T.G. Deliagina, A. G. Fel'drnan (A. G. Feldman), I. M. Gelfand and G. N. Orlovskii (G. N. Orlovsky),. "On the role of central program and afferent inflow in the ...
8.
9.
10. 11.
T.G. Deliagina, A. G. Fel'drnan (A. G. Feldman), I. M. Gelfand and G. N. Orlovskii (G. N. Orlovsky), "On the role of central program and afferent inflow in the control of scratching movements in the cat," Brain Res., 100, 297 (1975). V. IR. Edgerton, S. Grillner, A. Sj'ostr'om, and P. Zangger, "Central generation of locomotion in vertebrates," in: Neural Control of Locomotion, ed. R. M. Herman, S. Grillner, P. S. G. Stein, and D. G. Stuart, (Advances in Behavioral Biology, Vol. 18 ), New York- London ( 1976 ), pp. 439-464. E. Jankowska, M. G. M. Jukes, S. Lund, and A. Lundberg, "Reciprocal innervation through interneuronal inhibition," Nature, 206, 198 (1965). E. Jankowska, M. G. M. Jukes, and A. Lundberg, "The effect of DOPA on the spinal cord. 6. Half-centre organization of inter~leuronal transmitting effects from the flexor reflex afferents," Acta Physiol. Scand., 70, 389 (1967).
POSSIBLE AND
INTERACTION
PACEMAKER
ACTIVITY N.
IN
BETWEEN
MECHANISMS BURSTING
NEURONS
SYNAPTIC OF
ELECTRICAL OF
Helix
pomatia
I. Kononenko
UDC
577.352.5
Membrane hyperpolarization induced by short pulses of inward current, by stimulation of the anal nerve, which leads to the appearance of a long IPSP in the neuron, and developing during the appearance of spontaneous IPSPs in the neuron was investigated in neuron RPal of Helix pomatia. Short-term hyperpolarization of the neuron membrane by an inward current (I0 msec) led to the development of self-maintained (regenerative) membrane hyperpolarization lasting several seconds. The amplitude and duration of regenerative hyperpolarization increased ~v-ith an increase in amplitude and duration of the pulse of inward current. The time course of Il~SPs arising spontaneously in the neuron and in response to stimulation of the anal nerve was similar to that of regenerative hyperpolarization evoked by a pulse of inward current. It is suggested that regenerative hyperpolarization associated with activation of endogenous mechanisms of regulation of the bursting activity of the neuron may be due not only to short-term membrane hyperpolarization of the test neuron by the electric current, but also to hyperpolarization occurring during IPSP generation.
INTRODUCTION According to information in the literature postsynaptic potentials can exert some influence on endogenous electrical activity of molluscan neurons. As long ago as in 1958, Tauc postulated that long EPSPs can incorporate a pacemaker potential [ I0]. Later, Kandel et al. [6] studied the effect of activation of interneuron L10, which has an inhibitory input to bursting neuron L3, on modulation of endogenous activity of neuron L3 and reached the same conclusion. Many descriptions of activation of the pacemaker mechanism by PSPs can be found in a paper by Sokolov et al. [3]. The mechanisms of interaction between synaptic and pacemaker unit activity, however, still remain unexplained. This is largely because the ionic nature of the mechanisms of endogenous unit activity themselves are not fully understood, unlike the ionic nature of postsynaptic potentials [4]. In the investigation described below electrical responses induced by passage of a short hyperpolarizing pulse and also evoked by long-term inhibition [ 11], arising during spontaneous IPSPs and stimulation of the anal nerve, were studied in neuron RPal of Helix pomatia:
A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. I, pp. 67-74, January-February, 1981. Original article submitted October 16, 1979.
52
0090-2977/81/'1301-0052507.50
© 1981 Plenum Publishing Corporation
a
b
•
J
i
z~8 m V
m see (a, b)
see (c) Fig. 1. Action of inward pulse on electrical activity of neuron RPa]: a) electrical activity of neuron RPal; b) effect of inward current pulse (2.6 hA, 20 msec) on activity of same neuron; c) the same as b, but with faster recording speed. Time of pulse indicated by dot. 2
E X P E RII~f[E N T A
L IHE T H O D
Experiments were carried out on Helix pomatia in the fall and winter. Neuron R P a l and the anal nerve were identified on the basis of Sakharov's classification [2]. Depending on the subjects of the investigation, one or two microeleetrodes filled with potassium chloride solution (2.5 M) were inserted into n e u r o n / { P A L One microeleetrode was used to derive electrical activity, the other to pass the polarizing pulse. The resistance of the microelectrodes was 20-20 M~. In some experiments electrical activity of another neuron, possessing a common interneuron with RPa], also was recorded.
