Miniature synaptic potentials recorded intracellularly from Purkinje cell dendrites in guinea pig cerebellar slices

Brain Research, 311 (1984) 281- 287 Elsevier

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Miniature Synaptic Potentials Recorded Intracellularly From Purkinje Cell Dendrites in Guinea Pig Cerebellar Slices KOICHI OKAMOTO, HIDEO KIMURA and YUTAKA SAKAI Department of Pharmacology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359 (Japan) (Accepted March 6th, 1984) Key words: cerebellar slice - - Purkinje cell - - cerebellar interneuron - - synaptic transmission - - transmitter - intracellular recording - - synaptic potential - - antagonists

Intracellular recordings from Purkinje cell dendrites in guinea pig cerebeUar slices revealed the existence of miniature spikes with various amplitudes, which were firing apparently without any externally applied stimulations. These spikes were in a hyperpolarizing direction at a resting membrane potential, and their apparent reversal potential was at about -60 mV, indicating their inhibitory nature. Based on the blocking actions of high-Mg2+, tetrodotoxin and amino acid antagonists such as bicuculline etc., these spikes were suggested to be inhibitory synaptic potentials generated by cerebellar interneurons. INTRODUCTION In the course of intracellular electrophysiological studies with guinea pig cerebellar slices 3-6, it was often noticed that Purkinje cell dendrites were generating hyperpolarizing small spikes with various amplitudes (0.1-6 mV) under the condition with no externally applied electrical or chemical stimulations. Since preliminary studies on these spikes indicated a possibility that spontaneous or evoked synaptic inputs from cerebellar interneurons, such as basket or stellate cells, onto Purkinje cell dendrites were reflected by these spikes, the basic pharmacological properties of the spikes were investigated, and the results obtained are presented below. Statistical analyses aiming to demonstrate the quantal nature of the spikes are in progress, and the results will be reported elsewhere. MATERIALS AND METHODS Cerebellar slices were prepared by sagittal sectioning of isolated guinea pig cerebella using Vibratome to give a thickness of 150 ktm4,5, and 2 - 3 such slices

were placed on the bottom of the superfusion chamber 7 and superfused with K r e b s - R i n g e r bicarbonate (control) medium consisted of (in mM) 125 NaCI, 5 KC1, 2 CaC12, 1 MgCI2, 1 NaH2PO4, 24 N a H C O 3 and 11 glucose (pH = 7.4, oxygenated with 95%/5%O2/CO 2, 36.5 °C). A glass microelectrode filled with 3 M KC1 (60-100 Mr2) was impaled into a Purkinje cell dendrite (about 100 ~m apart from the Purkinje cell layer) to record a membrane potential and other electrical activities. Microelectrodes filled with potassium acetate or potassium citrate were not used because of high noise levels probably due to the higher capacitance of them compared with KCl-electrodes. The site of electrode impalement was identified to be the dendrite of a Purkinje cell by the appearance of spontaneous small Na + spikes1,2 (Fig. 2, top 3 records), large Ca2+ spikes2,5 which were not spontaneously firing but induceable by depolarizations, and of climbing fiber responses to white matter stimulationsl,2. The membrane potential of Purkinje cell dendrites was shifted, if necessary, to desired levels by passing DC currents through an impaled recording electrode using a high input-impedance bridge amplifier (MEZ-8202, Nihon Kohden, To-

Correspondence: K. Okamoto, Department of Pharmacology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.

282 kyo). The rates of rise and fall of individual spikes were directly measured from the record of each spike, which was enlarged (about × 50) by a data processor (ATAC-350, Nihon Kohden I. (+)Bicucutline (Sigma), strychnine nitrate (Wako Pure Chem., Tokyo), picrotoxin (Wako), A M B D ( 6a m i n o m e t h y l - 3 - m e t h y l - 4 H . 1.2.4-benzothiadiazine1,1-dioxidet. 10, a kind gift from Dr G. G. Yarbrough, Merck, U.S.A.) or tetrodotoxin (TTX, Sigma) was dissolved in the control medium and applied to the slice by superfusion at a flow rate of about l ml/min. L-Glutamate or L-aspartate (both from Wako) was applied onto a Purkinje cell dendrite by iontophoresis (S-5125B, Nihon Kohden) from a multi-barrel micropipette placed as closely as possible to an impaled recording electrode, the solution filled being 500 mM, p H 8.5 for each of the amino acids. When the medium containing high-Mg2÷ was required, the Mg 2÷ concentration in the control medium was simply raised. RESULTS

