0306-4522/92$5.00+ 0.00 Pergamon Press Ltd 0 1992IBRO

NeuroscienceVol. 48, No. 4, pp. 941-952, 1992 Printed in Great Britain

DEPOLARIZATION OF RAT LOCUS COERULEUS NEURONS BY ADENOSINE 5’-TRIPHOSPHATE L. HARMS,E. P. FINTA, M. T~CH~PL and P. ILLS* Department

of Pharmacology,

University of Freiburg, Hermann-Herder-Strasse F.R.G.

5, D-7800 Freiburg,

Abstract-Intracellular recordings were performed in a pontine slice preparation of the rat brain containing the locus coeruleus. The enzymatically stable P,-purinoceptor agonist a,/?-methylene ATP increased the firing rate without altering the amplitude or shape of action potentials; the afterhyperpolarization following a spike was not changed either. When locus coeruleus neurons were hyperpolarized by current injection in order to prevent spontaneous firing, a,/3-methylene ATP produced depolarization and a slight increase in the apparent input resistance. A combined application of kynurenic acid and bicuculline methiodide failed to alter the a$-methylene ATP-induced depolarization, and tetrodotoxin only slightly depressed it. A gradual shift of the membrane potential by hyperpolarizing current injection led to a corresponding decrease, but no abolition or reversal of the a,/?-methylene ATP effect. In the hyperpolarized region, the current-voltage curve of a$-methylene ATP came into close approximation with, but did not cross, the control curve. Elevation of the external K+ concentration, or the intracellular application of Cs+ by diffusion from the microelectrode, depressed the response to a,/?-methylene ATP, external tetraethylammonium was also inhibitory. External Ba*+ and Cs+ had no effect or only slightly decreased the a,/?-methylene ATP-induced depolarization. A low Na+, or a low Ca*+ high Mg2+ medium, as well as the presence of Co2+ in the medium, markedly reduced or even abolished the depolarization by a,/?-methylene ATP. ATP itself did not produce consistent changes in the membrane potential or input resistance. However, in the presence of the P,-purinoceptor antagonist 8-cyclopentyl-1,3_dipropylxanthine, ATP consistently increased the firing rate and evoked an inward current.

In conclusion, P,-purincceptor activation appears to depolarize locus coeruleus neurons by inhibiting a persistent potassium current, and at the same time opening calcium-sensitive sodium channels or calcium-sensitive non-selective cationic channels.

In the periphery ATP has been suggested to be a neurotransmitter in purinergic nerves as well as a co-transmitter of noradrenaline, acetylcholine and substance P in sympathetic, parasympathetic and sensory nerves, respectively.’ Although it is tempting to speculate on a similar function of ATP in the CNS, there are relatively few data to support this assumption. 27By contrast, the degradation product of ATP, adenosine, is an inhibitory neuromodulator in the CNS.L4*27 It is important to note that adenosine and ATP activate a separate set of receptors termed the Pi- and P,-types.* ATP has been shown to depolarize peripheral neurons;4 its mode of action was either the opening of non-selective cationic channels or the closure of potassium channels. In rat sensory neurons, ATP increased a cationic current,22 which is carried by sodium, calcium and potassium ions.’ In bullfrog sympathetic ganglion cells, ATP suppressed the Mtype potassium current,’ while in guinea-pig myen-

teric neurons it blocked a calcium-sensitive potassium conductance.i9 Central neurons, such as a subpopulation of rat dorsal horn cells were also depolarized by ATP; a sodium conductance increase was involved in this effect.‘* In the CNS a major group of noradrenergic neurons is concentrated in a pontine nucleus, the locus coeruleus (LC).i2sz4Recently, we have reported that enzymatically stable structural analogues of ATP increase the frequency of spontaneous action potentials in the rat LC.” Since ATP is degraded to adenosine, which inhibits the firing rate of LC neurons,35,36the excitatory effect of exogenous ATP could be demonstrated only after the blockade of P,purinoceptors.‘* Nevertheless, we concluded that endogenous ATP may be co-released with noradrenaline from dendrites or recurrent axon collaterals of LC cells’~*and produce excitation.38 The aim of the present work was to demonstrate the depolarization underlying the P,-purinoceptor-mediated excitation of LC neurons and to clarify the ionic mode of action.

