Neurotoxicologyand Teratology,Vol. 16, No. 1, pp. 11-15, 1994 Copyright©1994ElsevierScienceLtd Printedin the USA.All rightsreserved Pergamon 0892-0362/94 $6.00 + .00 Nerve Conduction Velocity Decreaseand Synaptic Transmission Alterations in Caffeine-Treated Rats ANGEL RAYA, ANA MARIA CUERVO, FERNANDO MACIAN, FRANCISCO JAVIER ROMERO AND JOAQUfN ROMA ~ Experimental Toxicology & Neurotoxicology Unit, Department o f Physiology, School o f Medicine and Dentistry, University of Valencia, A vda. Blasco Ibd~ez 17, E-46010 Valencia, Spain Received 19 D e c e m b e r 1992; Accepted 13 July 1993 RAYA, A, A. M. CUERVO, F. MACIAN, F. J. ROMERO AND J. ROMA. Nerveconduction velocitydecreaseand synaptic transmission alterationsin caffeine-treatedrats. NEUROTOXICOL TERATOL 16(1) 11-15, 1994.--The action of caffeine on peripheral neuromuscular function was studied by means of in vivo determinations of electrophysiological parameters, i.e., amplitude of extracellularly recorded muscle action potentials and nerve conduction velocity in the dorsal skeletal muscle and caudal nerve of the rat tail, respectively. Repeated exposure of the rats was carried out by adding caffeine to the drinking water for 10 days. Here we report the novel finding that motor nerve conduction velocity showed a significant decrease in caffeine-treated animals, whereas no change was observed in the amplitude of indirectly evoked extraceUular muscle action potentials. The physiological recovery of the amplitude of the compound muscle action potential observed in nonintoxicated rats after high-frequency stimulation (10 Hz) was not observed in intoxicated animals and is also discussed. Caffeine Neuromuscular toxicity Peripheral neuromuscular function Muscle action potential Synaptic fatigue Nerve conduction velocity distribution in nervous tissue have been proposed (1,8,10,14), that could explain the facilitating action of caffeine. This report studied the effects of chronic caffeine administration on peripheral neuromuscular function. In the first part of the study those effects were defined in terms of variation of two electrophysiological parameters, motor nerve conduction velocity (MNCV) and extracellular-recorded muscle action potential (MAP) amplitude, when MAPs were obtained by single electrical stimuli. In the second part, the effects of caffeine on MAP amplitude, when varying the stimulation frequency were determined to test the previously reported frequencydependent facilitating action of caffeine (19). CAFFEINE (1,3,7-trimethylxanthine) is a well known and extensively used drug with effects on motor activity. It has been widely used as a pharmacological tool in the study of excitation-contraction coupling in muscle physiology. The early descriptions that this xanthine could potentiate muscular activity (3) and induce contractures (4) gave rise to numerous studies to elucidate the main action mechanism of this drug on muscular function. A large number of reports studying caffeine's effects on skeletal and smooth muscle contraction have been published. However, the effects of caffeine on nerve conduction velocity, neuromuscular transmission, and muscle action potentials are less defined. It has been reported that caffeine facilitates neurotransmitter (NT) release in the synapse during nerve stimulation (5,6). This effect has been attributed to (a) modifications in NT release probability (2); (b) changes in the size of the NT available pool (6); or (c) alterations of NT mobilization on presynapse, which has been reported to be frequency-dependent (19). Moreover, several models of intracellular calcium re- METHOD Animals Forty male Wistar rats (weight and age controlled) were used. At the beginning of the experiments they had a body weight (Mean + SD) of 226.6 + 32.1 g and aged 83 + 8 Requests for reprints should be addressed to J. Rom~i,Experimental Toxicology & Neurotoxicology Unit, Department of Physiology, School of Medicine and Dentistry, University of Valencia, Avda. Blasco Ib~flez 17, E-46010 Valencia, Spain. 