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Calcium dependency of excitatory chemical synaptic transmission in the frog cerebellum in vitro
Brain Research, 114 (1976) 3546 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
CALCIUM D E P E N D E N C Y OF EXCITATORY C H E M I C A L TRANSMISSION IN T H E F R O G C E R E B E L L U M IN VITRO
35
SYNAPTIC
JOHN T. HACKETT Dept. of Physiology, School of Medicine, University of Virginia, Charlottesville, Va. 22901 (U.S.A.)
(Accepted February 9th, 1976)
SUMMARY Chemical synaptic transmission was studied with microelectrode techniques in isolated frog cerebella maintained in vitro. Purkinje cell (PC) EPSPs, elicited by selective monosynaptic electrical stimulation of both the parallel fiber (PF) and climbing fiber (CF) inputs, could be inverted by depolarizing (outward) current injections. Evoked synaptic transmission at both junctions was reduced by lowering the extracellular concentration of calcium ions ([Ca2+]) below 2 mM. Raising [Ca n+] above 2 m M to 8 m M did not further increase synaptic transmission. Mg 2+, Sr 2+, and Ba ~+ did not substitute for Ca n+ in the transmission process.
INTRODUCTION The isolation in vitro of identifiable synapses of the frog cerebellum would in principle permit alteration of the ionic composition of the extracellular media, which is of particular interest to the study of the mechanism of synaptic transmission in the vertebrate brain. In this regard, initial studies indicated that all chemical synaptic transmission in the cerebellum is blocked by Mg 2+, a Ca 2+ antagonist 13. It is well known that Ca 2+ is required for coupling depolarization to the release of acetylcholine from motor nerve terminals (see ref. 19). Ca n+ also appears to be required for chemical synaptic transmission in the vertebrate central nervous system 4,12,2°,29-31,35. However, intracellular records of cerebral cortical synaptic potentials were not altered by extracellular microiontophoretic application of Ca n+ (ref. 22), which probably reflects limitations of the technique. We conducted experiments to investigate the possibility that Ca ~÷ would be found necessary for cerebellar synaptic transmission and that transmission would be graded depending upon the concentration of Ca n+. Lass 25 has reported that the absence of Ca 2+ affects the electrical excitability of Purkinje cells and also blocks parallel fiber-Purkinje cell (PF-PC) transmission, but it was not clear if a decrease in electrical
36 excitability was indirectly responsible for the failure of synaptic transmission. The present report details our findings that Ca 2÷ appears to be required for both PF-PC and climbing fiber-Purkinje cell (CF-PC) transmission in the frog cerebellum. We have also investigated other divalent cations, and they were ineffective as substitutes for Ca 2÷. The Ca 2+ requirement of PF-PC and CF-PC synapses is similar to other junctions where transmission is chemically mediated. The inversion of PF-EPSPs and CF-EPSPs is also characteristic of chemical synaptic transmission. METHODS Frogs (Rana pipiens) were cooled in solution containing 250 mg/ml MS 222 and 2.5~ D M S O for 5-10 min and the cerebellum was removed rapidly. In those preparations where climbing fibers were electrically activated, part of the brain stem was included. The tissue was held on a gelatin base in such a manner that the molecular layer was oriented upwards. The preparations were superfused (Fig. 1A) at 21 °C with oxygenated (95~ O3 and 5 ~ COs) frog Ringer solution (100 m M NaC1, 2 m M KC1, 12 m M NaHCO3, 11 m M glucose) in which the [Ca 2+] was varied from 0.125 to 8.0 mM. Because a low [Ca ~+] depolarizes PCs, 1 m M [Mg 2+] was added which did not affect synaptic transmission but did prevent PC depolarization. Fig. 1B shows a schematic diagram of the neuronal circuit of the frog cerebellar
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Fig. 1. A: arrangement of experimental recording, stimulating and superfusion systems. The superfusion system consists of several reservoirs connected by polyethylene tubing to a common spout which is directed over the cerebellar surface. Solutions are changed by clamping the tubing near the reservoir3. Bipolar stimulating electrodes are made from fine wires. B: schematic diagram of the neuronal circuit of the frog cerebellum with two excitatory synaptic inputs to Purkinje cell (PC): parallel fiber (PF) and climbing fiber (CF); one hypothetical inhibitory interneuron, the stellate cell (SC); and one output, the Purkinje cell axon. Mossy fibers (MF) synapse on the granule cells (GC). All these neuronal elements can be clearly distinguished electrophysiologically except SC.
37
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Fig. 2. Chemical synaptic activation of PC recorded extracellularly in A - D and intracellularly in E-I. A: extracellular field potential evoked by PF stimulation and recorded just below the surface in the molecular layer. B : same stimulus as in A, but recording extracellular spike potential of a single PC about 200/~m deeper than A. C: unitary recording of PC in response to threshold stimulation of a single CF. Following 5 consecutive stimulations, the PC burst failed once in an all-or-nothing manner. D: as in C, but with stimulation intensity at two times threshold. E: intracellular records from PC showing EPSPs induced by increasing intensities of stimulation of the PFs. F: as in E, two EPSPs reach threshold for action potential initiation. G : EPSPs in PC evoked by activation of a single CF. CF activation was straddling threshold, 3 nA inward current hyperpolarized the cell. H: effect of polarizing DC currents on CF-EPSP, same cell as in G. Reversal potential was obtained at ÷ 2 mV by passing 2.0 nA depolarizing (Dep.) current. Number at end of trace indicates nA of injected current. I: effect of polarizing DC currents on PF-EPSPs (first stimulus) and CF-EPSPs (second stimulus) recorded in the same PC. The nullification of the PF-EPSP is seen at a more negative potential (1 nA Dep. current) than the reversal of the CF-EPSP (3 nA Dep. current). Note the slower rise time for the PF-EPSP. In this and subsequent illustrations a negative going 1 mV, 1 msec signal precedes each sweep, and arrows mark the beginning of the stimulus artifact. Time and voltage calibration and polarity are indicated by bars for G, H, and I, because the calibration signal was small.
cortex. T h e t w o e x c i t a t o r y i n p u t s to t h e P u r k i n j e cell (PC), p a r a l l e l fiber ( P F ) a n d c l i m b i n g fiber ( C F ) w e r e e v o k e d m o n o s y n a p t i c a l l y by selective electrical s t i m u l a t i o n , and the responses were recorded extracellularly or intracellularly with conventional m i c r o e l e c t r o d e t e c h n i q u e s . T h e m i c r o e l e c t r o d e s used for i n t r a c e l l u l a r r e c o r d i n g s w e r e filled w i t h 2 M K citrate, a n d t h e y h a d b e v e l e d tips to facilitate P C p e n e t r a t i o n .
