature (Lond.), 302 (1983) 432-~.34. 12 Hagiw~ra, S., Kusano, K. and Saito, N., Membrane changes of Onchidium nerve cell in potassium-rich media, J. PhysioL (Lond.), 155 (1961) 470-489. 13 Halliwell, J.V., Othman, I.B., Pelchen-Matthews, A. and Dolly, O., Central actions of dendrodotoxin, selective reduction of a transient K-conductance in hippocampus and
tribute further to epileptogenesis. T h e two phases of ictal-like events seen in the presence of 4 - A P mimic the tonic and clonic phases o f ictal seizures in whole animal experiments 2~4. This suggests that 4 - A P may provide a m o d e l for studying the mechanisms underlying the generation of ictai activity and the transition from interietal to ictai seizures.
ACKNOWLEDGEMENTS This work is supported by NS 24643 to P.S.G. and a K e m p n e r fellowship to P . - W . G . binding to localized acceptors, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 493-497. 14 Hermann, A. and Gorman, A.L.F., Effects of 4-aminopyridine on potassium currents in a molluscan neuron, 1. Gen. Physiol., 78 (1981) 63-86. 15 Hodgkin, A.L. and Huxley, A.F., A quantitative description of membrane currents and its application to conduction and excitation in nerve, J. Physiol, (Lond.), 117 (1952) 500-544. 16 Katz, B. and Miledi, R., A study of synaptic transmission in the absence of nerve impulses, J. Physiol. (Lond.), 192 (1967) 407-436. 17 Lancaster, B. and Adams, P.R., Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons, 1. Neurophysiol., 55 (1986) 1268-1282. 18 Lancaster, B. and NicoU, R.A., Properties of two calciumactivated hyperpolarizationsin rat hippocampal neurons, J. Physiol. (Lond.), 389 (1987) 187-203. 19 MacDermott, A.B. and Weight, F.F. Action potential repolarization may involve a transient Ca++-sensitive outward current in a vertebrate neuron, Nature (Lond.), 300 (1982) 185-188. 20 Madison, D.V. and Nicoll, R.A., Noradrenaline blocks accommodation of pyramidal cell discharge in the hippocampus, Nature (Lond.), 299 (1982) 636-638. 21 Madison, D.V. and Nicoll, R.A. Control of repetitive discharge of rat pyramidal neurons in vitro, J. Physiol. (Lond.), 354 (1984) 319-331. 22 Mad[~c,, D.V. and Nicoll, R.A., Actions of noradrenaline recorded intracellularly in rat hippocampal CA1 pyramidal neurons in vitro, J. Physiol. (Lond.), 372 (1986) 221-244. 23 Matsumoto, H. and Ajmone Marsan, C., Cortical cellular phenomena in experimental epilepsy: interictal manifestations, Exp. Neurol., 9 (1964a) 286-304. 24 Matsumoto, H. and Ajmone Marsan, C., Cortical cellular phenomena in experimental epilepsy: ictal manifestations, Exp. Neuroi., 9 (1964b) 305-326. 25 Nakajima, Y., Nakajima, S., Leonard, R.J. and Yamaguchi, K., Acetylcholine raises the excitability by inhibiting the fast transient potassium current in cultured hippocampal neurons, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 3022-3026. 26 NicoU, R.A., Cole, A.E., Madison, D.V. and Newberry, N.R., Norepinephrine and acetylcholine block a calciumactivated potassium hyperpolarization in hippocampal py~
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ramidal cells. In O. Creutzfeldt, R.F. Schmidt and W.D. Willis (Eds), Sensory-motor Integration in the Nervous System, Springer, Berlin, 1984, pp. 305-314. Pockett, S., Dopamine changes the shape of action potentials in hippocampal pyramidal cells, Brain Research, 342 (1985) 386-390. Rogawski, M.A., The A.current: how ubiquitous a feature of excitable cells is it? Trends Neurosci., 8 (1985) 214-219. Rutecki, P.A., Lebeda, F.J. and Johnston, D., 4-Aminopy. ridine produces epileptiform activity in hippocampus and enhances synaptic excitation and inhibition, J. Neurophysiol., 57 (1987) 1911-1924. Segal, M., Rogawski, M.A. and Barker, J.L., A transient potassium conductance regulates the excitability of cul-
31 32 33 34
tured hippocampal and spinal neurons, J. Neutosci., 4 (1984) 604-609. Shimahara, T., Modulation of synaptic output by the transient outward potassium current in Aplysia, Neurosci. Lett., 24 (1981) 139-142. Shimahara, T., Presynaptic modulation of transmitter release by the early outward potassium current in Aplysia, Brain Research, 263 (1983) 51-56. Storm, J.F., Action potential repolarization and a fast afterhyperpolarization in rat hippocampal pyramidal cells, J. Physiol. (Lond.), 385 (1987) 733-759. Voskuyl, R.A. and Albus, H., Spontaneous epileptiform discharges in hippocampal slices induced by 4-aminopyridine, Brain Research, 342 (1985) 54-66.