*To whom correspondence should he addressed.

a,,%meATI’, a$-methyleneadenosine 5’triphosphate; AHP, afterhyperpolarization following the spike; DPCPX, 8-cyclopentyl-1,3dipropylxanthine; I,,, inwardly rectifying current carried by both sodium and potassium ions; I-V curve, current-voltage curve; LC, nucleus locus coeruleus; TEA, tetraethylammonium;

Abbreviations:

TTX, tetrodotoxin.

EXPERIMENTAL PROCEDURES Brain slice preparation The preparation and maintenance of midpontine slices of the rat brain was similar to that previously described.‘6~35In brief, male Wistar rats (16&200 g) were anaesthetized with ether and decapitated. Slices of about 4OO/cm thickness,

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containing the caudal part of the LC were prepared in oxygenated medium at 14°C with a Lancer Vibratome. A single slice was placed in a recording chamber and was superfused at a rate of 2 ml/min with medium saturated with 95% 0, plus 5% CO, and maintained at 35 36°C. The medium was of the following composition (in mmol/l): NaCl 126, KC1 2.5, NaH,PO, 1.2, MgCI, 1.3, CaCI, 2.4, NaHCO, 25 and glucose 1 I. When KC1 or MgCI, was added, NaCl was reduced to maintain the same osmolarity. In some experiments, all NaCl was substituted by choline chloride. When CoCIZ was added, a slightly modified medium (NaCI 139, KC1 2.5, NaH, PO, 1.2,MgCI, 1.3, CaCl, 2.4, NaHCO, IO and glucose 11 mmol/l) was used in order to prevent the precipitation of Co2+. No osmotic compensation was made for BaC& or CsCI.

Idenrification of locus coeruleus neurons and intracellular recording LC neurons were identified under a binocular microscope at the ventrolateral border of the fourth ventricle. LC cells were distinguished from neighbouring mesencephalic trigeminal neurons by their electrophysiological properties including spontaneous firing at a frequency of 0.2-5 Hz, and by a hyperpolarizing response to noradrenaline.‘5,4z Recording of membrane potential and current injection was carried out with glass micro-electrodes filled either with KC1 2 mol/l or with potassium acetate 2 mol/l (tip resistance, 50-90 Ma) using a high impedance pre-amplifier and a bridge circuit (Axoclamp 2A). In some cases CsCl(2 mol/l) was used to fill electrodes and a$-methylene ATP (a,pmeATP) was added 3&50min after impalement. In most experiments LC cells were hyperpolarized (about 20 mV) by continuously passing current (- 70 to -260 PA) through the microelectrode. In addition, hyperpolarizing current pulses of constant amplitude and 200-250 ms duration were delivered at a frequency of 0.5 Hz. The current amplitude (-40 to - 110 PA) was chosen so as to obtain a shift of about 10 mV in the membrane potential. The duration of the current pulses was sufficient to fully charge the membrane capacitance and to reach a steady-state voltage deflection. The apparent input resistance was calculated from the peak potential change produced. Concentration--response curves of a,/?-meATP were obtained by the application of increasing concentrations of the purine to the same neuron. Hyperpolarizing current pulses of different amplitudes (see Fig. 6) were injected to non-spiking neurons; the resultant voltage changes were measured and current-voltage (I-V) curves were constructed. The membrane potential was determined on withdrawal of the microelectrode from the cell at the end of each experiment. The Axoclamp 2A single electrode voltage-clamp amplifier was used to record agonist-activated membrane currents. The microelectrodes were filled with KC1 2 mol/l (tip resistance 40-55 MR). The voltage at the amplifier headstage was monitored on a separate oscilloscope to ensure correct operation of the switch clamp (26 KHz, 30% duty cycle). Both the membrane potential and the membrane current were displayed on a Gould RS 3200 pen-recorder.