11 12 RAYA ET AL. days. Twenty-five animals were divided in 3 groups, caffeineintoxicated (10 rats), pair-fed (10 rats), and control (5 rats) and were used in the first part of the study. The other 15 animals were divided in three groups (5 in each as described above) and were used in the second part of the study. ad lib during 10 days. The mean dose of caffeine that each animal of the treated group received was 597.4 + 28.6 mg (n = 15), determined as the amount contained in the volume consumed. Care was taken to use a daily fresh prepared caffeine solution. Electrophysiological recordings were carried out in the three groups after the I0 days treatment. Treatment of A nimais Eiectrophysiologicai Studies The animals were fed a standard diet (Letica, Hospitalet, Spain). Caffeine (Merck, Darmstadt, Germany) was added to the drinking water in gradually increasing amounts: 2 g/l the first 3 days, 4 g/l the next 3 days, and finally 8 g/l the last 4 days, following a previously described procedure (7). Sucrose 50 g/1 was added to the caffeine solutions to mask the bitter taste. The weight of the animals was monitored daily throughout the experiment. Moreover, water and food consumed by the caffeine-treated group was quantitated once a day while replenishing the reservoires of the cages. Pair-fed rats were given those same amounts of chow and sucrose solution but without caffeine. The control group was fed the standard diet ® The method used allows in vivo determinations of electrophysiological parameters on the neuromuscular tall preparation on nonanesthetized rats (15). This method has shown to be adequate for the study of the peripheral neuromuscular toxicity (13,16). Rats were conveniently restrained by fixing them to a platform with elastic ribbons. The f'med tall was placed in a thermostated paraffin bath, to maintain the tall temperature constant at 270C (physiological temperature of the rat tall), and actual inner temperature of the tall was monitored by a needle thermopar inserted along the tall (Fig. IA). Foam rubber was placed between platform and Proximal stimulation electrode \ Recording electrode Base of the rat's tail / i i i i i l Needle thermopar Ground Distal stimulation electrode ~!~ Distance 2 60 mm Distance 1 ( ~ j E r-ms MAP2 Latency 1 - 1 FIG. 1. (A) Diagram of experimental technique for obtaining extracellular-recorded muscle action potentials (MAPs), showing electrodes placement on the rat tail. Each stimulation electrode consisted of two needles (+ = anode, - = cathode). (B) Typical plots of MAPs obtained by electric stimulation from proximal (MAP 1, lower trace) and distal electrode (MAP 2, middle trace). The intramuscular nerve action potential (INAP) indicated by the arrow in the middle trace is amplified in the upper trace. Latencies were defined as the intervals between time of stimulation and onset of MAPs. The point where the extrapolated baseline intersects with a straight line drawn on the ascending trace through the 10%0 and 90% of the maximal amplitude value, determines the onset of a MAP. Motor nerve conduction velocity was calculated as (Distance l-Distance 2)/(Latency l-Latency 2). CAFFEINE-INDUCED NEUROMUSCULAR TOXICITY animals where appropriate to avoid pain or discomfort to the animals. Evoked muscle action potentials were obtained using a standard technique (12) as follows. Square cathodal stimuli have been used throughout the experiments. The pulse length was 0.1 ms and its height was adjusted to 30070 over the minimum voltage required to evoke maximal response (supramaximal stimuli) for each experiment. These stimuli did not produce any signs of discomfort and were delivered by two pairs of electrodes (2 needles each) inserted in two positions, proximal and distal of the base of the tail and both at 5 mm depth. The extracellular compound muscle action potentials obtained, proximal (MAPI) and distal (MAP2), were recorded by one pair of recording electrodes inserted distally at 2 mm depth in the dorsal tall musculature, displayed on a singlebeam oscilloscope screen and photographed or alternatively digitized. Distance between recording and proximal stimulation electrodes was about 80 mm and around 20 mm from recording to distal stimulation electrodes (see Fig. 1A). In the first series of experiments the electrophysiological parameters studied were the amplitude and the latency of appearance of compound MAPs. MNCV was calculated by the indirect method (latency differences; see Fig. IB). In the second series of experiments, stimuli were delivered at 10 Hz frequency from the distal electrode for 1 min. Afterward, single stimuli were delivered, 1 every min during 15 min to study the recovery of evoked M A P amplitude. 