Miniature endplate potentials as a tool in neurotoxicology

Toxicology, 49 (1988) 121- 129 Elsevier ScientificPublishers Ireland Ltd.

MINIATURE E N D P L A T E POTENTIALS AS A TOOL IN NEUROTOXICOLOGY

M I C H A E L CSICSAKY, H E R B E R T WIEGAND, S T E F A N UHLIG, H O R S T L O H M A N N RENOS PAPADOPOULOS

and

Medizinisches Institut fftr Umwelthygiene an der Universitdt Dgsseldorf, Auf'm Hennekamp 50, D-4000 Diisseldorf I IF.R.G.)

SUMMARY The toxic effect of thallium added to the bath solution was studied with intra- and extracellular recordings from mammalian nerve-muscle preparations. To elucidate the target region, 3 different functional parameters were studied: (1) Post-synaptic endplate potentials (EPPs) resulting from evoked transmitter release; (2) Post-synaptic miniature endplate potentials (MEPPSs) resulting from spontaneous transmitter release; and (3) Presynaptic ion currents at the nerve terminal. At a concentration of 0.5 mMfl thallium acetate, EPP amplitudes were irreversibly decreased while MEPP amplitudes remained unaffected. MEPP frequencies were reversibly increased, indicating a presynaptic rather than a post-synaptic t a r g e t site of thallium toxicity. The subpopulation of small MEPPs (sub-MEPPs) behaved like the MEPP population, except that upon addition of 4-AP, the sub-MEPP population was augmented at the cost of the MEPP population. In view of the slow time course of the toxic effects (30 min for a 10-fold increase of MEPP frequency, 100--180 min for a 50°/0 reduction of EPP amplitudes), it is concluded that thallium needs to be transported across the cell membrane before it finally interferes with release mechanisms. It is hypothesised that thallium reduces the number of active sites recruited by one action potential (reduced EPP amplitude), while at the Address all correspondence and reprint requests to: Dr. Michael Csicsaky, Med. Institut f.

Umwelthygiene,Abt. Exper. Hygiene,Auf'm Hennekamp50, D-4000 Diisseldorf1, F.R.G. Abbreviations: EPP, endplate potential; MEPP, miniature endplate potential; sub-MEPP, sub-

miniature endplate potential; ACh, Acetylcholine; 4-AP, 4-amino-pyridine; TEA, tetraethylammonium;3,4-DAP, 3,4-diaminopyridine;Ca, Calcium;T1, Thallium. 0300-483X/88/$03.50 © 1988 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland

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same time the probability of transmitter liberation is enhanced (increased M E P P frequency). The rather indirect mode of action of thallium was also found when presynaptic ion currents were recorded using extracellular electrodes. In proportion to the decrease of the E P P amplitudes, a reduction of all inward and outward currents was observed. This effect was also irreversible. It is concluded that in spite of some similarities, thallium behaves quite differently from bivalent heavy metals like cobalt and cadmium, which act as competitive calcium antagonists at the presynaptic nerve terminal. In these toxic substances, the time course of intoxication is much faster, the required concentration is much lower, and the inhibition of the slow calcium current is reversible.

Key words: Thallium; Miniature endplate potentials; Evoked transmitter release; Spontaneous transmitter release; In vitro (mouse).

INTRODUCTION It appears now to be well established that the graded response of neuromuscular endplate potentials (EPPs) recorded post-synaptically is due to the recruitment of an excitation-dependent number of "active sites", at which clusters of vesicles filled with ACh liberate their contents into the synaptic cleft [1]. Next to the evoked release of ACh, however, some sort of "leakage" of ACh into the synaptic cleft exists; this phenomenon is also referred to as spontaneous transmitter release. Spontaneous transmitter release follows a stochastical time pattern, giving rise to socalled miniature endplate potentials (MEPPs). These, in turn, have graded amplitudes and seem to be composed of integer multiples of subunits termed sub-MEPPs. Whereas M E P P s are thought to reflect transmitter release from single synaptic vesicles [1], no morphological correlate of sub-MEPPs has been found as yet [2]. The interrelationship between M E P P s and sub-MEPPs, and, more specifically, the issue of non-vesicular transmitter release are still controversial [3,4]. When applied to toxicology, it is important that probably 3 independent release mechanisms exist at the neuromuscular endplate, namely 1 for evoked (EPPs) and 2 for spontaneous potentials (MEPPs, subMEPPs). While monitoring these mechanisms simultaneously, the probability of detecting differences in the neurotoxic potential of compounds under study is augmented. To demonstrate this, the influence of thallium (T1) upon EPPs, MEPPs, and sub-MEPPs will be reported in this paper. All kinds of endplate potentials mentioned so far are recorded postsynaptically. Therefore, only indirect evidence of toxic interference with presynaptic nerve terminals is obtained. Exploiting the fact that sodium channels are concentrated at the heminode whereas potassium and calcium