38 Current injections through the recording microelectrode were made using a W-P instrument precision electrometer. Counter balancing voltages were adjusted after impalement to remove the time independent component of electrotonic responses TM. RESULTS
Identification of Purkinje cell inputs There are two types of nerve terminals which functionally make excitatory contact to each cerebellar Purkinje cell (PC): the parallel fibers (PFs) and the single climbing fiber (CF)~, 26. These two synaptic inputs can be very easily identified electrophysiologically in the frog cerebellum in vitro as illustrated in Fig. 2. Investigation of both pre- and postsynaptic components of PF-PC synaptic transmission is most clearly accomplished by recording field potentials in the molecular layer evoked by surface stimulation of PFs. With the recording electrode on a beam of selectively excited PFs, a field potential can be recorded (Fig. 2A) which reflects the presynaptic compound action currents generated by PF (first positive-negative wave) followed by postsynaptic activation of the Purkinje cell (slow negative wave). The synaptic field potential corresponds to the average of many EPSPs in PCs activated by the PFs (Fig. 2E and F). As long as the PF volley and the PC passive properties and responsiveness to transmitter can be kept constant the amplitude of the synaptic field potential is a direct, quantifiable measure of the PF-PC synaptic transmission. A qualitative measure of PF-PC transmission can be obtained at the single cell level by recording the PF-evoked PC-spike potential (Fig. 2B). The presence of the all-ornothing spike potential can be a sensitive measure of the PF-PC synaptic transmission if the number of activated PFs evokes a summated PC-EPSP which is just threshold for spike generation (Fig. 2F). Under these conditions a slight decrease in EPSP amplitude below threshold will be signaled as a failure of spike potential occurrence. Of course, for this technique to be a valid indicator of a synaptic site of action for Ca 2+, it must be shown that non-synaptic effects on the PFs or the PCs are not responsible for the decrease in EPSP and subsequent failure of spike initiation. Climbing fiber activation of PCs has not been selectively identified from field potentials in the frog cerebellum. However, CF-PC synaptic transmission may be studied extracellularly (Fig. 2C and D) or intracellularly from an individual PC (Fig. 2G-I). The typical, identifiable response of a PC is a burst of spikes (Fig. 2C and D) or a large fast rising EPSP (Fig. 2G) both of which are all-or-nothing and invariant in shape with increasing intensity of CF stimulation. These characteristics of the CF-PC synapse were previously interpreted by Eccles et al. 9 and were discovered in the frog by Llin~s et al. 2n.
Mode of transmission The most important evidence that demonstrates that CF-PC synaptic transmission is chemically mediated has been given by Eccles et al. 9. They were able to reverse the direction of the CF-EPSP in the cat by depolarizing the PC with outward current injections through the recording electrode. This reversal property is exclusively
39
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Fig. 3. The effect of low [Ca 2+] to block PF-PC synaptic transmission. A: control extracellular field potentials recorded in the molecular layer (one sweep without stimulus). B: the time course of reduction in PF-PC synaptic potential; 5 traces were taken 0.25, 0.5, 1, 2, and 4 rain after the solution was changed to 0.125 [Ca~÷]. C: the effect of 0.125, 0.25, 0.5, 1 and 2 m M [Ca ~+] to decrease synaptic transmission. D : the effect of 0.125 m M [Ca 2+] to block the PF-PC synaptic transmission at the unitary level. Each stimulus evoked no more than one spike potential. Numbers refer to the order that photographs were taken. Calibration pulse of 1 mV and 1 msec begins each trace.
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40 related to the subsynaptic membrane of chemical synapses where neurotransmitterreceptor interaction results in a conductance increase essentially independent of the transmembrane potential (see ref. 7). Reversals of CF-EPSPs were obtained from frog cerebella (Fig. 2H and I). The reversal potential for the CF-EPSP in Fig. 2H was + 2 mV. The PF-EPSP reversals occur at a membrane potential more negative than that for the reversal potential for the CF-EPSP (Fig. 21, Dep. 1 nA). This result would indicate that the ionic mechanism for the two excitatory inputs are different, but that both are chemically mediated. It is also possible, however, that there is contamination of IPSPs in the PF-EPSP. Pharmacological evidence suggests that the neurotransmitter substance released for the PF and CF are probably different14.
Calcium dependence of PF-PC synaptic transmission Since the dependence of presynaptic nerve terminals on Ca 2+ for release of neurotransmitter substances has been so extensively documented (see ref. 32), blockage of synaptic transmission by low [Ca 2+] has become a useful criterion for identifying chemical synapses. A decrease of the [Ca 2+] reduced PF-PC synaptic transmission (Fig. 3). In Fig. 3B, several sweeps are superimposed that were taken after changing to 0.25 m M [Ca2+]. It can be seen that, compared to the control in Fig. 3A, low [Ca 2+] selectively blocks most of the PF-PC synaptic field potential within 4 min. The time course of this reduction in amplitude of PF-PC synaptic field potential is plotted in Fig. 4 (same experiment as Fig. 3A) along with the relatively small changes in the amplitude of PF. Slight changes in the amplitude of the PF volley were seen in many experiments (usually an increase) but apparently were mostly due to movement of the microelectrode. When these changes occurred, they could be compensated by adjusting the recording microelectrode or the intensity of stimulation• The blockage of synaptic transmission by a reduction of the [Ca 2+] was completely reversible. The effect of low [Ca ~+] to block PF-PC synaptic transmission was also measured at the single cell level• To record single PC spikes the microelectrode was
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Fig. 5. A: semilogarithmic plot of the dependence of PF-PC synaptic transmission on [Ca~+]. All data from the same experiment. Ordinate: amplitude of the PF-PC synaptic field potential in mV. The vertical bars give ~: 1 S.E. (6 trials). B: double logarithmic plot of the data in A. The slope of the dotted line is 1.61.