To stimulate the anal nerve it was drawn into a polyethylene tube filled with Ringer's solution. Stimulation was applied through a stimulator output isolated from ground, and two Ag-AgCl electrodes. One electrode was placed in the polyethylene tube and the other in the experimental chamber. The composition (pH 7.5) ]0. The experiments
of the Ringer's
solution was as follows (in mlV 0 : Na ]00, K 4, Ca 10, IVig 4, Tris-HCl
were carried out at a temperature EXPERIMENTAL
of 18-21°C. RESULTS
1~esp0nses of Neuron R P a I during M e m b r a n e Hyperpolarization by Electric Current. The characteristic picture of activity of bursting neurons R P a i is shown in Fig. in. The m e m b r a n e potential (MP) of this neuron showed a wavelike fluctuation. O n reaching a threshold value, bursts of action potentials (APs) were generated. M e m b r a n e hyperpolarization then took place, stopping A P generation. W h e n M P reached its most negative value, it began to change slowly until the threshold for A P generation was reached, and so on. The duration of the burst (and, con~sequentiy, the n u m b e r of AIDs in the burst) and the interval between bursts of neuron R P a l varied within very wide limits - from a few seconds to tens of seconds - in different preparations and even in the s a m e neuron under different conditions. After insertion of the mieroelectrode into the neuron and attainment of a stable picture of electrical activity (Fig. la) an inward pulse with amplitude of 2.6 n A and duration 10 m s e c was passed through the polarizing microelectrode during generation of a burst of A P (Fig. ]b). The analogous recording at higher winding speed is shown in Fig. It. It ~dll be clear that after a shift of M P evoked by the inward pulse a process of m e m b r a n e hyperpolarization began, m u c h longer in duration than the inward pulse itself. Evidently after the
53
JJjJjj / 0ITISeC " b
2~ msec
Imv 20 s e c
Fig. 2. Effect of duration of inward pulse on amplitude and duration of regenerative hyperpolarization in neuron RPal: a) pulse duration I0, b) 20 msec. Time of pulse indicated by dot. end of the inward pulse processes maintaining membrane hyperpolarization occurred in the neuron membrane, for which the shift of MP acted only as the trigger. This hyperpolarization was therefore described regenerative.
as
An increase in the duration (Fig. 2) or amplitude (not shown in this figure) of the inward pulse up to certain limits led to an increase in the amplitude and duration of regenerative hyperpolarization (in this case the increase in duration of the inward pulse was equivalent to an increase in amplitude of the pulse, for the time constant of the membrane of neuron RPal is much longer than the duration of the inward stimulating pulses, for it measured about 200 msec). If inward pulses were applied at different times after the end of endogenous inter-burst hyperpolarization, the amplitude and duration of regenerative h~perpolarization increased with an increase in the time elapsing after the beginning of the burst of APs (Fig. 3). A similar result was observed if each subsequent inward pulse was applied during generation of a single burst of APs. Inhibition in Neuron RPal Evoked by Anal Nerve Stimulation. The anal nerve was stimulated by pulses with a voltage of 4-5 V and a duration of 0.1-0.2 msec. This duration makes it impossible for more than one AP to be generated in a nerve fiber. As Fig. 4a, b shows, stimulation of the nerve in intervals between bursts of APs led to additional membrane hyperpolarization. After the end of the membrane hyperpolarization wave either a long depolarization wave accompanied by AP discharge appeared (Fig. 4a) or generation of ordinary bursts of APs was resumed (Fig. 4b). Stimulation of the nerve before a burst of APs developed cut short the existing burst and led to the formation of a hyperpolarization wave which was virtually indistinguishable in the kinetics of its development from the endogenous hyperpolarization wave (Fig. 4c). If the nerve was stimulated by a series of short pulses, the IPSP sequence which appeared underwent summation to evoke hyperpolarization of the neuron membrane with the highest possible amplitude. After stimulation ceased the ordinary process of depolarization began. In this case, however, the regenerative character of the hyperpolarization process could not be examined (Fig. 4d). Spontaneous Long-Lasting Inhibition in Neuron RPal. When electrical activity of neuron RPal was recorded long IPSPs could be seen to appear (what Tauc[ 11] called long-lasting inhibition). Such IPSPs in the same neuron, observed in different phases of development of bursting activity, are illustrated in Fig. 5a, b. If the IPSI~ appeared during the development of inter-burst hyperpolarization (Fig. 5a) it caused the development of an additional h}5oerpolarization wave of the neuron membrane; if, however, the IPSP appeared during development of a burst of APs (Fig. 5b) it cut short the burst and evoked a hyperpolarization wave similar in its kinetics to endogenous inter-burst hyperpolarization. The conductivity of the membrane during the development of these IPSPs could evidently be altered for a long time, thus considerably modulating the character of endogenous electrical activity of neuron RPal (Fig. 6).