General features of miniature spikes Fig. 1 shows the examples of miniature spikes recorded intracellularly from a Purkinje cell dendrite at two different m e m b r a n e potentials. - 3 0 and - 8 5 mV. Another set of the spikes recorded from another dendrite at various m e m b r a n e potentials is shown inFig. 2. At m e m b r a n e potentials such a s - 3 0 inV. some Purkinje cell dendrites showed spontaneous firing of Ca 2+ spikes together with Na ÷ spikes 1,2,4,5. The record shown in Figs. 1 and 2 are those only with Na ÷ spikes. As seen in Fig. 2. the spikes (arrow) at a resting m e m b r a n e potential (-55 m V in this case) were negatively directed and not easily distinguishable from spontaneously firing Na ÷ spikes (arrowheads for some of them) 2. Moreover. if Ca 2÷ spikes were also firing in some cases, miniature spikes could not be observed at all. Therefore. it is difficult to say how many Purkinje celt dendrites out of those studied so far exhibited these spikes. For finding the spikes, it was necessary to shift the m e m b r a n e potential to a hyperpolarized level such as - 8 0 m V or more negative (see Fig. 2, bottom record, for greater spike amplitudes). A m o n g 50 cases tested in this manner. 15 Purkinje cell dendrites were found to be generatmg miniature spikes. Such active dendrites usually ex-

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Fig. 1. Typical shapes of miniature spikes (arrows) recorded intraeellularly from a Purkinje cell dendrite in a guinea pig cerebellar slice. The upper and lower records were obtained at artificially shifted membrane potentials of-30 mV and -85 mV respectively. Potential fluctuations (arrowheads) in the upper record are Na ÷ spikes which are generated at the Purkinje celt soma and passively propagated to the recording dendritic site. In Figs. t and 2, the spikes recorded from the dendrite with nc spontaneous Ca2. spikes are illustrated.

hibited the spikes for at least l h without showing any noticeable changes in the firing pattern. Reversal potential of miniature spikes. Fig. 2 also shows changes in the shape, direction and amplitude of the spikes at varied m e m b r a n e potentials. When the dendritic m e m b r a n e potential was hyperpolarized below the resting level by injecting D C currents through an impaled recording electrode, the spikes were inverted to be positive and their amplitudes were increased by further hyperpolarizations (lower 4 records, Fig. 2). On the other hand, depolarizations above the resting level increased the amplitude of negatively directed spikes (upper 2 records. Fig. 2). Thus. the apparent reversal potential of the spikes appeared to be at the level slightly more negative than the resting potential (the mean resting p o tential from 45 dendrites was - 5 8 _+ I (+ S.E.M.~ mV). An apparent reversal potential may also be estimated from the change of the spike amplitude at varied m e m b r a n e potentials as shown in Fig. 3 (closed circles). The mean spike amplitude at a m e m b r a n e potential o f - 9 0 m V was 5.3 _+ 0.4 mV (n = 50), and it was linearly decreased as the m e m b r a n e potential was depolarized from - 8 0 m V toward - 6 0 m V (Fig. 3. closed circles). An extrapolation to the Xaxis (Fig. 3. broken line) gave a n apparent reversal potential of a b o u t - 4 8 mV, though this estimate was

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Fig. 2. Changes in the direction and amplitude of miniature spikes at different membrane potentials. Note that at the resting level (-55 mV in this dendrite), the spikes (arrows) are obscured by spontaneous Na spikes (some of them are marked with arrowheads) and become more distinguishable at depolarized potential levels, while they are inverted at hyperpolarized levels with increasing amplitudes.

thought to be biased toward a positive level by sprouting Na spikes (Fig. 2). Rates of rise and fall of miniature spikes. In Fig. 3 the mean time constant of the falling phase and the mean rate of rise of positively directed spikes at various membrane potentials are also shown. Variations in the membrane potential did not significantly change the mean time constant of the falling phase of the spikes, which was exponentially linear and was 5.4 _+ 0.4 ms (n = 9) at a membrane potential o f - 9 0 mV (Fig. 3, open circles). The mean rate of rise of the spikes, which was 2.6 _+ 0.2 V/s (n = 9) at -90 mV seemed to become slightly faster as the membrane potential shifted from -100 mV to -50 mV (Fig. 3, open squares), though a reason for this was unclear.