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channels are dispersed over the terminal ramifications, more direct evidence regarding possible interactions with neuronal ion currents can be obtained from presynaptic recordings [5]. Along this line, the effects to thallium upon the presynaptic ion currents as recorded by an electrode placed in the perineurium, will also be reported with special attention being given to the slow inward calcium current. This approach was chosen because of the known relevance of unimpeded calcium influx for evoked transmitter release. METHODS All experiments were performed at room temperature in a small perspex chamber. The exchange rate of oxygenated Ringer was kept at 2 - 3 ml/min; for intracellular recordings of E P P s and MEPPs, a high magnesium concentration (12 mM/1) was used to prevent muscle twitches. When presynaptic ion currents were recorded, d-tubocurarine (0.05 mM/1) was used to block post-synaptic receptors. Single neuromuscular endplates could be identified by use of a Zeiss microscope equipped with Nomarski optics and a water immersion front lens. A suction electrode was used for nerve stimulation, and recording electrodes (10--20 M•) were made from glass pipettes. During the E P P and M E P P recordings, resting membrane potential was monitored continuously. Stable recordings with no change of membrane potential could be obtained over time periods up to 6--7 h. Experiments with DC potential changes of more than _+ 10°/o were excluded from analysis. Stimulation parameters for E P P recordings were 0.1 ms pulse width, 1 pulse/s, amplitude at 3 times threshold. In presynaptic recordings, a 1-min interval between pulses was required to see the fully developed slow calcium inward current. After proper amplification and filtering (0.1--10 KHz in extracellular recordings, 0--10 KHz in intracellular recordings), signals were digitized at 20 KHz and subsequently processed off-line using a computer based system (DEC LSI-11). All E P P and M E P P amplitudes were corrected for non-linear summation [6]. As a measure of the average number of ACh quanta released per EPP, the mean quantal content was calculated as the average of the results from 3 different methods (direct, variance, and failures method [1]). Further technical details are described elsewhere [7,8]. E P P and M E P P recordings were carried out in phrenic nervehemidiaphragm preparations from young mice (NMRI strain). Presynaptic ion current recordings were performed in preparations of the m. triangularis sterni which is only 3 - 4 fibers thick; it allows for a better visual control during electrode impalement (for methods, see Ref. 5). RESULTS Figure 1 shows the time course of intoxication of a mouse phrenic nervediaphragm preparation with T1 applied to the bath solution at a concentration of 0.5 raM/1. While M E P P amplitude remained fairly constant, M E P P

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frequency was augmented. By contrast, E P P amplitude was reduced, as was the case for the mean quantum content. After 2.5 h, experimental Ringer was exchanged for normal Ringer to check for the reversibility of the observed effects. As can be seen from the graph at the bottom of Fig. 1, the rise in MEPP frequency is the only effect which is reversible. As mean frequency and mean amplitude are only crude measures of the toxic impact upon EPPs and MEPPs, amplitude histograms corresponding to

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amplitudes remained unchanged. However, MEPP frequency already elevated due to thallium was further augmented, though only for a short period of time. More detailed information is obtained from Fig. 4 which shows the amplitude distributions corresponding to the above experiment. As could be expected, the EPP amplitude augmentation observed in Fig. 3 at point 5 is reflected by a right-shift in the EPP amplitude distribution. The dramatic burst of the MEPP frequency induced by 4-AP is paralleled by a peculiar increase of sub-MEPP activity (arrow in Fig. 4, bottom left). In a preterminal motor nerve stimulated at very low rates (1 min-interval), the development of a slow Ca-current can be demonstrated if potassium channels are blocked by adding 10 mM/1 TEA and 250/~M/1 3,4-DAP to the

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Fig. 4. Amplitude histograms corresponding to the same experiment as in Fig. 3. T1 is added at point 1, and 4-AP at point 3. EPP amplitude augmentation is reflected by right-shift in the EPP amplitude distribution. Note the stability of the MEPP amplitude distribution which is in sharp contrast with the dramatic burst of sub-MEPP activity at points 6 and 7.

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Fig. 5. Even if applied at a high concentration (1 raM) and over a long period of time (top: time zero; middle: 20 rain after application; bottom: 60 rain after application), T1 does not interfere directly with the Ca-current. This is obvious from the unaltered overall appearance of the response curve after 20 rain of incubation. The late effect upon its size is rather a non-specific one, most probably related to compromised energy metabolism. s u p e r f u s i o n fluid. U n d e r t h e s e conditions, t h e influence of 1 mM/1 T1 upon t h e slow C a - c u r r e n t w a s studied. Only a f t e r a l a t e n c y of 1 5 - 2 0 min, a small d e c r e a s e of all signal c o m p o n e n t s b e c a m e noticeable (Fig. 5). E v e n o v e r a p r o l o n g e d e x p o s u r e period of 60 min, no effects o t h e r t h a n a slowly p r o g r e s s i n g loss of viability of t h e n e r v e t e r m i n a l could be o b s e r v e d . As t h e s e r e c o r d i n g s w e r e c a r r i e d out at t h e distal end of t h e p e r i n e u r i u m , t h e c o m p e n s a t i o n c u r r e n t w a s picked up b y t h e e l e c t r o d e i n s t e a d of t h e i n w a r d c u r r e n t caused b y calcium influx a t t h e distal end of t h e n e r v e t e r m i n a l . This p r o d u c e d an u p w a r d deflection in Fig. 5, m a k i n g the C a - c u r r e n t look reversed. DISCUSSION C o m p a r i n g our r e s u l t s o b t a i n e d with TI to t h o s e d e s c r i b e d b y C o o p e r et al. [8], one g e t s t h e i m p r e s s i o n t h a t , a p a r t f r o m d i f f e r e n c e s r e g a r d i n g t h e i r