54
l
f 40rnV 20sec
Fig. 3. Dependence of amplitude and duration of regenerative hyperpolarization in neuron !RPal on time of application of inward pulse. Pulse applied at different times after beginning of spontaneous depolarization wave. Times of pulse indicated by dots, a
b
C
d
.~]
40rnV
20 sec Fig. 4. Effect of anal nerve stimulation on electrical activity of neuron IRPaI: a, b) stimulation of nerve (5 V, 0. I msec) during development of inter-burst hyperpolarization; c) stimulation of nerve (5 V, 0. I msee) during generation of burst of action potential; d) stimulation of anal nerve (5 V, 0. I msec) by series of pulses with frequency of 2.5 Hz during development of inter-burst hyperpolarization (Ist series) and after beginning of generation of burst of APs (2nd series). Time of single nerve stimulation indicated by dots, time of stimulation by series of pulses shown by short horizontal lines. Simultaneous recording of electrical activity of neuron RPal and of an unidentified neuron, whose tion is shown on the diagram, is illustrated in Figs. 5 and 6. Synchronously the development of the [PSP neuron RPal a biphasic exeitatory-inhibitorypostsynaptic potential appeared in the neighboring neuron. is indirect evidence that long-lasting hyperpolarization in neuron RPal develops on account of the IPSP not on account of endogenous mechanisms of the neuron.
posiin This and
Considering" the similarity of the kinetics of changes in membrane potential during endogenous interburst hyperpolarization and also of regenerative hyperpolarization evoked by a pulse of hyperpolarizing currentj and also taking account of the kinetics of long-lasting inhibition, it can be postulated that the IPSP triggers the endogenous mechanism of generation of the hyperpolarization wave.
55
i
U
ii
b
140mV
20sec
Fig. 5. Spontaneous long-lasting inhibition in neuron RPaI: a) inhibition arising during development of hyperpolarization between bursts, b) during generation of burst of action potentials. Top trace from unidentified neuron (empty symbol on diagram of ganglion), lower trace from neuron RPal (filled symbol on diagram). Appearance of IPSPs in neuron RPal and of biphasic PSP in the other neuron shown by arrows. DISCUSSION What are the possible mechanisms of long-lasting hyperpolarization of the membrane of the bursting neuron evoked by short injection of an inward current? To answer this question we naturally need to know the properties of the membrane of bursting neurons and, in particular, properties connected with generation of endogenous bursting activity. However, despite increased interest in these phenomena in recent years, the membrane characteristics of bursting neurons are still inadequately studied. The main difficulty here arises when the voltage clamp method is used to analyze ionic currents in the bursting neuron: Even small shifts of holding potential toward depolarization induce A1 o generation in the axon, and this distorts the picture of ionic currents considerably. Furthermore, ionic currents connected with AP generation and usually recordable in a "silent" neuron evidently mask currents connected with generation of endogenous waves of membrane potential in the bursting neuron. It has been shown [ I, 9] that the phase of depolarization of MP waves in a bursting neuron is connected with the increased sodium steady-state potential-dependent membrane conductance. This conductance creates a constant tendency toward depolarization of the neuron membrane. Regarding the ionic nature of the hyperpolarization phase there are three hypotheses (disregarding the hypothesis of a role of the electrogenic sodium pump in the formation of the hyperpolarization wave in the bursting neuron which has now been almost completely abandoned. Some workers [9] consider that hyperpolarization is induced by ionic currents through special potassium channels which are activated during depolarization of the membrane, whereas others [ 5] consider that hyperpolarization is connected with activation of p o t a s s i u m c o n d u c t a n c e of the m e m b r a n e due to an i n c r e a s e in the i n t r a c e l t u l a r c o n c e n t r a t i o n of f r e e c a l c i u m , w h i c h e n t e r e d the n e u r o n d u r i n g A P g e n e r a t i o n . The w r i t e r p r e v i o u s l y [ 1, 7] p u t f o r w a r d a t h i r d h y p o t h e s i s on the i o n i c n a t u r e of the h y p e r p o l a r i z a t i o n p h a s e in t h e b u r s t i n g n e u r o n . It w a s found t h a t u n d e r v o l