Effects of high -Mge + and T T X on miniature spikes As shown in Fig. 4A, application of 10 mEq/l or 20 mEq/l Mg 2+ (in the presence of 2 mEq/l Ca 2÷) largely diminished both amplitude and frequency of minia-

ture spikes, suggesting that the spikes were mediated by synaptic transmission. The application of 10 or 20 mEq/1 Mg 2+ in the presence of low Ca 2+ such as 0.1 mEq/1 or in the absence of added Ca 2+ was found to cause gradual deterioration of the dendrite under recording. Therefore, complete and reversible blockade of miniature spikes by high-Mg2+ could not be achieved. Superfusion with TTX (3/~M) reversibly abolished spike firings (Fig. 4B), though the recovery took quite a long time such as 10 rain or longer.

Effects of amino acid antagonists on miniature spikes The findings shown in Fig. 2, namely that miniature spikes were negatively directed at a resting membrane potential and an apparent reversal potential was at about -60 mV, suggested that the spikes are inhibitory in nature. Therefore, the effects of amino acid antagonists were then examined. As shown in Fig. 5, application by superfusion of bicuculline (10pM) or strychnine (20/tM), and picro-

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were found to greatly and reversibly facilitate the generation of miniature spikes. This facilitation appeared not to be due to the depolarization of Pur-kinje celt dendrites. For example, in the case shown in Fig. 6, there occurred no noticeable depolarization. but miniature spikes were markedly facilitated. The rates of rise and fall of the spikes were little changed by this facilitation. Enlarged recordings of the spikes by means of the data processor indicated thal the large amplitudes of some of the facilitated spikes (Fig. 6) were due to the pile up of elementary spikes. The refore, a possiblity that L-glutamate or Laspartate created a new type of spike was unlikely

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Fig. 3. The time constant of the falling phase, the amplitude, and the rate of rise of positively directed miniature spikes at different membrane potentials. Open circles, mean time constants (ms, n = 9); closed circles, mean spike amplitudes (mV, n = 50); and open squares, the mean rates of rise (V/s, n -- 9). Vertical bars are + S.E.M. The broken line is an extrapolation to estimate an apparent reversal potential which is about -48 mV (see text for a biasing factor).

toxin (10 pM) or A M B D (200 pM) as well (data not shown for the latter two), was found to reversibly and rapidly abolish miniature spikes. The concentration used for these antagonists were those known to block the inhibitory effect of G A B A or taurine without affecting the membrane potential of Purkinje cell dendrites in guinea pig cerebetlar slices 6,8.

Effects of excitatory amino acids on miniature spikes For the purpose of testing the effect of amino acidinduced depolarization on miniature spikes, L-glutamate and L-aspartate were iontophoreticalty applied to the vicinity of the recording site. As shown in Fig. 6, these excitatory amino acids

Apparent reversal potentials estimated from the results shown in Figs. 2 and 3 were at about -60 and -50 mV. respectively. Since KCl-filled microelectrodes had to be used in this study, the equilibrium potential for C1- ions might be positively shifted by the leakage of CI- from the recording electrode. Therefore. it is likely that the true reversal potential might be at more negative potential levels, if miniature spikes were generated by the increase of a C1conductance. The Cl-equilibrium potential in Purkinje cell dendrites m guinea pig cerebellar slices was reported to be at about -70 mV~

Blockade by TTX The blockade by TFX of miniature spikes (Fig. 4B) may suggest two possibilities, one is that miniature spikes themselves are generated by TTXsensitive ionic currents such as Na + currents, and another is that TTX-sensitive action potentials are trans-synaptically generating miniature spikes. The former appeared to be unlikeIy, because the spikes were negatively directed at a resting membrane potential (Fig. 2), and also because the level of the apparent reversal potential seemed to be at about -60

Fig. 4. Blocking effects of 10 mEq/l and 20 mEq/l Mg2+ (A) and TTX (3 ~M, B) on miniature spikes. The effect of 20 mEq/l Mg 2. was partially reversible probably because of some deterioration of the slice due to the presence of high-Mg2÷ . A and B were obtained from different Purkinje cell dendrites. MP = -100 mV. Fig. 5. Blocking actions of bicucuUine (10/~M, applied by superfusion (A) and strychnine (20 tiM (B)) on miniature spikes. A and B were obtained from different Purkinje cell dendrites. Picrotoxin (t0/zM) and AMBD (200 pM) also showed similar blockades. MP = -100 mV.

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10ms Fig. 6. Facilitating effects of L-glutamate (A) and L-aspartate (B) on miniature spikes. Each of the amino acids was iontoph0retically applied onto the Purkinje cell dendrites as closely as possible to the recording site with the ejecting currents shown. A andB were recorded from the same dendrite. MP = -100 inV. mV (Fig. 2) which is far more negative than usual equilibrium potentials for Na + ions. It is therefore conceivable that miniature spikes may be generated by the release of transmitter(s) from some neurons whose synapses are formed on Purkinje cell dendrites.

