Modification of activity and ionic currents by intracellular injection of 2,4-DNP and CdCl2 in the bursting neuron of Helix pomatia L

Camp. Biochem. Physiol. Vol. 7lA. pp. 47 to 52. 1982 Printed in Great Britain. All rights reserved

0300.9629/82/010047-06103 CO/O Copyrrght 0 1982 Pergamon Press Ltd

MODIFICATION OF ACTIVITY AND IONIC CURRENTS BY INTRACELLULAR INJECTION OF 2,4-DNP AND CdC& IN THE BURSTING NEURON OF HELIX POA4ATZA L. I. v~Dkz* and J. S~Lhcr Biological Research Institute of the Hungarian Academy of Sciences, Tihany, Hungary 8237 (Receioed 26 May 1981) Abstract-l. Intracellular injection of 2,4-DNP decreased the amplitude and frequency of oscillation of membrane potential but did not eliminate spike generation. 2. During lengthened interburst intervals fast oscillation with low amplitude occurred. 3. Intracellularly injected CdCl, increased the amplitude of the slow oscillation after a depolarization then paroxismal activity appeared. 4. Speed of the inward current decreased significantly after 2,4-DNP injection while it influenced by CdCl, The amplitude of the inward current was decreased in both cases. 5. The initial value of the outward current was decreased strongly at 2,4-DNP injection steady state value at CdClz injection.

INTRODUCTION

the slow transient was not while its

cellular metabolism, namely, by injecting 2,4-dinitrophenol (2,4-DNP) being an inhibitor of oxidative phosphorillation and CdCl, known as a blocker of SH group of a number of enzymes into the cell. We examined also the effect of injection of these substances on current voltage relationship of the membrane.

It

was shown in earlier experiments that the characteristic bursting activity of the identified giant neuron of Helix pomutia called RPal (Sakharov & SalBnki, 1969) was eliminated at low or high temperatures (Salinki et al., 1973). The same effect of low temperature was described in Aplysia R15 neuron (Wachtel & Wilson, 1973) being homologous to the RPal neuron. This refers to the fact that the biomodal pacemaker mechanism is strongly metabolic dependent and can have an intimate relation to the internal metabolism of the cell. It has been suggested also by the results of other investigations (Barker et al., 1975; Ifshin et al., 1975; Treitsman & Levitan, 1976; Chaplain, 1976) that intracellular processes can play an important role in the initiation of the slow oscillation of the membrane potential which is a basic mechanism for the bimodal potential generation. For the explanation of the bimodal pacemaker activity of bursting neurons the Ca-hypothesis is widely accepted according to which Ca-induced permeability changes are responsible for the membrane oscillation (Meech, 1972). It is evident that membrane potential oscillation originates from the oscillation of membrane conductivity, however, it is not necessarily determined by Ca-ions flowing into the cell during action potential generation. Experiments showing that slow membrane oscillation can be present without spikes (Junge & Stephens, 1973; VadBsz & Sallnki, 1976) and also the fact that long series of evoked action potentials often do not cause hyperpolarization (Salknki et al., 1979) are against such a hypothesis. In the present investigations we wanted to clear the possibility of modification of the characteristic activity of the RPal neuron through influencing intra-

MATERIAL AND METHODS

Isolated ganglia of Helix pomatia was placed in a perfusion chamber with continuous flow of physiological saline stabilized at constant (22°C) temperature. Experiments were carried out in occasions only when bimodal activity of the RPal neuron was well expressed. This neuron was impaled with two glass microelectrodes filled with KC1 (2.5mol/l) for recording membrane and action potentials a well as for measuring ion currents. The resistance of the voltage and current electrodes was 8-10 and 3-5 Mohm, respectively. Current and voltage curves were photographed by using Tektronix storage oscilloscope, action potentials were also visualized on a Brush recorder. For intracellular injection micropipettes with l-2 pm tip diameter filled with 100 mmol/l solution of 2,4-dinitropheno1 (2,4-DNP) or CdC12 were used. Injection was performed by pressure. Before each experiment the value of the hydrostatic pressure-necessary for passing 0.1-0.2 ml solution from the pipette in 1 set-was determined. The duration of pressure time was regulated electronically. The uhvsiological solution was as follows: NaCl 3.0g: KC1 6.3Jg; MgCl,.6H,O 2.4g; CaCl,.2H,O 1.5g; NaHCO, 0.2 g in 1000 ml distilled water. RESULTS

(1) Effect of intracellularly injected substances on the spontaneous activity of the RPal neuron Injection of 2,4-DNP

Alterations of the spontaneous activity starting a few minutes after intracellular introduction of

* Present address: Institute for Heavy Chemistry Industries, VeszprBm, Hungary 8200. 47

1. VADASZ and J. SALANKI

48 conrrol

f

t 5 min

liulll

IO

mln

50 ml/

l

b

0

IO

set

+ IO min

5mV

d

Fig. I. Effect of 2,4-dinitrophenol

2 see

injection on the spontaneous neuron RPal.