41
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Fig. 6. The effect of 0.125 mM [Ca2+] to reduce CF-PC synaptic transmission. Numbers at the end of each series of records indicates the time in minutes after changing to a Ringer solution containing 0.125 mM [Ca~+].At 4 rain the all-or-nothing properties of CF-activation are shown. lowered 200 # m into the PC-layer. A typical example of the blockage produced by lowering [Ca 2+] from 2 to 0.125 m M is given in Fig. 3D. Single traces are on the left and superimposed traces, taken immediately after each single trace, are on the right. Synaptically evoked PC-spike potentials were blocked within 5 min and recovered upon returning to 2 m M [Ca2+]. Extrasynaptic effects of low [Ca 2+] were minimal as judged by the absence of a change in PC-spike potentials in solutions in which [Ca 2+] varied from 0.125 to 8 m M (1 m M [Mg ~+] always present). For example, Fig. 3D 4 shows the occurrence of one spontaneous spike potential in one out of four consecutive superimposed sweeps which was not affected by 0.125 m M [Ca~+]. In agreement with Lass 25, the frequency of occurrence of PC-spike potentials increased and then decreased to zero after all Ca 2+ was removed. There was also a decrease in amplitude and eventual failure of orthodromic, antidromic and spontaneous PC-spike potentials in low [Ca2+]. To avoid these effects on electrically excitable membranes, we never used less than 0.125 m M [Ca 2+] and always included 1 m M [Mg2+]. The Ca 2+ dependency of PF-PC synaptic transmission was examined at several [Ca2+]. Five superimposed records, each taken at different [Ca 2+] are shown in Fig. 3C. As with the time course experiment, the different [Ca z+] selectively affected only the PF-PC synaptic transmission. PF-PC transmission was increased as [Ca ~+] was raised from 0.125 to 2 m M [Ca2+]. In 3 other preparations further increase of [Ca 2+] to 3.6 m M
42 0.125 Ca
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Fig. 7. Time course of the failure of CF-PC synaptic transmission in 0.125 mM [Ca~+]. The [Ca2+] was lowered from 2 mM between the arrows. further enhanced synaptic transmission, but there was no difference between 3.6 and 7.2 mM. The dose-dependent action of Ca 2+ for 6 trials is plotted on semilogarithmic coordinates (Fig. 5A) and on double logarithmic coordinates (Fig. 5B). The maximum initial slope for the double logarithmic graph was 1.61. Similar data was obtained in two other experiments. Further analysis of the data by Lineweaver-Burke plots gives an apparent dissociation constant for Ca 2+ of 1.1 mM. This value is similar to those obtained at the vertebrate neuromuscular junction1, 5. Calcium dependence o f CF-PC synaptic transmission
One good long-term measure of the CF-PC synaptic transmission is the number of extracellular spikes (CF-spikes) in an evoked response. The number of spikes elicited in a PC is constant as long as the CF and the PC are not damaged, and this reflects the size of the CF-EPSP. Fig. 6 shows that as the [Ca 2+] is lowered, the number of spikes in the CF-response decreases. This reduction of spike number is reversible (Fig. 8A). The time to half-maximum blockage is 5-10 min, the maximum effect occurring within 20 min. The dose-response curve for the dependence of CF-PC synaptic transmission on [Ca 2+] (Fig. 8B) is similar to that for PF-PC transmission. The initial slope of the log (number of CF-spikes) as a function of low [Ca 2+] is 1.49 which is similar to that obtained for the PF-PC synaptic transmission. The effects of low [Ca ~+] on electrically excitable membranes are probably not responsible for disruption of transmission at the CF-PC synapse. Extracellular spike amplitude and time course are not affected by 0.125 m M [Ca 2+] for up to 20 min (Fig. 6). Failure o f Mg 2+, Sr 2+and Ba 2+ to substitute for Ca 2+
Although Mg 2÷ blocks chemical synaptic transmission in the frog cerebellum
43 0.125 Ca
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Fig. 8. A: time course of the effect of 0.125, 0.25 and 0.5 mM [Ca 2+] on CF-PC synaptic transmission. The holding [Ca ~÷] was 2mM. B : semi-logarithmic plot of the dependence of CF-PC synaptic transmission on [Ca z+] (same cell as in A).
in vitrola, TM, it m a y reduce transmission t h r o u g h the effects on the electrical excitability o f the pre- a n d p o s t s t y n a p t i c n e u r o n a l elements at n o n - s y n a p t i c sites. This is d e m o n s t r a t e d in the specimen records in Fig. 9. F o l l o w i n g the superfusion o f 10 m M [Mg2+], there is a r e d u c t i o n in the a m p l i t u d e o f the P F volley and, as w o u l d be expected f r o m the effects o f high [Mg 2+] on other tissues, b l o c k a g e o f synaptic transmission. Ba z+ p r o d u c e d a similar effect (Fig. 9B and C). W i t h i n 6 min synaptic transmission was blocked, b u t this was partly due to the decrease in the P F volley. I n Fig. 9C synaptic transmission was b l o c k e d by reducing the [Ca 2+] to 0.5 m M , b u t a d d i t i o n o f 1.5 m M [Ba 2+] was n o t able to restore transmission a n d even b l o c k e d the P F volley.
Contr
C'
i
Recovery mV Fig. 9. Effect of 10 mM [Mg2+] (A), 2 mM [Ba2+] (B), and 1.5 mM [Ba~+] ÷ 0.5 mM [Ca ~+] (C) to block the PF-PC synaptic transmission• Columns A, B, and C are from 3 different experiments for which the top, middle and bottom rows contain records of control, the maximum effect and recovery, respectively. In the recovery for C a second record was superimposed at higher stimulus intensity.