Application of drugs Drugs were applied by changing the superfusion medium by means of three-way taps. At the constant flow rate of 2 ml/min about 30 s was required until the drugs reached the bath. With a few exceptions, the contact times of agonists were 3 min. When ATP or a$-meATP was applied more than once, the drug-free interval was at least 10min. The I-V curves were determined both in the absence and in the presence of a$-meATP (measurements were started 3 min after the beginning of drug-application). S-Cyclopentyl-1,3-dipropyixanthine (DPCPX) was added 15-20 min before ATP and was present throughout the experiment. Tetrodotoxin (TTX; 0.5 pmol/l) was in contact

with the tissue for 10 min. All changes m the lomc ~crrnposition of the medium were maintained for 10 min bcfole a$-meATP (30~molil) was applied the second tonne

Materials The following drugs were used: (-)-noradrenaline hydrochloride (Hoechst. Frankfurt am Main. F.R.G I: ATP disodium ‘salt. a#-meATP lithium salt, (-)-bicuculline methiodide, tetraethylammonium chloride, TTX (Sigma. Deisenhofen, F.R.G.): DPCPX. kynurenic acid (RBI. Natick, MA, U.S.A.). Stock solutions (l--IO mmol/l) of all drugs were prepared with distilled water; only DPCPX was dissolved in &methylsulfoxide (Sigma. Deisenhofen. F.R.G.). Further dilutions were made with medium. Equivalent quantities of the solvents had no effect. Statistics Means f S.E. are tailed Student’s r-test means and for the probability level of statistically significant.

given throughout. The paired, twowas used for the comparison of the comparison of means with zero. A 0.05 or less was considered to be

RESULTS

Eflects of adenosine S-triphosphate

Since we have found previously that ATP increases the firing rate of LC neurons only after a preceding blockade of adenosine receptors,3* a first series of experiments was performed in the presence of DPCPX (0.1 pmol/l). Under these conditions, ATP (100 pmol/l) increased the spontaneous firing of the neurons, while noradrenaline (100 pmol/l) inhibited it and, in addition, produced hyperpolarization (Fig. 1; n = 3). Both effects were reversible on washout. When the cells were voltage-clamped at a holding potential of -60 mV, ATP (100 pmol/l) produced an inward current and noradrenaline (lOO~mol/l) evoked an outward current (Fig. 1). In most subsequent experiments, spontaneous firing was prevented by continuously passing current through the recording electrode; the cells were hyperpolarized by approximately 20mV. The apparent input resistance was determined by injecting hyperpolarizing current pulses of constant amplitude at regular intervals. In the absence of DPCPX, ATP (100 pmol/l) failed to produce consistent changes of the membrane potential or input resistance. In one out of six cells, ATP caused hyperpolarization and a conductance increase on the first application; both responses disappeared when the purine was applied a second time (Fig. 2A). In another four cells, ATP (100 pmol/l) caused a small, but significant depotarization of 5.0 + 0.6 mV (P < 0.01; resting membrane potential, - 57.8 f 1.1 mV), without an accompanying change of the input resistance tire-drug value, 190.6 f 28.6 M52; 1.2 + 0.8% decrease; P > 0.05). The depolarizing effect of ATP slightly decreased on repeated application (23.2 f 4.0%; P < 0.01: Fig. 2B). Finatly, in one cell Al? (100 ymol/l) caused a biphasic response; a predominant but transient hyperpolarization was followed by a depolarization.

943

ATP effects on locus coeruleus neurons

DPCPX 0.1 pmol/l 1 min

ATP 100 pmol/l

14 min Noradrenaline 100 pmol/l

2 min

ATP 100 pmol/l

Noradrenaline 100 pmol/l

Fig. 1. Effects of ATP and noradrenaline in a rat LC neuron. potential and firing rate. The full height of action potentials (lOO~~mol/l) increased the firing rate, while noradrenaline hyperpolarized the cell. Lower trace, current recording from

Upper two traces, recording of the membrane was not reproduced by the pen-recorder. ATP (lOO~mol/l) abolished it and, in addition, the same neuron voltage-clamped at -60 mV.