13 Motor nerve conduction velocity (m/s) 30 20 10 0 CONTROL PAIR-FED CAFFEINE FIG. 2. Effect of chronic caffeine administration on motor nerve conduction velocity (MNCV). Extracellular-recorded muscle action potentials were obtained and MNCV was calculated as described in Fig. 1. Results are expressed in m/s and are the Means + SD, F(2, 22) = 9.29, p < 0.01; *p < 0.01, significantly different from pairfed and control values. Statistical Analysis Results are expressed as Mean + SD. Kolmogorov:Smirnov test and analysis of variance (ANOVA) were used to evaluate the results. When A N O V A was significant, the significance between groups was assessed by means of unpaired Student's t test; p values less than 0.01 were considered to be significant. RESULTS AND DISCUSSION Our results show a decrease in body weight after treatment (10 days) in both caffeine-treated (33.2 + 7.3%) and pair-fed rats (18.1 + 8.3%). This decrease is statistically significant, F(2, 37) = 67.82, p < 0.01, when compared to the control group (17 < 0.01), which showed a 12.7 +_ 2.8% increase in body weight. Therefore, the electrophysiological findings we report cannot be attributed to weight loss. The study of MNCV revealed a significant decrease of this parameter in caffeine-treated rats (20.1 + 1.9 m/s) when compared to both control (26.3 + 3.4 m/s) or pair-fed (26.3 + 4.6 m/s) groups (see Fig. 2). There was no significant difference between MNCV values in control and pair-fed groups. The ionic changes in nerve fiber caused by this drug described in many in vitro studies could account for the MNCV decrease found in our study. Calcium appears to be the main ion involved in these changes. This ion may act on different ionic channels regulating membrane excitability (9,17). Previous reports show that caffeine could increase calcium concentration in nerve fibers (10), this fact and the ability of calcium to activate potassium channels (18) could be responsible for the membrane hyperpolarization and consequently for the reduced excitability that might lead to MNCV decrease. No significant difference in the value of M A P amplitude was found between caffeine-treated ( M A P I , 2.7 + 1.9 mV; MAP2, 3.7 + 2.2 mV), pair-fed (MAP1, 3.2 + 1.2 mV; MAP2, 4.1 + 2.1 mV) and control groups ( M A P I , 3.1 + 0.9 mY; MAP2, 4.5 +_ 1.8 mV) when MAPs were evoked by single stimuli, F(2, 22) = 0.31, p > 0.5, for M A P 1 and, F(2, 22) = 0.19, p > 0.5 for MAP2. Although these results seem to contradict previous in vitro findings showing that caffeine facilitates NT release at the neuromuscular junction (2), it must be taken into account that we study compound action potentials, while those in vitro studies record excitatory postsynaptic potentials (EPSPs). The increase in the amplitude of EPSPs may become inappreciable when recording compound muscle action potentials, which are constantly and invariably generated once EPSPs rise above a certain threshold. Our technique is useful to test synaptic fatigue by means of high-frequency stimulation. The experiments carried out at 10 Hz frequency stimulation for 1 min in control animals, show a decrease of MAPs amplitude probably due to a reduction in the number of muscle fibers depolarized. This is basically the result of the progresive exhaustion of the NT available pool in the neuromuscular synapse, i.e., synaptic fatigue. After the high-frequency stimulation is finished, this NT pool is restored, and M A P amplitude returns to the initial values in control rats (see Fig. 3). Interestingly, while in the control and pair-fed groups the subsequent physiological recovery of MAPs amplitude occurs inmediately after high-frequency stimulation is finished, caffeine-treated animals do not exhibit such a recovery, at least within the first 15 min after highfrequency stimulation (see Fig. 3). Statistical analysis reveals significant differences of percentual MAPs amplitude between caffeine-treated (38.0 + 13.9070) and control (69.5 + 2.0°70) groups 1 min after high-frequency stimulation is finished, and after 3 min when comparing caffeine-treated (36.5 _+ 16.2070) and pair-fed (75.6 + 12.6070) animals. These differences remain statistically significant until the end of the experiment. A possible explanation for this impairment of synaptic transmission recovery after high-frequency stimulation could 14 RAYA ET AL. Amplitude of muscle action potential (%) 120 ,I/ 100 8O 60 20 0 0 10 20 30 40 50 60 Time (s) 4 8 12 16 Time (min) 10 Hz S T I M U L A T I O N D U R I N G 60 SECONDS SINGLE STIMULI EVERY MINUTE FIG. 3. Caffeine-induced suppression of the recovery of muscle action potential (MAP) amplitude after highfrequency stimulation. Stimulation at 10 Hz frequency for 60 s was followed by single stimuli every minute for 15 rain. Note that MAP amplitude values in caffeine-treated animals (O) after high-frequency stimulation tend to block whereas both control (O) and pair-fed (D) ones return to initial values. Results are expressed as percentage of initial values and are the Mean 4= SD, F(2, 162) = 271.38, p < 0.01. p < 0.01, significantly different from control values. *p < 0.01, significantly different from control and pair- fed values. also be ascribed to calcium m o