44 This Ca2+/Ba2+-Ringer had deleterious effects on the synaptic transmission that were not fully reversible. Thus Mg 2+ and Ba 2+ have 'local anesthetic' effects on the PFs and furthermore do not restore synaptic transmission in low [Ca2+]. Although Sr 2+ can substitute for the role of Ca 2+ at motor nerve terminals 6, this cation did not affect cerebellar synaptic transmission in two preparations. DISCUSSION The results of this paper indicate that transmission at PF-PC and CF-PC synapses is chemically mediated, because EPSPs in frog PC have reversal potentials (see ref. 7) and that transmission at these synapses is dependent upon Ca 2+. This Ca 2+ dependency is probably a reflection of the presynaptic role of Ca 2+ in depolarizationsecretion coupling (see ref. 32). Other properties of these synapses that indicate chemically mediated synaptic transmission are: (i) presence of presynaptic vesicles and synaptic cleftsXL3a, (ii) pharmacological properties 14 at the PF-PC synapse and (iii) occurrence of miniature postsynaptic potentials 15. These results are pertinent to the eventual identification of the cerebellar neurotransmitters. Although we have not accurately determined membrane potential, it is apparent that the reversal potential at CF-PC synapses is about zero, and at PF-PC synapses it is at a more negative value (Fig. 2I). The difference in the rate of rise of the EPSPs evoked by these two inputs would suggest, in accordance with the anatomical connections t7, that a CF innervates the proximal dendrites and the surface-activated PFs innervate the distal dendrites. The time courses of the EPSPs may also differ because of the dispersion of presynaptic volley, the rate at which transmitter is released, and the kinetics of receptor activation. It would be expected that PF-EPSPs on the distal dendrites would require more injected current to reach the reversal potential, but this reversal potential is lower than that for CF-EPSPs (Fig. 2I), and less current was needed. The difference in reversal potentials indicates that the ionic mechanism or electrochemical gradients underlying the generation of the two types of EPSPs are different, though there may be some contribution of IPSPs to the PF-EPSP. Furthermore, the synaptic transmitters are probably not the same 14 and can be separated on the basis of their reversal potential. It is clear from Figs. 3 and 6 that Ca 2+ is required for synaptic transmission at both the CF-PC and PF-PC synapses. The narrow range of [Ca 2+] we used was necessary, because [Ca 2+] lower than 0.125 m M probably depolarizes the Purkinje cell (see ref. 23) and higher than 8 m M probably increases the threshold (see ref. 20). Thus, we used a range of [Ca 2+] which selectively affected synaptic transmission. This dependency on Ca 2+ appears to be exclusive of the other alkaline earth cations, Mg 2+, Sr 2÷ and Ba 2+. The site of action for Ca 2+ at cerebellar synapses cannot be concluded from the present results. However, a physiological role for Ca 2+ at subsynaptic membranes has not been demonstrated. In fact, increased [Ca a÷] depresses responses to transmitter of the subsynaptic membrane2,n,23,24,2s, ~4. This depression is in the opposite direction to what we have seen in the frog cerebellum with 0.125-8 m M [Ca~+]. On the other hand, the values for the slope of the double logarithmic plots of synaptic
45 responses vs. [Ca 2+] w o u l d indicate t h a t cooperative action o f several C a 2+ is necessary for e v o k e d release o f n e u r o t r a n s m i t t e r from P F and C F as has been d e m o n s t r a t e d for m o t o r nerve terminals3,G, is. Thus our results, which give slopes o f 1.61 a n d 1.49 c o m p a r e d to 2.5 o b t a i n e d in the guinea-pig olfactory cortex, m a y be due to different requirements o f the p r e s y n a p t i c n e u r o s e c r e t o r y systems for C a 2+. Differences in the m e c h a n i s m b y which Ca 2+ couples d e p o l a r i z a t i o n to the final secretory steps c o u l d a c c o u n t for the differences in slopes between p r e p a r a t i o n s . T h e detection o f C a 2+ entry into the p r e s y n a p t i c nerve terminal during release o f transmitter 21,27 is s t r o n g s u p p o r t o f a p r e s y n a p t i c site for the physiological role o f C a 2+ in synaptic transmission. ACKNOWLEDGEMENTS I wish to t h a n k Dr. N. Sperelakis for critically r e a d i n g an earlier d r a f t o f this m a n u s c r i p t a n d also Mrs. S a n d r a Cahill for her technical assistance. This research was s u p p o r t e d by N S F G r a n t B M S 74-01423 A01 a n d U S P H S N I D A R e s e a r c h Scientist D e v e l o p m e n t A w a r d 1 K02 D A 00009-01.