DPCPX (0.1 pmol/l) was added 15min before ATT (lOO~mol/l) and was present throughout the experiment. The contact times to ATP and noradrenaline are indicated by the horizontal bars. The intervals between the traces are shown. Effects of u, $-methyladenosine S-triphosphate on the action potential In view of the inconsistency of the ATP effect, we decided to use the enzymatically stable structural analogue, a,/?-meATP. The spontaneous firing of LC neurons was increased by a$-meATP (30 pmol/l) even in the absence of DPCPX (Fig. 3Aa). The amplitude and duration of the action potentials, as well as the afterhyperpolarizations (AHPs) were unchanged by the purine (Fig. 3Ab,c; n = 3). In three experiments, constant current was passed through the electrode to hyperpolarize LC neurons by about 20 mV and action potentials were evoked by depolarizing current injection (Fig. 3B). Then a$-meATP (30 pmol/l) was applied and the membrane potential was clamped manually to its pre-drug level; action potentials were evoked again in the presence of the purine. Under these conditions, prominent and shortlasting AHPs followed each spike, which were practically identical before and after the addition of a$-meATP (30 pmol/l). Effects of cc,@methyleneaa’enosine S-triphosphate the membrane potential and input resistance

on

The results of the following experiments were obtained in 61 cells whose membrane potentials and input resistances were - 58.2 f 0.8 mV and 171.7 f

9.5 MR, respectively. a$-meATP (30 pmol/l) caused a depolarization of 9.2 f 0.4 mV and increased the input resistance by 6.0 + 1.3% (P < 0.01 in each case; Fig. 4A,B). In 12 out of 61 cells, the depolarization was preceded by an initial hyperpolarization (4.0 + 0.5mV) accompanied by a reduction of the input resistance (7.6 + 2.4% decrease; P < 0.05 in each case) (Fig. 4A). The depolarizing effect of a,/?-meATP was concentration-dependent; it amounted to 0.6 f 0.5 mV at 3 pmol/l, 8.2 + 1.5mV at lOpmol/l and 12.7 + 2.9 mV at 30 pmol/l a,/3-meATP (n = 4; P < 0.05 at 10 and 30 pmol/l). Subsequently we confirmed that even the highest concentration (30 pmol/l) of a$meATP did not produce desensitization; the compound was applied twice with a drug-free interval of 10 min. In three out of five cells there was an initial hyperpolarization followed by a depolarization; when a$-meATP was given the second time, there was no hyperpolarizing response (Fig. 4A). However, in none of the five LC neurons did the depolarization decrease from the first to the second application; by contrast, a small increase of 11.0 f 2.4% (P < 0.05) was observed. Figure 4B demonstrates that the increase of the input resistance by a$-meATP (30 pmol/l) persisted, when the membrane potential was temporarily restored to its pre-drug value with

L. HARMSel ul

944

hyperpolarizing current. TTX (0.5 pmol/l) depressed the effect of a$-meATP (30 pmol/l) by 19.7 + 5.0% (n = 9; P < 0.01). In contrast, the combined application of kynurenic acid (500 pmol/l) and bicuculline methiodide (100 pmol/l) did not change the depolarization by a$-meATP (30 pmol/l) [8.5 -I_10.2% potentiation; n = 4; P > 0.051. The involvement of K+ in the a#-methyleneadenosine 5’-triphosphate eflect

The amplitude of the depolarizing response to a$-meATP (30 pmol/l) decreased with membrane hyperpolarization (Fig. 5). A stepwise current injection into the cells elevated the resting membrane potential ( - 55.1 f 1.7 mV; n = 7) by - 20, - 40 and -60 mV; this resulted in a corresponding reduction of the a$-meATP effect (9.9 + 1.OmV, 6.0 + 0.6 mV and 3.9 + 0.5 mV); the percentage inhibition in comparison with the original depolarization by a$-meATP (30pmol/l) was, 36.6 f 7.5% and 58.4 + 5.7% (n = 7; P < 0.01 each). It is noteworthy that even at a markedly hyperpolarized membrane potential of about - 120 mV there was a residual response to a,/?-meATP (30pmol/l; Fig. 5). The relationship between the a$-meATP-induced de-

polarization and the membrane potential was the same both in the presence and absence oi‘ TTX (0.5 gmol/l) [n = 21. One out of three similar experiments in which I-V curves were determined both before and after the application of cc,/?-meATP (30pmol/l) are shown in Fig. 6. This particular neuron did not fire spontaneously; in the presence ot a, /?-meATP (30 flmol/l), the injection of tO0 pA current evoked an action potential, although under control conditions 200pA current was needed to obtain this response. Moreover, the slope of the I- V curve was increased by a$-meATP only between - 70 and - 110 mV; at a more hyperpolarized region the two curves failed to cross each other, demonstrating that there is no reversal of the cl,/?-meATPinduced depolarization. An increase in the K+ concentration in the medium from 2.5 to 10 mmol/l depressed the response to a,/?-meATP (30 pmol/l) by 56.6 f 5.6% (n = 7; P < 0.01); this effect was completely reversible on returning to the original K+ concentration (Fig. 7). In contrast, the depolarizing effect of ;c,j?-meATP (30pmol/l) was not changed in a medium containing Ba2+ (2 mmol/l) [6.3 t_ 5.6% inhibition; II = 4; P > 0.051. When recording was with a microelectrode