2,4-DNP are time dependent. In about 5 min the number of spikes within a burst does not change, however their amplitudes were reduced. The reduction of amplitudes is about 20% at the beginning and about 10% at the end of of the burst. Interspike intervals were lengthened by 2>30% and the duration of interburst intervals was increased by 50%. The amplitude of the slow oscillation of the membrane potential was reduced to l/2-1/3 as compared to the control (Figs la and b). After 15 min the number of spikes within the burst decreased by 40% and a further decrease in the amplitude of the first spike could be observed. Interspike intervals increased about 4 times (Fig. lc). Interburst period consisted of a short hyperpolarization with low amplitude and a longer prolonged depolarization. In the second phase of depolarization a fast (1.5 cps) oscillation of low amplitude (2mV) occurred usually. The activity of the neuron was strongly reduced in 25 min after injection (Fig. Id) and groups of action potentials appeared only rarely. Parameters of the action potentials were practically the same as before. The fast oscillations of the membrane potential occurring during the 40-80 set long interburst periods were not followed in every case by spikes, but the group of spikes was always preceded by such an oscillation (Figs Id and e). Injection of CdCl, As it has been shown previously (SalBnki et cd., 1979) intracellular injection of CdClz strongly influenced the activity of the RPal neuron: for a short period bursting activity was suspended, the membrane became depolarized and at this time monomoda1 pacemaker activity was observed. The frequency of spiking raised with increased depolarization. After 20-25 set a hyperpolarizing wave appeared that was followed by the returning of the bimodal pacemaker activity. The amplitude of the slow oscillation was

activity of the bimodal pacemaker

higher as compared to the control. 8-10 min following the injection of CdCl, the amplitude and number of spikes decreased gradually and only a paroxismal activity could be observed. (2) E&t of intracellularly injected substances on the ionic currents of the RPal neuron Ionic currents recorded in control conditions, after injecting 2,4-DNP and CdClz into the neuron are shown on Fig. 2. In the control the slope of the maximum inward current was 51.4.10-6A/sec and 20.0. lo- 6 A/set for the ascending and descending part, respectively. After 2,4-DNP these parameters were 36. 1O-6 A/set and 6.25. 10e6 A/set while after CdClz 49. 10e6 A/set and 18. 10m6 A/set, respectively. The inward conductivity plotted against voltage is shown on Fig. 3a. Its peak value was 4.33. 10s6 ohm-’ in the control, while after injecting 2,4-DNP and CdC12 it was 1.83.10-6 and 1.05.10-6 ohm-‘, respectively, referring in both cases to a considerable reduction. At the same time the peak values were shifted by 5 and 15 mV towards negative voltage region. In case of outward currents two conductivities were determined: g0Ut,p)marks the conductivity during the peak of the current (Fig. 3b) while gOU,cs,refers to the conductivity measured during steady state outward current (Fig. 3c) plotted against voltage. It was found that outward conductivity was markedly reduced after injecting either of the substances. After 2,4-DNP the reduction of the peak and steady state values (1 and 2) of the outward current proved to be about the same : gout(p) control

= 1.4

g<,,,(p)2,4-DNP g,,, ,s) control g ,>u,(s)2>4-DNP

- 1.6.

-__I 200 nA

200 m%ec

Fig. 2. tonic currents of the RPal neuron. a-control; &after injection of Z,4-DNP; c--after injection of Cd& Measurements were carried out 2630 min after injection; &,,,., = - %ImV CAP. 71,in--Y

LVADASZ

and J.

SAL~NKI

(b)

(a)

(c) Qoy, ( 10e6ahme’ )

I

IO-

-50

-10

IO

U (mV)

Fig. 3. Voltage dependence of the RPal neuron conductances. t*, control; O--O, after 2,4-DNP injection; A---A, after CdClz injection a-inward conductance; b-outward peak conductance; c-outward steady state conductance Calculation of conductance equations: ,g = 1/U = 1/U_, - U,_,, equilibrium potential was + 30 mV for inward and - 60 mV for outward current.

However, after CdC12 the peak value (3) of the outward conductivity was reduced only slightly but the steady state value (4) was strongly reduced: goutcp)control

“’

gou,(p) CdClz

=

gauccs)control ____gout(s)CdCl2

= 3.6.