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45 17 Hillman, D. E., Morphological organization of frog cerebellar cortex: a light and electron microscopic study, J. Neurophysiol., 32 (1969) 818-846. 18 Hubbard, J. I., Jones, S. F. and Landau, E. M., On the mechanism by which calcium and magnesium affect the release of transmitter by nerve impulse, J. Physiol. (Lond.), 196 (1968) 75-86. 19 Katz, B., The Release of Neuronal Transmitter Substance, University Press, Liverpool, 1969, pp. 22-35. 20 Katz, B. and Miledi, R., A study of spontaneous miniature potentials in spinal motoneurones, J. Physiol. ( Lond. ) , 168 (1963) 389-422. 21 Katz, B. and Miledi, R., Tetrodotoxin-resistant electrical activity in presynaptic terminals, J. Physiol. ( Lond. ) , 203 (1969)459--487. 22 Kelly, J. S., Krynevi6, K. and Somjen, G. G., Divalent cations and electrical properties of cortical cells, J. Neurobiol., 2 (1969) 197-208. 23 Koketsu, K., Calcium and the excitable cell membrane, Neurosci. Res., 2 (1969) 1-39. 24 Lambert, D. H. and Parsons, R. L., Influence of polyvalent cations on the activation of muscle end plate receptors, J. gen. PhysioL, 56 (1970) 309-321. 25 Lass, T., Calcium and Purkinje cells in a perfused frog cerebellum, Brain Research, 72 (1974) 337-339. 26 Llin~is, R., Bloedel, J. R. and Hillman, D. E., Functional characterization of the neuronal circuitry of the frog cerebellar cortex, J. NeurophysioL, 32 (1969) 847-870. 27 Llin~s, R. and Nicholson, C., Calcium role in depolarization-secretion coupling: an aequorin study in squid giant synapse. Proc. nat. Acad. Sci. (Wash.), 72 (1975) 187-190. 28 Nastuk, W. and Liu, J. H., Muscle postjunctional membrane changes in chemosensitivity produced by calcium, Science, 156 (1966) 266-267. 29 Richards, C. D. and Sercombe, R., Calcium, magnesium and the electrical activity of guinea-pig olfactory cortex in vitro, J. Physiol. (Lond.), 211 (1970) 571-584. 30 Rovainen, C. M., Physiological and anatomical studies on large neurons of central nervous system of the sea lamprey (Petromyzon marinus). II. Dorsal cells and giant interneurons, J. NeurophysioL 30 (1967) 1024-1042. 31 Rovainen, C. M., Synaptic interactions of reticulospinal neurons on nerve cells in the spinal cord of the sea lamprey, J. comp. Neurology, 154 (1974) 207-224. 32 Rubin, R. P., The role of calcium in the release of neurotransmitter substances and hormones, Pharmac. Rev., 22 (1970) 389-417. 33 Sotello, C., Ultrastructural aspects of the cerebellar cortex of the frog, In R. Llin~is (Ed.), Neurobiology of Cerebellar Evolution and Development, Amer. Med. Ass. Educ. and Res. Found., Chicago, I11., 1969, pp. 327-371. 34 Takeuchi, N., Effects of calcium on the conductance change of the endplate membrane during the action of transmitter, J. Physiol. (Lond.), 167 (1963) 141-155. 35 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro, Exp. Brain Res., 14 (1972) 423-435.
CALCIUM D E P E N D E N C Y OF EXCITATORY C H E M I C A L TRANSMISSION IN T H E F R O G C E R E B E L L U M IN VITRO
35
SYNAPTIC
JOHN T. HACKETT Dept. of Physiology, School of Medicine, University of Virginia, Charlottesville, Va. 22901 (U.S.A.)
(Accepted February 9th, 1976)
SUMMARY Chemical synaptic transmission was studied with microelectrode techniques in isolated frog cerebella maintained in vitro. Purkinje cell (PC) EPSPs, elicited by selective monosynaptic electrical stimulation of both the parallel fiber (PF) and climbing fiber (CF) inputs, could be inverted by depolarizing (outward) current injections. Evoked synaptic transmission at both junctions was reduced by lowering the extracellular concentration of calcium ions ([Ca2+]) below 2 mM. Raising [Ca n+] above 2 m M to 8 m M did not further increase synaptic transmission. Mg 2+, Sr 2+, and Ba ~+ did not substitute for Ca n+ in the transmission process.
INTRODUCTION The isolation in vitro of identifiable synapses of the frog cerebellum would in principle permit alteration of the ionic composition of the extracellular media, which is of particular interest to the study of the mechanism of synaptic transmission in the vertebrate brain. In this regard, initial studies indicated that all chemical synaptic transmission in the cerebellum is blocked by Mg 2+, a Ca 2+ antagonist 13. It is well known that Ca 2+ is required for coupling depolarization to the release of acetylcholine from motor nerve terminals (see ref. 19). Ca n+ also appears to be required for chemical synaptic transmission in the vertebrate central nervous system 4,12,2°,29-31,35. However, intracellular records of cerebral cortical synaptic potentials were not altered by extracellular microiontophoretic application of Ca n+ (ref. 22), which probably reflects limitations of the technique. We conducted experiments to investigate the possibility that Ca ~÷ would be found necessary for cerebellar synaptic transmission and that transmission would be graded depending upon the concentration of Ca n+. Lass 25 has reported that the absence of Ca 2+ affects the electrical excitability of Purkinje cells and also blocks parallel fiber-Purkinje cell (PF-PC) transmission, but it was not clear if a decrease in electrical
36 excitability was indirectly responsible for the failure of synaptic transmission. The present report details our findings that Ca 2÷ appears to be required for both PF-PC and climbing fiber-Purkinje cell (CF-PC) transmission in the frog cerebellum. We have also investigated other divalent cations, and they were ineffective as substitutes for Ca 2÷. The Ca 2+ requirement of PF-PC and CF-PC synapses is similar to other junctions where transmission is chemically mediated. The inversion of PF-EPSPs and CF-EPSPs is also characteristic of chemical synaptic transmission. METHODS Frogs (Rana pipiens) were cooled in solution containing 250 mg/ml MS 222 and 2.5~ D M S O for 5-10 min and the cerebellum was removed rapidly. In those preparations where climbing fibers were electrically activated, part of the brain stem was included. The tissue was held on a gelatin base in such a manner that the molecular layer was oriented upwards. The preparations were superfused (Fig. 1A) at 21 °C with oxygenated (95~ O3 and 5 ~ COs) frog Ringer solution (100 m M NaC1, 2 m M KC1, 12 m M NaHCO3, 11 m M glucose) in which the [Ca 2+] was varied from 0.125 to 8.0 mM. Because a low [Ca ~+] depolarizes PCs, 1 m M [Mg 2+] was added which did not affect synaptic transmission but did prevent PC depolarization. Fig. 1B shows a schematic diagram of the neuronal circuit of the frog cerebellar
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Fig. 1. A: arrangement of experimental recording, stimulating and superfusion systems. The superfusion system consists of several reservoirs connected by polyethylene tubing to a common spout which is directed over the cerebellar surface. Solutions are changed by clamping the tubing near the reservoir3. Bipolar stimulating electrodes are made from fine wires. B: schematic diagram of the neuronal circuit of the frog cerebellum with two excitatory synaptic inputs to Purkinje cell (PC): parallel fiber (PF) and climbing fiber (CF); one hypothetical inhibitory interneuron, the stellate cell (SC); and one output, the Purkinje cell axon. Mossy fibers (MF) synapse on the granule cells (GC). All these neuronal elements can be clearly distinguished electrophysiologically except SC.