Brain Research, 480 (1989) 160-169 Elsevier
BRE 14249
The transient potassium current, the A-current, is involved in spike frequency adaptation in rat amygdala neurons Po-Wu Gean and Patricia Shinnick-Gallagher Department of Pharmacology and Toxicology, The Universityof Texas Medical Branch, Galveston, TX 77550 (U. S.A. ) (Accepted 26 July 1988)
Key words: A-current; 4-Aminopyridine(4-AP); Action potential accommodation; Basolateral amygdala
The possible functional roles of the transient K + current, 1A, in basolateral amygdala (BLA) neurons were studied using a rat brain slice preparation and conventional intracellular recording techniques. Conditioning depolarization, which inactivates 1A, slowed the action potential repolarization while conditioning hyperpolarizationaccelerated the action potential repolarization. 4-Aminopyridine (4-AP, 100/JM), a specific I A antagonist, also caused a clear delay in spike repolarization similar to the effect of conditioning depolarization suggesting that I Ais involved in the action potential repolarization. When BLA neurons were excited by injecting long depolarizing ctn'rent pulses (500 ms), they responded with an initial rapid discharge of action potentials which slowed or ac~mmodated; an afterhyperpolarization (AHP) followed the depolarizing current pulses. Superfusion of 4-AP (100/~M) blocked accommodation restdting in an increase in action potential discharge in 74% (32 out of 43) neurons tested. The remaining l l cells responded with an increased frequency of discharge of the first few action potentials. Unlike the effect of cadmium (Cd2+, 100/~M), a calcium channel blocker, 4-AP did not reduce the AHP. In the presence of norepinephfine (NE, 10#M), a neurotransminer which has been shown to block calcium-activated potassium conductance, 4-AP caused a further increase in the number and frequency of action potential discharge. In addition, in BLA neurons, spontaneous interictal and ictal-like events were observed at low and high concentrations of 4-AP, respectively. We conclude that I Ais involved in the action potential repolarization as well as spike frequency adaptation in BLA neurons and that these actions may contribute to the convulsant effect of 4AP.
INTRODUCTION The A-current (IA) is a fast, transient outward K +current that is activated at subthreshold m e m b r a n e potentials by depolarizing voltage steps from potentials more negative than the resting potential 6. The A-current was first discovered in molluscan neurons ~2and has now been found in a variety of cells iaeluding m a m m a l i a n sympathetic ganglia and hippocampal neurons (for review, see ref. 28). The A-current, like other potassium currents, has a stabilizing effect on the membrane potential. Functionally, it has been shown to play an important role in determining the repetitive firing pattern of the cells6. Activation of the A-current in the subthreshold region of the m e m b r a n e potential lengthens the
interspike interval and slows the spike frequency 7"8-30. Similarly, I A can modulate the efficacy of synaptic transmission by its short-circuiting effect on depolarizing current in the subthreshold range 31. M o r e o v e r , recent evidence from mammalian neurons show that I A is significantly de-inactivated at the resting m e m b r a n e potential and is activated by the spike upstroke 3. Thus, I A appears to be involved in and contribute to the repolarizing phase of the action potential s,33 In the present study, we examine the functional role of I a in the rat basolateral amygdaloid neurons by using a specific antagonist, 4-aminopyridine (4AP). W e found that IA is involved in the regulation ot spike frequency adaptation as well as the action potential repolarization and suggest that these action~,
Correspondence: P. Shinnick-Gallagher, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77550, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
161 may in turn contribute to the convulsant effect of 4AP.