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r e l a t i v e p o t e n c y , all toxic m e t a l s t e n d to d e c r e a s e e v o k e d t r a n s m i t t e r r e l e a s e and to i n c r e a s e s p o n t a n e o u s t r a n s m i t t e r r e l e a s e . H o w e v e r , one has to b e a r in m i n d t h a t " i n c r e a s e d t r a n s m i t t e r r e l e a s e " in E P P s is m e a s u r e d in a m p l i t u d e units, w h e r e a s it is e x p r e s s e d in f r e q u e n c y units in M E P P s and s u b - M E P P s . T h e u n d e r l y i n g m e c h a n i s m s are quite different: h i g h e r E P P a m p l i t u d e s t r a n s l a t e e i t h e r an i n c r e a s e in t h e a m o u n t of s y n c h r o n i s a t i o n and/or t h e a m o u n t of r e c r u i t e d active sites, w h e r e a s higher M E P P f r e q u e n c i e s r e s u l t f r o m an i n c r e a s e d p r o b a b i l i t y of t r a n s m i t t e r r e l e a s e in t h e t i m e domain. If one c o m p a r e s E P P a m p l i t u d e s with M E P P a m p l i t u d e s -- which would be m o r e a p p r o p r i a t e - one will notice t h a t the f o r m e r d e c r e a s e u n d e r T1, while t h e l a t t e r do not change. E P P and M E P P f r e q u e n c i e s cannot be c o m p a r e d to each other, b e c a u s e t h e f o r m e r is i m p o s e d by the experimentator. A p a r t f r o m t h e s e clarifications, t h e r e is enough r e a s o n to believe t h a t the s i m i l a r i t y b e t w e e n t h e m o n o v a l e n t m e t a l T1 and t h e b i v a l e n t m e t a l s studied b y Cooper [8] is only a superficial one. Within t h e o r g a n i s m , T1 b e h a v e s as a " s u p e r p o t a s s i u m " , i.e. it is a c t i v e l y p u m p e d into t h e cell b o d y w h e r e it displaces p o t a s s i u m f r o m its binding sites b e c a u s e of its higher affinity. M i t o c h o n d r i a s e e m to be 1 m a j o r t a r g e t (for r e v i e w , see Ref. 9). T h e one m e c h a n i s m of i n t e r f e r e n c e with t r a n s m i t t e r r e l e a s e is not y e t fully u n d e r s t o o d , b u t f r o m t h e neurotoxicological kinetics one can conclude t h a t g a t i n g m e c h a n i s m s a r e not t h e p r i m a r y t a r g e t . T a k e n t o g e t h e r with t h e fact t h a t T1 lacks an a n t a g o n i s t i c p r o p e r t y with r e s p e c t to Ca, as w a s disclosed b y p r e s y n a p t i c ion c u r r e n t monitoring, the e v i d e n c e p r e s e n t e d h e r e does not s u p p o r t t h e notion t h a t thallium b e h a v e s like o t h e r h e a v y m e t a l s , a conclusion which o t h e r w i s e could h a v e b e e n d r a w n f r o m t h e o b s e r v a t i o n of E P P s and M E P P s alone. REFERENCES

1 2 3

4 5 6 7 8 9

J.I. Hubbard, R. Llinas and D.M.J. Quastel, Electrophysiological Analysis of Synaptic Transmission, Edward Arnold Publ., London, 1969. J.E. Heuser, T.S. Reese, M.J. Dennis, Y. Jan, L. Yah and L. Evans, Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J. Cell Biol., 81 (1979) 275. M. Csicsaky, R. Papadopoulos and H. Wiegand, Detection of sub-miniature endplate potentials by harmonic analysis. J. Neurosci. Methods, 15 (1985) 113. L. Tauc, Non-vesicular release of transmitter. Physiol. Rev., 62 (1982) 857. R. Penner and F. Dreyer, Two different presynaptic Ca currents in mouse motor nerve terminals. Pflfiegers Arch., 406 (1986) 190. S.S. Kelly, The effect of age on neuromuscular transmission. J. Physiol., 274 (1978) 51. H. Wiegand, H. Lohmann and Satya V. Chandra, The action of thallium acetate on phasic transmitter release in the mouse neuromuscular junction. Arch. Toxicol., 58 (1986) 265. G.P. Cooper, J.B. Suszkiw and R.S. Manalis, Presynaptic Effects of Heavy Metals, in T. Narahashi (Ed.), Cellular and Molecular Neurotoxicology, Raven Press, New York, 1984, p. 1. J.J.G. Prick, Thallium poisoning, in P.J. Vinken and G.W. Bruyn (Eds.), Handbook of Clinical Neurology, Elsevier, Amsterdam, 1979, p. 239.

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