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Fig. 2. Chemical synaptic activation of PC recorded extracellularly in A - D and intracellularly in E-I. A: extracellular field potential evoked by PF stimulation and recorded just below the surface in the molecular layer. B : same stimulus as in A, but recording extracellular spike potential of a single PC about 200/~m deeper than A. C: unitary recording of PC in response to threshold stimulation of a single CF. Following 5 consecutive stimulations, the PC burst failed once in an all-or-nothing manner. D: as in C, but with stimulation intensity at two times threshold. E: intracellular records from PC showing EPSPs induced by increasing intensities of stimulation of the PFs. F: as in E, two EPSPs reach threshold for action potential initiation. G : EPSPs in PC evoked by activation of a single CF. CF activation was straddling threshold, 3 nA inward current hyperpolarized the cell. H: effect of polarizing DC currents on CF-EPSP, same cell as in G. Reversal potential was obtained at ÷ 2 mV by passing 2.0 nA depolarizing (Dep.) current. Number at end of trace indicates nA of injected current. I: effect of polarizing DC currents on PF-EPSPs (first stimulus) and CF-EPSPs (second stimulus) recorded in the same PC. The nullification of the PF-EPSP is seen at a more negative potential (1 nA Dep. current) than the reversal of the CF-EPSP (3 nA Dep. current). Note the slower rise time for the PF-EPSP. In this and subsequent illustrations a negative going 1 mV, 1 msec signal precedes each sweep, and arrows mark the beginning of the stimulus artifact. Time and voltage calibration and polarity are indicated by bars for G, H, and I, because the calibration signal was small.
cortex. T h e t w o e x c i t a t o r y i n p u t s to t h e P u r k i n j e cell (PC), p a r a l l e l fiber ( P F ) a n d c l i m b i n g fiber ( C F ) w e r e e v o k e d m o n o s y n a p t i c a l l y by selective electrical s t i m u l a t i o n , and the responses were recorded extracellularly or intracellularly with conventional m i c r o e l e c t r o d e t e c h n i q u e s . T h e m i c r o e l e c t r o d e s used for i n t r a c e l l u l a r r e c o r d i n g s w e r e filled w i t h 2 M K citrate, a n d t h e y h a d b e v e l e d tips to facilitate P C p e n e t r a t i o n .
38 Current injections through the recording microelectrode were made using a W-P instrument precision electrometer. Counter balancing voltages were adjusted after impalement to remove the time independent component of electrotonic responses TM. RESULTS
Identification of Purkinje cell inputs There are two types of nerve terminals which functionally make excitatory contact to each cerebellar Purkinje cell (PC): the parallel fibers (PFs) and the single climbing fiber (CF)~, 26. These two synaptic inputs can be very easily identified electrophysiologically in the frog cerebellum in vitro as illustrated in Fig. 2. Investigation of both pre- and postsynaptic components of PF-PC synaptic transmission is most clearly accomplished by recording field potentials in the molecular layer evoked by surface stimulation of PFs. With the recording electrode on a beam of selectively excited PFs, a field potential can be recorded (Fig. 2A) which reflects the presynaptic compound action currents generated by PF (first positive-negative wave) followed by postsynaptic activation of the Purkinje cell (slow negative wave). The synaptic field potential corresponds to the average of many EPSPs in PCs activated by the PFs (Fig. 2E and F). As long as the PF volley and the PC passive properties and responsiveness to transmitter can be kept constant the amplitude of the synaptic field potential is a direct, quantifiable measure of the PF-PC synaptic transmission. A qualitative measure of PF-PC transmission can be obtained at the single cell level by recording the PF-evoked PC-spike potential (Fig. 2B). The presence of the all-ornothing spike potential can be a sensitive measure of the PF-PC synaptic transmission if the number of activated PFs evokes a summated PC-EPSP which is just threshold for spike generation (Fig. 2F). Under these conditions a slight decrease in EPSP amplitude below threshold will be signaled as a failure of spike potential occurrence. Of course, for this technique to be a valid indicator of a synaptic site of action for Ca 2+, it must be shown that non-synaptic effects on the PFs or the PCs are not responsible for the decrease in EPSP and subsequent failure of spike initiation. Climbing fiber activation of PCs has not been selectively identified from field potentials in the frog cerebellum. However, CF-PC synaptic transmission may be studied extracellularly (Fig. 2C and D) or intracellularly from an individual PC (Fig. 2G-I). The typical, identifiable response of a PC is a burst of spikes (Fig. 2C and D) or a large fast rising EPSP (Fig. 2G) both of which are all-or-nothing and invariant in shape with increasing intensity of CF stimulation. These characteristics of the CF-PC synapse were previously interpreted by Eccles et al. 9 and were discovered in the frog by Llin~s et al. 2n.
Mode of transmission The most important evidence that demonstrates that CF-PC synaptic transmission is chemically mediated has been given by Eccles et al. 9. They were able to reverse the direction of the CF-EPSP in the cat by depolarizing the PC with outward current injections through the recording electrode. This reversal property is exclusively
39
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Fig. 3. The effect of low [Ca 2+] to block PF-PC synaptic transmission. A: control extracellular field potentials recorded in the molecular layer (one sweep without stimulus). B: the time course of reduction in PF-PC synaptic potential; 5 traces were taken 0.25, 0.5, 1, 2, and 4 rain after the solution was changed to 0.125 [Ca~÷]. C: the effect of 0.125, 0.25, 0.5, 1 and 2 m M [Ca ~+] to decrease synaptic transmission. D : the effect of 0.125 m M [Ca 2+] to block the PF-PC synaptic transmission at the unitary level. Each stimulus evoked no more than one spike potential. Numbers refer to the order that photographs were taken. Calibration pulse of 1 mV and 1 msec begins each trace.
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40 related to the subsynaptic membrane of chemical synapses where neurotransmitterreceptor interaction results in a conductance increase essentially independent of the transmembrane potential (see ref. 7). Reversals of CF-EPSPs were obtained from frog cerebella (Fig. 2H and I). The reversal potential for the CF-EPSP in Fig. 2H was + 2 mV. The PF-EPSP reversals occur at a membrane potential more negative than that for the reversal potential for the CF-EPSP (Fig. 21, Dep. 1 nA). This result would indicate that the ionic mechanism for the two excitatory inputs are different, but that both are chemically mediated. It is also possible, however, that there is contamination of IPSPs in the PF-EPSP. Pharmacological evidence suggests that the neurotransmitter substance released for the PF and CF are probably different14.