superfused with ACSF of the following composition (in raM): NaCI 117, KCi 4.7, CaCI2 2.5, MgCI2 1.2, NaHCO 3 25, NaH2PO 4 1.2 and glucose 11 saturated with 95% 0 2 - 5 % CO2. Intracellular recordings were obtained from neurons of basolateral amygdala nucleus using conventional intracellular recording techniques. Microelectrodes were pulled from microfiber-filled 1.0 mm capillary tubing (Frederick Haer) on a Brown-Flaming electrode pulier (SuRer). The electrodes were filled with 4 M potassium acetate with resistances ranging from 80 to 180 MfL Electrical signals were amplified using an Axoclamp II amplifier and recorded on a Gould 2400s chart recorder. Fast transient potentials which could not be adequately resolved with the chart recorder were stored on tapes using a Video Cassette Format Instrumentation Recorder (Model 420 B, A.R. Vetter) and digitized using a Data-6000 digital osciiloscope (Data Precision). All data are expressed as
MATERIALS AND METHODS In this study intracellular recordings were made from brain slices of the amygdala complex of male albino rats (125-200 g). The preparation and the maintenance of the brain slices was similar to that previously reported s°. Briefly, rats were decapitated using a guillotine and the brain rapidly removed from the skull and placed in cold oxygenated artificial cerebrospinal fluid (ACSF). Transverse slices of 500 p m thickness were cut and the appropriate slices placed in a beaker of ACSF gassed with 95% 0 2 - 5 % CO 2 at room temperature for at least 1 h before recording. A single slice was then transferred to a recording chamber where it was held submerged between two nylon nets. The slice was maintained at 32 _+ 1 °C and
A -55 mV
-62 mV
__3-'-1.
-74mV
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-74 mV Fig. 1. Effect of conditioning depolarization and hyperpolarizationon action potential repolarization. A: action potentials were elicited at different holding potentials by injection of cathodal or anodal DC current. B: records in A were superimposed at a fast time base to show that a smallbut distinct accelerationof the spike repolarizationfollowingthe conditioning hyperpolarization.Dash line is the action potential elicited at the resting membrane potential of -62 inV. Current monitor is positioned belowthe voltage trace.
162 mean +S.E.M. Statistical analysis was performed using the paired Student's T-test. Membrane resistance was measured by passing currents of 100 ms duration and various intensities across the membrane and recording the resultant electrotonic potentials. The input resistance of the nemon was determined from the slope of the linear (-60 to -90 mV) portion of the current-voltage Curve.
RESULTS The results presented here were obtained from 53 stable intracellular recordings from basolaterai amygdala (BLA) neurons having resting membrane potentials more negative than-55 mV and action potential amplitude greater than 70 mV. The resting membrane potential and neuronal input resistance were-67 _+4 mV (n = 53) and 46 _ 14 MO (n = 48), respectively.
1A and spike repolarization One of the distinct characteristics of 1A is its voltage-dependency of activation and inactivation. For example, in cultured hippocampal cells, IA is activated upon stepping from hyperpolarized levels to - 5 0 to -40 mV and is inactivated at potentials mo:e positive than - 5 0 mV 3°. Thus, the participation of I A in spike repolarization can be tested by altering membrane potential with constant current injection, since IA is enhanced by conditioning hyperpolarization. The effect of varied membrane potentials on the spike repclarization was tested in 10 BLA neurons. The main observation was thai the rate of spike repolarization progressively increased as the holding level was shifted toward hyperpolarized potentials. This behavior is illustrated in Fig. 1, where the time course of the action potential elicited from different holding levels is shown. The spikes shown in Fig. 1A are superimposed at a faster sweep speed in Fig. 1B to demonstrate more clearly the voltage-dependency of the falling phase. In 10 neurons, the duration of action potential (measured from one-third of the peak amplitude) is 0.93 _+ 0.12 ms when membrane potential was held at - 7 4 + 2 mV and 1.08 _+ 0.15 ms when held a t - 5 5 + 2 mV (P < 0.001). To test further the hypothesis that i A contributes to spike repolarization, the effect of 4-AP on the action
potential o~onfiguration was analyzed in 40 BLA neurons. Action potentials, occurring spontaneously or elicited by intraceilular depolarizing current injection, rapidly repolarized to a particular membrane potential termed the point of rapid repolarization 27 (Fig. 2A) and were followed by a slow depolarizing afterpotential (DAP) in BLA neurons. In 40 neurons, the point of rapid repolarization ranged from - 4 2 mV to - 6 4 mV (mean _+ S.E.M.: -53 _+ 5 mV). Superfusion of 4-AP (100 ~M) invariably caused a clear de!ay in the spike repolarization and shifted the point of rapid repolarization an average of 4 mV (-49
A
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4-AP
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Fig. 2. Effect of 4-AP on the spike repolarization. A: action potentials were evoked by current injection (Ai) or occurred spontaneously (A2) in the same BLA neuron. Current monitor is positioned below the voltage recording. Superfusionof 4-AP (100 /~M, 7 min) shifted the point of rapid repolarization (marked by stars) to a less negative potential and resulted in the initiation of second spike riding on the DAP. Resting membrane potential (RMP) was -63 mV. B: action potentials obtained in A2 were superimposed to show the spike-broadening effect of 4-AP. Note the uniform broadening of the spike from the peak to the base.