A

1 min

I ATP 100 pmol/l

20 mV 6 min

ATP 100 pmol/l

6 min ATP 100 pnol/l

..__._._._,_.

---

--

ATP 100 pmoi/l Fig. 2. EEects of ATP on the membrane potential and apparent input resistance. Constant current was passed through the electrode to hypeqolarize the cells by about 20 mV. Downward de&ctions represent electrotonic potentials caused by hypcrpolarizing current pulses of constant amplitude. These electiotonic potentials are directly proportional to the input resistance. (A) Desensitization to the hyperpoltiaing effect of ATP (100 pmol/I) on repeat& application. (B) Slightly decreasing response-sto ATP (100 _tmol/l) on repeated application. The contact times are in&at& by the horizontal t#us. The intwvals between the traces arc shown. The broken lines mark the maximum drug effects. Two different brain sli were used in A and B.

945

ATP effects on locus coeruleus neurons

a,B-meATP 30 pmol/l

B

b

Control

c

a,LhneATP 30 (3 mid

a

Control

b

a,B-meATP 30 (3 mid

_,

-_

I---___- --_-

A

1 2 nA

40 . mV

I

Fig. 3. Effects of a,/?-meATP on various parameters of action potentials. (Aa) Recording of the membrane potential and firing rate. The contact time to a&meATP (30 rmol/l) is indicated by the horizontal bar. (Ab,c) Recording of action potentials at two different time-scales both before and 3 min after the beginning of a&meATP (30 rmol/l) application. Each tracing shows the average of four spontaneous spikes. (Ra,b) Action potentials evoked by depolarizing current injection; a constant hyperpolarizing current was passed through the electrode in order to raise the membrane potential by about 20 mV. During the application of a,/?-meATP (30 pmol/l) for 3 min the membrane potential was manually clamped to its pre-drug value. Note the failure of a,/?-meATP to alter the AHP following each spike. Upper traces, applied current; lower traces, membrane potential. filled with CsCl (2 mol/l), the duration, but not the amplitude of the action potentials increased; this improved the reproduction of the spike amplitude by the pen-recorder (Fig. 8). Moreover, under these conditions the pronounced AHP following each spike almost completely disappeared. Intracellular Cs+ depressed the response to a$-meATP (3Opmol/l) by about 80% [CsCl (2 mol/l), 2.1 f 0.9 mV; n = 5;

P < 0.051, when compared with time-matched controls [KC1 (2 mol/l), 9.6 + 0.9 mV; n = 51. Extracellular Cs+ (2 mmol/l) [43.4 f 8.8% inhibition; n = 4; P c 0.051 and tetraethylammonium (TEA; 10 mmol/l) I55.0 + 11.9% inhibition; n = 8; P < 0.011 approximately halved the depolarizing effect of a,flmeATP (30 pmol/l). The involvement of Na+ and Ca2+, but not Cl- in the a-P-methyleneaaknosine 5’-triphosphate efect The depolarization evoked by a$-meATP (30 pmol/l) was markedly depressed when the Na+ concentration in the bath was reduced from 152.2mmol/l to 26.2 mmol/l (Fig. 9; 77.4 + 8.7% inhibition; n = 6; P < 0.01). A similar but still larger depression was observed, when the normal medium containing Ca’+ (2.4 mmol/l) and Mgr+ (1.3 mmol/l)

was changed to a low Ca2+ (0.3 mmol/l) high Mgr+ (20 mmol/l) medium; the inhibition of the a$meATP effect was reversible on returning to the normal concentration of Ca*+ and Mg2+ ions (Fig. 10; 95.4 f 2.7% inhibition; n = 4; P < 0.01). Co2+ (2 mmol/l) also reversibly reduced the depolarizing response to a$-meATP (30 pmol/l) [96.5 + 3.5% inhibition; n = 4; P < 0.011. When the microelectrodes were fllled with potassium acetate instead of potassium chloride, a$meATP continued to produce depolarization (6.8 + 1.2 mV, n = 3), which did not decrease from the tirst to the second application (7.0 + 4.2% increase; P > 0.05).