Calcium dependence of PF-PC synaptic transmission Since the dependence of presynaptic nerve terminals on Ca 2+ for release of neurotransmitter substances has been so extensively documented (see ref. 32), blockage of synaptic transmission by low [Ca 2+] has become a useful criterion for identifying chemical synapses. A decrease of the [Ca 2+] reduced PF-PC synaptic transmission (Fig. 3). In Fig. 3B, several sweeps are superimposed that were taken after changing to 0.25 m M [Ca2+]. It can be seen that, compared to the control in Fig. 3A, low [Ca 2+] selectively blocks most of the PF-PC synaptic field potential within 4 min. The time course of this reduction in amplitude of PF-PC synaptic field potential is plotted in Fig. 4 (same experiment as Fig. 3A) along with the relatively small changes in the amplitude of PF. Slight changes in the amplitude of the PF volley were seen in many experiments (usually an increase) but apparently were mostly due to movement of the microelectrode. When these changes occurred, they could be compensated by adjusting the recording microelectrode or the intensity of stimulation• The blockage of synaptic transmission by a reduction of the [Ca 2+] was completely reversible. The effect of low [Ca ~+] to block PF-PC synaptic transmission was also measured at the single cell level• To record single PC spikes the microelectrode was
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Fig. 5. A: semilogarithmic plot of the dependence of PF-PC synaptic transmission on [Ca~+]. All data from the same experiment. Ordinate: amplitude of the PF-PC synaptic field potential in mV. The vertical bars give ~: 1 S.E. (6 trials). B: double logarithmic plot of the data in A. The slope of the dotted line is 1.61.
41
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Fig. 6. The effect of 0.125 mM [Ca2+] to reduce CF-PC synaptic transmission. Numbers at the end of each series of records indicates the time in minutes after changing to a Ringer solution containing 0.125 mM [Ca~+].At 4 rain the all-or-nothing properties of CF-activation are shown. lowered 200 # m into the PC-layer. A typical example of the blockage produced by lowering [Ca 2+] from 2 to 0.125 m M is given in Fig. 3D. Single traces are on the left and superimposed traces, taken immediately after each single trace, are on the right. Synaptically evoked PC-spike potentials were blocked within 5 min and recovered upon returning to 2 m M [Ca2+]. Extrasynaptic effects of low [Ca 2+] were minimal as judged by the absence of a change in PC-spike potentials in solutions in which [Ca 2+] varied from 0.125 to 8 m M (1 m M [Mg ~+] always present). For example, Fig. 3D 4 shows the occurrence of one spontaneous spike potential in one out of four consecutive superimposed sweeps which was not affected by 0.125 m M [Ca~+]. In agreement with Lass 25, the frequency of occurrence of PC-spike potentials increased and then decreased to zero after all Ca 2+ was removed. There was also a decrease in amplitude and eventual failure of orthodromic, antidromic and spontaneous PC-spike potentials in low [Ca2+]. To avoid these effects on electrically excitable membranes, we never used less than 0.125 m M [Ca 2+] and always included 1 m M [Mg2+]. The Ca 2+ dependency of PF-PC synaptic transmission was examined at several [Ca2+]. Five superimposed records, each taken at different [Ca 2+] are shown in Fig. 3C. As with the time course experiment, the different [Ca z+] selectively affected only the PF-PC synaptic transmission. PF-PC transmission was increased as [Ca ~+] was raised from 0.125 to 2 m M [Ca2+]. In 3 other preparations further increase of [Ca 2+] to 3.6 m M
42 0.125 Ca
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Fig. 7. Time course of the failure of CF-PC synaptic transmission in 0.125 mM [Ca~+]. The [Ca2+] was lowered from 2 mM between the arrows. further enhanced synaptic transmission, but there was no difference between 3.6 and 7.2 mM. The dose-dependent action of Ca 2+ for 6 trials is plotted on semilogarithmic coordinates (Fig. 5A) and on double logarithmic coordinates (Fig. 5B). The maximum initial slope for the double logarithmic graph was 1.61. Similar data was obtained in two other experiments. Further analysis of the data by Lineweaver-Burke plots gives an apparent dissociation constant for Ca 2+ of 1.1 mM. This value is similar to those obtained at the vertebrate neuromuscular junction1, 5. Calcium dependence o f CF-PC synaptic transmission
One good long-term measure of the CF-PC synaptic transmission is the number of extracellular spikes (CF-spikes) in an evoked response. The number of spikes elicited in a PC is constant as long as the CF and the PC are not damaged, and this reflects the size of the CF-EPSP. Fig. 6 shows that as the [Ca 2+] is lowered, the number of spikes in the CF-response decreases. This reduction of spike number is reversible (Fig. 8A). The time to half-maximum blockage is 5-10 min, the maximum effect occurring within 20 min. The dose-response curve for the dependence of CF-PC synaptic transmission on [Ca 2+] (Fig. 8B) is similar to that for PF-PC transmission. The initial slope of the log (number of CF-spikes) as a function of low [Ca 2+] is 1.49 which is similar to that obtained for the PF-PC synaptic transmission. The effects of low [Ca ~+] on electrically excitable membranes are probably not responsible for disruption of transmission at the CF-PC synapse. Extracellular spike amplitude and time course are not affected by 0.125 m M [Ca 2+] for up to 20 min (Fig. 6). Failure o f Mg 2+, Sr 2+and Ba 2+ to substitute for Ca 2+
Although Mg 2÷ blocks chemical synaptic transmission in the frog cerebellum
43 0.125 Ca
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Fig. 8. A: time course of the effect of 0.125, 0.25 and 0.5 mM [Ca 2+] on CF-PC synaptic transmission. The holding [Ca ~÷] was 2mM. B : semi-logarithmic plot of the dependence of CF-PC synaptic transmission on [Ca z+] (same cell as in A).