163 -4- .5 mV, n = 40, P < 0.0001) to less negative potential. This effect was usually accompanied by initiation of a second spike riding on the DAP. 4-AP at a concentration of 100/~M has no significant effect on resting membrane potential or neuronal input resistance. The resting membrane potential and input resistance w e r e - 6 7 + 4 mV and 47 +_ 14 Mf~ before a n d - 6 6 _+ 4 mV and 44 _+ 10 MQ after application of 4-AP. These results suggest that 1A contributes to rapid repolarization of the action potential in B L A neurons.
IA and spike frequency adaptation An electrophysioiogical characteristic of many CNS neurons is that the frequency of spike discharge declines during a constant depolarizing pulse. This accommodation or adaptation in B L A neurons, similar to that of hippocampal cells 21, is due largely to a calcium-activated potassium conductance. This can
A
control
B
be demonstrated by the application of cadmium (Cd 2+) or norepinephrine (NE). Fig. 3 shows that, on injecting a long depolarizing current pulse (500 ms), B L A neurons responded with an initial rapid action potential discharge which slowed or accommodated after 100-250 ms (Fig. 3At,3Bt). The end of the catelectrotonic pulse was followed by an afterhyperpolarization (AHP) (Fig. 3Ct). Superfusion of Cd 2+ ( 1 0 0 / z M , n = 8) or NE-containing solution ( 1 0 ~ M , n = 8) reduced accommodation resulting in an increased frequency and duration of action potential discharges (Fig. 3A2,B2). The A H P was also decreased in amplitude (Fig. 3C2). The effect of 4-AP on spike frequency adaptation was tested in 43 B L A neurons. Normally, intracellular injection of long depolarizing current pulses (500 ms) elicited trains of action potentials which rapidly accommodated (Fig. 4Al). 10 min after superfusing C Cont;og
Control
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3 Cadmium and noreoineDhrine block accommodation ~nd the AHP in BLA neurons. At,B~: control responses of BLA neurons
* pulses . (500 ms) . which elicit . . . tram of action . tog" long" depolarizing current accommodatmg potentials. A,,B,: m Cd-'+ (100pM) or NE (l 0 /.tM) solution, the accommodation was reduced such that the frequency of action potential discharge increascd and the duration of this discharge was prolonged. A3,B3: responses after washing preparations with control solution for 20 rain. RMP was -68 mV in A and -67 mV in B. C1: chart records of response of the neuron to injection of a 70 msec depolarizing current pulse. Membrane potential was held at -60 inV. C2:10 min after the superfusion of NE (10pM), the slow AHP was blocked. B and C were recorded from the same neurons.