DISCUSSION

Inconsistent effect of aaknosine 5’-triphosphate in the absence of a PI-(A,-)purinoceptor antagonist In a previous extracellular study we have shown that enzymatically stable structural analogues of ATP, such as a$-meATP increase the firing rate of rat LC neurons.38 ATP itself had inconsistent effects; in some cells it facilitated, while in other cells it

946

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HARMS

et al.

cx,B-meATP

30 pmol,‘l

oc,S-meATP

30 pmoh’l

cx,D-meATP

30 pmol/l

B

a,S-meATP 30 pmolil Fig. 4. Effects of a$-meATP on the membrane potential and apparent input resistance. (A) Biphasic response to a$-meATP (3O~mol/l). On repeated application there was a desensitization to the hyperpolarixing but not to the depolarizing effect. (B) Stable depolarizing et%sotof a,&meATP(30 pmol/l) on repeated auction. The slight &rease in input resistance persisted, when the ~~e potential was manually clamped to its pm-drug value. The contact times to a$-meATP are indicated bj the horizontal bars. The intervals between the traces are shown. The broken lines mark the mum drug effects. Two different brain slices were used in A and B.

I min

,I

20 mV

_”__________ _” -77

mV

9 min cx,B-meATP 30 pmol/l

-97

mV 10 min a,/.%meATP 30 pmol/l

-117

mV cx,B-meATP 30 pmolll Fig. 5. Relationship between the a$-meATP-induced depolarization and the membrane potential. When inward current (shown at 1eFt)was injected to move the membrane to more hyp@polarixed levels, the response to a$-meATP (3Opmol) decmased but did not disappear. The co&act-times to a&meATP are indicated by the horizontal bars. The intervals between the traces are shown. The broken line.+mark the maximum drug effects.

ATP effects on locus coeruleus neurons

inhibited the discharge of action potentials. However, in the presence of the adenosine A,-receptor antagonist DPCPX,23 ATP always produced excitation.‘* In the present intracellular study we confirmed these findings. After blockade of Pi-(A,-) purinoceptors by DPCPX, ATP produced an inward, depolarizing current with a concomitant increase in firing. It is interesting to note that the main transmitter of LC cells, noradrenaline, caused an outward, hyperpolarizing current and inhibited the firing.1’~42 In the absence of DPCPX, ATP failed to alter the membrane potential consistently. Most neurons were depolarized, but in some cells a hyperpolarization, or a predominant hyperpolarization followed by a depolarization was observed. Bath-applied ATP may be enzymatically degraded to adenosine within a short period of time; in the LC, adenosine causes hyperpolarization and a decrease of the input resistance.35T36 In accordance with the recently reported finding that P,-purinoceptors of LC neurons strongly desensitize,35 the hyperpolarizing response to ATP disappeared on repeated application. By contrast, it is difficult to explain why the depolarizing response to ATP did not increase when the purine was applied a second time. a

b

Control

a,BmeATP

941

Direct &polarizing effect of CL,kmethyleneadenosine S-triphosphate

In some experiments, a,/?-meATP also produced an initial hyperpolarization, when applied the first, but not the second time. Since a,/?-meATP is more resistant to dephosphorylation than ATT’,” it is unlikely that the classic P,-purinoceptors of LC cells are activated by the P,-agonist a$-meATP. However, recently a common inhibitory receptor for adenosine and ATP has been found at the terminals of various postganglionic sympathetic nerves;‘3s3’ similar receptors may also exist on some LC neurons. In contrast to ATP, a#-meATP, in the absence of DPCPX, also caused a predominant depolarization in the LC. The depolarizing response did not decline on repetitive application, confirming the lack of desensitization demonstrated in our previous extracellular study. ” The increase in firing produced by a$-meATP could be counteracted by the P,antagonist suramin,” proving the existence of a P,purinoceptor. 38 Most of the a,/?-meATP effect persisted after the inhibition of all synaptic inputs by TTX. Thus, it may be concluded that only a small part of the a,/?-meATP-induced change in membrane