(3)

To investigate the time relations of the outward currents the current values were plotted in a half logarithmic scale (Fig. 4). This way several lines were obtained and their time constants (t) could be determined. In case of the control, time constant of the current curve (7 control) was 820msec and nearly the

control =820 msec

Fig. 4. Kinetics of Uholdins= -50 mV, u,,,, = 0----O, after 2,4-DNP CdC& injection. ‘T,,.,~~,= TI,,,,z = 370ms;

outward current. the 40 mV. M, control; injection; A-A, after 820 ms; t2,+oNp = 830 ms; Q,,~,~ = 830ms

same was found after the injection of 2,4-DNP (72,LDNP = 830 msec). However, after CdClz injection two lines were obtained. The first one, between 50 and 600 msec, corresponding to the beginning of the inactivation was characterized by TV = 370 msec, while the second line, over 9OOmsec by ~2 = 830msec. Between 600 and 900 msec a transient phase occurred. This means that the outward current has only one time constant in control conditions and after 2,4-DNP, but after the injection of CdC12 it is composed of two well differentiated time constants (Fig. 4). DISCUSSION

As it is known, 2,4-DNP inhibits ATP synthesis by blocking oxidative phosphorylation necessary to energetical background of cell functioning. In case of neurons which possess an active ATP-dependent Napump 2,4-DNP treatment causes-as a result of inhibition of ATP synthesis-the decrease of the membrane potential (Nakajima & Takahashi, 1966; Ayrapetyan, 1969; Godfraid et al., 1971). In Aplysia R15 neuron 2,4-DNP applied in superfusion evoked first a depolarization, then increase of resting potential and inhibition of bursting activity occurred (Junge & Stephens, 1973). In our experiments intracellularly injected 2,4-DNP modified the spontaneous activity pattern of the RPal neuron of Helix pomatia by depressing slow oscillation. On the other hand, preceding the rarely occurring bursts, a comparatively fast membrane oscillation with low amplitude was generated. It is remarkable that the frequency of spikes within the burst was similar to the control. On the basis of these results one can suppose that 2,4-DNP inhibits those intracellular processes, which are responsible for the periodic appearance of the bursts. A similar phenomenon was observed in the pattern of the neuron at cooling the preparation (Wachtel & Wilson, 1973; Carpenter, 1973; Salanki et al., 1973; Vadasz & Vtr6, 1974). Both 2,4-DNP and cooling influenced the generation of action potentials to a small degree only, after 2,4-DNP the change of speed

Intracellular injection of 2,4-DNP and CdCl, in snail neuron

of the inward current and amplitudes of inward and outward currents were similar to that described in case of cooling (VadBsz & V&6, 1975). The decrease in the conductivity of the membrane can be partly ascribed to inhibition of intracellular metabolism, partly to a direct effect of 2+DNP on the membrane. At intracellular injection of CdClz the change of activity pattern was similar to that evoked by extracellular application of CdC& (SalBnki et al., 1979; Vadasz & SalBnki, 1976). Since the characteristic pattern was eliminated, this effect can be explained by inhibition of intracellular processes, however, the changes of current curves refer also to a direct membrane effect of CdC12. The latter is supported by the results of experiments carried out on isolated soma membranes (Kostyuk et al., 1977). Experiments using voltage clamp technics also showed that intracellular injection of CdCl,-similar to the effect of CoCl, (Eckert & Lux, 1976; Vadasz & Salinki, 1976bauses a considerable decrease in the slow inward (Ca2+) and the late outward currents but depressed the initial peak value of the outward current only slightly. It seems that the characteristic activity of the bursting neuron can be influenced at two different targets by intracellular injection : indirectly through influencing intracellular metabolism and through changing directly the ionic permeability of the membrane. The role of intracellular free Ca’+ in the change of membrane potential was shown by Meech (1974), Lux et a/. (1976) and others, and recently a similar function was demonstrated for anions (Morita et al., 1980). The change of ionic concentrations can directly be dependent on intracellular metabolic cycles, the effect of which is reflected in periodic changes of membrane conductivity. Ca2 + may play a dominant role in this process (Meech, 1972), but further experiments are needed to support this theory. The kinetics of the outward current was characterized by similar time constants in the case of the control and after injecting 2,4-DNP. After CdC12 injection the decay of the outward current was characterized by two time constants, showing that CdC12 has a more profound effect on the membrane characteristics than 2,4-DNP. ~~~~~~~ clearly differs from the control and refers to a fast inactivation of the outward current, while tZCdCl, characteristic for the steady state outward current was similar to the control. These components refer to the different states of the K-channels, but the presence of two different channel populations cannot be either excluded (Meech & Standen, 1975; Heyer & Lux, 1976). In any case it can be concluded that the direct membrane effect of the CdCl, is much more pronounced than that of the 2,4-DNP.

51

endogenous discharge of Aplysia neurones. In Neurobiology of Invertebrates. Mechanisms of Rhythm Regulation (Edited by SALANKIJ.) pp. 35-58 Akadtmiai

Kiad6, Budapest. CHAPLAINR. A. (1976) Metabolic regulations of the rhythmic activity in pacemaker neurons. II. Metabolically induced conversions of beating to bursting pacemaker activity in isolated Aplysia neurons. Brain Res. 106, 307-319.