v e m e n t s . C a f f e i n e increases stimulus-mediated calcium release f r o m the s m o o t h endoplasmic reticulum o f presynaptic nerve endings (11). M o r e o v e r , previous reports have p r o p o s e d t h a t caffeine m a y reduce the stimulus-evoked calcium influx f r o m the extracellular comp a r t m e n t in a n e u r o m u s c u l a r p r e p a r a t i o n (14). T h e latter aut h o r hypothesized t h a t the large increase in cytosolic calcium caused b y the caffeine-mediated release f r o m the s m o o t h end o p l a s m i c reticulum w o u l d be controlled b y m i t o c h o n d r i a l sequestration (14). If such a decrease in the intracellular cytosolic calcium p o o l would occur, the s u b s e q u e n t effect o n N T release could explain the fact t h a t caffeine-treated a n i m a l s are u n a b l e to recover its M A P a m p l i t u d e a n d could also explain its t r e n d to n e u r o m u s c u l a r blockade after a high-frequency stimulation. W h e t h e r acetylcholine release is affected or n o t in this experimental m o d e l is currently being investigated in our laboratory. ACKNOWLEDGEMENTS We are thankful for the advice and suggestions of G. T. Sdez and the late Dr. A. Jordd. We also thank B. Sofia for his helpful scientific criticism and to C. Avellaneda for his expert technical assistance. This work was partially supported by Grant No. 92/0403 from the FISS and PM92/0146 from the DGICYT (Spain). AR is a research fellow of the Conselleria de Cultura, Educaci6 i Ci~ncia de la Generalitat Valenciana. A.M.C. and F.M. are research fellows of the Ministerio de Educaci6n y Ciencia. REFERENCES 1. Christesen, B. N.; Martin, A. R. The end-plate potential in mammalian muscle. J. Physiol. 210:933-945; 1970. 2. Elmqvist, D.; Feldman, D. S. Calcium dependence of spontaneous acetylcholine release at mammalian motor nerve terminals. J. Physiol. 181:487-497; 1965. 3. Foltz, E.; Ivy, A. C.; Barborka, C. J. The use of double work periods in the study of fatigue an the influence of caffeine on recovery. Am. J. Physiol. 136:79-86; 1942. 4. Goffart, M.; Ritchie, J. M. The effect of adrenaline on the contraction of mammalian skeletal muscle. J. Physiol. 116:357-371; 1952. 5. Golberg, A. L.; Singer, J. J. Evidence for a role of cyclic AMP in neuromuscular transmission. Proc. Natl. Acad. Sci. USA 64: 134-140; 1969. 6. Hoffmann, W. W. Caffeine effects on transmitter depletion and mobilization at motor nerve terminals. Am. J. Physiol. 50:21092128; 1969. 7. Jordd, A.; Portol~s, M.; Guasch, R.; Bernal, D.; Sdez, G. T. Effect of caffeine on urea biosynthesis and some related processes, ketone bodies, ATP and liver amino acids. Biochem. Pharmacol. 38:2727-2732; 1989. 8. Lee, W.; Anwyl, R.; Rowan, M. Caffeine inhibits post-tetanie potentiation but does not alter long-term potentiation in the rat hippocampal slice. Brain Res. 426:250-256; 1987. CAFFEINE-INDUCED NEUROMUSCULAR TOXICITY 9. Meech, R. W. Calcium dependent potassium in nervous tissues. Ann. Rev. Biophys. Bioeng. 7:1-18; 1978. 10. Neering, I. R.; McBurney, R. R. Role for microsomal Ca 2+ storage in mammalian neurones? Nature 309:158-160; 1984. 11. Ohta, Y.; Kuba, K. Inhibitory action of Ca 2+ on spontaneous transmitter release at motor nerve terminals in a high K solution. Pflfigers Arch. 386:29-34; 1980. 12. Petajan, J. H. Changes in rat ventral caudal nerve conduction velocity during cold exposure. Am. J. Physiol. 214:130-132; 1968. 13. Raya, A.; Gallego, J.; Hermenegildo, C.; Puertas, F. J.; Romero, F. J.; Felipo, V.; M~ana, M. D.; Grisolia, S.; Rom~, J. Prevention of the acute neurotoxic effects of phenytoin on rat peripheral nerve by H7, an inhibitor of protein kinase C. Toxicology 75:249-256; 1992. 14. R0ed, S. Caffeine-induced blockade of neuromuscular transmis- 15 15. 16. 17. 18. 19. sion and its reversal by dantrolene sodium. J. Pharmacol. 83:8390; 1982. Rormi, J.; Soria, B. Isonicotinic acid hydrazide: Early effects on peripheral nerve conduction velocity. Experientia 40:378-380; 1984. Rom~t, J.; Cuervo, A. M.; Maci~n, F.; Raya, A.; GaUego, J.; Llopis, J. E.; Romero, F. J. Temperature dependence of the toxic effects of phenytoin on peripheral neuromuscular function of the rat tall. Neurotoxicol. Teratol. 12:627-631; 1990. Schwarz, W.; Passow, H. Calcium activated potassium channels in erythrocytes and excitable cells. Ann. Rev. Physiol. 45:359364; 1983. Smith, S. J.; McDermott, A. B.; Weight, F. F. Detection of intracellular transits in sympathetic neurones using arsenazo Ill. Nature 304:350-352; 1983. Wilson, D. F. Effects of caffeine on neuromuscular transmission in the rat. Am. J. Physiol. 225:862-865; 1973.