Inhibitory effects of antagonists The blocking actions of all of bicuculline, strychnine. picrotoxin and A M B D on miniature spikes (Fig. 5) indicate that postsynaptic receptors for inhibitory amino acids, such as those for G A B A . taurine. glycine etc.6-8 are involved in the generation of miniature spikes. Postsynaptic actions of these inhibitory amino acids on Purkinje cells in guinea pig cerebellar slices were reported to be blocked by any one of these antagonistsS, though A M B D (at 200 ~ M but not at 400 fzM) shows a weak specificity to the taurine action 6. Therefore. it is premature to specify the inhibitory amino acid as that mediating miniature spikes. However. since G A B A is believed to be the transmitter of

basket interneuronsg, while taurine may be that of stellate interneurons 6 either one of them. or both. might be involved in the generation of miniature spikes.

Facilitation by excitatory amino acids As shown in Fig. 2. the depolarization of a Purkinje cell dendrite by injecting D C currents, namely postsynaptic depolarizations, did not increase the frequency of miniature spikes, while L-glutamate or L-aspartate markedly facilitated spike firing without causing depolarization (Fig. 6). Therefore. it is most likely that these excitatory amino acids facilitated miniature spike firing by exciting presyrmptic structures such as the somata and/or axon of basket or steUate interneurons, and consequently increasing the release of inhibitory neurotransmitter(s). In conclusion, it may be suggested, based on the present findings, that miniature spikes are generated by inhibitory interneurons, basket and/or stellate cells. Whether these interneurons are directly generating spontaneous TTX-sensitive action potentials or

287 TTX-sensitive depolarizations, or are stimulated by the activities of parallel fibers cannot be concluded at present. In any case, observed miniature spikes may be thought to represent the stimulus-induced release of inhibitory neurotransmitter(s) from cerebellar interneurons, namely they are equivalent to IPSPs. Since it is technically very difficult to selectively stimulate a single interneuron with a stimulating electrode, or to stimulate basket and stellate cells sepa,rately, to obtain IPSPs even with the use of cerebellar

slice preparations, the miniature spikes r e p o r t e d above are expected to provide useful information about the m o d e of the synaptic release of inhibitory neurotransmitters in m a m m a l i a n central nervous system. It may also be expected that the presynaptic actions of centrally acting drugs can be studied by utilizing miniature spikes as a presynaptic index. F o r these purposes, investigations on the statistical properties of miniature spikes are in progress.

REFERENCES 1 Llin~s, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 171-195. 2 Llimts, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 197-213. 3 Okamoto, K., Kimura, H. and Sakai, Y., Taurine-induced increase of the Cl-conductance of cerebellar Purkinje cell dendrites in vitro, Brain Research, 259 (1983) 319-323. 4 Okamoto, K., Kimura, H. and Sakai, Y., Effects of taurine and GABA on Ca spikes and Na spikes in cerebeUar Purkinje cells in vitro: intrasomatic study, Brain Research, 260 (1983) 249-259. 5 Okamoto, K., Kimura, H. and Sakai, Y., Ionic mechanisms of the actions of taurine on cerebellar Purkinje cell dendrites in vitro: intradendritic study, Brain Research, 260 (1983) 261-269. 6 Okamoto, K., Kimura, H. and Sakai, Y., Evidence for tau-

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rine as an inhibitory neurotransmitter in cerebellar stellate interneurons: selective antagonism by TAG (6-aminomethyl-3-methyl-4H,1,2,4-benzothiadiazine-l,l-dioxide), Brain Research, 265 (1983) 163-168. Okamoto, K., Quastel, D. M. J. and Ouastel, J. H., Actions of amino acids and convulsants on cerebellar spontaneous action potentials in vitro: effect of deprivation of CI-, K ÷ or Na ÷, Brain Research, 113 (1976) 147-158. Okamoto, K. and Sakai, Y., Localization of sensitive sites to taurine, ~'-aminobutyric acid, glycine and fl-alanine in the molecular layer of guinea pig cerebellar slices, Brit. J. Pharmacol., 69 (1980) 407-413. Woodward, D. J., Hoffer, B. J., Siggins, G. R. and Oliver, A. P., Inhibition of Purkinje cells in the frog cerebellum. II. Evidence for GABA as the inhibitory transmitter, Brain Research, 33 (1971) 91-110. Yarbrough, G. G., Singh, D. K. and Taylor, D. A., Neuropharmacological characterization of a taurine antagonist, J. Pharmacol. exp. Ther., 219 (1981) 604-613.