30 (3 mid

I

200

I

100 ms 40

~1

VL

100

e

-J

-100

-v

---

‘Y--

---L{--

-300 -cc-

-Lf-

-400 -CA-

-u-

-ziii_

Current (pAI

mV -500

-250

0 . -60

/ /

-200--k.___r

-

C

u-

Fig. 6. The effect of a&meATP on the I-V relation. In an LC neuron which did not fire spontaneously, electrotonic potentials were evoked by injection of current pulses of the indicated amplitude. (a) At the resting membrane potential of -7OmV, 200-pA current evoked an action potential. (b) Three minutes after the application of a$-meATP (30 rmol/l), the membrane potential was depolarized by 7 mV to -63mV, and already NO-pA current evoked an action potential. (c) In the hyperpolarized region, the I-V curve of a&meATP comes into close approximation with, but does not cross the control curve.

L. HARMS et ul

948

potential arises indirectly via transmitter release onto LC neurons. These experiments have some limitations. TTX interferes with action potential conduction in the axons of non-LC neurons (effects at somatic receptors are excluded), but does not inhibit the release of an excitatory transmitter from the terminals of these neighbouring neurons (effects at presynaptic receptors may persist). If this release were Ca2+-dependent, it should be abolished in a low Cat-, high Mg2+containing medium or after the addition of Co’+. Although the effect of a$-meATP was indeed inhibited under such conditions, this does not necessarily mean that the P,-agonist acts primarily on neighbouring cells; it may also indicate that the response to a$-meATP is dependent on external Ca’+. Such an explanation is favoured by the following findings. In the LC, kynurenic acid blocks the depolarization caused by all types of excitatory amino acid agonists,’ and bicuculline methiodide inhibits the responses to GABA.” Moreover, the concomitant application of kynurenic acid and bicuculline methiodide was shown to almost abolish the electrically-evoked depolarizing synaptic potentials.’ Since in our experiments a combination of kynurenic acid and bicuculline methiodide did not alter the cc,/?-meATP effect, it is unlikely that this compound acts via the release of glutamate and/or GABA. Ionic mechanism of P,-purinoceptor-mediated

effects

The depolarization by a$-meATP was accompanied Iby a slight increase in input resistance,

K’

which may be due to the blockade of a restmg outward current. The involved ionic species appears to be potassium as suggested by the following evidence. (1) The response to cqfl-meATP decreased when it was evoked at hyperpolarized resting membrane potentials. However, it was not abolished at the potassium equilibrium potential of - 110 mV.“’ even in the presence of TTX. Hence, the release of an

excitatory transmitter does not explain the residual depolarization, which may be due to the opening of non-selective cationic channels (see below). The slope of the I-V curve was increased by q?-meATP only between - 60 and - 110 mV; at a more hyperpolarized region the two curves failed to cross each other. demonstrating that there was no reversal of the o$-meATP-induced depolarization. (2) An increase from 2.5 to in the external K+ concentration 10 mmol/l decreased the a$-meATP-induced change in membrane potential. (3) Intracellular Cs+ applied by diffusion from the recording electrode filled with CsCl(2 mol/l) depressed but did not abolish the effect of a$-meATP. (4) TEA (10 mmol/l) also reduced the response to a$-meATP. Both intracellular Cs’ and extracellular TEA block various potassium conductances.2’ We suggest that the persistence of a residual r,(imeATP-induced depolarization near to EK and in the presence of intracellular Cs+ may be caused for the following reason. ?u,/?-meATP probably decreases a resting potassium conductance and at the same time increases a sodium conductance or a non-selective cationic conductance. Such an idea is supported by I min

2.5 mmol/l

I 20

1 mv 12 mm oc,B-meATP

30 pmol/l

K’ 10.0 mmol/l .“,.._.. _ .._.,._“_._,, 11 min

K*

c@meATP

30 pmolil

oc,P-meATP

30 pmol/l

2.5 mmob’l

Fig. 7. The dependence of the response to a,~-meATP on the external K+ concentration The depolarizing effect of c@-meATP (3Opmol/l) beenme smaller, when the K+ concentration in the PnffisiDB medium was increased from 2.5 to 10 mmol/l (shown at left). This depression was compk.tely reversible on retuming to the original K+ concentration. The contact times to a$-meATP are indicated by the horizontal bars. The intervals between the traces are shown. The broken lines mark the maximum drug effects.