ECKERTR. & Lux H. D. (1976) A voltage-sensitive persistent calcium conductance in neuronal somata of He/ix. J. Physiol. Lond. 254, 129-151.

GODFRAIDJ. M., KAWAMURAH., KRNJEVICK. & PUMAIN R. (1971) Actions of dinitrophenol and some other metabolic inhibitors on cortical neurons. J. Phy.siol., Lond. 210, 897-917.

HEYE;RC. B. & LUX H. D. (1976) Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia. J. Physiol., Lond. 262, 349-382. IFSHINM.

S., GAINERH. & BARKERJ. L. (1975) Peptide factor extracted from molluscan ganglia that modulates bursting pace-maker activity. Nature, Lond. 254, 72-73. JUNGE D. & STEPHENSC. L. (1973) Cyclic variation of potassium conductance in a burst-generating neurone in Aplysia. J. Physiol., Land. 235, 155-181. KOSTYUKP. G. & KRISHTAL0. A. (1977) Separation of sodium and calcium currents in the somatic membrane of mollusc neurones. J. Physiol., Lond. 270, 545-568. Lux H. D., ECKER~R., HOFMEYER C. & HEYERC. (1976) Intracellular Ca” depresses net late outward current in snail neurones. Biophys. J. 16, 23a. MEECHR. W. (1972) Intracellular calcium injection causes increased potassium conductance in Aplysia nerve cells. Comp. Biochem. Physiol. 42A, 493-499.

MEECHR. W. (1974) The sensitivity of He/ix aspersa neurones to injected calcium ions. J. Physiol., Lond. 237, 259-277.

MEECH R. W. & STANDENN. B. (1975) Potassium activation in Helix aspersa neurones under voltage clamp: a component mediated by calcium influx. J. Physiol.. Lond. 249, 21 l-239. MORI~AK., KOKETSUK. & KUBA K. (1980) Oscillation of Cal+ i-linked K+ conductance in bullfrog sympathetic ganglion cell is sensitive to intracellular anions. Nature, Lond. 283, 204205.

NAKAJIMAS. & TAKAHASHIK. (1966) Post-tetanic hyperpolarization and electrogenic Na+-pump in stretch receptor neurone of crayfish. J. Phqsiol., Lond. 187, 105-127. SAKHAROVD. A. & SAL,&NKIJ. (1969) Physiological and pharmacological identification of neurons in the central nervous system of Helix pomatia L. Acta physiol. hung. 35, 19-30.

SALANKIJ., VAD~SZ I. & VL?R~M. (1973) Temperature dependence of the activity pattern in the Br-type cell of the snail Helix pomatia L. Acta physiol. hung. 43, 115-124. SALP;NKIJ., S.-R~ZSA K. & VAD~SZI. (1979) Synaptic and metabolic modulation of the bimodal pacemaker activity in the RPal neuron of Helix pomatia L. Comp. Biochem. Physiol. 64, 265-271.

REFERENCES

AYRAPE~YAN S. N. (1969) Mechanism of regulation of the spontaneous activity of the giant neurones of the snail Biojizika 14, 866872. BARKERJ. L., IFSHINM. S. & GAINERH. (1975) Studies on bursting pacemaker potential activity in a molluscan neurons-III. Effects. of hormones. Brain Res. 84, 501-513. CARPENTER D. 0. (1973) Ionic mechanisms and models of

TREITSMAN S. N. & LEVITANI. B. (1976) Alteration of electrical activity in molluscan ne&ones by cyclic nucleotides and peptide factors. Nature, Land. i61; 62-64. VAD~SZ I. & Vk~d M. (1974) Effect of temperature transition on the activity parameters of Br-type neurone of Helix pomatia L. Annls Biol., Tihany, 41, 81-89. VADASZI. & VI?R~ M. (1975) Ion current temperature dependent of Br-type neuron of Helix pomatia L. Annls biol., Tihany, 42, 129-137.

VADASZI. & SAL~NKIJ. (1976) Mechanisms of spike and burst generation in the bimodal pacemaker RPal neuron

52

I. VADASZand J. SALANKI

of Helix pomatia L. In Neurobiology of Invertebrates. Gastropoda Brain (Edited by SAL~~NKI J.) pp. 371-380. Akadtmiai Kiad6, Budapest. WACHTELH. & WILSONW. A. (1973) Voltage clamp analy-

sis of rhythmic slow wave generation in bursting neurones. In Neurobiology of Invertebrates. Mechanisms oj Rhythm Regulation. (Edited by SAL~NKI J.) pp. 59-80 AkadCmiai Kiad6, Budapest.