164 100pM 4-AP, this behavior changed radically in 74% (32 out of 43) cells, such that the same intensity current injection now produced sustained trains of spike firing at frequencies up to 30 H z (Fig. 4A2). The remaining 11 cells responded with an increased frequency of discharge of the first few action potentials during the pulse (Fig. 5). In all cells studied, 4-AP slowed the action potential throughout the entire repolarization phase beginning at the peak of the spike. Additionally, in the presence of 4-AP, action potentials were triggered at a more hyperpolarized level. In 15 neurons, 4-AP (100/~M) lowered the threshold for triggerine an action potential from -50.7 +_ 5.1 mV to Z,3.5 + 4.1 mV (P < 0.005). Action potentials in the presence of 4-AP are larger in amplitude and
A 1
8
Control
_
longer in duration (Figs. 4B and 5B). Spike amplitude and duration (measured at the midpoint between the threshold and the peak) were 85 _+ 7 m V and 0.73 _ 0.15 ms before and 88 _+ 7 m V (n = 16, P < 0.0005) and 0.87 + 0.24 ms (n ffi 16, P < 0.0005) after superfusion with 100pM 4-AP. The next question to be tested was whether 4-AP exerted its effect on accommodation through blocking a Ca2+-dependent K+-conductance. We therefore examined the effect of 4-AP on the amplitude and duration o f the A H P following a cathodal current injection. In all cells tested, 4-AP blocked the spike frequency adaptation without affecting the A H P (Fig. 4D). In 18 neurons, the amplitude and duration of the A H P were 3.4 _+ 1.3 m V and 1.6 + 0.4 s before
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Fig. 4.4-AP blocks spike frequency adaptation without reducing the amplitude and duration of the AHP. A: responses of a BLA neuron to long depolarizing current injections. The strong adaptatior_~,'~,~.,:sreversibly suppressed resulting in repetitive firing in 100/~M4AP. RMP -- -63 mV. B: superimposed action potentials taken from the beginning of each train of spikes in A~ and A 2 to show that 4AP increased amplitude and duration of the action potential. The initial part of action potentials indicated (star) in B are shown at a higher gain in C to show that 4-AP also lowered the action potential threshold. Dash fine represents the control action potential; the sofid line, the effect of 4-AP. D: traces of AHPs taken from A~ and A 2were superimposed to show that 4-AP blocked spike frequency adaptation without ,'educing the amplitude or the duration of th:~.AHP.
165 A 1
2
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-
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20mV '2 ms Control
Fig. 5. Partial blockade of spike frequency adaptation by 4-AP in some BLA neurons. A l: control response of a BLA neuron to a long depoiarizing current injection. A2: in 11 of 43 neurons tested, superfusion of 4-AP (100/¢M, 10 min) only increased the first few action potentials during the pulse. A3: the effect of 4-AP was reversible after 20 min of washing. RMP = -64 InV. B: action potentials taken. from the beginning of each train in A I and A 2 were superimposed to show that, in all cells studied, 4-AP increased amplitude and duration of the action potential. A
Control
B
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Fig. 6.4-AP caused a further increase in the number of action potentials in NE-containing solution. All records taken from the same ,euron. A: response of a BLA neuron to a depolarizing current stimulus. B: 10 min after the application of NE (10 gM). NE caused a slight hyperpolarization (3 mV) and the membrane potential was returned to control level by a continuous passing cathodal current through the recording electrode. C: as in B, but 7 min after the addition of 4-AP (100gM). D and E: 3 and 20 min after the 4-AP was washed from the recording chamber. F: 10 min after returning to control solution. RMP = -69 inV.
166 and 3.5 _+ 1.3 mV and 1.8 + 1.0 s (n = 18) after the application of 4-AP (100-200/~M). Neurotransmitters such as norepinephrine (NE) have been shown to block the Ca2+-activated K +conductance and reduce spike frequency adaptation in hippocampai pyramidal cells22. To minimize the
i " c
ilOm
CaZ+-activated K+-conductance, a NE (10/~M) containing solution was applied to the preparation. As expected, application of NE caused an increase in repetitive tiring (Fig. 6B). Addition of 4-AP (100/~M) to the medium resulted in a further increase in the number of action potentials and the frequency of action potential discharge (Fig. 6C, n = 4) elicited by the stimulus of the same intenaity. These results together with a lack of an effect on the A I I P suggest that the effect of 4-AP on the spike frequency adaptation is not mediated through a blocking of a Ca z+dependent K+-conductance. This timing is consistent with observations in other neuronal membranes which suggest that 4-AP at concentration of 100 # M did not block Ca2+-dependent K+-conduc tance 3°. In some cases, it has been reported that 4-AP actually enhanced a Ca2+-dependent K+-conduc tance ~4~3°. Thus, it is probable that, in addition to a cae+-activated K+-conductance, IA is involved in the regulation of spike frequency adaptation.
Convulsant effect of 4-A7
l
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C
0
120
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Fig. 7. Spontaneous interictal-like and ictal-like activity induced by 4-AP. A: intracellular recording of typical spontaneous interictal-likebursts in a BLA neuron 20 rain after the superfusion of 1001~M4-AP. RMP = -66 inV. B: chart recording of a spontaneous ictal-like event seen after 15 rain in 2 mM 4AP solution. RMP = -72 mV. C: exparided record of the tonical bursting phase of the ictal-likeevent shown in B. D: following the tonic phase, the frequency of bursting decreased, similar to the clonic phase of seizures.