ATP effects on locus coeruleus neurons

A

KC1 2 mol/l

10 5

949

1 min

I

20 mV

6 min Noradrenaline 100 pmol/l

,.._. ,,

..........

..........*,,,..,,.,,,,.

oc,B-meATP 30 pmol/l B

CsCl 2 mol/l

10 s

40 mV 4 min 1 min

I mV oc,B-meATP 30 pmol/l Fig. 8. Effect of a,/?-meATP in the absence and presence of intracellular Cs+. (A) When recording was with a microelectrode tilled with KC1 (2 mol/l: shown at left). a marked hyperpolarization followed each spike. This LC neuron was identified by its sensitivity to noradrenaline; a,~-;nekTP (30 pmol/l) produced depolarization. (B) When recording was with a microelectrode filled with CsCl (2 mol/l; shown at left), the duration, but not the amplitude of the action potentials increased; this improved the reproduction of the spike amplitude by the pen-recorder. Moreover, there was almost no AHP following the spikes and a,j-meATP (30 pmol/l) was inactive. The contact times to a$-meATP are indicated by the horizontal bars. The intervals between the traces are shown. The broken line marks the maximum effect of a,fl-meATP.

the finding that the a$-meATP effect was strongly reduced or even abolished when the external Na+ concentration was decreased. The involvement of an inwardly rectifying current carried by both Na+ and K+ (Z,,)‘o35 may be excluded, since in LC cells, only a K+ specific inward rectifier was shown to occur.29c3 In other types of neurons, agonist effects mediated by Is disappear in the presence of low concentrations (l-2 mmol/l) of extracellular caesium;26*32 this was certainly not the case in our experiments. Moreover, the a,/?-meATP-induced depolarization was not due to the efflux of Cl- from the cells, since it did not change when the microelectrodes were filled with potassium acetate instead of potassium chloride. Finally, it is unlikely that the P,-agonist inhibits a Ca*+-dependent K+-current (Z,,): which is active near the resting potential in LC neurons.4’ Although the a$-meATP effect was abolished in a low Ca*+ high Mg*+ medium or in the presence of external Co*+, the purine did not decrease the AHP following an action potential, which is due to the activation of Z,,." Of course this argument would not be valid if a,/?-meATP

inhibited a Ca*+-dependent K+-conductance, which is different from that mediating the AHP. However, at the present time we have no experimental evidence for such a mechanism of action and, therefore, the involvement of a calcium-sensitive sodium conductance, or a calcium-sensitive nonselective cationic conductam@ is tentatively suggested. In LC neurons the activation of a,-adrenoceptors,” adenosine A,-receptors,35*36 opioid ZI-receptors, 28,39*40*43 and somatostatin-receptors” GABAs-receptors” opens inwardly rectifying potassium channels. The resulting hyperpolarization reverses polarity at about -1lOmV in normal external K+, and the reversal potential of these responses at various follows EK. Moreexternal K+ concentrations over, the effects of the respective agonists are depressed by both intra- and extracellular Cs+ as well as by extracellular Ba*+. The actions of opiaid?,” and baclofengo persist in the absence of external Ca*+, whereas the response to a$-meATP was abolished in a low Ca*+- high M$+-containing medium. Hence, these results as a whole show that

L. HARMSet al

950 Na+

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12 min cc,.&meATP

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Fig. 9. The dependence of the response to a&meATP on the external Na+ concentration. The depolarizing effect of a,j-meATP (30 amol/l) was almost abolished, when the Na+ concentration in the perfusing medium was decreased from 152.2 to 26.2 mmol/l (shown at left). This inhibition was completely reversible on returning to the original Na+ concentration. The contact-times to a&meATP are indicated by the horizontal bars. The intervals between the traces are shown. The broken lines mark the maximum drug effects. Ca2+

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c

Depolarization of rat locus coeruleus neurons by adenosine 5'-triphosphate.

Intracellular recordings were performed in a pontine slice preparation of the rat brain containing the locus coeruleus. The enzymatically stable P2-pu...
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