t a g e c l a m p c o n d i t i o n s a t e s t i n g s h i f t of h o l d i n g p o t e n t i a l on the m e m b r a n e of a b u r s t i n g l ~ P a l n e u r o n a t t h e l e v e l o f b e t w e e n - 3 5 a n d - 4 5 m V t o w a r d h y p e r p o l a r i z a t i o n l e a d s to t h e d e v e l o p m e n t of a t i m e - and p o t e n t i a l - d e p e n d e n t c u r r e n t Ib. T h i s c u r r e n t w a s o u t w a r d in d i r e c t i o n . Having r e a c h e d i t s m a x i m u m i t w a s i n a c t i v a t e d with a t i m e c o n s t a n t of a b o u t 500 m s e c . The r e v e r s a l p o t e n t i a l of c u r r e n t Ib w a s a b o u t - 55 inV. The a m p l i t u d e of c u r r e n t Ib w i t h i n the r a n g e of p o t e n t i a l s s t u d i e d e x c e e d e d the a m p l i t u d e of the s t e a d y - s t a t e c u r r e n t Is ( l e a k a g e c u r r e n t ) .
56
CJ ~l~z~ rnV 2~sec Fig. 6. Spontaneous PSP in n e u r o n R P a ! . Bottom t r a c e (filled s y m bol on d i a g r a m ) f r o m n e u r o n R P a l , top t r a c e (unfilled s y m b o l ) f r o m u n i d e n t i f i e d neuron. PS1° i n d i c a t e d by a r r o w s .
The presence of this current evidently converts the membrane of the bursting neuron into a system with positive feedback during hyperpolarization. Short-term hyperpolarization of the neuron membrane ought to induce a process of self-maintained (regenerative} membrane hyperpolarizatio~ the kinetics of which will depend on the properties of the current Ib and on the passive electrical properties of the neuron membrane. It has been suggested [7] that the current Ib is connected with potassium conductance, activated on hyperpolarization of the membrane. The more positive reversal potential for current Ib (-55 mV) than for the potassium equilibrium potential in the bursting neuron (-70 mV) [8] can be explained on the assumption that other ions (chlorine and/or sodium) participate in the transport of current Ib. In the present writer's opinion, the third hypothesis corresponds most closely to the truth and well explains the mechanism of formation of regenerative hyperpolarization. The question arises why, despite the fact that the hyperpolarization wave is a regenerative process its amplitude and, correspondingly, its duration depend on the shift of membrane potential. The position is evidently that values of membrane potential at the most positive and most negative points of the wave differ considerably from the sodium and potassium equilibrium potentials, although they are determined by them. For that reason, variation in amplitude of the stimulating shift of inward current voltage under the given conditions leads to the changes observed in the level of regenerative membrane hyperpolarization. As Fig. 3 shows, the amplitude and, correspondingly, the duration of regenerative hyperpolarization evoked by single pulses of inward current, but at different phases of development of a burst of AP~ increases with an increase in the time after generation of endogenous hyperpolarization and after the beginning of the AI D burst. This result can be explained as follows. It has been shown [I], that depolarization of the membrane abolishes the inactivation of potassium conductance connected with the current lb. It can be tentatively suggested that the longer the depolarization lasts the more the inactivation of channels of the current Ib will be abolished. The results of the present experiments are evidence in support of the view that the IPSP in a bursting neuron can trigger the process of generation of a wave of membrane potential which differs onJy a little in its kinetic characteristics from the endogenous hyperpolarization wave. Time- and potential-dependent conductance connected with the current Ib is evidently the ionic basis both of the hyperpolarization phase in generation of the endogenous MP wave and (partly) of long-lasting inhibition in the bursting neuron. It must be pointed out, however, that changes in MP induced by a polarizing current are in general not fully identical with changes in the properties of the membrane evoked by I~SP. %~,~ereas in the first case orgy MP changes, in the second case membrane conductance also changes, and it is this which determines the change in MID. This absence of complete identity probably plays a minor role when the neuron is 'TsilentT~ or when the characteristic time of the PSP is much shorter than the duration of the interspike interval~ but in other cases it cannot be disregarded.