During the superfusion of 4-AP (100/~M-2 mM), we often (66%, 33 out of 50 neurons) observed the appearance of spontaneous epileptiform activity in BLA neurons. In a low concentration of 4-AP (100-200/~M), this spontaneous epileptiform activity resembled spontaneous interictal-like bursting activity consisting of a number of spikes riding on a large depolarizing potential termed a paroxysmal depolarizing shift (PDS) 23 and followed by an AHF (Fig. 7A). At higher concentrations of 4-AP (500 g M - 2 mM, n = 5), in addition to interictal-like activity, spontan e o l i g ;e'tal-l;lre . . . . . . . . . . . .h'~mv~';nr* . . . . . 6 a. v. e. n.t e were s¢en. These ictal-like events consisted of 20-50 recurrent bursL, tiding on long-duration, large depolarizing potentials (Fig. 7B). During ictal-like activity, there was usually an initial increase in burst frequency (Fig. 7C) for a period of 20-30 s which resembled the tonic phase ot seizures. Following the tonic phase, the frequency ot the interictal-like burst decreased (Fig. 7D), these are similar to the clonic Fhase of seizures. DISCUSSION The primary finding of this work is that a transienl outward K-current (IA) is involved in the regulatior
167 of spike frequency adaptation as well as action potential repolarization in BLA neurons. This conclusion is based on the following observations: (1) Conditioning hyperpolarization accelerates the rate of action potential repolarization. (2) 4-AP at concentration of 100/~M broadened the spike and the spike-broadening effect of 4-AP showed the same characteristics as that caused by conditioning depolarization: a uniform broadening from the peak to the base of the spike. (3) 4-AP blocked spike frequency adaptation without affecting the amplitude and duration of the AHP. (4) In the presence of NE (10/zM), a near-maximal concentration needed to reduce the calcium-activated potassium conductance in hippocampal pyramidal cells26, 4-AP was still effective and caused a further increase in the number and frequency of action potential discharge elicited by the same intensity stimulus. Furthermore, in unpublished experiments using the single-electrode voltage-clamp we have found that 4-AP (1 mM) blocks the fast transient but not the sustained TEA-sensitive (10 mM) outward current suggesting that the effect of 4-AP on accommodation and spike repolarization is due to its effect on the Acurrent. Traditionally, the ionic currents reponsible for the repolarization of the action potential in squid axon were thought to be the delayed rectifier K+-current, I K, and Na+-current inactivation 15. However, subsequent studies in vertebrate neurons have revealed that currents other than I x may be important. For example, in frog sympathetic ganglia, spike repolarization is mainly due to Ca2+-activated K+-currents ~'~9. A fast transient ,outward current (/.~.) has been shown to participate in the spike repolarization in rat superior cervical gang!iona. Recent evidence from rat hippocampal Ca-1 cells indicated that a Ca2+-depen dent outward current, probably the fast TEA-sensitive K+-current, I c, contributes substantially to spike repolarization. In addition to I c, a 4-AP sensitive presumably I A also seems to contribute to spike repolarization 33. The present study suggests that I A contribute to spike repolarization in BLA neurons. The effect of the conditioning membrane potential on the action potential falling phase is a clear indication for the presence of this transient outward current (IA). Trains of action potentials in BLA neurons, as in
hippocampal pyramidal cells21, are followed by an AHP. This AHP characteristically has two components, one fast and one slow, both of which are due to Ca2+-activated K+-conductance and are blocked by calcium channel blockers 17as. There is general agreement that the slow component (slow AHP, IAHP) plays a role in spike frequency adaptation. Neurotransmitters such as norepinephrine n~°, histamine n and acetylcholine4.5 block the slow AHP, reduce spike frequency adaptation and increase neuronal excitability. In the present study~ we show that a Ca2+-activated K+-conductance, the slow AHP, does not appear to be the only factor that controls accommodation in BLA neurons. After exposure to norepinephrine at a concentration of 10/~M which has been reported to be a near-maximal concentration of norepinephrine necessary to block the slow Ca2+-activate~ K+-conductance, some residual accommodation was still present. Previous studies3° in cultured hippocampal neurons have reported that the presence of 1A would tend to retard action potential generation and thus dampen excitability. Application of 4-AP lowered the threshold for triggering action potential and the frequency of repetitive firing was increased 3°. Furthermore, it has been suggested that the mechanism underlying the acetylcholine block of accommodation is mainly through the inhibition of IA25. We tested the possible involvement of IA in spike frequency adaptation by applying 4-AP in the presence of norepinephrine. 