in vitrola, TM, it m a y reduce transmission t h r o u g h the effects on the electrical excitability o f the pre- a n d p o s t s t y n a p t i c n e u r o n a l elements at n o n - s y n a p t i c sites. This is d e m o n s t r a t e d in the specimen records in Fig. 9. F o l l o w i n g the superfusion o f 10 m M [Mg2+], there is a r e d u c t i o n in the a m p l i t u d e o f the P F volley and, as w o u l d be expected f r o m the effects o f high [Mg 2+] on other tissues, b l o c k a g e o f synaptic transmission. Ba z+ p r o d u c e d a similar effect (Fig. 9B and C). W i t h i n 6 min synaptic transmission was blocked, b u t this was partly due to the decrease in the P F volley. I n Fig. 9C synaptic transmission was b l o c k e d by reducing the [Ca 2+] to 0.5 m M , b u t a d d i t i o n o f 1.5 m M [Ba 2+] was n o t able to restore transmission a n d even b l o c k e d the P F volley.
Contr
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Recovery mV Fig. 9. Effect of 10 mM [Mg2+] (A), 2 mM [Ba2+] (B), and 1.5 mM [Ba~+] ÷ 0.5 mM [Ca ~+] (C) to block the PF-PC synaptic transmission• Columns A, B, and C are from 3 different experiments for which the top, middle and bottom rows contain records of control, the maximum effect and recovery, respectively. In the recovery for C a second record was superimposed at higher stimulus intensity.
44 This Ca2+/Ba2+-Ringer had deleterious effects on the synaptic transmission that were not fully reversible. Thus Mg 2+ and Ba 2+ have 'local anesthetic' effects on the PFs and furthermore do not restore synaptic transmission in low [Ca2+]. Although Sr 2+ can substitute for the role of Ca 2+ at motor nerve terminals 6, this cation did not affect cerebellar synaptic transmission in two preparations. DISCUSSION The results of this paper indicate that transmission at PF-PC and CF-PC synapses is chemically mediated, because EPSPs in frog PC have reversal potentials (see ref. 7) and that transmission at these synapses is dependent upon Ca 2+. This Ca 2+ dependency is probably a reflection of the presynaptic role of Ca 2+ in depolarizationsecretion coupling (see ref. 32). Other properties of these synapses that indicate chemically mediated synaptic transmission are: (i) presence of presynaptic vesicles and synaptic cleftsXL3a, (ii) pharmacological properties 14 at the PF-PC synapse and (iii) occurrence of miniature postsynaptic potentials 15. These results are pertinent to the eventual identification of the cerebellar neurotransmitters. Although we have not accurately determined membrane potential, it is apparent that the reversal potential at CF-PC synapses is about zero, and at PF-PC synapses it is at a more negative value (Fig. 2I). The difference in the rate of rise of the EPSPs evoked by these two inputs would suggest, in accordance with the anatomical connections t7, that a CF innervates the proximal dendrites and the surface-activated PFs innervate the distal dendrites. The time courses of the EPSPs may also differ because of the dispersion of presynaptic volley, the rate at which transmitter is released, and the kinetics of receptor activation. It would be expected that PF-EPSPs on the distal dendrites would require more injected current to reach the reversal potential, but this reversal potential is lower than that for CF-EPSPs (Fig. 2I), and less current was needed. The difference in reversal potentials indicates that the ionic mechanism or electrochemical gradients underlying the generation of the two types of EPSPs are different, though there may be some contribution of IPSPs to the PF-EPSP. Furthermore, the synaptic transmitters are probably not the same 14 and can be separated on the basis of their reversal potential. It is clear from Figs. 3 and 6 that Ca 2+ is required for synaptic transmission at both the CF-PC and PF-PC synapses. The narrow range of [Ca 2+] we used was necessary, because [Ca 2+] lower than 0.125 m M probably depolarizes the Purkinje cell (see ref. 23) and higher than 8 m M probably increases the threshold (see ref. 20). Thus, we used a range of [Ca 2+] which selectively affected synaptic transmission. This dependency on Ca 2+ appears to be exclusive of the other alkaline earth cations, Mg 2+, Sr 2÷ and Ba 2+. The site of action for Ca 2+ at cerebellar synapses cannot be concluded from the present results. However, a physiological role for Ca 2+ at subsynaptic membranes has not been demonstrated. In fact, increased [Ca a÷] depresses responses to transmitter of the subsynaptic membrane2,n,23,24,2s, ~4. This depression is in the opposite direction to what we have seen in the frog cerebellum with 0.125-8 m M [Ca~+]. On the other hand, the values for the slope of the double logarithmic plots of synaptic
45 responses vs. [Ca 2+] w o u l d indicate t h a t cooperative action o f several C a 2+ is necessary for e v o k e d release o f n e u r o t r a n s m i t t e r from P F and C F as has been d e m o n s t r a t e d for m o t o r nerve terminals3,G, is. Thus our results, which give slopes o f 1.61 a n d 1.49 c o m p a r e d to 2.5 o b t a i n e d in the guinea-pig olfactory cortex, m a y be due to different requirements o f the p r e s y n a p t i c n e u r o s e c r e t o r y systems for C a 2+. Differences in the m e c h a n i s m b y which Ca 2+ couples d e p o l a r i z a t i o n to the final secretory steps c o u l d a c c o u n t for the differences in slopes between p r e p a r a t i o n s . T h e detection o f C a 2+ entry into the p r e s y n a p t i c nerve terminal during release o f transmitter 21,27 is s t r o n g s u p p o r t o f a p r e s y n a p t i c site for the physiological role o f C a 2+ in synaptic transmission. ACKNOWLEDGEMENTS I wish to t h a n k Dr. N. Sperelakis for critically r e a d i n g an earlier d r a f t o f this m a n u s c r i p t a n d also Mrs. S a n d r a Cahill for her technical assistance. This research was s u p p o r t e d by N S F G r a n t B M S 74-01423 A01 a n d U S P H S N I D A R e s e a r c h Scientist D e v e l o p m e n t A w a r d 1 K02 D A 00009-01.
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