57
LITERATURE I. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11.
CITED
N.I. Kononenko, "Investigation of ionic mechanisms of bursting activity in Helix pomatia neurons, ~' Neirofiziologiya, 10__,193 (1978). D.A. Sakharov, The Genealogy of Neurons [in Russian], Nauka, Moscow (1974), 183 pp. E.N. Sokolov et al., The Pacemaker Potential of the Neuron [in Russian], Metsniereba, Tbilisi (1975), 216 pp. D.O. Carpenter, "Ionic mechanisms and models of endogenous discharge of Aplysia neurones," Neurobiol. Invert., Tihany, Hungary (1973), pp. 35-58. A.L.F. Gorman and M. V. Thomas, "Changes in the intracellular concentration of free calcium ions in a pacemaker neurone measured with the metallochrome indicator dye Arsenazo Ill," J. Physiol. (London), 275, 357 (1978}. E.R. Kandel, Cellular Basis of Behavior, San Francisco (1978), 727 pp. N.I. Kononenko, "Modulation of the endogenous electrical activity of the bursting neuron in the snail Helix pomatia," Neuroseience, 4, 2047 (1979). D.L. Kunze, J. L. Walker, and H. M. Brown, "Potassium and chloride activities in identifiable Aplysia neurons," Fed. Proc., 30, 255 (1971). T.G. Smith, J. L. Barker, and H. Gainer, "Requirements for bursting pacemaker potential activity in molluscan neurones," Nature, 253, 450 (1975). L. Tauc, "Physiology of the nervous system," in: Physiology of Mollusca, New York (1966), pp. 387-454. L. Tauc, "Transmission in invertebrate and vertebrate ganglia," Physiol. Rev., 47, 32 (1967).
EFFECT
OF
ACTIVITY IDENTIFIED N.
THEOPHYLLINE OF
BURSTING Helix
pomatia
ON TYPE
ELECTRICAL IN
AN
NEURON UDC 577.352.5:615.015
I. K o n o n e n k o
The effect of theophylline, an inhibitor of cyclic nueleotide phosphodiesterase, on e l e c t r i c a l activity of bursting neuron R P a l of' Helix pomati.a was investigated. In a concentration of 1 mM theophylline, when added to the external solution, i n c r e a s e s the frequency and n u m b e r of action potentials in the b u r s t and also the duration of the i n t e r - b u r s t interval and the amplitude of m e m b r a n e potential waves. In concentrations of 2.5 and 5.0 mM theophylline leads to r e v e r s i b l e inhibition of bursting activity. During rinsing this activity r i s e s to a higher level and then r e t u r n s to the original value. The action of theophylline develops and d i s a p p e a r s (as a r e s u l t of rinsing) in the c o u r s e of 1-5 rain, depending on concentration of the inhibitor. It is suggested that electrical activity of the molluscan bursting neuron is controlled through the cyclic nucleotide s y s t e m .
INTRODUCTION It was shown p r e v i o u s l y [3, 6, 9, 10, 12, 15] that a compound of peptide nature (modulating f a c t o r ) , which c a u s e s an i n c r e a s e in amplitude of m e m b r a n e potential (MP) waves in bursting neurons o r initiates bursting activity in those neurons which did not p r e v i o u s l y generate such activity, is p r e s e n t in the CNS of mollusks of different species. Application of the fraction obtained f r o m a homogenate of Helix pomatia ganglia, containing modulating factor, d i r e c t l y to the soma of the bursting neuron also gives s i m i l a r effects [2]. On the basis of the r e s u l t s of these and other eleetrophysiotogical experiments [ 1, 8 ] it was concluded that modulating factor is s e c r e t e d by an unidentified interneuron, that it acts on the s o m a of the bursting neuron, and induces activation of potential- and time-dependent ionic channels which participate in the generation of MP waves in the bursting neuron. A. A. Bogomolets Institute of Physiology, A c a d e m y of Sciences of the Ukrainian SSR, Kiev. Translated f r o m Neirofiziologiya, Vol. 13, No. 1, pp. 75-79, J a n u a r y - F e b r u a r y , 1981. Original article submitted J a n u a r y 239 1980. 58
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