4-AP caused a further increase in the number and frequency of action potential discharge elicited by same stimulus sttggesting that 1A does play a role in accommodation. Accommodation appeared to be due to at least two potassium currents, the calcium-activated potassium current, and the A-current. Thus, the A-current in amygdala neurons exerts a braking action on action potential discharge. In conclusion, we have shown that the transient K +'current (IA) is involved in the action potential repolarization and regulation of spike requency adaptation in BLA neurons. 4-AP slows the spike repolarization and blocks accommodation. These findings are particularly interesting in light of the recent reports that 4-AP is a potent convulsant9"29"34and that dendrotoxin, a neurotoxin whose convulsive action can be attributed to a reduction of I A, binds to synaptic region of the hippocampus 13suggesting that potas-
168 sium channels affected by dendrotoxin and 4 - A P m a y be present presynaptically, A n enhancement o f action potential firing b y reducing accommodation and a prolongation o f individual spikes would exert a powerful excitatory action if these changes t o o k place at presynaptic nerve terminals, where small changes of action potential duration and frequency produce remarkable changes in the amount o f transmitter released t6.32. Thus, blocking of presynaptic I A by 4-AP may predispose the neurons to a situation in which transmitter could be released synchronously. In addition, postsynaptic neurons responding to excitatory stimuli without accommodation would conREFERENCES 1 Adams, P.R., Constanti, A., Brown, D.A. and Clark, R.B., lntraceUular Ca ++ activates a fast voltage-sensitive K + current in vertebrate sympathetic neurons, Nature (Lend.), 296 (1982) 746-749. 2 Ayala, G.F., Matsumoto, H. and Gumnet, R.J., Excitability changes and inhibitory mechanisms in neocortical neurons during seizures, 1. Neurophysiol., 33 (1970) 73-85. 3 Belluzzi, O., Sacehi, O. and Wanke, E., A fast transient outward current in the rat sympathetic neurons studied under voltage-clamp conditions, J. Physiol. (Lond.), 358 (1985) 91-108. 4 Benardo, L.S. and Prince, D.A., Cholinergic excitation of mammalian hippocampal pyramidal cells, Brain Research, 249 (1982) 315-331. 5 Cole, A.E. and Nicoll, R.A., Characterization of a slow cholinergic post-synaptic potential recorded in intro from rat hippocampal pyramidal cells, 1. Physiol. (Lond.), 352 (1984) 173-188. 6 Connor, J.A., and Stevens, C.F., Prediction of repetitive firing behavior from voltage clamp data on an isolated neuron soma, I. PhysioL (Lond.), 213 (1971) 31-53. 7 Connur, J.A., Neural repetitive firing: a comparative study of membrane properties of crustacean walking leg axons, Z Neurophysiol., 38 (1975) 922-932. 8 Connor, J.A.. Slow repetitive activity from fast conductance changes in neurons, Fed. Proc., 37 (1978) 2139-2145. 9 Galvan, M., Gra.fe, P. and Ten Bruggencate, G., Convu!sant actions of 4-aminopyridine on the guinea-pig olfactory cortex slice, Brain Research, 241 (1982) 75-86. 10 Gean, P.W. and Shinnick-Gallagher, P., Picrotoxin induced epileptiform activity in amygdaloid neurons, Neuresci. Lett., 73 (1987) 149-154. 11 Haas, H.L. and Konnerth, A., Histamine and noradzenaline decrease calcium-activated potassium conductance in hippocampai pyramidal cells, Nature (Lond.), 302 (1983) 432-~.34. 12 Hagiw~ra, S., Kusano, K. and Saito, N., Membrane changes of Onchidium nerve cell in potassium-rich media, J. PhysioL (Lond.), 155 (1961) 470-489. 13 Halliwell, J.V., Othman, I.B., Pelchen-Matthews, A. and Dolly, O., Central actions of dendrodotoxin, selective reduction of a transient K-conductance in hippocampus and
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31 32 33 34
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